Indian Journal of Experimental Biology Vol. 40, April 2002, pp. 486-506

Equine gonadotropins as models for glycosylation studies

George R Bousfield & Vladimir Y Bulnev

Department of Biolog ical Sciences, Wichita State University, Wichita, KS 67260, USA

Tel: 00 1-3 16-978-6088; Fax: 001-316-978-3772; email: george.bousfield @wichita.edu

Gonadotropins require carbohydrate for their normal physiological function, however, determining the mechan isms in­volved is complicated by the existence of several glycosylation sites decorated with a variety of oligosaccharide structures that contribute to various aspects of gonadotropin structure and function . Partially overlapping function s of these o ligosac­chari des provide addit ional complications. N-linked oligosaccharides contribute to prote in folding, heterodimer stability , circu latory survival, receptor-binding affinity, and cellular activation. O-linked oligosaccharides influence N-linked oli go­saccharide processing and extend circulatory survival. Equ ine LH and eCG provide useful subjects for studying the influ­ence of carbohydrate on gonadotrop in action because they differ only in their carbohydrate moieti es, which produce sub­stantial effects on their activities. Moreover, because these hormones bind FSH receptors, as well as LH receptors, they can be used to study the role of carbohydrate in hormone activation of both receptors .

The gonadotropins are the most abundant members of the classical glycoprotein hormone family . Two of the three pItuItary glycoprotein hormones, follicle­stimulating hormone (FSH) and luteinizing hormone (LH), are gonadotropins. The third, thyroid­stimulating hormone (TSH), is the single non­gonadotropin member. Two groups of mammals, pri­mates and equids, elaborate a placental glycoprotein hormone, chorionic gonadotropin (CG)I -4. These het­erodimeric molecules are structurally rel ated because they possess a common a subunit combined with a hormone-specific ~ subunit. The crystal structures of hCG and hFSH revealed that despite less than 10% sequence homology between them, both a and ~ sub­units possess a cystine-knot motif in common5-8. This makes the glycoprotein hormones members of the cysti ne knot growth factor superfamill and identifies the basis for the structural relationship between the a and ~ subunits first suggested by Dayhoft~). Other cystine knot growth factor superfamily members in­clude the nerve growth factor family , the platelet­deri ved growth factor family, and the transforming growth factor ~ superfamily' °- '2. Gl ycoprotein hor­mone subuni ts derived from LH, FSH, and TSH are N-glycosylated at one or two conserved sites. Chori­onic gonadotropin ~ subunits and eLH~ are O-glyco­sylated in the C-terminal extension unique to these hormones. A review of the vast variety of ro les car­bohydrates play in glycoprotein hormone structure and funct ion was appropriately entitled: "Biological roles of oligosaccharides: all of the theories are cor-

rect"l3, could be applied to the glycoprotein hormones alone. Glycosylation influences gonadotropin subunit folding, subunit association, carbohydrate processing, heterodimer stabil ity, secretion, circulatory surv ival, receptor binding affinity, signalling, internali zation, and sensitivity to proteolytic digestion.

Glycoprotein hormones require carbohydrate to be functional and their activities are modulated by the structures of the attached 0ligosaccharidesl 4. The lit­erature is replete with studies show i. ng retention of substantial receptor-binding activity, but almost com­plete loss of biological act ivity resulting from elimination of all or virtuall y all of the carbohydrate from gonadotropins '5-25 . Classical chem ical and enzy matic deglycosylation experiments indicated a subunit carbohydrate was more important than ~

b · bid ?O 22 ?3?6 S· d· d su unIt car 01Y rate-· .-.- . Ite Irecte mutagene-sis provided valuable informati on regard ing the functiona l roles of individuai glycosy lation sites. For example, Asn52 was identified as the site of the a subunit oligosaccharide that was critical for hCG and hFSH biological activit/7

-29 . However, the impact of

altering the structures of the oli gosaccharides, other than removing terminating sialic ac id res idues, is virtually unexplored. This is a curious defici ency because glycoprotein hormone preparations consist of a co llection of isoforms that possess the same primary structure, but differ in the structures of the oligosac­chari des attached to three or more glycosy lation sites. The carbohydrate structures are believed to change under different physiological conditions, implying a

BOUSFIELD & BUTNEV: EQUINE GONADOTROPINS AS MODEL FOR GLYCOSYLATION 487

functional significance3o.33 . However, the structural changes are so poorly defined that hypotheses put forward to test structure-function relationships are dismi ssed as too speculative and stud ies proposed to clari fy carbohydrate structures are dismissed as too descri pti ve.

N-Glycosylation patterns in the glycoprotein hor­mones

The common a subun it possesses two N­glycosy lati on sites at Asn56 and Asn82 (Fig. I. Note the corresponding sites in human glycoprotein hor­mones will be designated as haAsn52 and haAsn78

because its numbering differs from all other mammal­ian a subunits due to an amino terminal deletion of fou r ami no acid residues that results from altern ati ve splicing34 .) A va ri ab le number of glycosylation sites located at conserved pos itions ex ist on the hormone­spec ific ~ subunits (Fig. 2). LH~ and CG~ subunits are N-glycosylated at Asn l3 or Asn30, or both. FSH~ subu nits are potenti all y N-glycosylated at Asn7 and Asn24 (homologous to LH/CG~ Asn l3 and Asn30, re­spectively), while TSH~ subunits are glycosy lated at Asn23 (homologous to LH/CG~ Asn30) 3. Parti ally gly­cosy lated hCG~ and FSH~ isoforms have been 'd . f" d ' 35·39 I ent l Ie I n recent years .

Alpha Subunit Glycosylation

Non-human, mammalian 56 82 a subunit i ~

52 78

Human a subunit i ~ 56 82

Avian a subunit i ~ 57 83

Amphibian a subunit i ~ 52- 57 77-83

Fish a subunit i ~ Free, mammalian pituitary 56 82 a subunit ~ ~ ~

Fig. I-Alpha subunit glycosy lat ion sites. N·glycosy lation si tes are indicated by the "tuning fork"-like symbols while O·glyco­sylation sites are indicated by the "loll ipop" sy mbols . The position of each Asn and Thr residue is indicated above the sy mbol.

N-linked oligosaccha ride structures found in gly­coprotein hormones

The number of different oligosaccharides attached to a si ngle glycosy lation site varies considerably. For example, on ly 2-4 oligosaccharide structures were obtained from each of the four hCG N-glycosylation sites40 while as many as 18 eLH~ Asn l3 oligosaccha­rides were characterized representing 940/0 of the total carbohydrate isolated from this site41. The -l inked oligosaccharides derived from glycoprotein hormones belong to all three major classes, high man nose, com­plex, and a hybrid class characterized by a hi gh man­nose al-6 branch and a complex al-3 branch42 . Hi gh man nose oligosaccharides vary in the number of Man residues they possess, generally around 5-6 (Fig. 3). Complex oligosaccharides exhibit the widest range of va ri ation in terms of numbers of oligosaccharide branches (2 to 4) as well as in the structures of indi­vidual branches. Many exhibit the classical complex structure of Sia(a l-3 or 6)Gal(~I-4)G lcNAc(~1-2 or 4) . Man, however, an unusual characteristic of pi tui­tary glycoprotein hormones in volves hybrid and com­plex oligosaccharides which are terminated with su l­fate -4-GaINAc(~ I -4)GlcNAc43.45, wh ile eCG oli go­saccharides possess lactosamine repeats (Ga l (~ 1-4)GlcNAc)11 where n= 2-441. The sulfated carbohy­drate motif has been conserved over vertebrate evolu­tion46 and has been postulated to contribute to the pul­satile pattern of LH secretion by providing a mecha­nism for rapid clearance of newly secreted hormone47 .

O-gJycosyJa tion pa tterns in equid a nd primate gly­coprotein hormones

O-glycosylation has been less well characterized even though it is often invoked to explain the func­tional differences between hCG and oLH48.51. In pri­mates, the four O-glycosy lation sites observed in hCG~ are reduced to three in baboon CG~ and one in macaque CG~, although a potential N-glycosy lat ion site is substituted in the macaque at one of the hCG~ O-glycosylation sites (Fig. 4). Greater conservation of O-glycosylation sites is observed among the eq uids, as the bulk of the twelve O-glycosylation sites identi­fied in both eLH~ and eCG~ are present in donkeys and zebras as we1l52 . Two possible exceptions include a predicted Cys residue in place of Serl1 8 in the don­kei3 and a potenti al N-glycosylation site at pos ition 138 in the zebra sequence. The greater conservation of O-glycosy lation in the equid species could be re­lated to the use of carbohydrate to distinguish between

488 INDIAN J EXP BIOL, APRIL 2002

Luteinizing Hormone IJ

ovine, bovine. porcine. rabbit. mouse, rat , wnalc, salmon

y 13

human

equine

y 13

y

Foll icle-Stimulating Hormone 1.1

60: 7 23 o r 24

y 230: 24

Thyroid-Stimulating Hormone I)

y

Chorionic Gonadotropin 11

human y y

13 30

baboon

V V 13 30

macaque

V 30

horse

y 13

donkey y 13

zebra

13

l 118 123 127· 13 133 1371 ': ' 1-1 9

eFSHI.I

I I I I

hFSH IJ __ J

12 1 1271 32 138

? ?? 121 1211 32

y? 1?6 131

116 123 127·1 3113313714 11-19

111.i 123 127·13 1 133137 1·;1 1·1 9

11 6 1231 27· 13 1 133 137 1 ·~1 149

Fig. 2-Beta subunit glycosylation sites. N- and O-glycosylation sites are indicated with the same symbols as in Fig. I. Incomplete ly glycosylated FSH~ isoforms patterns are indicated for the naturally occuring hormone. The glycosy lation sequons exist, but no carbohy­drate is attached. An insect cell-expressed hFSH~ isoform bearing only Asn7 oligosaccharide has been reported (vide infra).

BOUSFIELD & BUTN EV: EQU INE GONADOTROPINS AS MODEL FOR GLYCOSYLATION 489

A

( '\ ) 1-3 ('\ ) 1-4 - -1f

~ A~~ A n A n A n A n

B 4 2 2 2

50% ~j 61 % 79%~ ~ 91% J; ););J; hCGa hCG~

52 78 13 30 121127132138

13 13 13 Key

s~ 1 1 S~4 S04-4-GaINAc

18% 12% S,94 S04-3-Gal

A~U _ · 15%

". u2,6-NANA hLHa hLH~ 52 78 30 " u2,3-NANA

11 11 11 , ~1.4-GaINAc

~~ 1 , ~1.4-Gal

25% 29% P ~ 1,6-GlcNAc

hTSH~

24% ~ ~ 1,2-GlcNAc

hTSHa 52 78 23 P u 1,6-Man

1% 1% 5% 3% 15% 16% 16% 20% 18% Cl.. u1,3-Man

~r'tr St~ {, )1 - 2

() )1-2 () )1 -3 t. )1-3 t. )1-3 t. )1-4

~ ,~J:, Q ~t 1. !y}~ ~t I Man~1.4-GlcNAc

1 J ? l? 9 ~1.4-GlcNAc A nAn An -(> U 1,6-Fuc

hFSHa a!a a~c hFSH~ !~D 6~D ~ u 1,3-Fuc 52 78 7 24

Fig. 3-Seq uences of CG~ subunits. Primary structures determined for cLH/CG~ and hCG~ along with the pred ictcd amino acid sc­quences for donkey, zebra, baboon, and macaque CG~ subunits. Glycosy lation sites are indicated by open boxes enc losing thc N­glycosy lati on sequon or O-glycosylated Tllr or Ser residue. Potential glycosy lati on sites are indicated by shading.

490 INDIAN J EXP BIOL, APRIL 2002

pituitary Ll-I and placental CG in these species. Free a subunit isolated from the anterior pituitary is 0-glycosylated at Thr43

.54 . In the horse, the relative abundance of O-glycosylated pituitary a subunit is 50%55, while less than 10% free hCGa is modified in this manner 56.

Gonadotropin glycoforms Largely as a result of oligosaccharide structural

heterogeneity, it is difficult to isolate enough of a ho­mogeneously glycosylated, glycoprotein hormone isoform preparation for extensive characterization. For example, human pituitary FSH was separated into 20 fractions by a combination of isoelectric focusing and anion exchange chromatograph/7, yet provided only enough material for sialic acid measurements, leaving the remainder of the oligosaccharide unchar­acterized58. Moreover, individual isoforms often ex­hibit small differences in biological activity that make it difficult to define the contribution of each glycosy­lation site to the overall activity of the hormone. In

i ~.,

<v

this regard, equine LH and CG offer a unique oppor­tunity to study substantial differences in biological activity that result from variations in carbohydrate structure. Both hormones display both LH and FSH activities59.62 , possess identical amino acid se­quences63.65; yet differ significantly in their carbohy­drate moieties37.4 1.66.7t . Therefo re, all functional dif­ferences that are observed between eLH and eCG ill vivo and in vitro are due only to glycosylation. In the rat, the apparent affinity of eLH for either the LH or FSH receptor is 5-10 times that of eCG37,52,n, while in the horse, the affinity of eLH for the LH receptor is 50- to 75- fold greater than that of eCG73 . In vivo, the plasma halflife of eCG is 6 days, while that of eLH is 5 hours, almost a 30-fold difference. A 5.7-fold dif­ference in clearance rates for eLH and eCG was noted in the raeo.

