254

Biochimica et Biophysica Acta, 354 ( 1 9 7 4 ) 2 5 4 - - 2 6 3 © Elsevier Scient i f ic Publ i sh ing C o m p a n y , A m s t e r d a m - - P r in ted in The Ne the r l ands

BBA 2 7 4 3 9

THE TIMECOURSE OF COLLAGEN CROSS-LINKING

J E F F R E Y S. PINTO* and J. P E T E R B E N T L E Y

Biochemistry Department, University of Oregon Medical Scholl, Portland, Oreg. 97201; (U.S.A.) (Rece ived F e b r u a r y 4 th , 1974)

Summary

The incorporation of [3 H]proline into all major denaturation subunits of acid-extractable skin collagen was followed in vivo over a period of 30 days in young male rats. The radioactivity was incorporated into the al and a2 sub- units to an equal extent in less than 1 day and disappeared from these subunits at an equal rate. It was then transferred to the ~ subunits, the ratio of specific activity of a:~ becoming unity at about 17 days. No transfer of radioactivity from ~ to 7 collagen was seen during the 30 days of the experiment indicating that ~ units do not cross-link further to 7 during this time. A separate group of rats made lathyritic with ~-aminopropionitrile showed a similar rate of collagen cross-linking as reflected by the return of a:~ ratios to normal values within about 15 days following the cessation of ~-aminopropionitrile administration. In the normal rats radioactive proline was incorporated very rapidly into a fraction of collagen eluted from CM-cellulose with 0.5 M NaC1 following the salt gradient. It is proposed that this fraction may represent a fibrogenesis nucleation factor.

Introduct ion

Collagen molecules are composed of three polypeptide chains. The col- lagen of rat skin consists of two identical a l chains and one dissimilar a2 chain and under mild denaturat ion conditions, the molecule can be dissociated into its three const i tuent chains. With increasing time, after the biosynthesis of the molecule, covalent cross-links are introduced between specific residues on the monomer (~) polypeptide chains giving rise to dimers and trimers. Dependent upon which monomer chains are cross-linked, the resultant dimers (/3) and trimers (7) will differ; thus, cross-link formation between two a l chains will give rise to a fll 1 unit, and between two al and one a~ will give rise to a 7112

* Present address: S t i e fe l Research Ins t i tu te , Oak Hill, N.Y. 12460, U.S.A.

255

unit. Cross-links arising within a single molecule are referred to as intramolecu- lar cross-links, while those involving a chains of two adjacent molecules are referred to as intermolecular cross-links. The mechanism of formation of these cross-links has been fairly well characterized, and it is known that intramolecu- lar cross-links arise by aldol condensation of two lysine-derived aldehydes present near the N-terminal end of each a chain. These aldehydes are produced by oxidative deamination of the e-amino groups of specific lysine residues in the protein chain, this reaction being catalyzed by a specific connective tissue lysyl oxidase. By contrast, lysine-derived intermolecular cross-links are formed from one allysine (the lysine-derived aldehyde) and the e-amino group of a lysine residue in an adjacent chain (for a recent review of collagen cross-linking mechanisms see Tanzer [1 ]). Condensation of two such residues gives rise to an unsaturated Schiff base which is very slowly reduced in vivo to a stable cross- link. Bailey [2] has shown that this reduction process takes place over a period of several months, and during this period the unsaturated Schiff base is labile under dilute acid conditions. In contrast to this very slow formation of stable intermolecular cross-links, very little is known about the time course of intra- molecular cross-link formation. In vivo pulse labeling experiments by Martin et al. [3] and by Orekhovich et al. [4] suggest that radioactivity initially incor- porated into a subunits is gradually transferred to ~ subunits. However, since the studies of Martin et al. [3] were carried out for only 7 days, and those of Orekhovich et al. [4] for only 6 days, the complete time course of intra- molecular cross-linking has not been established. It is to this problem that we have addressed ourselves. We chose to extract collagen from rat skin with dilute acetic acid since it has been shown [2] that this reagent labilizes unreduced intermolecular cross-links even at 4 ° C, whereas the aldol condensation products are unaffected. The more recently described intermolecular cross-link aldol-- histidine [26] is also labile under the conditions used (Mechanic, G., personal communication). The study would therefore not be complicated by the pres- ence of intermolecular bonds.

