an immunochemical approach to the identification of the mbta binding site of the nicotinic...

Vol. 122, No. 2, 1984

July 31, 1984

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 602-608

AN ~4UNOCHI~[ICAL APPROAC~I TO THE IDENTIFICATION OF THE MBTA BI~DING SITE OF THE NICOTINIC ACETYICHOLINE RECEPTOR OF TORPI~30 CALIFORNICA

Sean Cahill and Jakob Schmidt

Department of Biochemistry, State University of New York at Stony Brook, Stony Brook, NY 11794-5215

Received June 15, 1984

SUMMARY Monospecific anti-[4-(N-maleimidobenzyl)trimethylanmonit~n] (~TA) antibodies were prepared frc~ sera of rabbits irmlunized with an albtm/n-~TA conjugate and used to synthesize an MBTA-specific irsntmosorben~. Torpedo californica acetylcholine receptor was affinity labeled with [ H]-~TA and proteolyzed extensively with pronase, and the peptide fraction of the digest chrcmatographed on the anti-5~rfA resin. The amino acid ~mposition of the purified 5STA-peptide fraction was compared with the sequences fl~ing the seven cysteinyl residues of the ~-subunit. The best fit was observed with the segment containing cysteine 142.

The nicotinic acetylcholine receptor (AcChR)1 from Torpedo californica

electric tissue is coni0osed of 5 subunits, arranged in a rosette-like

fashion, in the molar ratio ~2 ~76, all of which have recently been sequenced

by application of recc~binant DNA technology (1-4). %~lile the pentameric

conplex contains the ~lete signal transduction machinery including the

cation channel (5), the ~-subunit is believed to contain part or all of the

acetylcholine (AcCh) binding site (6). Much of this evidence derives fr~

affinity labeling studies with radioactive compounds (tritiated 4-(N-

maleimido-benzyl) trimethylarr~onium (MBTA) and bromoacetylcholine) that

contain a moiety highly reactive toward free thiols. Such a thiol function

can be generated near the AcCh binding site by treatment with dithiothreitol

(7). Affinity labeling with reagents of varying dimensions has suggested

that the distance between the negative subsite that binds the quaternary

anmonium group and the sulphydryl function formed upon reduction is about 0

i0 A. Thus it is reasonable to assume that identification of the cysteinyl

i Abbreviations: AcCh, acetylcholine; AcChR, acetylcholine receptor; MBTA, 4- (N-maleimidobenzyl) trimethylammonium.

0006-291X/84 $1.50 Copyright © 1984 by Academic Press, Inc. All rights of reproduction in any form reserved. 602

Vol. 122, No. 2, 1984 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

residue susceptible to affinity labeling will provide additional information

on the AcCh binding site, as amino acyl residues close by in the primary

structure are likely themselves to be constituents of the site.

The Torpedo californica AcChR ~-subunit contains only seven cysteinyl

residues. Since the sequence surrounding each of them is known, it should

be possible to identify the MBTA-reactive residue by determining the amino

acid ccr~position of the MBTA peptide(s) isolated from a proteolytic digest

of affinity-labeled AcChR. Here we report on the results of such an

experiment.