Carbohydrate and biosynthesis Carbohydrate plays a facilitating role in glycopro­

tein hormone biosynthesis as evidenced by reduced

tCq\ <I-,CG{\ zCQl tiX3(\ txCQl m:CQi

S !'. , ; :" L F: ? I.. C ? ':"" I ~ ,".. ' :' L .:" ,:\ £ K E :~'. (' r' 1 C i T F T -:' S I C ..... (; '! C l ' S :< \ ' R 'J :.: P .-\ .:" : . r) :,' :; ;.' ',; :' :4 E .: ) ~ C R ;. : ;N A 'I\ L ;" ..... =: :< E ... ·. C ;"' I ': : ';' :-' ':' T S ! ~.: .:.. ~ ';' ~ R. S :.: .... :< ': :-: : ' .:" .:" :...;:- ')-, ~~ ;:- ~ ? : . ? ! :... C ~ P !: ::N . .(\....Ti L ,:0., .. \ :.: " E ,:.... ( . = C 1 T F '": ' T S I t,' .:.... .. ; .!. C :.-' :-; ~< ',f ::: ',. " :-: r; ,:', ..... L :J ~; ~: E :.' :"';-': :~ : ' C Po P : r: .:" T i L.-\ ': ::::< E C . :'- :~ ~.r '.' : T ~.' '-~IT-r : :' ... \ :.; ':' ~_.:.I -:':-: ': ' ?, -: ~ r.~' '"; '::, r' , , ',

s :" !': :.- ~~ : ~ ? : ~ ,_' ?. i~ : NAT: L .:., :\ :.: : : E :\ C ? ' / ::: \ . "!' ": !l ,T ~TI 1 :.,' ,:.. . ..; ':' C !' .:- :-: :': ::. -: L I:. ,:', ': :.:) :,'; ~j ~ : E ? !. ~ : ' :., C' R P ' / l.tt.A.~~ L .:'. :'.. =: ;"', E ,~ C ~ ': ~; ' : i '. F .N.tft :T; ! .- ' ,:', ''; .!. ,,,: :; s :-: ': p ' .- :... G'!' 1 :... ?

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cK:Xi!~ ? !. ? Q ? ;/ C T '!' R E L R ? G S IR LPG C ? ? G \' :J P :-: '/ S F P ' ,'. :\ : .. :~ C H C ~; P C ~. :.. : : ':- 7 :; C _ 0: ze.::q~ ? :: p r..: ? 'J C T '{ F: E :... R F "'''. S I R ~ ? G C : .. p rj V :; r 1,: ',j S F P .... :"'. L S (' H C G I" C R :.. i< T ';:' :J ~ : ,~;i

t~f) :\ L P Q ',," ... ~ (' :! 'I' ~ D \ ' p, F E ~; ! :t ~ P G C ? P. G ~l ~: P 'J ',J So "1' r. 'J ;., L S C ':., C ..... L \ .. : P. ? S ~ T :) ,'" _ ..... ta:.GlS ? ./ ? Q ',J 'i C :! Y ? E "i R F £ S r R :.. f-' C C po p ~ , ~/ 0 P i·: v ~; v P ""l .: ... L ~~ e R e ;... L C F: R S T S J C ~ 1,-"

n~ ::> ~ ? ) 2 ~.: r: t: '{ ~i E L R. F T S ' / P. L ? ....; C F. ? G \ ' 0 P -..' I} ~; :.j ? ',i .:'. :.. 5 ~ ;.: c: G L C R ::: S y ~: !) c: _ I '"

110 E O I I ! _--1-

G .: ;:- !\ ~ t) P L :\ C ,:,. r' - - Q ,:, s S S [S! r'. D ? !"' l-S; 0 p :. ~T. ,i:SrT~:S - ':.: !) :7J P G ,7" ~ :). :< R r .'~~fs . :-! ? :.. ? I :~ ' ~ ' ~~;~ _ ' I ~.' r~ ; P R D E P : , :\ C ':" :' - - - 0 7 S ss e :: D ? ? S 0. !'"' L T S T S T ? T P G :'" S R :< S S :~ :> L i-' : : 1 T S 1.::0 '''''; G ? R D Ii P L /, C .· .... ? - - - 0 .:.. S S S S K :: ? p S ~ ? :. T S T S T ? T P G "", S i'l :, S S ~ ::' : .. :-- : :: ':' S ! · ~ 9

G \"'; ? :: D H ? LT C:> u P ~ F Q ~ s s s : s ~ :~ .. \ p ? ? .5::: L ? S P ~ R :... ? G :~ 'sl u ':-- .. G ( ; P r'. D Ii i.l !. 'J' C D P t ! L Q ; .. :l S S S i': D P P ? S ;-' ? S P S R L :.. E P .:.. G '!' -

G ~l :... !\ :-! E ? L c: CD ',' S T F (:y D S :-; S - i-: D !-' F P ~c:.t] S P S Q L ~ E ? .; D 1) -

Fig. 4-0ligosaccharide heterogeneity in gonadotropins. A. Examples of the variety of oligosaccharide structures found in gonadotropin preparations. All three major classes are represented. Only a few high mannose structures have been reported, but others are probably uncharacterized due to low yield. Hybrid complex/high man nose are commonly attached to aAsn56 (haAsn52

) . Complex oligosaccharides represent the most diverse class. Biantennary oligosaccharides are the most abundant, although FSH and TSH possess more extensively branched oligosaccharides. Many pituitary gonadotropin oligosaccharides are terminated with sulfated GalNAc residues. Fucosylation of the proximal GIcNAc residue is subunit-specific. Less than 25% of a subunit oligosaccharides are fucosylated while more than 90% of ~ subunit oligosaccharides are fucosylated. B. Carbohydrate heterogeneity in human glycoprotein hormones. Site-specific studies from Renwick 's laborator/o.171 .i72 determined the distribution of oligosaccharide structures at each N-glycosylation site in tree human glyco· protein hormones, hLH, hTSH, and hCG. The structure of the most abundant oligosaccharide at each site is shown. The percentage indio cates the relative abundance of that structure at that site and the number above the oligosaccharide indicates the number of different oli· gosaccharide structures characterized by NMR. Positions of the O-linked glycosylation sites in hCG~ are in icated along with an oligo· saccharide structure proposed by Bahl's laboratory 173. Structures of hFSH oli9,0saccharides are shown along with thei r abundance in one studyi2J, however, these were obtained from all four N-glycosylation sites l23 ,1 4.

BOUSFIELD & BUTNEV: EQUINE GONADOTROPINS AS MODEL FOR GLYCOSYLATION 49 1

yields of recombinant glycosylation mutants. Muta­tion of any of the three TSH glycosy lati on sites re­sulted in a reduced yie ld of recombinant hormone7

.J .

Reduced yie lds of recombinant hCG were noted when either a or ~ subunit glycosy lati on sites were elimi­nated with the effects of a subunit glycosy lation greater than ~ subunit glycosylation35.75 . Reduced se­cretion of hFSH glycosy lation mutants, particularly when a subunit glycosy lation sites were eliminated, was reported29. When all four glycosy lation sites were mutated, no secreted hormone and little intracellular hormone were detected29 . The greater impact of a subunit glycosy lation was probably rel ated to the con­tribution of its carbohydrate, particul arly haAsn78

, to subunit folding . Bovine and human a subunits have been denatured under reducing cond itions, yet effi­ciently refolded when the reducing and chaotrophic agents were removed76. Bovine a. subunit expressed in bacterial cells, which lacked the N-glycosylation ma­ch inery , folded to such a low extent «1 %) that only a sensitive, conformation-dependent RIA could detect the presence of folded molecules77

. In vivo, the fold­ing of the hCGa. proceeded so rapidly that after a two­minute pulse labeling experiment, the di sulfide bonds were completely oxidized78

. The carbohydrate at­tached to haAsn78 appears to participate in a. subunit fold ing. Located between hairpin loops Ll and L3 its proximal GIcNAc residue forms hydrogen bonds with several amino acid res idues in both loops. In contrast, the ~ subunit N-linked oli gosaccharides are located in two ·antiparallel ~ strands, Pros-Val IS and lIe27

_ Tyr6.36

and do not appear to interac t with the polypeptide moiety. Indeed, folding of hCG~ lacking both N­linked oli gosaccharides has been obtained2o.79. Rota­tion of the haAsn78 Man (~1-4)GIcNAc(~1 -4) GlcNAc W I-N) tri saccharide is restri cted due to interaction with a loops Ll and L3, while the presence of oligo­saccharide distal to GlcNAc I contributes to hCGa stabilit/o.

Oligosaccharide attached to haAsn52 is located in loop L2 and does not appear to interact with the ex subunit polypeptide81 , although it seems to pl aya role in stabili zing the heterodimer82. It is located in the part of aL2 that is embraced by the seatbelt 100p5.6 and it is differenti all y glycosy lated71 due to the diffe r­ent ~ subunit context47. Since hCG heterodimer fo r­mation appears to occur prior to latching of the seat­belt in viv083 and because agents that promote partia l di sulfide bond reducti on facilitate hCG subunit asso-

.. . . 8485 I A 5? I' I ' d clatlon III vllro . , 10. sn - 0 Igosacc laJ'l e may limit the time when dimer formati on can occur. In hCG biosynthes is, thi s appears to be the interval be­tween formation of the 93- 100 disulfide bond, when dimerization competence is obtained, until format ion of the 26- 11 0 seatbelt latch di sulfide, when steric hin­drance on the part of aAsn52, GlcNAc2Man<)Glc3 hi gh mannose ol igosaccharide blocks dimer fo rmat ion86

.

For in vilro studies, the fact that the oligosaccharide must be threaded through the seatbelt 100ps7 selects for smaller oligosaccharides such as the hybrid oli go­saccharides attached to hCG and oLH40

.88. Indeed, we

have noted that the increased size of equine a subunit . Asn56 oligosaccharides is accompanied by a reduction

in dimer form ation effi ciency that is largely rel ieved by eliminating the oligosaccharide from eLH a and eFSHa with peptide-N-glycanase, but only partially relieved in the case of eCGa 72

. Free hCGa prepara­tions exhibited impaired ability to associate wi th hCG~ because of increased oli gosaccharide branch­ing56 and this appears to reflect the relati ve abundance of triantennary oligosaccharides attached to free hCGa Asn52

,S9 . The corollary that triantennary oligo­saccharides stabilize the heterodimer by preventing denatured subunits from completely di ssociating, thus permitting the native conformation to be recovered has not been demonstrated. Although di ssoc iation­resistant forms of oLH , hFSH , eLH, and eFSH are known to exist, the structures of the oligosaccharides in these preparations have not been charac terized. Whether or not the oli gosaccharide contributes to the stability of the heterodimer depends on the particu lar gonadotropin under investigation. A recent report claimed that the hCGa Asn52 oligosaccharide stabi­li zed the heterodimer and the loss of biological ac tiv­ity attendant upon eli mination of thi s glycosy lat ion site by mutagenes is was due to hetereodimer di ssoc ia­tion into inacti ve subunits rather than the caJ'bohy­drate participating in hCG ac ti ons2. This report is in contrast to the increased thermal stability of chemi­ca ll y deglycosy lated oLH, oFSH, and hCG prepara-

. 90-9? M . I 56 tlons -. oreover, III our lands, an N dg-eLHa:eLH~ preparation ex perienced no loss of recep­tor-binding activity fo llowing 72 hr incubation at 37°C. Despite the greater stability of thi s preparati on th an N52a -hCG mutants, the N56dg-eLHa:eLH~ preparation ex hibited an 89% reducti on in LH bi o­logica l activity , while retaining 100% LH receptor binding activity. Moreover, recombinant eLH/CG ex pressed in human embryonic kidney 293 ce ll s

492 INDIAN J EXP BIOl, APRil 2002

remained stable even when the seatbelt latch disulfide 26-110 was eliminated by mutation93. Biological ac­tivity of stabilized N52dg-rechCG was measured in a cell line overexpressing the LH receptor. If oligosac­charide is involved in ligand-occupied self­association94, then the greater than lO-fold higher re­ceptor concentration may obviate the need for oligo­saccharide in this process. In the case of eFSH, the exAsn56 oligosaccharide definitely contributed to het­erodimer stability as 85 % receptor-binding activity of the latter was lost after 24 hr incubat ion at 37°C, while the intact eFSH preparation required was only 50% dissociated after 72 hr95 . However, heterod imer stability is not a good predictor of biological a tivity. Equine FSH is less stable than pFSH and hFSH96

, yet is significantly more active than both hormones in the rat gran ulosa cell bioassa/7.

O-glycosylation of hCG and eCG, as well as eLH~, is characterized by only a fraction of the potential gly-

I · . b' I' h 'd h ' 375) 98 cosy atlon sites eanng 0 Igosacc an e c alI1s . -. . Automated Edman degradation indicated partial gly­cosylation at 10 of the 12 sites in eLH~ and eCG~ preparations ranging from a low of 10-20%, respec­tively, to 100%52. Mass spectrometry of hCG peptides indicated mutuall y exclusive glycosylation of Ser residues 127 and 13298 . O-glycosylation of hCG~ was reported to occur during the last phase of post­translational modification prior to secretion35.99, how­ever, recent studies have indicated that at least one of these sites influences processing of the N-linked oli­gosaccharides 100.101.