Materials and Methods

25 young male Long Evans rats (200--300 g) were each injected intra- peritoneally with a single dose of 100 #Ci [U -3 H] proline. At 1, 5, 10, 20 and 30 days following administration of the labeled proline, five animals were killed by decapitation, shaved, and the entire back skin removed. The skin was scrap- ed free of subcutaneous fat and was frozen in liquid nitrogen. It was then crushed in a stainless steel mortar and extracted with 0.5 M acetic acid at 4°C as described previously [5] without prior neutral salt extraction.

Each skin was processed separately. The acetic acid extracts were clarified by centrifugation and the collagen precipitated by the addition of NaC1 to 5%. The precipitate was redissolved in 0.01 M acetic acid and reprecipitated with NaC1. It was again dissolved in 0.01 M acetic acid and the solution dialyzed against 0.01 M disodium phosphate. The precipitate which formed was dissolv- ed in 0.01 M acetic acid and the solution was clarified by centrifugation for 1 h at 40 000 × g. Carboxymethyl-ceUulose chromatography of the collagen sub- units was carried out by a slight modification of the method of Piez et al. [11] .

256

Aliquots containing 0.5 mg of purified collagen were heated to 40°C for 30 min to denature the collagen. These were applied to jacketed columns of CM-cellulose (Whatman CM 32) 2.5 cm diameter and 15 cm in height heated at 40°C and eluted at a rate of 120 ml/h with a linear gradient of NaC1 from 0 to 0.0775 M superimposed over 800 ml of sodium acetate buffer, pH 4.8, I = 0.06. The column effluent was continuously monitored at 230 nm in a Zeiss PMQ II spec t rophotometer and 10-ml fractions were collected. Following the linear salt gradient, stepwise elution was continued first with 80 ml of sodium acetate buffer, pH 4.8, I = 0.06, containing 0.5 M NaC1 followed by sodium acetate buffer, pH 4.8, I = 0.06, made 6.0 M in urea and 1.0 M in NaC1 accord- ing to the technique of Veis and Anesey [7] . These two latter steps eluted additional peaks of 230-nm absorbing material. Those fractions under each peak were collected as shown in Fig. 1, pooled, dialyzed salt free, hydrolyzed and were assayed for hydroxyprol ine on a Technicon Auto Analyzer by the method of Grant [8] . They were assayed for radioactivity in a Packard Tri- Carb liquid scintillation spect rophotometer employing a Scintisol--toluene-- PPO fluor and correcting for quenching by automatic external standardization. Specific activities of each collagen fraction were calculated for each animal and expressed as dpm/pmole hydroxyproline.

Recovery from lathyrism In order to determine whether the rate of cross-linking is influenced by

excess amounts of uncross-linked collagen, we made 25-age-matched rats lathy- ritic by a daily intraperitoneal injection of fi-aminopropionitrile fumarate (Al- drich} in physiological saline at a dosage of 100 mg/100 g body weight. 25 control rats were injected with physiological saline only. Following administra- tion of fl-aminopropionitrile for 17 days, the t reatment was discontinued and the animals kept on a normal diet for 0, 4, 8, -12 and 16 days at which time five control and five fi-aminopropionitrile-treated rats were killed. The content of 0.45 M neutral salt~extractable collagen from skin was moni tored throughout the experiment. The content of neutral salt-soluble collagen from aorta and the ~:/3 ratios of collagen extracted from skin with 20% lactic acid were monitored during the recovery from lathyrism. The neutral salt and lactate extractions were performed as described previously [9] and the separation of ~,/3 and 7 subunits was carried out by polyacrylamide disc gel electrophoresis using the technique of Nagai et al. [10] . Quantification of the subunits was performed using a Gilford recording gel scanner following staining of the gels for 1 h in amido black (0.5% amido black in 7% acetic acid), and destaining overnight in 7% acetic acid.

