A short chain (Pro)collagen from aged endochondral chondrocytes. Biochemical characterization

THEJOURNALOF BIOLOGICAL CHEMISTRY Vol. 258. No. 15,Issue of August 10, pp. 9504-9509,1983 Printed in U.S.A.

A Short Chain (Pro)collagen from Aged Endochondral Chondrocytes BIOCHEMICAL CHARACTERIZATION* (Received for publication, February 14, 1983)

Thomas M. SchmidS and ThomasF. Linsenmayer From the Developmental Biology Laboratory, Departments of Medicine and Anatomy, Harvard Medical School and the Massachusetts General Hospital, Boston, Massachusetts 02114

by different mitotic indices, morphologies, and rates of macromolecular synthesis (1-5). In uiuo, the majority of chondrocyte proliferation occurs in a thin band (zone 1)which separates the tarsus (epiphysis) from the rest of the tibia (3, 5). Proceeding proximally to zone 2, some of the cells stop dividing andacquire an elongatedmorphology. The maximal rates of collagen and chondroitin sulfate synthesis are found in the cells of the proximalhalf of this zone. In the next most proximal zone (zone 3), thecells begin to hypertrophy with a concomitant decrease in macromolecular synthesis. Thecells replaced by in the proximal half of zone 3 degenerate and are bone and marrow. For experimentalpurposesit is possible to cleanly and reproducibly dissect the developing tarsus into three zones, thusenriching for differentpopulations of chondrocytes. These regions can either be cultured intact as organ cultures or dissociated and grown as cell cultures. Under both typesof in vitro conditions the cells of the different regions seem to maintain their characteristicdifferences (6-8). With progressive time in cell culture, they temporally progress in development andbegin to exhibit characteristicsindicative of cells in the nextzone or stage of development (8-10). We have been studying the collagens synthesized by chondrocytes from these regions. In previous radiolabeling studies (9, 10) we observed the production of a collagenous molecule which, by a number of criteria, appeared to be unrelated to any others previously described. In addition to thismolecule, Limb development requires precise the temporal and spatial the chondrocytes also synthesized type I1 collagen and la-, coordination of growth and remodeling of the skeletal ele- 2a-, and 3a-collagen(s) (9), as would be expected. One of the ments. Suchprocesses must involve the de nouo synthesis and most distinctive and interesting biochemical characteristics deposition of new matrix molecules within certain regions of of this new collagen is its composition of chains less than two-thirds the lengthof a normal a chain. We have termed it the expanding skeleton, with concomitant degradation and removal of pre-existing elements in other regions. Cellular SC’ collagen. Several independent lines of evidence suggest that its short chains are not artifactual products resulting changes must also occur. For example, during the morphogenesis of long bones, the chondrocytes proliferate, deposit from abnormal proteolysis (see Ref. 10 and “Discussion”).By an extensive cartilage matrix, hypertrophy, and eventually SDS-PAGE analysis of radiolabeled material, we observed that the largest molecular weight form of SC collagen is disappear from regions of the tissue. These processes can be advantageously studied in the or- composed of nondisulfide bonded chains each having an apganizedepiphysealgrowth plate, that region of developing parent molecular weight of 59K. We have termed this the long bones inwhich elongation is achieved through endochon- 59K form. The molecule has both helical (bacterial collagenand nonhelical (pepsin-sensitive) domains. dral bone formation. Within theepiphyseal growth region of ase-sensitive) of native molecules reduces the size of Limited pepsinization the 12-day chick tibiotarsus, differentiated chondrocytes pass through a continuum of stages of their life cycle, characterized the chains to 45K; the molecule containing these chains is termed the 45K form. Developmentally, SC collagen is of interest since its syn* This work was supported by National Institutes of Health Grants thesis seems to be initiated exclusively by older chondrocytes AM03564, EY02261, and Training Grant HD07092. This is Publication 922 of the Robert W. Lovett Group for the Study of Diseases Causing Deformities. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Fellow of the Arthritis Foundation.

‘The abbreviations use are: SC, short chain; SDS-PAGE, Na dodecyl sulfate-polyacrylamide gel electrophoresis; B-APN, 2-aminopropionitrile fumarate; BSA, bovine serum albumin; 59K and 45K, molecules with chains of 59,000 and 45,000 molecular weight, respectively.