While both ex subunit N-glycosylation sites as well as LH, CG, and TSH ~ subunit N-glycosylation sites are always decorated with carbohydrate in naturally occurring hormone preparations, N-glycosylation of FSH~ is not quantitative. Partial glycosylation of re­combinant bFSH~ was reported in abstract form, with no indication of the particular glycosylation site36. We reported that eFSH~ consisted of two isoforms, one glycosylated at both Asn7 and Asn24, while the other was partially glycosylated37. N-terminal sequence analysis indicated that Asn7 was partially glycosy­lated, while no conclusion could be made regarding the glycosylation status of Asn24 due to N-terminal heterogeneity. Automated Edman degradation re­vealed the presence of phenylthiohydantoin (PTH)­Asn at position 7, which indicated that carbohydrate had never been attached to this position. The rela­tively low yield of PTH-Asn initially suggested that

partial glycosylation also occurred at Asn24, however, analysis of tryptic peptides released from eFSH~ by mass spectrometry revealed only a non-glycosylated peptide containing Asn7 (unpublished data). More­over, automated Edman degradation of the partially glycosylated eFSH~ band separated from deglycosy­lated eFSH~ and eFSHex by SOS-PAGE and blotted on PYOF also indicated a low yield of PTH-Asn at cycles 5 and 7. Oias and colleagues reported that Asn24 was only partially glycosylated in insect cell­expressed recombinant hFSH~38 . We observed a gly­cosylation isoform of hFSH~ derived from human pituitaries that was present in all hFSH charge iso­forms derived from chromatofocusing experiments39. Mass spectrometry suggested that this hFSH~ isoform was not glycosylated and automated Edman degrada­tion corroborated the absence of carbohydrate and indicated that it had not been attached ro the protein because PTH-Asn was observed for both glycosyla­tion sites. Because N-glycosy lation followed by car­bohydrate removal by peptide-N-glycanase produces PTH_Asp I02, the relatively greater abundance of this PTH-amino acid derivative associated with Asn7 and Asn24 than that assoc iated with Asn I raised the possi­bility that some of the non-glycosylated hFSH~ re­sulted from this process. As in the case of eFSH~ above, the yields of PTH-Asn7 and PTH-Asn24 were both low for the non-glycosylated hFSH~ isoform derived from human pituitary extracts l03. Electropho­retic mobility of pFSH bands provided preliminary evidence that similar ~ subunit isoforms exist for this hormone '04 . Because Coomassie blue and silver­stained gels did not reveal the presence of hFSH~ iso­forms, western blotting with FSH~-.;pecific mono­clonal antibodies appears to be a more reliable method to detect FSH~ isoforms lO3 . The initial reports indicating changing patterns of FSH glycosylation were based on changes in the elution volumes during gel filtration 105.106. Deletion of hFSH~ N­glycosylation sites produced altered gel filtration pat­terns for recombinant hFSH I07 , suggesting that elimi­nation of one or more FSH~ oligosaccharides oc­curred under physiological conditions. Taken to­gether, these results suggest that the activity of the enzyme that initiates N-glycosylation, oligosaccharyl transferase, is regulated in gonadotropes. A recent report indicated that protein kinase C inhibited the glycosylation activity of yeast oligosaccharyl trans­ferase lO8

• As the gonadotropin releasing factor, GnRH, regulates protein kinase C activity in gonadotropes 109,

r -,

7 •

BOUSFIELD & BUTNEV: EQUINE GONADOTROPINS AS MODEL FOR GLYCOSYLATION 493

this suggests a possible mechanism for producing par­tially glycosylated or non-glycosylated FSH~ iso­forms. It also raises an interesting question, why don ' t we observe partial glycosylation of LH~ and the common a subunit?

Carbohydrate regulation of metabolic clearance rates

Studies with TSH II O and FSH III have indicated that the ~ subunit N-linked oligosaccharides modulated metabolic clearance rates. Oligosaccharides termi­nated with S04-4-GaINAc- have been the focus of attention fo r regul ating LH clearance by glycosylation because these are the most abundant in the more read­il y studied oLH, bLH, and pLH preparationsll2. A liver lectin that binds oligosaccharides terminated with sulfated GalNAc was discovered ll3

. This appears to be a Cys-rich carbohydrate recognition domain of the macrophage mannose receptorI14.11 7. The exis­tence of thi s lectin provides a mechanism to explain the relatively short halflife of pituitary LH, in which sulfated oli gosaccharides are the most abundant type, as compared with placental gonadotropins, which are exclusively sialylated47 . Studies on hLH isoforms suggested that the location of the sulfate moiety was critical for determining metabolic c1eaJ·ance I1 8. How­ever, studies on LH have been conducted on a hor­mone-wide scale rather than at the level of individual glycosylation sites l12. TSH hybrids incorporating pi­tuitary-derived subunits possessing sulfated oligosac­chari des and the complementary recombinant subunits possessing only sialylated oligosaccharides indicated that ~ subunit glycosylation rather than a subunit gly­cosylation dictated metabolic clearance rates llO. Stud­ies with hCG have focused on the role of the C­terminal extension, ignoring the potential contribution of the N-linked oligosaccharides. When the hCG~ C­terminus was coupled to recombinant bLH~ , a long­acting LH derivative was produced that had a 2- to 3-fold longer halflife than recombinant bLH lacking the C-terminal extension 11 9. A 3- to 5-fold increase in FSH potency in vivo was noted when the hCG~ C­terminal extension was coupled to recombinant hFSH~1 2o . However, the role of the ~Asnl3 oligosac­charide in modulating metabolic clearance rates should not be ignored. Almost 30% of the 125I_eLH injected into rats was found in the liver 30 min later, while only 10% of 125I_eCG was present in this tissue and 75% remained in the circulation7o. Binding of 1251_eLH to isolated hepatic endothelial cells was

demonstrated and , while cold eLH could compete with 125I_eLH for these binding sites, cold eCG could not. For exclusively sialylated glycoprotein hormones, such as hCG and hFSH, the liver does not appear to be the site for metaboli c clearance. Despite the text­book model for sialylated glycoprotein clearance in­volving desialylation by peripheral neuraminidase to expose galactosy l residues that are cleared by the liver asialoglycoprotein receptor l21, this mechani sm has been shown to be invalid for hCG 122

. The same may be true for hFSH, which possesses only 7% sulfated oligosaccharides, unlike the 30-40% sulfated oligo­saccharide content of bovine, porcine, ovine, and equine FSH preparations89.123

. Studies with recombi­nant mutant hFSH indicated that elimination of either ~ subunit N-glycosylation site produced a 2- to 3-fold increase in metabolic clearance rate (MCR), while elimination of both sites resulted in a lO-fold in ­crease ili . No statistically significant effect of a sub­unit N-linked oligosaccharides was observed, al­though elimination of aAsn78 glycosylation appeared to increase MCR. The crystal structure of hFSH has revealed a molecule similar in shape to hCGs. The ~ subunit oligosaccharides protruded outward, effec­tively doubling the narrow diameter (the molecular dimensions of deglycosylated hCG have been re­ported to be 75 x 35 x 30 A 6, while an N-Iinked oligo­saccharide can extend as much as 30A 124). This change in shape could affect ultrafiltration rates in the kidney, thereby altering clearance rates for the hor­mone. Baenziger47 proposed that sulfated oligosaccha­rides were responsible for the rapid clearance of LH, which resulted in an episodic pattern of LH secretion . The longer haltlife and lower levels of circulating FSH have made it more difficult to demonstrate epi­sodic release of this hormonel25. While episodic re­lease of GnRH is required for proper pituitary func­tion 126, demonstration that the accompanying episodic gonadotropin release is necessary for proper ovarian function has not yet been obtained. FSH is responsible for several different activities in the ovary, including pre-antral stage granulosa cell proliferation, antrum formation, induction of aromatase, induction of LH receptor, and resumption of meiosis by the oocyte 127.

The pre-ovulatory LH rise provides the ovulatory sig­nal, although the two-cell model for estradiol synthe­sis assumes LH stimulation of theca cells to provide the necessary androgen precursors to the granulosa cells l28. The hFSH~ double glycosylation mutants were completely inactive in the classical weight gain

494 INDI AN J EX P S IOl, APR IL 2002

assay for FSH, suggesting that granulosa cell prolif­erati on and antrum formation were not supported by I · . fi ll . . . t l iS ISO orm . However, the acti vation of cumulus-

oocyte complexes was more effec ti vely stimulated by the least acidic hFSH isoforms l29 , whi ch possess onl y the non-glycosy lated ~ subunit isoform. These iso­fo rms are relat ively rare during the fo llicul ar and I utea l phases of the human menstrual cycle, but ap­pear during the preovulatory ri se in FSH 33. If a peri­ovulatory function, such as acti vation of meiosis re­quires an epi sodic pattern of FSH stimulati on, the rapid clearance of the non-glycosylated hFSH~­

containing hFSH isofo rm may provide such a signal. Alternati vely, thi s structure is located at the greatest di stance from the vasculari zed theca layer and a more rapid delivery of hFSH may be required for acti vities associated with ovulation that necess itates the ri se in less acidic hFSH isofo rms that occurs prior to ovula­tion. Chemi call y deg lycosy lated oFSH is accumulated to a greater extent by rat ovaries than intact oFSH during the first 30 min fo llowing injecti onI3o

. More rapid clearance in the kidney may be an unavoidable consequence of the need fo r more effecti ve FSH de­livery to target cells.

Carbohydrate inhibits receptor-binding Once gonadotropins reach their target cell s, both

receptor-binding and cellular activati on are influenced by glycosy lation. Chem ical and enzy matic deglycosy­!ation revealed that cellular ac tivation was affected to a greater extent than receptor-bindingIS-25. The latter was ei ther unchanged or modestly increased, while the fo rmer was signi fica ntly reduced. The absence of carbohydrate increased the assoc iation rate fo r deg ly­cosy lated oLH, but had no effect on its di ssociati on raten . In contrast, a recombinant mutant hCG lacking all fo ur N-linked oligosaccharides exhibited essen­tia ll y irreversible binding to both human and rat LH receptorsl31. Deglycosy lati on of individual subunits demonstrated th at ex subunit carbohydrate was more important fo r signal transd uction than the ~ subunit carbohydrate, but carbohydrate fro m both subunits had to be eliminated to obtain maximu m loss of bio-I . I .. 20 ?? 1 3 16 S . d' d . oglca acti vity '--'- '- . Ite- Irecte mutagenes Is identi fied hexAsns2 as the critical oligosaccharide for signall ing in the hCGex subunit and ~Asn l 3 as a sec­ondary criti cal site in the hCG~ subunit27. Interest­ingly, the effect of mutating the Asnl3 glycosy lati on site was not detectable until after the hCGexAsn52 site had been eliminated27. Thi s combinati on ~f ex and ~ subunit mutants has not been replicated with the other

glycoprotein hormones . However, the requirement fo r chemical deglycosy lati on of both oLH ex and oLH ~ subunits for max imum loss of biolOGical acti vity sUG­gested ~Asn l 3 oligosaccharide is in:olved in LH bi~­logical ac ti vity, as oLH~ possesses onl y the Asn l3

glycosy lati on site23.26. The part ial loss of FSH ac ti vity fo ll owing remova l of exAsnS6 carbohydrate with PNGase in N56dg-eLHex:eFSH~ hybri d preparati ons suggests FSH ~ oli gosaccharides also part icipate in FSH ac tion. While carbohydrate deleti on stud ies were va luable for providing ev idence that carbohydrate was in vo lved in the biological ac ti vi ty of the gonadotro­pi ns, they were limited by the fac t that the ex subunit is always decorated with carbohydrate. (A lthough the presence of hFSH inhibitory acti vity in serum samples deri ved from women treated with GnRH antagoni sts suggested that FSH lacking one or more ex subunit oligosaccharides mi ght exist I32

, these have not yet been isolated. ) Composition analysis demonstrated hormone-specific diffe rences in glycosy lation of por­cine glycoprotein hormones i33

. Th is suggested that the effects of altered glycosy lati on could be studied by isolating ex subunits from gonadotropin preparati ons, combining them with the same ~ subun it preparati on, and comparing the biological acti vities of the result­ing hybrid preparations.

We employed gonadotropins deri ved from the horse because it was possible to obtain large amounts of each gonadotropin from thi s species I34.I35

. The plan was to prepare all possible combinations of equine gonadotropin subunits, perfo rm receptor-b inding as­says to confirm fo rmation of fun Ct ional heterodimers, and proceed to functi onal bioassays. Because it was somewhat cont roversial as to whether or not deglyco­sy lated LH and hCG were somewhat more ac ti ve, somewhat less ac ti ve, or as ac tive as the intact hor-

. t b' d' 15-25 . . d mone 111 recep or- In Ing assays , we anti cipate that the receptor-binding phase of the investi ga ti on would be brief. Instead, we found was a wide range of receptor-binding potencies wi th a 600-fold di fference in potency between the most act ive and least ac ti ve preparations. These resulted fro I both ex subunit and Bsubunit glycosy lation affec ting subunit assoc iat ion as well as receptor-binding acti vi ty72. Oligosaccharide mapping of exAsns6 carbohydrate selectively removed by PNGase digesti on of intact ex subuni t preparati ons had revealed a hormone-spec ific pattern of glycosyla­tion at thi s site71. The major strLlc tural va ri ati on bet­ween eLH ex, eFSHex, and eCGa. Asn56 oligosaccha­rides consisted of diffe rences in the size of the Man(ex l- 6)Man branch (Fig. 5). In cLHex this consisted

BOUSFIELD & BUTNEV: EQU IN E GONADOTROPINS AS MODEL FOR GLYCOSYLATION

0.4

0.3

0.1

.- -.-o ~--~'-------------------------------~---+O

2.1

1.4

,-...