Results and Discussion

CM-cellulose separation Fig. 1 shows a representative separation of the denaturation subunits of

collagen on CM-cellulose at 40°C. The elution profile is characteristic of that seen by other workers in acid extracts of skin collagen [7 ,11,12,13] . The componen t fi22 which contains an intermolecular cross-link is absent, probably reflecting the lability of the aldimine cross-link in 0.5 M acetic acid. fi22 is

257

0.50

~ 0.40

~o.~o

t 0.20

0.10

0 m I00

al p i 2

A p..~ / \ / \ .2

2~" " ~ - - ~ - ~;o-- Elution (mr)

L~/2~1.06 6. u._~l

[1"o ~_~.---~ _ - 6OO 7OO

F i ~ 1. Fract ionat ion of denatured col lagen by chromatog~raphy on CM*cellulose. The bars o n the absc issa represent por t ions o f the c o l u m n ef f luent used for r a d i o a s s a y .

normally found in collagen extracted under milder conditions and elutes imme- diately after ~2 [13] . The component 711 ~ which also contains an intermo- lecular cross-link was also absent in these chromatograms. This component usually elutes as a shoulder on the trailing edge of the ~ ~ subunit [7,14] . The minor componen t 7~ ~ 2 was seen directly between the two subunits, as depict~ ed in Fig. 1, or as a shoulder on the leading edge of 13t ~. Following the salt gradient, elution with acetate buffer, pH 4.8, containing 0.5 M NaC1 always brought of f an additional component , the salt (S) fraction, which appeared as either a double peak, shown in Fig. 1, or a single peak with a shoulder. Further elution with the same buffer containing 6.0 M urea and 1.0 M NaC1, produced an additional component , the urea peak (U) which appeared either as a single peak with a shoulder as shown, or a double peak. Pre-~t seen here as an unretarded componen t eluting near the void volume at the beginning of the salt gradient, should be distinguished from the pre-a~ fraction of Bellamy and Bornstein [15] which is thought to be a metabolic precursor of a~, and which elutes as a shoulder on the leading edge of a 1. The designation "pre-~ 1 " in the present s tudy refers only to the elution position of the component and not to its metabolic fate.

Amino acid analysis of the S, U and pre-a~ peaks (Table I) shows that whilst the S and U peaks have a composit ion typical o f collagen, the pre-a~ peak does not. Piez et al. [11] have reported that the p r e ~ component increases with more drastic denaturation conditions and is dialyzable. Our stud- ies confirm this and furthermore we found that this fraction is unretarded on polyacrylamide gel electrophoresis carried out in sodium dodecylsulfate.

Rate of cross-linking Fig. 2 shows the average specific activities of the a l , a2, fi] 1, and f3~ 2

collagen subunits, during the 30-days post-labeling. Five animals were used for each t ime point. Individual CM-cellulose separation and radioactivity assays

258

T A B L E I

A M I N O ACID C O M P O S I T I O N OF R A T SKIN C O L L A G E N S U B U N I T S

(expressed as r e s i d u e s / 1 0 0 0 residues)

Pre-a 1 S U

H y d r o x y p r o l i n e 62 .5 111 .8 109.3 Aspar t ic acid 53.2 46.6 53.1 T h r e o n i n e 23.2 21 .5 21.7 Serine 80 .7 40.1 41.7 G l u t a m i c acid 91 .5 74.6 77.9 Prol ine 126 .3 121.9 129.1 Glyc ine 309.1 339 .8 317 .3 Alanine 101.1 98 .5 102 .5 Cys te ine 0 0 0 Valine 19.5 20.9 21 .0 Meth ion ine Trace Trace 5.7 I so leuc ine 12.6 11.3 10.3 Leuc ine 22.6 21.9 23.6 Tyros ine 6.6 2.5 3.2 Pheny la l an ine 11 .4 7.9 11 .5 H y d r o x y l y s i n e 2.6 4.4 4.1 Lys ine 36.6 25 .4 21.9 His t id ine 4.6 6.4 3.7 Argin ine 35.9 44 .4 42 .4

were performed on the collagen from each animal. As can be seen, the specific activities of the two a subunits are very similar and decay in a parallel manner. Piez [16] had earlier suggested that al and a2 differed in cross-linking ability. Piez reasoned that, since the theoretical concentration ratio of ~ 2 to ~ , ,