~TERIALS AND ME~{ODS

(I) Preparation and quantitation of AcC~/~: Frozen T0rpedo californica electric tissue (Pacific Bicr~arine, Venice, CA) was thawed in 3 volumes of homogenization buffer (I0 mM sodium phosphate, 10 n~M sodium azide, 5 mM sodium ~3TA pH 7.0), hcmogenized in a Waring blendor, and centrifuged briefly at low speed. A receptor-enriched membrane preparation was obtained by ultracentrifugation of the resulting supernatant, and resuspended in a small volume of homogenization buffer containing 1% Triton X-100. After brief agitation detergent-insoluble matter was removed by c~rifugation. The concentration of solubilized AcChR was determined with ---I-~- bungarotoxin using the DEAE-cellulose disk technique (8). (2) Affinity labeling of AcChR: Affinity alkylation of the detergent- solubilized receptor was carried out by the method of Karlin (9). Briefly, the sarsple was reduced with dithiothreitol at pH 8.0 and separated from excess reducing agent by chromatography on Bio-Gel P-6. The reduced receptor was then treated at pH 7.0 ~ith a slight excess of tritiated MBTA and rechr _~_~atographed on P-6. The [ HI-MBTA used was a mixture of (methyl-[ H] ) -~3TA obtained from New England Nuclear (batch 1784-070) and non-radioactive MBTA ~ynthesized by the method of Karlin (9). The specific activity of reacted [~H]-MB~I~ was estimated by dividing the amotmt of H, incorporated specifically into AcChR (as defined by co-migration with the 15~ubunit in SDS polyacrylamide gel electrophoresis), by twice the number of -~I-~-bungarotoxin binding sites. (3) Proteolysis of labeled AcChR: Labeled receptor was dialyzed against 0.1% SDS in 1 mM Tris-HCl pH 7.2, lyophilized, and redissolved in a small vol~ne of 50 mM ammonium acetate, 5 mM CaCl 9 pH 7.5. It was then digested, at 37°, by repeated additions of pronase (S£reptcrmyces griscus protease VI, Sigma) to the concentrated sanple solution at an enzyme to protein ratio of approximately I:I0, and the progress of proteolysis over several days monitored by chrcmatographing small aliquots on Sephadex G-50. The digest was finally fractionated on G-25, and all radioactive material eluting after the void volume peak was pooled ("peptide fraction"). (4) Construction of anti-MBTA ~osorbent: Rabbit se/nan alb~md_n was isolated frcm normal rabbit "serum by a ccmbination of ammonium sulfate precipitation and DEAE-cellulose chromatography. The albtmtin was thiolated by the N-acetylhcmocysteine-thiolactone procedure of White (ref. I0, protocol A) and after adjusting the pH of the reaction mixture to ~. 1, reacted with MBTA, which had been doped with a small quantity of [ H]-MBTA. MBTA-albtm~in was subsequently separated frGm ~nall molecular weight cc~s by gel filtration on Sephadex G-50. Six mg of carrier protein containing about 15 MBTA moieties per molecule were obtained. Four rabbits were immunized with emulsions of MBTA-albt~in in cc~plete Freund's adjuvant

603


4

Ld z_

I

I0 20 30

FRACTION NUMBER

Fig. 1 Preliminary test of anti~TA immunosorbent.

One ml of anti-MBTA IgG Affigel-10 (open circles) was placed in a Pasteur pipet colunm (equilibrated in 150 ~4 sodit~n chloride, 10 mM sodi~ phosphate, 0.02%~sodium azide pH 7.4 - 'PBS'), and incubated with 0.4 ~Ci (i0 piccmoles) [JH]-MBTA (hydrolyzed by exposure to PBS to prevent reaction with protein sulphydryl groups) overnight. The coltmm was washed with PBS, and strongly retained material eluted with 1% acetic acid (arrow). Fractious of 1.5 ml were collected, and 0.5-ml aliquots counted after mixing with 3.5 ml Biofluor (N~q) . The eluted resin was removed frGm the pipet, mixed with Biofluor and counted (single data point in parenthesis). Filled circles indicate data obtained with a l-ml sample of control resin (nonspecific rabbit IgG coupled to Affigel-10; elution with acetic acid indicated by V). Values are given in units of 100,000 and 250,000 cpm for experiment and control, respectively.

by repeated intrade_r~al injection. Two of the immunized animals developed titers exceeding i0 ~ M over a per~ of 6 weeks; titers were determined by double immune precipitation using---I-labeled MBTA albumin as antigen and goat anti-rabbit immunoglobulin antibodies as precipitant. In~une precipitation was inhibited 50% by MBTA; 4-(N-maleamido)benzyldimethylamine (precursor in MBTA synthesis); tetramethylammoni~n; and carbamoylcholine at 0.05; 2; I0; and i0 mM, respectively. Monospecific antibodies were isolated on a resin containing ~TA-albumin coupled to agarose via an azo linkage (ii). Fifty mg (ca. 300 nancmoles) of IgG with an antigen binding capacity of ca. 100 nancmoles were obtained. Anti-MBTA antibody (ca. 25 rag) was coupled to 8 ml Affigel-10 (Bio-Rad) following the instructions of t~e manufacturer. The resulting affinity resin was capable of binding [~H]-MBTA (Fig. i). Alternatively, anti-MBTA antibody was coupled to Sepharose by the CNBr procedure (12). (5) Amino acid analysis: Affinity-purified samples were hydrolyzed in 6 N HCI at ii0 ° for 22 hours prior to analysis.

RESULTS AND DISCUSSION

Extracts of electric tissue were prepared and labeled with [3H]-~TA of

low specific activity, resulting in 45 and 30 nanc~oles, respectively, of

affinity-alkylated sites in two separate experiments. Electrophoretic

604


o

0.~

0.1

t ~ (L r t

I0 2 0 3 0

FRACTION NUMBER

5

?

Fig. 2 Affinity chromatography of t~TA-peptide(s).