~ 0.7 '-'" <l.l C/)

c o 0.. C/)

<l.l ~ o g:

-

0.8-

1.0

0.5

o o

B

c

D

20

eLHa Asn56 oligosaccharides f-1.0

f- 0.8

/ ; 0.6

.' --0.4

0.2

o eeGa Asn56 oligosaccharides

1.0

0.8

r-0.6

f-0.4

r-0.2

o eFree-a Asn56 oligosaccharides

f-1.0

r-0.8

f-0 .6

0.4

0.2

40 60 80 100

Time (min)

495

Fig. 5-Structures of equine ex subunit Asn56 o ligosaccharides. Oligosaccharides released from Asn56 by PNGase digestion of intact ex subunit preparations are illustrated, These are large ly based on composition and average mass data. Two oligosaccharide structu res were characterized by NMR analys is of the indicated peaks in panels Band D. A cautionary note was served when the hybrid complex su l­fa ted/high mannose o li gosaccharide believed to populate eLHex Asn56 was revealed to be high mannose o li gosaccharide when the purified o ligosaccharide fractions were characte ri zed.

496 INDI A.N J EXP BIOL, APR IL 2002

of two Man residues derived from exoglycosidase processing of the Glc3Man9GlcNAc2 precursor. In eFSHa either su l fate-4-Ga INAc(~1--4)GlcNAc (~1-2)

Man(al-6) or NeuNAc(a2-3)Gal (~I--4) GlcNAcMan (a l-6) were present, while in eCGa, the NeuNAc (a2-3) Gal (~1--4)GlcNAcMan(al-6) structure was extended by lactosamine repeats. Since it was not possible to determine the masses of individual oli go­saccharides due to the negative charges provided by terminal sulfate and sialic acid moieties, the differ­ence in mass between each a subunit preparation and its Asn56-deglycosy lated derivative was determined

"C Q)

E .... 0 u. .... Q)

E Q ~ 0

20 A

5

o

100 90 80 70 60 50 40 30 20 10 0

~ r I

" ~ r I

'"

B

1250

• 0

.. " (J) I

" <1> r I ",

<1> () G)

" <1> r I ",

<1> r I

" <1>

" (J) I ",

<1>

" (J) I

" <1>

" (J) I ",

<1> () G)

" <D

" (J) I ",

Hybrid preparation

~ ~

Intact ex N56dg-ex

1500 1750 2000

<1>

" (J) I

" '" 8 ",

~

2250

Average N56_CHO mass (amu)

250

Fig. 6-EITccts of aAsn56 oli gosaccharide size on recovery of biologica l acti vity and dimer formati on efficiency. A. Rat io of FSH receptor-bind ing ac tivity recovered following reassoc iation of intac t a subunit preparations with eLH ~, eFS H~, or eCG~ vs that recovered foll owing reassoc iation of Asn56-deglycosy lated a subunit preparations with the same ~ subunit preparat ions, as ind icated. B. Dimerizati on efficiency as a fun cti on of aAsn56

oligosaccharidc mass determined by mass spectrometry. Increased oli gosaccharide size was corre latcd wit h red uced dimeri zation effi ciency (P < 0.05) of eFS H~ hybrids (c losed circles). The rela­ti onship was no longer signi ficant (P> O.OS) when aAsn56 carbo­hydrate was removed by PNGase diges ti on (open circles).

using mass spectrometry . Th is anal ys is revealed a progressive increase in the average Asn56 oli gosaccha­ride mass ranging from 1482 mass units for eLHa to 2327 for eCGa. Increased size of the Mana l-6 branch was responsible for the increased mass of eFSHa and eCGa oligosaccharides and produced sig­ni ficant (P < 0.05) correlati on between carbohydrate size and reduced dimerizat ion efficiency leading to an eventual 23 % reduction (Fig. 6B). Removi ng the aAsn56 oligosaccharide with PNGase72 elimi nated the correlation between increased oligosaccharide size and reduced dimer formation (P> 0.05) . Both eFSH~ and eCG~ combined less efficiently with the a sub­unit preparations than did eLH(beta) by revealing an inhibitory effect of ~ subunit carbohydrate on dimer fo rmation . The inhibitory effects of a and ~ subunit carbohydrate on dimer form ation compli cated inter­pretation of results obtained with eLH~ and eCG~ hybrids because the heterod imer and ~ subunits co­purified in every avail able chromatographic method. We then removed the C-terminal 29 residues from eLH~ and eCG~, making it possible to purify the het­erodimer fraction . Experiments with des( 12 1-1 49)­eLH~t and -eCG~t hybrids confirmed the inhibitory effects of increasing aAsn56 oli gosaccharide size on both LH and FSH receptor-binding affinity (Fig . 7). However, they also revealed that eCGa Asn82 oligo­saccharide inhibited receptor-binding activity of hy­brids possessing eCGa. Comparison of the inhi bitory effects of a subunit carbohydrate on eLH~t and eFSH~ hybrids revealed an attenuated influence of oligosaccharide size on FSH receptor inding. Thus, eLHa:eFSH~ was more acti ve than eCGa:eFSH~, however, the magnitude of the difference between these hybrids was only 2-fo ld, while th ~ difference in FSH receptor-binding activity between eLHa:eLH~t

and eCGa:eLH~t was 5-fold.

We proposed two explanati ons for the greater magnitude of the inhibitory response to increased aAsn56 oligosaccharide size in eLH~ hybrids as com­pared with eFSH~ hybrids72

• The first wag steric hin­drance on the part of the carbohydrate accompanied by a difference in the orientat ion of eLH~ hybrids and eFS H~ hybrids in the FSH receptor. The increased size of the more flexible Man(a l-6) branch inhibits receptor binding by steric hindrance because it is located closest to the putative receptor-binding site and simply gets in the way . The problem with thi s rati onale is the differenti al effect observed in LH~

BOUSFIELD & BUT EV: EQU INE GO ADOTROPINS AS MODEL FOR GLYCOSY LATION 497

and FSH~ hybrids. It is not simply a matter of steri c hindrance on part of the carbohydra te, as the same oli gosaccharides produ ce a bigger effect in the fo rmer than in the latter. A model for the interac ti on of de­glycosy lated hCG with a model of the LH/CG recep­tor extracellul ar domain placed the GlcNAc residues of the hcxA sn52 oli gosacchari de core close to the re­ceptor, illustrating how large oli gosaccharides at­tached to th is position coul d get in the wa/ 36

. The other N- li nked oligosaccharides were located on the opposite side of the hormone, fac ing away from the

100-

75 A

50 -

C'I 25

.5 '0 c

CD .5:1 0 == CJ 100-a> c. en ~ 0

75- B

50 -

25-

putat ive hormone-receptor interface. The C-term inal portion of the cystine knot, which is important for FSH binding to its receptor, was also directed away from the receptor. Rotation of the hormone to orient thi s region toward the putati ve hormone-binding site of the receptor, moved the hcxAsn52 oli gosaccharide away fro m the receptor, potenti all y reducing its steric infl uence. We suggested th at eLH and eCG interact with both LH and FSH receptors in the same orienta­tion that LH and hCG engage the LH receptor. This has the consequence that cxAsn56 oligosaccharides

---.- eLHu·eLHp

--- eFSHa·eLHp

---- eCGa·eLHp

-A- eLHa·eLHpl

--- eFSHa·eLHpt

---- eCGa·eLHpl

- .6. - eLHa·eCGpl -.-eFSHu' eCGpl

- e - eCGu'eCGpl

~ eLH

-0- eCG-L

---.- eLHa-eLHp

--- eFSHu·eLHp

---- eCGu'eLHp

-A- eLHa·eLHpl

--- eFSHu'eLHpl

---- eCGu·eLHpt

- .6. - eLHu-eCGpl -.-eFSHa-eCGpl

- e- eCGu'eCGpl

~ eLH

eCG-L

O -r-~~~~~~~~~ry-~~~~~--~--~~~~~~~

0.01 0.1 10

[Hormone] nM

100 1000

Fig. 7- Receptor-bi nd ing ac ti vitics of pu rified equine gonadotropin hybrid _hormone prepara tions. A. LH rad iol igand assay using 1251_ hCG tracer and rat testi s homogenate. B. FSH rad ioli ga nd assay cmploy ing 12' I_eFS H tracer and ra t tes ti s homongenate

498 INDIAN J EXP BIOl, APRil 2002

inhibit eLH~ hybrid binding to both receptors to the same degree. Moreover, the partial activation of FSH target cells by eLH, eCG, and hybrids possessing their

~ subunits, is consistent with a partially inappropriate interaction with the FSH receptor. The relative po­tency of eLH and eLH~ hybrids in a rat testis recep­tor-binding assay ranged from 5% to 19% that of eFSH. In the granulosa cell assay the relative poten­cies were all lower, ranging from 1 % to 7%, averag­ing 3.8-fold lower biological potency than FSH recep­tor-binding potency 72.

The second explanation was that increases in oligo­saccharide size altered the conformation of the pro­tein, thereby reducing receptor-binding affinity. In

this model, the conformation of eLH~ hybrids changed

Cll c e Cll Ul o Ul Cll I-

350

300

250

200

150

~ 100

50

1000

~ 800 o .... Cll U; 600 o Ul Cll I- 400 Ol Q.

(lJ c e Cll Ul

200

80

70

60

50

40 Cll Ol e 30 a.. Ol 20 Q. 10

o

A

c

-_.LHa ... LH~

- _ eFSHu·eLH~

__ eCGu·eLH~

-o-eLH

__ eLHa·eCG~1

__ eFSHu·.CG~1

___ eCG,,-eCG~1

- :J-eLH

-+-eFSH -o-eU< -o- eCG _ hCG - .. . el Ho-eFSHfJ - Ir . eFSHa4SH~ -. . eCGQ·.FSH~ ___ eLHo-eLHp -.- eFSHu-elHp ___ eCGn-.LHfJ

0.0001 0.001 0.01 0.1 1 () 100

nM Hormone

Fig. 8-11'1 vitro bioassays of equine gonadotropin hybrids . A. Diethylstilbestrol-primed rat granulosa cell FSH bioassay. The endpoint was progesterone synthesis after 72 hr incubation . Band C. Rat testis Leydig cell steroidogenesis lH bioassay. Because of the short-term incubation invol ved in this assay, fewer samples cou ld be accommodated at one time.

more than that of FSH~ hybrids producing a more pronounced reduction in receptor binding affinity for the former set of hybrid preparations than for the lat­ter. Preliminary data supporting a conformational change in eLH~ hybrids have been obtained using circular dichroism and more extensive studies involv­ing both eLH~ and eFSH~ hybrid preparations are in progress. Since the two explanations are not mutually exclusive, the inhibitory effects of carbohydrate struc­ture on hormone activity might reflect both steric hin­drance by the carbohydrate moieties as well as con­formational changes in the peptide moiet ies. The crys­tal structure of hFSH suggested the nature of the con­formational change was likely to consist of variations in the positions of the ex and ~ subunits relative to each otherS. Two conformational isomers of hFSH were observed in crystals of insect cell·-expressed re­combinant hFSH. While each ex subunit could be overlaid without significant differences in the posi­tions of the backbone atoms, the complementary ~ subunits did not align well and vice versa. A compari­son of the 3D structures for chemically deglycosy­lated hCG and desialylated hCG complexed with an­tibody Fv regions revealed changes in backbone con­formation in both subunits, however, it was not clear whether the presence of virtually complete oligosac­charide chains or antibody binding were responsible for the changes in conformation. Based on changes in affinity of monoclonal antibodies for various hCG and LH derivatives, Moyle and colleaguesl37-139 have ar­gued that a change in hormone conformation occurs with receptor binding. Since the epitopes recognized by these antibodies remain largely undefined, it is not yet known what sort of conformational changes are involved. If changes in the relative posi tions of the ex and ~ subunits are important elements in hormone­receptor activation, then the role of the exAsn56 oligo­saccharide may indeed consist primarily of a stabiliz­ing effect, permitting subunits to shift in their orienta­tion relative to one another without falling apart and terminating cellular activation. This would support the model proposed by Moyle and colleagues that a minimal oligosaccharide structure is necessary for biological acti vitl5

. Too small an oligosaccharide, such as the GlcNAc2 remnant following chemical de­glycosylation, could only parti ally activate LH recep­tor-mediated responses. HO\vever, in this model , the oligosaccharide interacts with the receptor to alter its conformation, thereby activating it. The stability model, suggests that be.low a mini mum size, oligosaccharide

BOUSFIELD & BUTNEV: EQUINE GONADOTROPINS AS MODEL FOR GLYCOSYLATION 499

cannot prevent subunit dissociation during hormone­receptor interaction .