3000

2500

2000

,soo

5 I0 20 30

\ / •

5 I0 20 30

OOys Post Labeling

Fig. 2. Specif ic ac t iv i ty o f ~ a nd ~ subuni t s at va ry ing t imes a f t e r a d m i n i s t r a t i o n o f labeled prol ine. Means + 2 S.E. are presentecL (A) o- . . . . . • , c¢ i ;o o, c~2 ; (B) o- - . . . . • , ~ i i ; o o , ~ l 2.

259

2.0

'5

1.0

2.5

I I I I 0.5 5 I0 20 30

Oay~ Post Lebeling

1.5

~, 0.5

I

5 lIO 20 Days Post Labeling

3'0

Fig. 3. The specific activity ratio o f ~ and ~ (separa ted by CM-cel lulose c h r o m a t o g r a p h y ) a t va ry ing t imes fo l lowing a d m i n i s t r a t i o n of l abe led prol lne , Po in t s r ep resen t m e a n s + 2 S.E. Fig~ 4. The specific activity ratio o f 7 and ~ a t val, y ing t imes a f te r a d m i n i s t r a t i o n of l abe led pro l ine . Means + 2 S.E. are presented.

which should be exactly 2, is actually closer to 4, there may be a greater reactivity of a2 then of ax. If this were the case, it would be expected that the specific activity of a~ would show a more rapid rate of decay than would that of a l • Since, as seen here, that is not the case, it is unlikely that these subunits differ in reactivity. The pulsing phenomenon (Fig. 2) has been previously re- ported from this laboratory [17] and by other workers [25]. Attempts to explain it have been made [17,18,25] but it remains a poorly understood observation.

Two previous studies have shed light on the overall rate of cross-linking in collagen. Orekhovich et al. [4] administered [14C] glycine to rats and studied the incorporation of label into skin collagen extracted with citrate buffer. The collagen was fractionated into a and ~ components by an (NH4)2 SO4 precipi- tation procedure which has subsequently been superseded by the more specific CM-cellulose technique [6]. These authors found a rapid (3 h) incorporation of label into both a and ~ components. At 24 h, the ratio of radioactivity of a :/3 was 3.3, in approximate agreement with our findings. However, at the final time point in this experiment (6 days), the a:~ ratio was 2.1 in contrast to our ratio of 1.3 at this time (Fig. 3), possibly indicating incomplete separation of and/3 components in the (NH4)2 SO4 fractionation used by these investigators.

A study carried out by Martin et al. [3] followed the incorporation of [14 C] glycine label into the a and ~ subunits of acetic acid-soluble skin collagen of normal and lathyritic rats over a total period of 7 days. These investigators found that whereas the formation of ~ components in lathyritic animals was markedly retarded, the a:~ ratio in normal controls approached unity (ratio-- 1.12) at 7 days. These data are in close agreement with those of the present experiment which shows (Fig. 3) that, following a pulsed label, a ratio of 1.2 is

2 6 0

reached at 7 days but the time required for the ratio of the specific activities of and ~ to become unity is approx. 17 days. The initial rate of cross-linking is

very high (Fig. 3) but slows markedly within 14 days. By 17 days, 50% of the subunits have been conjugated through an acid-stable intramolecular cross-link with another ~ subunit to form ~ subunits.