The peptide fraction from a [3H]-MBTA receptor hydrolysate was isolated by chrcmatngraphy on Sephadex G-25, and ca. 20 nanomoles of MBTA-peptide loaded onto an ~osorbent column (35 ml of Sepharose 4B-CL containing covalently coupled ca. 35 mg anti-MBTA IgG) in i0 mM sodi~n phosphate, 0.02% sodium azide pH 7.4 ('start buffer'). After several hours elution was begun with start buffer; ll-ml fractions were collected and assayed for absorbance at 280 nm (O) and tritium (cpm per 0.05 ml,~. At fractions 5 and 20 elution was started with PBS and 1% acetic acid, respectively (arrows).

analysis revealed that about 50% of the total incorporated 3H migrated to

the position of the ~-subunit suggestive of specific labeling . The

pronase-digested receptor preparation was fractionated on Sephadex G-25 from

which the bulk of the labeled material eluted in a broad peak between

neurotensin and angiotensin II (peptides of 13 and 8 amino acids,

respectively), i.e. like a mixture of oligopeptides averaging ten residues

in size. The peptide fraction was subjected to immttnosorbent chromatography

(Fig. 2). Rechromatography of the specifically retained material yielded

only marginal further purification. After desalting on Sephadex G-10, the

final product was subjected to amino acid analysis whose results are shown

in Table I.

Pronase digestion obviously does not yield a unique ~BTA-peptide and

was neither expected nor intended to do so. Since the primary structure of

the ~-subunit is known, knowledge of the amino acid composition of a

labeled peptide or even a mixture of labeled peptides may suffice to

identify the alkylated site. There are only seven cysteine residues in the

mature ~-subunit and the site of MBTA binding can therefore be established

by comparison of an ~imentally observed amino acid cenloosition with the

605


Table I Amino acid composition of affinity-purified peptide(s)

Residues per mole ~BTA

Asp, Asn 1.71 Thr 0.72 Ser 2.28 Glu, Gln 3.15 Pro 0.82 Gly 4.69 Ala 1.64 Cys, cysteic acid Val 1.30 Met 0.23 Ile 0.63 Leu 1.10 Tyr 0.42 Phe 0.50 His 0.52 Lys 0.48 Arg 0.40

Fractions 12 to 24 and 27 to 29 of Fig. 2 were pooled, desalted, lyophilized, and repurified by ~osorbent chromatography. The desalted preparation was subjected to acid hydrolysis followed by amino acid analysis.

amino acid environments of the seven cysteines in the primary structure of

the polypeptide chain. Such a comparison is presented in Fig. 3. It

reveals that cysteine 142 and the sequence surrounding it best fit the

composition of the peptide mixture isolated on an affinity label-specific

immunosorbent.

The high background observed is not unexpected. There are several

factors contributing to it: (a) one obvious reason is that there are amino

acyl residues common to two or more candidate sequences. Thus the single

residue of isoleucine observed in the MBTA-peptide hydrolysate n~st be

credited to any one of the seven candidate sequences, all of which carry it

within nine positions from the cysteine residue. Similarly leucine,

phenylalanine, aspartate/asparagine, and valine occur in the majority of the

regions under investigation. Upon elimination of multiple assignments the

signal-noise ratio improves significantly (see parenthetical figures in

Fig. 3) . In addition (b) nonspecifically reacted MBTA (estimated to account

for about half of all incorporated affinity label) will result in an over-

representation of abundant amino acids such as alanine and valine.

Finally (c), the disproportionate amounts (with respect to receptor

606


AMINO ACID SEQUENCE CYSTEINE "LONGEST BEST DEKA- POSITION ~ _ ' _ PEPTIDE" PEPTIDE FiT

%

128 3 6~9 (LI (401 WTPPAI[KSYCEIIVTHFPFD

142 VTHFPFDQQNCTMKLGIWTYD

192

8 81.4

2 65.0 RGWK HWVYYTCC P D T P Y L D I T (I) (16)

193 I m I ~ m J ~ 4 63,0 (L51

2Z2 4 70.5 L Y F V V N V I I P C L L F S F L T G L V { ) (3i)

M V I O H I L L C V F M L I C I I G T ( ) (37)

418 HILLCVFMLICIIGTVSVFAG

5 69.5

Fig. 3 Matching the observed amino acid ~sition with candidate sequences.