Surprisingly small effect of altered receptor-binding affinity on ill vitro biological activity of equine go­nadotropin hybrids

While the effects of carbohydrate on receptor­binding affinity revealed by comparison of equine gonadotropin hybrid preparations were larger than expected, the differences in steroidogenic potency between the preparations were reduced to the extent that they were no longer significant. This may have resulted from amplification of the ligand-dependent receptor activation as well as the variability intro­duced by the two-assay nature of the steroidogenesis assay; target cell incubation to induce steroidogenesis followed by steroid RIA to measure the product. One possible conclusion was that oligosaccharide structure at aAsn56 beyond a minimal basic structure was ir­relevant for biological activity. However, when the ratios of receptor-binding IC50 to steroidogenic EC50 were calculated for each preparation, a different pat-

[ 140 d d· tern emerged. Mendeleson et a . emonstrate m rat testis Leydig cells that maximum hCG-stimulated cAMP accumulation occurred at 50% LH receptor occupancy, while testosterone synthesis reached its maximum level at I % receptor occupancy. Greater than 100-fold amplification was observed with the hybrid LH preparations that appeared to increase with larger aAsn56 oligosaccharide size. The ratio of the ID50 for LH receptor binding to ED50 for steroidogen­sis was 500 for eLHa:eLH~, 800 for eFSHa:eLH~, and 2800 for eCGa:eLH~. The corresponding ratios for the truncated hybrid preparations were 250 for eLHa:eCG~t, 400 for eFSHa:eCG~t, and 1200 for eCGa:eCG~t72. Our results suggested that the pres­

ence of the Man(al-6)Man branch in eFSHa and increased length of this branch in eCGa was associ­ated with enhanced cellular activation, largely offset­ting the reduced receptor-binding activity that may be due to the same structural feature. Since hCGa Asn52

oligosaccharides for the most part possess only one complex branch, the apparent enhanced biological activity due to the addition of a second branch to oli­gosaccharides attached to this site may provide an interesting corollary to oligosaccharide size model85 : increased oligosaccharide size or branching produces enhanced biological activity. Whether this phenome­non is also observed with more extensively branched aAsn56 oligosaccharides will be interesting to deter-

mine. However, the fact that the binding assay was performed with 25 mg testis homogenate (from which 1,200,000 interstitial cells can be obtained), while the bioassay measured the steroidogenic response of 60,000 interstitial cells, suggests further studies with comparable numbers of cells are necessary.

The mechanism for carbohydrate involvement in cellular activation remains elusive. Seth and Bahl 141 reported that G protein activation was compromised by elimination of hCG carbohydrate, suggesting par­tial receptor activation. The nature of gonadotropin receptor activation is unknown. For the prototype of G protein coupled receptors, the ~ radrenergic recep­tors, ligand-binding in the transmembrane helical re­gion alters the relative positions of helices 3 and 6142 . This appears to alter the conformations of cytoplasmic loops 2 and 3, permitting G protein activation by ex­posing buried residues in these loops. The decapep­tide releas ing factor, GnRH, appears to bind the ex­tracellular loops of its receptor to achieve the same rearrangement of transmembrane helices l43. The gly­coprotein hormone receptors differ in that high affi n­ity binding occurs in an enlarged N-terminal domain that comprises roughly half the receptor l44 . Activation of the receptor has been suggested to involve a low affinity site in the extracellular 100pS 145. Alternatively , co-expression of cDNAs encoding the extracellular domain and transmembrane domain of the LH recep­tor produced a functional receptor l46. Changes in ex­tracellular domain resulting from hormone binding could alter the conformation of the transmembrane domain via interactions between the extracellular do­main and the extracellular loops of the transmembrane domain. In contrast to other G protein coupled recep­tors, no glycoprotein hormone antagonists have been developed. Chemically and genetically deglycosylated

. . I b· I . I .. '02 127147 W hCG retam reSidua looglca actlVlty-· . . . e prepared an interesting FSH inhibitor consisting of Asn56-deglycosylated eLHa combined with des(l21-149)eLH~ (N56dg-eLHa:eLH~t) that exhibited no FSH activity in the rat granulosa cell bioassay, al­though it did exhibit 5% LH activity in the rat Leydig cell bioassay' 48 . N56dg-eLHa:eLH~t completely in­hibited the steroidogenic response to 300 pg eFSH or 2000 pg hFSH. However, when other variants of this derivative were tested for FSH inhibitory activity, it was either reduced lO-fold or else completely elimi­nated. These results illustrate a complicating factor in studies involving gonadotropin carbohydrate, although aAsn52 plays a primary role in coupling receptor-

500 INDIAN J EXP SIOL, APR IL 2002

binding to cellular ac ti vati on, other oligosacchari des, such as ~Asn 13 pl ay secondary roles. Thcse prov ide part ial compensati on fo r the absence of aAsn52 o ligo­sacchari de and imply that carbohydrate-mediated re­ceptor aggregation is involved in cellul ar ac ti va tion.

A model for gonadotropin receptor act iva ti on pro­posed over 20 years ago, but inadeq uately tested was that carbohydrate was in volved in receptor aggrega­tion, as labeled hCG appeared to be clustered at the surface of target cell sI49.'50. Recently, Roess and col-I 151 eagues presented the resul ts of fluorescence reso-nance energy transfer studies in volvi ng indi vidual cells expressing LH receptor-green fluorescent protein or -yell ow flu orescent protein chimeras that indicated that oLH and hCG binding induced ligand-receptor self-associati on. Earlier studies showing antiserum directed against hCG restored biological ac ti vity to chemically deglycosylated hCG were interpreted as res toring a native confo rmation to the deglycosyl ated molecule'52. '54 . Indeed, the determinant loop of hCG complexed with recombinant Fv fragments of a sub­uni t- and B subunit-directed monoclonal anti bodi es di spl ayed an altered conformation, which might re­presen t the functional confo rmation of the hormone? On the other hand, wheat germ agglutinin (WGA ) could also restore the activity of deg lycosyl ated­hCG155. As WG A was used in the isolati on of the LH receptorl56, it could probably aggregate cell surface receptor on its own. However, WGA by itself had no effect on cellular acti vity , therefore, association of I igand-acti vated receptors appears to be necessary fo r target cell activation. In support of thi s, constitutively ac ti vated receptors onl y partiall y acti vate target cells. While inacti ve LH receptor-GFP chimeras failed to self-associate '51, constitutively ac ti vated receptors were not tes ted. Ligand-mediated receptor di meriza­ti on has been known to be a mechanism for receptor ac ti va tion ever since the crystal structure of growth hormone bound to two extracellular domains was re­ported I5? The standard textbook model for G protein coupled receptor action predicted isolated receptors should be full y fun ctional. However, GnRH antago­ni sts were reported to become agoni sts when crosslinked by antibodies '58 , and this has subsequentl y been confirmed by reports that GnRH antagonists do not initi ate fluorescence energy transfer by GnRHR­GFP, GnRHR-YFP chimeras expressed in the same cel\ 159. The premier system for GPCR studies, the ~r adrenergic receptor provides a progressi ve model for hormone-receptor induced receptor phosphoryl ation

leading to Gs to G; switching fo ll owed by GRK phos­phorylati on which is fo ll owed by ~-a rrestin-med i a ted desensiti za tion and internali za tion, which by clu ster­ing with growth facto r receptors leads to act ivati on of MA P kin ase path waysl60. Although the dimerizati on determinants of the gonadotrop in receptors do not appear to reside in the transmembrane domai ns 161 , ~­arrestin-medi ated desensiti zation and internali za ti on occur, albeit largely in the absence of receptor phos­phorylation'62 . Studies involving cell s ex pressing LHR-GFP chimeras indicated that concentrat ion of the LH receptor was di stributed over a large porti on of the cell l5 1, rather th an the punctate pattern observed fo r the Bradrenergic receptorl63 Studies with hCG glycopeptides ind icated th at carbohycl raL, especially that obtained from hCGa, could inhibit hCG­stimulated adenylyl cyc lase acti vation at doses that had no effect on 12sI_hCG binding l64. Indeed, the amount of G-protein associated with the LH receptor was greater when cell s were treated with intac t hCG than when treated with chemically deglycosy lated hCG 141. If ligand-receptor self assoc iation represents the mechanism for gonadotropi n receptor acti vation, then the secondary ro le for ~Asnl 3 oli gosaccharide in hCG biological activity might stem from its ability to participate in self aggregati on. The partial compensa­ti on may result from hormone-receptor complexes oriented improperl y that may need reorientation fo r functional acti vation.

O-gJycosyJation effects on gonadotropin activity Deletion of the C- terminal extension from eLH ~

and hCG~ by mi Id ac id hydrolysis 165. 16C1 and by muta­ti on of recombinant hCGB '6? '68 had littl e effect on the in vitro biological activiti es of the eLH and recombi­nant hCG deri vat ives. This led to the conclusion that the C-termin al ex tension only affected these hor­mones in vivo. Enhanced in vivo potency following introduction of the CTP into both LH and FSH has been experimentally confirmed" 9.120. However, trun-cation of the hCG~ C-terminus by site -directed mutagenesis revealed its influence on N--l inked oligo­saccharide processing, thereby ex tend ing the list of functional acti vities associate with this structural I 100 101 R I" I ' CG ' f e ement . . ecent stuc les 111 VO v111g e ISO orms

revealed that the high degree of O-glycosy lation asso­ciated with eCG-H inhibited LH and FSH receptor­binding acti vity'69. Because reduced receptor-binding ac ti vity was not compensated by enhanced ability to stimulate cellular acti vity, red ced LH steroidogenic

BOUSFIELD & BUTNEV: EQUINE GONADOTROPINS AS MODEL FOR GL YCOS YLATION SOl

potency was also observed (Butnev and Bousfield, unpublished) . Removal of residues 121-149 by mild acid hydrolysis of ASp1 20_pro 121 virtually eliminated the difference in receptor-binding activity between eLH~t and eCG~t hybrids sharing a common ex sub­unit, despite the fact that the mass of eLH~t was 1053 mass units less than that of eCG~t. This result was obtained with all three equine ex subunit preparations. Recombinant eLH/CG mutated to eliminate the exAsn56 glycosylation site was reported to lose LH biological activity, but not FSH biological activity suggesting that O-glycosylation can selectively affect the latter 170.

Acknowlegements This research is based upon work supported by

NIH grants AG 15428 and DK52383 and by NSF un­der EPS-9874732 with matching support from the State of Kansas.

References Murphy B D & Martinuk S D, Equine chorionic gonad­otropin , Endocri/le Re v. 12 (1991 ) 27.

2 Bousfield G R, Perry W M & Ward D N, Gonadotropins: Chemistry and biosy nthesis, in The Physiology of Repro­duction (Raven Press, New York) 1994, 1749.

3 Moy le W R & Campbell R K, Gonadotropin s, in Reproduc­tive Endocrinology, Surgery and Technology (Lippincott -Raven, Hagers town) 1996,683.

4 Hearn M T W & Gomme P T, Molec ul ar architecture and biorecognition processes of the cysti ne knot protein super­family: part I. The glycoprotein hormones, J Mol Recog/lil, 13 (2000)223. .

5 Lapthorn A J, Harri s D C, Littlejohn A, Lustbadcr J W, Canfield R E, Machin K J, Morgan F J & Isaacs N W, Crystal structure of human chorion ic gonadotropin , Nalllre, 369(1994)455.

6 Wu H, Lustbader J W, Liu Y, Canfield R E & Hendrickson W A, Siructure of human chorionic gonadotropin at 2.6 A resolution from MAD analysis of the selenomethionyl pro­tein , SIrI{ClUre, 2 ( 1994) 545.

7 Tegoni M, Spinelli S, Verhoeyen M, Davis P & Camillau C, Crystal structure of a ternary complex between human chorionic gonadotropin (hCG) and two rv fragmcnts spe­cifi c for the a and ~-subunits, J Mol Bioi, 289 (1999) 1375.

8 Fox K M, Dias J A & Van Rocy P, Three-d imensional structurc of human folli cle-s timul ating hormone, Mol EII­docrinol, IS (200 I) 378.

9 Dayhoff M 0, Atlas of Protein Sequence and Structure, (National Biomedical Research Foundation ,Washington, DC) 1976, 122.

10 McDonald N Q, Lapallo R, Murray-Rust J, Gunning J, Wlodawe r A & Blundell T L, New protein fold revealed by a 2.3-A resolution crystal structure of nerve growth factor , Nal II re, 354 ( 199 1) 411.

II Murray-Rust J, McDonald N Q, Blundcll T L, Hosa ng M, Oefner C, Winkler F & Bradshaw R A, Topo logica l simi-

larities in TGF-~2, PDGF-BB and NGF define a super­family of polypeptide growth factors, SlruelUre, I (1993) 153.

12 McDonald N Q & Hendrickson W A, A structural super­family of growth factors containing a cystine knot motif, Cell, 73 (1993) 421.

13 Varki A, Biological roles of oli gosaccharides: all of the theori es are correct, Glycobiology, 3 (1993) 97.

14 Thotakura N R & Blithe D L, Glycoprotein hormones: gly­cobiology of gonadotropins, thyrotropin and free a subunil , Glyeobiology, 5 (1995) 3.

IS Moyle W R, Bahl 0 P & Marz L, Role of the carbohydrate of human chorionic gonadotropin in the mechanism of hormone acti on, J Bioi Cirem, 250 ( 1975) 9163 .

16 Sairam M R & Schi ll er P W, Receptor binding, bi ological, and immunolog ical properties of chemically deglycosy lated pituitary lutropin , Arch Bioehem Biophys, 197 (1979) 294.

17 Sai ram M R, Effects of carbohydrate removal on the Slruc­ture and activity of bovine lutropin , Biochilll Biophys AeIQ, 717 (1982) 149.