The s tudy leaves unresolved the relationship between fl and % One would expect that if ~ were a terminal cross-link, the radioactivity from ~ would be transferred to 7 and the 7 specific activity would steadily increase while specific activity would decrease. Similarly, if fl were a terminal cross-link, the specific activity of this fraction should increase steadily. However, neither seems to be the case as seen from Figs 2 and 4. Fig. 2 shows that the specific activity of fl is no t different at the beginning and end of the experiment. This could be explained by a dilution of fl radioactivity through contributions to that pool from the rupture of hydroxymerodesmosine cross-links in preformed % However, this is very unlikely due to the small size of the ~ pool and the similarity of its specific activity to that of ~. Fig. 4 shows that the ~:~3 specific activity ratio, rather than increasing during the course of the experiment, actu- ally decreases slightly. This indicates either a lack of transfer of radioactivity from/3 to 7 or else a very rapid transfer f~om ~ through 3' to a higher acid-stable polymer. Since the specific activity of 7 in the early time points is the same as that of fi, either the transfer of ~ to ~ is as fast as the transfer of a to/3 or some trifunctional intramolecular cross-links are formed at once. If the transfer from

to ~/ was slow in the face of a rapid transfer of activity from a to ~ one would expect a buildup of radioactivity in/3 which is not seen here.

Recovery from lathyrism After 17 days of ~-aminopropionitrile t reatment the rats were seen to

exhibit a lathyritic response as evidenced by (1) a 200% increase in the levels of

\ \ \\

\

\x

\

x x

x x x

D0ys Recovery from ~APN

Fig. 5. T h e r a t i o o f (~ t o /3 s u b u n i t s in skin col lagen of n o r m a l ra ts (e e) a n d r a t s at v a r y i n g t imes a f t e r c e s s a t i o n o f a d m i n i s t r a t i o n o f ~-aminopropioni t~i le (~APN) (o- . . . . . o). Means -+ 2 S.E. are p r e s e n t - ed .

261

~ 3ooo ~)t~

2500 '~

2000 ~ .~,

,500 '¢.,' ; i % " ' . . . A a l 0 \ " . , . . . . . . . . . . ~ ~ 2

tO00 I I I

5 I0 2O 30 Time (doys)

Fig. 6. Mean specific activity of ~I, a2, and S fractions at varying times after administration of labeled proline.

0.45 M neutral salt~extractable collagen in the skin, (2) a 600% increase in the salt~extractable collagen from aorta and, (3) a 4-fold increase in the a:/~ ratios of total extractable collagen from skin. Following cessation of/3-aminopropio- nitrile therapy, the a:~ collagen ratios fell rapidly {Fig. 5). Fig. 3 shows that in normal rats the a:~ specific activity ratio reflected a similar time course of cross-linking. In Fig. 3 the ratio of specific activity of a and ~ are presented while Fig. 5 compares amounts of the subunits separately by gel electrophore- sis. The absolute ratios will therefore differ but both methods of presentation reflect the kinetics of the cross-linking process. Comparison of the two sets of data suggest that the quantity of uncross-linked collagen is not rate determining in intramolecular cross-linking.

The S and U fractions The collagen eluted from CM-cellulose with 0.5 M NaCI, (the S peak),

showed a very high initial specific activity (Fig. 6). This specific activity was in fact higher than the specific activity of either a, or a2 24 h after administra- tion of the label. The rate of fall of specific activity was very rapid for the S peak. Clark and Veis [121 reported that a similar peak which they eluted from CM-cellulose columns with urea also demonstrated a high incorporation of radioactivity, but these authors did not do a time study on the rate of loss of the radioactivity from this peak.

In the present experiment the S and U peaks (Fig. 1) were rechromato- graphed on CM-cellulose at 45°C after denaturation at 60°C. This high tem- perature denaturation and elution is necessary to prevent S and U from precipi- tating upon dialysis against the sodium acetate starting buffer. The results are presented in Fig. 7 and it can be seen that whereas the urea peak gave rise to all the subunits normally seen on a CM-cellulose fractionation of denatured col- lagen, the S peak gave only S and U upon rechromatography. This would

2 6 2

0.50

~ 0.40

~0,30

.~020

~0.10

r/2= 1,06 6M UREA

['/:)=0.56

al BII " .