On the left the position numbers and primary structure enviror~ents of the seven cysteinyl residues of the Torpedo californica AcChR e-subunit are shown. Observed amino acid stoichicmetries are indicated as bars. The bar graph is constructed by filling amino acid positions to the maximal extent from observed molar ratios (see Table I), starting at the centrally positioned cysteine (which is pre~ to be present in the hydrolysate, although MSTA-cysteine is not eluted off polystyrene sulfonate in the automated amino acid analysis procedure) and going 10 residues in either direction. Cross-hatched bars indicate that although a specific amino acid was present in the hydrolysate, it should not be counted twice in a given matching procedure. Two matching protocols were Employed: (a) establishing the longest possible uninterrupted peptide surrounding a cysteinyl residue. Molar fractions are rounded to the nearest integer (i.e. values of 0.50 and higher are counted as 1.0, while lower values are ignored). Such peptides are indicated by a double arrow immediately above the bar graph - (b) selecting the cysteine decapeptide best matched by the observed amino acid cQmposition and calculating the fractional fit (long double arrow). The values in parenthesis show how reserving available amino acids for the leading candidate (cysteine 142) affects the candidacy of the other cysteines. The values are obtained by first filling hexapeptide sequences on either side of cysteine 142 with residues from the peptide hydrolysate (Table I) and then matching, one at a time, pairs of hexapeptides flanking the other cysteines with the remaining amino acids.

cc~0osition) of glycine, alanine, and serine may represent contam/_nating

free amino acids in the buffers and media used.

The peptide ~ t surrounding cysteine 142 has previously been

suggested as a candidate for the AcCh binding site by Noda et al. (I).

Their argument can be summarized as follows: Based on the hydrophilicity of

607

Voh 122, No. 2, 1984 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the polypeptide chain only four of the seven cysteinyl residues are likely

to be located on the surface of the receptor, namely cysteines 128, 142,

192, and 193. Application of the Chou and Fa~an (13) rules predicts that

the region of cysteine 128 and 142 encc~passes a B-turn structure. In

addition, a carboxylate side chain as required for a choline binding subsite

is present (glutamate 129 or aspartate 138). Asparagine 141, the immediate

neighbor of cysteine 142, is the only possible site of N-glycosidic linkage

and therefore likely to be located on the extracellular surface of the

receptor molecule where AcCh binding must occur.

In agreement with these considerations, the results presented here

strongly suggest cysteine 142 as a component of the AcCh binding site. With

the advent of microscale amino acid analysis and peptide sequencing

techniques the immunochemical approach described in this c~L~fdnication may

prove fruitful in the characterization of the binding sites of other

proteins for which specific covalent labels are available.

ACKNOWIZIXIMENTS

This research was supported in part by PHS grant NS 18839. We thank

Dr. M. Elzinga and Mr. N. Alonzo, Dept. Biology, Brookhaven National

Laboratory, for performing the amino acid analysis.

REFERENCES

I. Noda, M., Takahashi, H., Tanabe, T., Toyosato, M., Furutani, Y., Hirose, T., Asai, M., Inayama, S., Miyata, T., and Nt~na, S. (1982) Nature 299, 793-797.

2. Noda, M., Takahashi, H., Tag~ibe, T., Toyosato, M., Kikyotani, S., Hirose, T., Asai,M., Takashima, H., Inayama, S., Miyata, T., and Numa, S. (1983) Nature 301, 251-255.

3. Noda, M., Takahashi, H., Tanabe, T., Toyosato, M., Kikyotani, S., Furutani, Y., Hirose, T., Takashima, H., Inayama, S., Miyata, T., and Numa, S. (1983) Nature 302, 528-531.

4. Claudio, T., Ballivet, M., Patrick, J., and Heinemann, S. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 1111-1115.

5. Changeux, J.P. (1981) The Harvey Lectures 75, 85-254. 6. Damle, V.N. and Karlin, A. (1978) Biochemistry 17, 2039-2045. 7. Karlin, A. (1980) in Cell Surface Reviews Vol. 6, eds. Co,nan, C.W.,

Poste, G., and Nicolson, G.L. (North Holland, Amsterdam), pp.191-260. 8. Schmidt, J. and Raftery, M.A. (1973) An alyt. Biochem. 52, 349-353. 9. Karlin, A. (1977) Methods Enzymology 4_66, 582-590. I0. White, F.H. (1972) Methods Enzymology 25, 541-546. ii. Cohen, L.A. (1974) Methods Enzymology 34, 102-107. 12. Porath, J. (1974) Methods Enzymology 34, 13-30. 13. Chou, P.Y. and Fasman, G.D. (1978) Annu. Rev. Biochem. 47, 251-276.

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an immunochemical approach to the identification of the mbta binding site of the nicotinic...

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