18 Manjunath P & Sairam M R, Biochemical, biologica l, and immunological properties of chemica ll y deglycosy lated human choriogonadotropin, J Bioi Chelll , 257 ( 1982) 7109.

19 Manjunath P, Sairam M R & Sairam J, Studics on pitu ita ry follitropin. X. Biochemical, receptor binding and im mu ­nological properties of deglycosy lated ovine hormone, /vlol Cell Endocrinol, 28 (1982) 125 .

20 Governman J M, Parsons T F & Pierce J G, Enzy matic de­glycosy lation of the subunits of chorionic gonadotropin: ef­fects on formation of tert iary structure and biological act iv­ity , J Bioi Chelll , 257 (1982) 15059.

2 1 Kalyan N K & Bahl 0 P, Role of carbohydrate in human chorionic gonadotropin: effect of deglycosy lation on the subunit interact ion and on its in vilro and ill vivo biologica l properties, J Bioi Ch elll , 258 ( 1983) 67.

22 Keutmann H T, Mcilroy P J, Bergen E R & Ryan R J, Chemicall y deg lycosy lated human chorionic gonadotropin subunits: characterization and biological properties, Bio­chelllistry, 22 ( 1983) 3067.

23 Liu W K, Young J D & Ward D N, Deglycosy lated ov inc lutropin: preparation and characterization by ill vitro bind­ing and steroidogenesis, Mol Cell Endoerinol, 37 ( 1984) 29.

24 Berman M I, Thomas C G, Jr. , Manjunath P, Sairam M R & Nayfeh S N, The role of the carbohydrate moi ety in th y­rotropin action, Bioehem Biophys Res Co 111 111 un, 133 ( 1985) 680.

25 Calvo F 0, Keutmann H T, Bergert E R & Rya n R J, De­glycosy lated human follitropin: charac terization and effects on adenosine cycli c 3',S'-phosphate production in porcine granulosa cell s, Biochemistry, 25 (1986) 3938.

26 Sairam M R, Deglycosy lation of ov ine pituitary lut rop in subunits: effects on subunit interaction and hormone act iv­ity, Arch Bioehel1l Biophys, 204 ( 1980) 199.

27 Matzuk M M, Keene J L & Boime I, Site specifi ci ty of thc chorionic gonadotropin N-linked oligosaccharides in signal transduct ion, J Bioi Chelll , 264 ( 1989) 2409.

28 Flack M R, Bennet A P, Froehlich J, Anasti J N & Ni sula B C, Increascd biological activity due to basic isoforms in rc­combinant human follicle-stimulating hormone produced in a human cell line, J c/ill Endoerillol Me/ab, 79 (1994) 756.

502 INDIAN J EXP BIOL, APRIL 2002

29 Bishop L A, Robertson D M, Cahi r N & Schofi eld P R, Spec ifi c rol es for the asparagine-linked carbohydrate resi­dues of recombinant human follicle stimul ating hormone in receptor binding and signal transducti on, J Mol EndocrinoL, 8 (1994) 722.

30 Ulloa-Aguirre A, Miller C, Hyland L & Chappel S, Produc­tion of all follicle-stimul ating hormone isohormones from a puri fied preparation by neuraminidase digest ion, Bioi Re­prod, 30 (1984) 382.

3 I Wide L, The regulat ion of metaboli c clearance rate of hu­man FS H in mice by va ri ation of the molec ular structure of the hormone, Ac/a Elldocrillol, 11 2 (1986) 336.

32 Wide L & Bakos 0, More bas ic forms of both human foll i­cle-stimul ati ng hormone and luteini zing hormone in serum at midcyc le compared wi th the follicul ar or luteal phase, J Ciin Endocrillol Merab, 76 (1993) 885.

33 Zambrano E, Oli vares A, Mendez J P, Guerrero L, Diaz­Cueto L, Ye ldhu: J D & Ulloa-Agu irre A, Dy nam ics of basal and gonadotropin -releasing hormone- releasab le se­rum folli cle-stimulating hormone charge isoform distri bu­tion th roughout the human menstrual cyc le, J Ciin Elldo­erillol Metab, 80 ( 1995) 1647.

34 Chin W W, Maize l J Y, Jr. & Habener J F, Diffe rence in sizes of human compared to murine a-subuni ts of the gly­coprotein hormones ari ses by a fo ur-codon gene deleti on or insert ion, Enducrinology, I 12 ( 1983) 482 .

35 Matzuk M M & Boime I, Site-specific mutagenesis defi nes the intracellular role of the asparagi ne-l inked oligosaccha­rides of chori onic gonadotropin ~ subu nit, J Bioi Chem, 263 ( 1988) 17 106.

36 Chappel S, Bcek A, Nugent N, Hyman L, Zabrecky J & Maurer R, Productioll of buvill e f ollicle stimulating hor­mOlle (FSH) by recombinallt DNA technology, 69th Annu al Meeting of the Endocrine Soc iety, Indianapol is, 1987.

37 Bousfie ld G R, Butnev Y Y, Gotschall R R, Baker Y L & Moore W T, Struetural fea tures of mammalian gonadotro­pins, Malec Cell Ellducrillol, 125 ( 1996) 3.

38 Dias J A, Lindau -Shepa rd B, Hauer C & Auger I, Huma n fullic le-stimulati ng hormone structure-activity relation­ships, Bioi Reprod. 58 (1998) 133 1.

39 Walton W J, Nguyen Y T, But nev Y Y, Singh Y, Moore W T & Bousfield G R, Charac teriza ti on of huma n follicle­stimul ating hormone isoforms reveals a non-glycosy lated ~-subu nit in addition to the conventional glycosy lated ~­subun it. , J Ciill Ellducrillol Metab , 86 (200 I) 3575.

40 Weisshaar G, Hiya m:J J & Renwick A G C, Site-spec ific N­glycosylation of human chorionic gonadot ropin-structural analys is of glycopept ides by one- and two-dimensional I H NM R spec troscopy, ClycoiJ iology, I ( 199 I) 393 .

41 Matsui T, Mi zouchi T, T ita ni K, Okinaga T, Hosh i M, Bousfie ld G R, Sugino H & Ward D N, Structura l analysis of N- lin ked oli gosacchari des fro m equ ine chori on ic go­nadotropin and lutropin ~-su bu nit s, Biuchemistry, 33 ( 1994) 14039.

42 Qasba P K, In volvement of sugars in prote in-protein inter­actions, CariJohyd PolYIII , 4 1 (2000) 293.

43 Parsons T F & Pi erce J G, Oligosaccha ride moieties of gly­coprotein hormones: bov ine lutrop in resists enzy mati c de­glycosy lati on because of terminal O-sul fa ted N­;Jccty lhexosa mines, Proc Nat l Acad Sci USA, 77 (1980) 7089.

44 Gree n E D, van Halbeek 1-1 , Boime I & Baenziger J U, Structural elucidation of the disul fa ted oligosaccharide from bovine lutropin , J BioL Chem, 260 (1985) 15623.

45 Green E D & Baenziger J U, Asparagine-linked oli gosac­chari des on lut ropin, follitropin , and thyrotropin: I. struc­tural elucidati on of the sulfated and sialYlated oligosaccha­rides on bov ine, ovine and human pituitary glycoprote in hormones, J Bioi Chem, 263 ( 1988) 25.

46 Manze lla S M, Dharmesh S M, Beranek M C, Swanson P & Baenziger J U, Evolutionary conservat ion of the sul fa ted oligosaccharides on vertebrate glycoprote in hormones that cont ro l circulatory half-li fe, J Bioi Chem, 270 (1995) 2 1665 .

47 Baenziger J U & Green E D, Pitu itary glycoprotein hor­mone oligosaccharides : structure, synthesis and functi on of the asparagine- lin ked oligosaccharldes on lu tropi n, follit ro­pin and thyrotropin , Biochim Biophys Acta, 947 (1988) 287.

48 Garfink J E, Moyle W R & Bahl 0 P, A I'apid, sensiti ve bioassay for luteinizing hormone-li ke gonadotropins, Cen COIl1P EndocrinoL, 30 ( 1976) 292.

49 Mock E J & Niswender G D, Di fferences in the ra tes of in ­ternali za tion of 1251-labe led human chorion ic gonadotropin, lutein izi ng hormone, and epiderma l growth faclor by ov ine luteal cells, Elldocrill ology, 11 3 (1983) .

50 Mock E J, Papkoff H & Niswendcr G D, Interna li za ti on of ov ine lutein izing hormone/hu man chorion ic gonadotropin recombinants: diffe renti al effects of the a - and ~-subu n i t s, Endocrinology, 1 13 (1983) 265.

5 1 Niswender G D, Roess D A, Sawyer H R, Sil via W J & Ba­ri sas B G, Differences in the latera l mobil ity of receptors fo r luteini zing hormone (LH ) in the luteal cell plasma membrane when occupied by ov ine LH \ 'erS l/S human chori oni c gonadotropi n, Elldocrinology, 116 ( 1985) 164.

52 Bousfield G R, Butnev Y Y & Butnev Y Y, Ide nt ifi ca ti on of twelve-O-glycosy lati on sites in eCG~ and e LH ~ by so lid-phase Edman degradation, Bioi Reprod, 64 (200 1) 136.

53 Le igh SEA & Stewart F, Part ial cDNA seque nce for the don key chori onic gon ado trop i n-~ - subun l t suggests evolu­tion from an ancestral LH-[3 gene, J Mol Endocrinol, 4 (1990) 143.

54 Parsons T F, Bloomfield G A & Pierce j G. Pu rifi ca ti on of an alte rnate form of the a subu nit of the glycoprotein hor­mones fro m bovine pit uitari es and ident ifi cati on of its 0-linked oli gosaccharide, J Bioi Clz elll , 258 ( 1983) 240.

55 Bousfield G R & Ward D N, Reduct ion and reoxidat ion of equine gonadot ropin a subunits, EI:docrillo logy. 13 1 ( I 9l)2) 2986.

56 Blithe D L, N- linked oligosaccharides on free a in terfere with its ab ility to combine with hu man chori on ic gonad­o t ropin-~ subu nit, J Bioi Cli elll , 265 ( 1990) 2 195 1.

57 Stanton P G, Robertson D M, Bu rgon P G, Schmauk-White B & Hearn M T W, Isolati on and physicochemica l charac­teri za ti on of human fo lli cle-sti mu lat ing hormone isoforms, Enducrillology, 130 ( 1992) 2820.

58 Stanton P G, Shcn Z, Kecori us E A, Bergon P G, Robe rson D M & Hearn M T, Appli cat ion of a sensitive HPLC-based nuorometric assay to de termine the sial i acid content of human gonadotropin isoforms, J Bioclielll Biophys Methot/s. 30( 1995) 37.

BOUSFIELD & BUTNEV: EQUINE GONADOTROPINS AS MODEL FOR GLYCOSYLATION 503

59 Christakos S & Bahl 0 P, Pregnant mare serum gonadotro­pin. Purification and physicochemical, biological , and im­munological charac terizati on, J Bioi Chem, 254 ( 1979) 4253.

60 Moore W T, 1r. & Ward D N, Pregnant mare serum gonad­otropin: an in vitro biological charac teri zati on of the lut ro­pin-follitropin dual activity, J Bioi Chem, 255 (1980) 6930.

6 1 Combarnous Y, Guillou F & Martinat N, Compari son of ill vitro follicle-stimulating hormone (FSH) ac tivity of equine gonadotropins (luteini zing hormone, FSH and chorionic gonadotropin) in male and female rats, Elldocrinology, 115 (1984) 1821.

62 Bousfield G R & Ward D N, Direct demonstration of in­trin sic follicle-stimulating hormone receptor-binding ac ti v­ity in acid-treated equine luteini zing hormone, Biochim Biophys Acta, 885 ( 1986) 327.

63 Sugino H, Bousfi eld G R, Moore W T, 1r. & Ward D N, Structural studies on equine gonadotropins: amino acid se­quence of equine chori onic gonadotropin ~-subunit , J Bioi Chem, 262 (1987) 8603.

64 Bousfield G R, Liu W-K, Sugino H & Ward D N, Struc­tural stud ies on equine glycoprotein hormoncs: amino acid sequcncc of equ ine lutropin ~ subunit, J Bioi Chem, 262 ( 1987) 86 10.

65 Sherman G B, Wolfe M W, Farmeri e T A, Clay C M, Thrcadg ill D S, Sharp D C & Nil son J H, A single gene en­codes the ~-su bunits of eq uine luteini zing hormone and chori onic gonadot ropin , Mol Elldoerillol, 6 (1992) 95 I.

66 Bousfi cld G R, Sugino H & Ward D N, Demonstration of a COOH-terminal ex tension on equine lutropin by mcans of a co mmon acid- lab ile bond in equine lutropin and equine chorion ic gonadotropin , J Bioi Chem, 260 (1985) 953 1.

67 Kammcrling J P, Damm J B L, Hard K, van Dedcm G W K, de Boer W & Vli egc nthart J F G, Differences in struc­tu re of carbohydrate chains of human and eq ui ne chori onic gonadot ropins, in Glycoprotein Horrnones: Structure, Syn­thesis. and Biologic Functi on (Serono Sympos ium, USA. Norwe ll, MA) 1990, 123.

68 Damm J B L, Hard K, Kammerling J P, van Dedem G W K & Vliegen thal1 F G, Structure dctermi nat ion of the major N- and O- linked carbohydrate chains of the ~ subunit from equ ine chorionic gonadotropin, EliI' J Biochelll. 189 (1990) 175.