I00 200 300 400 500 600 Elulion (ml)

1.00

0.50

. 0.10

700

Fig~ 7 . R e c h r o m a t o g r a p h y o n C M - c e l l u l o s e o f t h e S ( . . . . . . ) a n d U ( ) f rac t ions f r o m rat s k i n c o l l a g e n .

suggest that the collagen represented by the S peak is either highly thermo- stable or demonstrates extremely rapid reaggregation properties, and indeed, when the S fraction was dialyzed against water, a fibrous precipitate was always seen. Such enhanced renaturation properties have recently been described by Veis et al. [21] for a material eluted from CM-cellulose with urea, their so- called U, peak. Examination of this renatured product with the electron micro- scope showed the typical formation of fine filaments with native collagen registration [21]. Veis and his co-workers [12,21,22] have postulated that this material represents a species of collagen important for nucleation during fibro- genesis. They proposed that this nucleation collagen was an intermediate spe- cies between the procollagen with its registration peptide [15], the immediate biosynthetic product of the cell, and the tropocollagen found in the collagen fibril. They referred to this intermediate as TCa and suggested that only part of the registration peptide had been removed upon secretion from the cell.

The S peak is clearly collagen on the basis of its amino acid composition (Table I), although the absence of cysteine indicates the absence of an intact registration peptide which is known to contain this amino acid [23]. The very rapid decline in radioactivity associated with this peak is consistent with its rapid transfer out of the acid-soluble pool, perhaps into the insoluble fibrous pool.

In 1972, Bornstein et al. [24] showed that the enzyme responsible for the removal of the registration peptide is inhibited at low pH. The acid conditions used in the present experiment would thus be expected to favor retention of the registration peptide on procollagen. The absence of cysteine in the S frac- tion suggests that prior cleavage had occurred and that S does not consist of an aggregate of intact procollagen molecules. It may, however, represent the inter- mediate proposed by Veis et al. [21]. The enhanced renaturation properties of S as evidenced by its precipitation upon dialysis against water together with its rapid decrease in specific activity make this an attractive suggestion.

263

In contrast to the S peak, the material eluted with urea (Fig. 1) referred to as the U peak does not demonstrate a high rate of incorporation of radioactivi- ty nor does the incorporated radioactivity fall rapidly as seen for the S peak. Although this material possesses the amino acid composition typical of col- lagen, it appears to be somewhat heterogeneous since it can be denatured at 60°C and rechromatography (Fig. 7) shows that only a portion is thermostable or capable of reaggregation while the remainder is eluted as a and ~ subunits. This fraction also shows the enhanced renaturation properties upon dialysis against distilled water.

Acknowledgements

We are grateful to Mrs Kathryn K. Johnson and Mr Albert N. Hanson for technical assistance.

Supported in part by N.I.H. grant HL 14126 and by a grant from the Medical Research Foundation of Oregon. Jeffrey S. Pinto was in receipt of an N.I.H. post~doctoral fellowship.

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Press, New York 17 Bentley, J.P. and Jackson, D.S. (1963) Biochem. Biophys. Res. Commun. 10, 271--276 16 Goodwin, B.C. (1963) Temporal Organization in Cells, Academic Press, New York 19 Grassman, W., Hannig~ K. and Engel, J. (1961) Hoppe-Seylers Z. Physiol. Chem. 324, 284--288 20 Piez, K.A. (1965) Biochemistry 4, 2590--2596 21 Veis` A., Anesey, J., Yuan, L. and Levy, S.J. (1973) Proc. Natl. Acad. SoL U.S. 70, 1464-1487 22 Veis~ A., Anesey, J., GaJ~in, J.E. and Dimuzio, M.T. (1972) Biochem. Biophys. Rcs. Commun. 48,

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1311--1315