69 Matsui T, Sugino 1-1 , Mi ura M, Bousficld G R, Ward D N, Titani K & Mizuochi T, ~-S ubu n i ts of eq ui ne chori onic go­nadotropi n and lut eini zing hormone with an iden tical amino ac id sequence have different aspa ragine-li nked oli­gosaccharide chains, Biochem Hiophys Res COIIII III/ll . 174 ( 199 1) 9-10.

70 Silli th P L, Bousrield G R, Kumar S. Ficte \) & l3acn/.ig.:r J U, Equine lutropin and chorioni c gonadotropin bear oligo­saechari dcs terminating wit h SO.I-4- GaINAc :md siaa2.3Ga l respec tivc ly. J Bioi Ch em. 268 ( 1993) 795.

71 Gotschall R R & Bousfic ld G R, Oli gosacc hari de m:Jpping reveals hormonc-speci ri c glycosyl:.ll ion patte rn s on cquine gonadotropin a-subuni t Asn56

, Elldocrillology, 137 ( 1996) 2543.

72 But ncv V Y, Gotschall R R, Butncv V y, Baker V L. Moore W T & Bous fi eld G R, Honnonc-spcc ific inhibitory innuence of a-subun it Asn56 oligosaccharide on ill vitro subuni t :Jssociati on and FS H receptor bindin g or equ inc go­nadotropins, Bioi Reprod, 58 ( 1998) 458.

73 Stewart F & Allen W R, The binding of FSH, LH, and PMSG to equine gonadal ti ssues, J Reprod Fertil Suppl, 27 (1 979) 431.

74 Grossmann M, Szkudlinski M W, Tropea 1 E, Bishop L A, Thotakuara N R, Scholfield P R & Weintraub B D, Ex pres­sion of human thyrotropin in cell lines with di ffe rent glyco­sy lation pallerns combined with mutagenesis of specific glycosyl ation sites : characteri za tion of a novel rol e fo r the oligosaccharides in the in vitro and in vivo bioactivity, J Bioi Chel1l, 270 (1995) 29378.

75 Matzuk M M & Boime I, The role of the asparagine-l in ked oligosaccharides of the a subunit in the sec retion and as­sembl y of human chori onic gonadotropin, J Cell Bioi, 106 ( 1988) 1049.

76 Giudice L C & Pierce 1 G, Gl ycoprotein hormones: some aspects of secondary and teritary st ructure, in Structure and Functi on of the Gonadotropins (Pl enum, New York) 1978, 8 1.

77 Strickland T W, Thomason A R, Nil son J & Pierce J G, The common alpha-subunit of bov ine glycoprotein hor­mones: limited formati on of nati ve structure by the tota ll y nonglycosy lated polypeptide chain , J Cell Biochelll, 29 (1985) 225.

78 Ruddon R W, Krzesicki R F, Norton S E, Beebe J S, Peters B P & Perini F, Detecti on of a glycosy lated , incompletely fo lded form of chori onic gonadotrop in ~ subunit that is a precu rsor of hormone assembly in trophoblasti c ce ll s. J Bioi Chelll , 262 ( 1987) 12533 .

79 Huth J R, Norton S E, Lockridge 0 , Shikone T, Hsueh A J W & Ruddon R W, Bacterial expression and ill vil ro fold­ing of the ~-s ubunit of hu ma n chori on ic gonadotropin (hCG~) and fun ctional assembly of recombi nant hCG~ with hCGa, Elldocrillology, 135 ( 1994) 9 11.

80 van Zuylen C W E M, Kamerling J P & Vli egenthart J F G, Glycosy lati on beyond the Asn7H-linked GlcNAc res iduc has a signi fica nt enhancing effect on the stab il ity of the a sub­unit of human chori oni c gonadotropi n, Biochelll Biophys Res Co m III II II, 232 (1997) I 17.

8 1 Weller C T, Lustbader J, Seshadri K, Brown J M, Chadwick C A, Kolt hoff C E. Ramnarai n S, Poll ak S. Can­field R & Hom:lns S W, St ruc tu ral and conformational an alys is of glyca n moieties ill silll on isotopically IJC,I S -cnriched recombi nalll human chori onic gonadotropin, Bio· chelllistrv. 35 ( 1996) 88 15.

32 \-l cikoop J C, Va n den Boogaan P, Dc Lccuw R, Rose U M, Mu ldc rs J W M & Grootcnhuis P D J, Part ially ckglycosy­lated human chori onic gonadot ropin , stabilized by intcr­subun it di su lfi de bonds, shows ful l bioactivit y, EliI' J Rio· ('helll, 253 ( 1998) 354.

83 Huth J R, Moun tjoy K, Perini r. Bedows E & Ruddon R W, Domain- dependcnt protein folding is indicatcd hy the intracellu lar kinet ics of disu lfide bond format ion of human chorion ic gonadotropin ~ sub un it. J Bioi Clielll , 267 (1992) 2 1396.

11-+ \-l uth J R, Fcng W & Ruddon R W, Rcdox conditions for sti mu lation of ill vill'U fo lding and assc mbly of the glyco­protein hormonc chorion ic gonadotropin, 8iOleclllIOI Bio· (, lIgilleer, 44 ( 1994) 66.

85 Moyle W R, Campbc ll R K, Rao S N V, Ayad G, Ber­nard M P, Han Y & Wa ng Y, Mode l of hum:1I1 chorionic gonadotropin and lutropin receptor intcraction that explains

504 INDIAN J EXP BIOL, APRIL 2002

signal transduct ion of the glycoprotein hormones, J Biol Chem, 270 (1995) 20020.

86 Huth J R, Perini F, Lockridge 0, Bedows E & Ruc1don R W, Protein folding and assembly in vitro parallel intrace llu­lar folding and assembly: catalysis of folding and assembly of the human chorionic gonadotropin a~ dimer by protein disu lfide isomerase, J Biol Chem, 268 (1993) 16472.

87 Xing Y, Williams C, Campbell R K, Cook S, Knoppers M, Addona T, Altarocca V & Moyle W R, Threading of a gly­cosylated protein loop through a protein hole: implications for combination of human chorionic gonadotropin su units , Prot Sci, 10 (200 I) 226.

88 Weisshaar G, Hiyama J & Renwick A G C, Site-specific N-glycosy lation of ov ine lutropin : structural ana lys is by one- and two- dimensional I H-NMR spectroscopy, Eur J Biochem, 192 (1990) 741.

89 Bousfield G R, Baker V L, Gotscha ll R R, Butnev V Y & Butnev V Y, Carbohydrate analysis of glycoprotein hor­mones, Meth ods, 21 (2000) IS.

90 Sairam M R & Manjunath P, Comparison of the thermal stability characteristics of native and deglycosy lated ovine pituitary lutropin, Int J Pept Protein Res, 19 (1982) 3 15 .

91 Sairam M R & Manjunath P, Studies on pituitary follitropin XII. Enhanced thermal stability induced by chemica l de­glycosy lat ion, Mol Cell Endocrillol, 28 (1982) 151.

92 Manjunath P & Sairam M R, Enhanced thermal stability of chemically deglycosylated human choriogonadotropin, J Bioi Chem, 258 ( 1983) 3554.

93 Galet C, Chopincau M, Martinat N, Combarnous Y & Guil­lou F, Expression of an in vitro biologically active equine LH/CG withou t C-terminal peptide (CTP) and/or ~26-11 0 di sulphide bridge, J Endocrinol. 167 (2000) 117.

94 Roess D A, Brady C J & Barisas B G, Biological function of the LH receptor is associated wit h slow receptor rota­tional diffusion, Biochilll Biophys Acta, 1464 (2000) 242.

95 Bousfield G R & Butnev V Y, Therlllal stability of FSH preparations. 34th Annual Meeting of the Society for the Swdy of Reproducti on , Oltawa, 200 I.

96 Gu ill ou F & Combarnous Y, Purification of equine gonad­otropins and comparative study of their acid-di ssoc iation and receptor-binding spec ificity, Biochilll Biophys Acta, 755 ( 1983) 229.

97 Liu W-K, Bousfield G R, Moore W T, Jr. & Ward D N, Priming procedure and hormone preparations influence rat granulosa cel l response, Endocrinology, 11 6 ( 1985) 1454.

98 Liu C & Bowers L D. Mass spect rometric characteri zation of the beta-subunit of human chorionic gonadotropin. J Mass Spectrolll, 32 ( 1997) 33 .

99 Hanover J A, Elting J, Mintz G R & Lennarz W J, Tempo­ral aspects of the N- and O-glycosy lation of human chori ­onic gonadotropin , J Bioi Chelll, 257 (1982) 10 172.

100 Muyan M, Furuhashi M, Sugahara T & Boime I, The car­boxy-termina l region of the ~- subunit s of luteini zing hor­mone and chori onic gonadotropin differentially influence secretion and assembly of the heterodimers, Mol Endocri­nol. 10 (1996) 1678.

10 1 Muyan M & Boime I, The carboxy l-terminal region is a de­terminant for the intracellular be havior of the chori onic go­nadotrop in ~ subunit: effects on the processing of the Asn­linkcd oli gosaccha rides, Mol Endocrinol. 12 ( 1998) 766.

102 Tarentino A L, Gomez C M and Plummer T H, Jr. , Degly­cosy lat ion of asparagine-linked glycans by peptide-N­glycosidase F, Biochemistry, 24 ( 1985) 4665.

103 Walton W 1, Nguyen V T, Dias .J A, Bidart J M & Bous­field G R, Glycosylation patterns in fou ... groups of hUlllan FSH isoforms, 81st An nual Meeting of the Endocri ne Soci­ety, Toronto, 2000.

104 Gordon W L, Bousfield G R & Ward D N, Comparative binding of FSH to chicken and rat testi s, J Endocrinol In ­vest, 12 (1989) 383.

105 Peckham W D, Yamaji T, Dierschke D J & Knobil E, Go­nadal function and the biological and physicochemical properties of follicle st imulating hormone , Endocrinology, 92 (1973) 1660.

106 Bogdanove E M, Campbe ll G T & Peckham W D, FSH pleomorphism in the rat--regulat ion by gonadal steroids, Endocr Res COI/1/1/un. I (1974) 87.

107 Flack M R, Froehlich J, Bennet A P, Anasti J & Nisul a B C, Site-d irected mutagenesis defines the individual roles of the glycosylat ion sites on folli cle-stimulating hormone, J Bioi Chem. 269 ( 1994) 14015.

108 Park H & Lennarz W J, Evidence for interac tion of yeast protein kinase C with several subunits of oligosaccharyl transferase, Glycobiology, 10 (2000) 737.

109 Braden T D, Brevig T & Conn P M, Protein kinase-C acti­vat ion stimul ates sy nthesis of gonadotropin- releasing hor­mone (G nRH) receptors, but does not mediate GnRH­stimulated receptor sy nthesi s, Endocrinology. 129 (1991 ) 2486.

110 Szkudlinski M W, Thotakura N R, Tropea J E, Grossmann M & Wei ntraub B D, Asparag ine- lin ked ol igosaccharide structures determine clearance and organ distribution of pi ­tuitary and recombinant thyrotropin , Endocrinology. 136 ( 1995) 3325.

III Bishop L A, Nguyen T V & Schofi eld P R, Both of the ~­subunit carbohydrate residues of folli cle-stimulating hor­mone determine the metabolic clearance rate and in vivo potency , Endocrinology, 136 ( 1995) 2635.

11 2 Baenziger J U, Kumar S, Brodbeck R M, Smith P L & Ber­anek M C, Circulatory half-life but not inte,action with the lutropin/chorionic gonadotropin receptor is modulated by sulfation of bovine lutropin oligosaccharides, Proc Nail Acad Sci USA. 89 ( 1992) 334.

11 3 Fiete D, Srivastava V, Hindsgau l 0 & Baenziger J U, A hepatic reti culoendothelial cell receptor specific for SOr 4Ga I NAc~ 1 ,4GlcNAc~ I,2Mana that mediates rapid clear­ance of lutropin , Cell. 67 ( 1991) 11 03.

11 4 Fiete D & Baenziger J U, Isolati on of the S04-4-GaINAc~ I ,4GlcNAc~ 1,2Mana-spec ifie receptor from rat li ver, J Biol Chelll , 272 (1997) 14629.

115 Fiete D, Beranek M C & Baenziger 1 U, The macro­phage/endothelial ce ll man nose receptor cDNA encodes a protein that binds oligosaecharides terminating with S04-4-GaINAc~ I ,4G lcNAc~ or Man at independent sites, Proc Natl Acad Sci USA, 94 ( 1997) 11256.

116 Fiete D J, Beranek M C & Baenziger J U, A cysteine- ri ch domain of the "mannose" receptor mediates Ga INA c-4-S04 binding. Proc Natl Acad Sci, 95 (1998) 2089.

117 Liu Y, Chir ino A J, Misulovin Z, Leteux C, Feizi T. Nus­senzwe ig M C & Bjorkman P J, Crystal structure of the cysteine- rich domain of mannose receptor cO ll1 plcxccl with

BOUSFIELD & BUTNEY: EQUINE GONADOTROPINS AS MODEL FOR GLYCOSYLATION 505

a sulfated carbohydrate ligand, J Exp Med, 191 (2000) 1105.

118 Burgon P G, Stanton P G & Robertson D M, In vivo bioac­tivities and clearance patterns of highly purified human lu­teinizing hormone isoforms, Endocrinology, 137 (1996) 4827 .

119 Risma K, Clay C M, Nett T M, Wagner T, Yun J & Nilson J H, Targeted overexpression of luteinizing hormone in transgenic mice leads to infertility, polycystic ovaries, and ovarian tumors, Proc Natl Acad Sci USA, 92 (1995) 1322.

120 Fares F A, Suganuma N, Nishimori K, LaPolt P S, Hsueh A J W & Boime I, Design of a long-acting follitropin agonist by fusing the C-terminal sequence of the chorionic gonad­otropin ~ subunit to the follitropin ~ subunit, Proc Natl Acad Sci USA, 89 (1992) 4304.

121 Drickamer K, Molecular structure of animal lectins, in Mo­lecular Glycobiology (Oxford University Press, New York) 1994,53.

122 Lefort G P, Stolk J M & Nisula B C, Evidence that desialy­lation and uptake by hepatic receptors for galactose­terminated glycoproteins are immaterial to the metabolism of human choriogonadotropin in the rat, Endocrinology, 115 (1984) 1551.

123 Green E 0 & Baenziger J U, Asparagine-linked oligosac­charides on lutropin, follitropin, and thyrotropin II. Distri­butions of sulfated and sialylated oligosaccharides on bo­vine, ovine, and human pituitary glycoprotein hormones, J Bioi Chem, 263 (1988) 36.

124 Rudd P M, Wormald M R, Stanfield R L, Huang M, Mattson N, Speir J A, DiGennaro J A, Fetrow J S, Dwek R A & Wilson I A, Roles for glycosylation of cell surface re­ceptors involved in cellular immune recognition, J Mol Biol, 293 (1999) 351.

125 Reame N, Sauder S E, Kelch R P & Marshall J C, Pulstatile gonadotropin secretion during the human menstrual cycle: evidence for altered frequency of gonadotopin-releasing hormon secretion, J Clin Endocrinol Metab, 59 (1984) 328.

126 Knobil E, Patterns of hypophysiotropic signals and gonad­otropin secretion in the rhesus monkey, Biol Reprod, 24 (1981) 44.

127 Robker R L & Richards J S, Hormonal control of the cell cycle in ovarian cells: proliferation versus differentiation, Bioi Reprod, 59 (1998) 476 .

128 Gore-Langton R ~ & Armstrong D T, Follicular steroido­genesis and its control, in The Physiology of Reproduction (Raven Press, New York) 1994, 571.

129 Andcrsen C Y, Leonardsen L, Ulloa-Aquirre A, Barrios-de­Tomasi J, Moore L & Byskov A G, FSH-induced resump­tion of meiosis in mouse oocytes: effect of different iso­forms, Mol Hum Reprod, 5 (1999) 726.

130 Sebok K, Sairam M R, Cantin M & Mohapatra S K, Distri­bution of follitropin and deglycosylated follitropin in the rat: a quantitative radioautographic study, Molec Cell En­docrinol, 52 (1987) 185.

131 Dunkel L, Jia X C, Nishimori K, Boime I & Hsueh A J, Deglycosylated human chorionic gonadotropin (hCG) an­tagonizes hCG stimulation of 3',S'-cyclic adenosine mono­phosphate accumulation through a noncompetitive interac­tion with recombinant human luteinizing hormones, Endo­crinology, 132 (1993) 763.

132 Dahl K D, Bicsak T A & Hsueh A J W, Naturally occurring anti hormones: secretion of FSH antagonists by women treated with a GnRH analog, Science, 239 (1988) 72.

133 Maghuin-Rogister G, Closset J & Hennen G, Differences in the carbohydrate portion of the u subunit of porcine lutro­pin (LH), follitropin (FSH) and thyrotropin (TSH), FEBS Lett, 60 (1975) 263.

134 Moore W T, Jr. & Ward 0 N, Pregnant mare serum gonad­otropin: rapid isolation procedures for the purification of intact hormone and isolation of subunits, J Bioi Chem, 255 (1980) 6923.

135 Bousfield G R & Ward 0 N, Purification of lutropin and follitropin in high yield from horse pituitary glands, J Bioi Chem, 259 (1984) 1911.

136 Jiang X, Dreano M, Buckler 0 R, Cheng S, Ythier A, Wu H, Hendrickson W A & Tayar N E, Structural predictions for the ligand-binding region of glycoprotein hormone re­ceptors and the nature of hormone-receptor interactions, Structure, 3 (1995) 1341 .

137 Moyle W R, Pressey A, Dean-Emig 0, Anderson 0 M, Demeter M, Lustbader J & Ehrlich P, Detection of confor­mational changes in human chorionic gonadtropin upon binding to rat gonadal receptors, J Bioi Chem, 262 (1987) 16920.

138 Cosowsky L, Rao S N Y, Macdonald G J, Papkoff H, Campbell R K & Moyle W R, The groove between the u­and ~-subunits of hormones with lutropin (LH) activity ap­pears to contact the LH receptor, and its conformation is changed during hormone binding, J Bioi Chem, 270 (1995) 20011.

139 Wang Y, Bernard M P & Moyle W R, Bifunctional hCG analogs adopt different conformations in LH and FSH re­ceptor complexes, Mol Cell Endocrinol, 170 (2000) 67 .

140 Mendelson C, Dufau M & Catt K, Gonadotropin binding and stimulation of cyclic adenosine 3':5'-monophosphate and testosterone production in isolated Leydig cells, J Biol Chem, 250 (1975) 8818.

141 Seth P K & Bahl 0 P, Human choriogonadotropin-induced coupling of receptor and Gs protein, are the effect of hor­mone deglycosylation, Mol Cell Endocrinol, 80 (1991 ) 105 .

142 Gether U & Kobilika B, G protein-coupled receptors II. Mechanism of agonist activation, J Bioi Chem, 273 (1998) 17979.

143 Ji T H, Grossmann M & Ji t, G protein-coupled receptors I. Diversity of receptor-ligand interactions, J Bioi Chem, 273 (1998) 17299.

144 Segaloff D L & Ascoli M, The lutropinichoriogonadotropin receptor. . .4 years later, Endocrine Rev, 14 (1993) 324.

145 Ji I & Ji T H, Human choriogonadotropin binds to a lutro­pin receptor with essentially no N-terminal extension and stimulates cAMP synthesis, J Bioi Chem, 266 (1991) 13076.

146 Remy J J, Bozan Y, Couture L, Goxe B, Salesse R & Gar­nier J, Reconstitution of a high-affinity functional lutropin receptor by coexpression of its extracellular and membrane domains, Biochem Biop!Jys Res Commun, 193 (1993) 1023.

147 Sairam M R, Hormonal antagonistic properties of chemi­cally deglycosylated human choriogonadotropin, J Bioi Chem, 258 (1983) 445.

506 INDIAN J EXP BIOL, APRIL 2002

148 Butnev V Y, Singh V, Nguyen V T & Gray C M, Trun­cated beta, a~paragine56 deglycosylated equine luteinizing hormone alpha is a potent FSH antagonist, 33rd Meeting of the Society for the Study of Reproduction, Madi son, WI, 2000.

149 Anderson W, Kang Y-A, Perotti M E, Bramley T A & Ryan R J, Interaction of gonadotropins with corpus luteum membranes. III. Electron microscopic localization of [1251]_ hCG binding to sensitive and desensitized ovaries seven days after PMSG-hCG., Bioi Reprod, 20 (1979) 362.

150 Luborsky J L, Slater W T & Behrman H R, Luteinizing hormone (LH) receptor aggregation: modification of fer­ritin-LH binding and aggregation by prostaglandin F2a and ferritin-LH, Endocrinology, 115 (1984) 2217.

151 Roess D A, Horvat R D, Munnelly H & Barisas B G, Lu­teinizing hormone receptors are self-associated in the plasma membrane, Endocrinology, 141 (2000) 4518 .

152 Rebois R V & Fishman P H, Antibodies against human chorionic gonadotropin convert the deglycosylated hor­mone from an antagonist to agonist, J Bioi Chem, 259 (1984) 8087.

153 Rebois R V & Liss M T, Antibody binding to the I)-subunit of deglycosylated chorionic gonadotropin converts the an­tagonist to an agonist, J Bioi Chem, 262 (1987).

154 Hattori M, Hachisu T, Shimohigashi Y & Wakabayashi K, Conformation of the 13 subunit of deglycosylated human chorionic gonadotropin in the interaction at receptor sites, Mol Cell Endocrinol, 57 (1988) 17.

155 Hattori M A, Shimohigashi Y & Wakabayashi K, Reversal of the antagonism of deglycosylated human chorionic go­nadotropin by aggregation with wheat germ agglutinin, Mol Cell Endocrinol, 66 (1989) 207.

156 McFarland K C, Sprengel R, Phillips H S, Kohler M, Rosemblit N, Nickolics K, Segaloff D L & Seeburg P H, Lutropin-choriogonadotropin receptor: an unusual member of the G protein-coupled receptor family, Science, 245 (1989) 494.

157 Cunningham B C, Ultsch M, de Vos A M, Mulkerrin M G, Clauser K R & Wells J A, Dimerization of the extracellular domain of the human growth hormone receptor by a single hormone molecule, Science, 254 (1991) 821.

158 Blum J J & Conn P M, Gonadotropin-releasing hormone stimulation of luteinizing hormone release: a ligand­receptor-effector model , Proc Natl Acad Sci USA , 79 (1982) 7307 .

159 Nelson S, Horvat R D, Malvey J, Roess D A, Barisas B G & Clay C M, Characterization of an intrinsically fluores­cent gonadotropin-releasing hormone receptor and effects of ligand binding on receptor lateral diffusion, Endocrinol­ogy, 140 (1999) 950.

160 Lefkowitz R J, G protein-coupled receptors: III. New roles for receptor kinases and b-arrestins in receptor signalling and desensitization, J Bioi Chem, 273 (1998) 18677.

161 Sanchez-YagUe J, Rodriguez M C, Segaloff D L & Ascoli M, Truncation of the cytoplasmic tail of the lutro­pin/choriogonadotropin receptor prevents agonist-induced uncoupling, J Bioi Chem, 267 (1992) 7217 .

162 Lamm M L & Hunzicker-Dunn M, Phosphorylatior· independent desensitization of the luteinizing hor­mone/chorionic gonadotropin receptor in porcine follicular membranes, Mol Endocrinol, 8 (1994) 1537.

163 von Zastrow M & Kobilika B, Ligand-regulated internali ­zation and recycling of human 132 -adrenerg ic receptors be­tween the plasma membrane and endosomes containing transferrin receptors, (1992).

164 Calvo F 0 & Ryan R J, Inhibition of adenylyl cycl ase activity in rat corpora luteal tissue by glycopeptides of human chorionic gonadotropin and the a -subunit of human chorionic gonadotropin, Biochemistry, 24 (1985) 1953.

165 Bousfield G R & Ward D N, Biologic activity of eLH and lice following removal of the C-terminal glycopeptide, 68th Annual Meeting of the Endocrine Society, Anaheim, CA,1986.

166 Bousfield G R, Liu W-K & Ward D N, Effects of removal of carboxy-terminal extension from equine luteinizing hormone (LH) 13-subunit on LH and fol licle-stimulating hormone receptor binding activi ties and LH steroidogenic activity in rat testicular Leydig cells., Endocrinology, 124 (1989) 379.

167 EI-Deiry S, Kaetzel D, Kennedy G, Nil son J & Puett D, Site-directed mutagenesis of the human chorionic gonad­otropin 13-subunit: bioactivity of a hetero logous hormone, bovine a-human des-( 122-145)13, Mol Endocrinol, 3 (1989) 1523.

168 Matzuk M M, Hsueh A J W, Lapoh P, Tsafriri A, Keene J L & Boime I, The biological role of the carboxyl-terminal extension of human chorionic gonadotropin 13-subunit, En­docrinology, 126 (1990) 376.

169 Butnev V Y, Gotschall R R, Baker V L, Moore W T & Bousfield G R, Negative influence of O-linked oligosac­chari des of high molecular weight equine chorionic gonad­otropin on its luteinizing hormone and follicle-stimulating hormone receptor-binding activities, Endocrinology, 137 (1996) 2530.

170 Min K-S, Hattori N, Aikawa J-I , Shiota K & Ogawa T , Site-directed mutagenesis of recombinant equine chorionic gonadotropinlluteinizing hormone: differential role of oli­gosaccharides in luteinizing hormone- and follicle­stimulating hormone-like activities, Endocr J, 43 (1996) 585.

171 Hiyama J, Surus A & Renwick A G C , Purification of hu­man pituitary LH and thyrotropin by hy rophobic chroma­tography, J Endocrinol, 125 (1990) 493 .

172 Weisshaar G, Hiyama J, Renwick A G C & Nimtz M, NMR investigations of the N-linked oligosaccharides at individual glycosylation sites of human lutropin, Eur J Bio­chem, 195 (1991) 257.

173 Kessler M J, Mise T, Ghai R D & Bahl 0 P, Structure and location of the O-glycosidic carbohydrate units of human chorionic gonadotropin, J Bioi Chem, 254 ( 1979) 7909.

174 Renwick A G C, Mizuochi T, Kochibe N & Kobata A, The asparagine-linked sugar chains of human follicle­stimulating hormone, J Biochem, 101 (1987) 1209.