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After 5 weeks in secondary culture, chondrocytes derived from specific regions of the embryonic chick tibiotarsus secrete >90%of their culture medium collagen as a short chain (SC) collagen. Quantities of the molecule, sufficient for biochemical characterization, were isolated without proteolytic treatment from the medium of such mass cell cultures. The chains, M , = 59,000, of SC collagen are cleaved to M, = 45,000 by limited pepsin digestion of the native molecule. These two forms of SC collagen are referred to as the 59K form and the 45K form. The CD spectrum of the 59K form confirms the presence of a triple helical domain within the molecule. The amino acid composition of the two forms of SC collagen show it to be different from any other known collagen, including the short chain collagens that have been isolated by the proteolytic extraction of cartilages. The most characteristic features of SC collagen are its highcontent of methionine, low levelof arginine, and a lack of cysteine. The amino acid composition of the 45K form shows it to be the collagenous domain, while the differences between the 45K and 59K form presumably reflect thecomposition of thenonhelical domain of the59K form. The nonhelical domain contains greatly elevated levels of aromatic amino acids whichcontribute to thehydrophobic character of this domain.

A Chain Short

(Pro)collagen fromChondrocytes Aged

undergoing the process of hypertrophy ( i e . those in zone 3). We havedetectedsynthesis of SC collagen in short term organ cultures of pieces of zone 3 tissue, but not in similar cultures of tissue pieces from zones 1and 2. The cell derived from zones 2 and 3 synthesize SC collagen after a week in primary cell culture, where it may constitute up to20% of the collagen in the culture medium. The cells isolated from zone 1initiate the synthesis of SC collagen onlyafter serialpassage in culture. In the present study, we have observed that in long term mass cultures of such passaged chondrocytes, the relative synthesis of SC collagen increases with the duration of the cells in culture. After 6 weeks in cell culture, SC collagen accounts for greater than 90% of the collagen secreted into the medium. This has provided a source of sufficient quantities of SC collagen to perform the biochemical characterization of the molecule presented in this report. MATERIALSANDMETHODS

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Cell Culture-Primary cultures of chondrocytes were prepared from zones 2 and 3 (5) of the distal end of the tibiotarsus of 12-day chick embryos as previously described (6, 11).The chondrocytes, isolated from a dozen embryos, were plated in 100-mm Falcon tissue culture dishes a t a density of lo5 cells/lO ml of medium. The medium in which the cells were plated consisted of Dulbecco's modified Eagle's medium (Gibco) adjusted to pH 7.0 (11) containing 2 g/liter of Dglucose, penicillin (100 units/ml), streptomycin (100 pg/ml), and 10% RESULTS fetal calf serum (Sterile Systems). The medium was replaced every otherdaywithfresh medium at a p H of 7.4. The cells reached Quantities of SC collagen sufficient for biochemical charconfluence after a week of primary culture and were passaged into secondary culture in 150-mm dishes. The secondary cultures reached acterization were obtained from the culturemedium harvested confluence in a week and we began the daily collection of culture from secondary passage chondrocyte cell cultures. The primedium. On odd number days of harvest the medium was supple- marycultures fromwhich these were passaged had been mented with 2% fetal calf serum, 100 pg/ml of @-APN, and100 pg/ initiated with cells liberated from epiphyseal zones 2 and 3. ml of ascorbate. On the even number days of harvest the fetal calf The mass secondary cultures could be maintained, and the serumconcentration was raisedbackto10%and no 8-APNor culture medium harvested, for up to 6 weeks. Such long term ascorbate was added. The medium was collected from these cultures maintenance requireda specific feeding regimen inwhich for 5 weeks and that of the odd and even numbered days separately days of low serum ascorbate-supplemented medium were alpooled. In the analyses to be described, only the material that had been synthesized in the presence of ascorbate, @-APN, and low serum ternated with onesof high serum ascorbate-deficientmedium was ut.ilized. (see under "Materials andMethods"). It is known that under Collagen Isolation-The culture medium from all plateswas pooled in vitro conditions exogenous ascorbate is required for the each day and made 5 mM in EDTA, 1.0 mM N-ethylmaleimide, and efficient hydroxylations of proline and lysine in newly syn0.3 mM in phenylmethanesulfonyl fluoride. The pooled medium was clarified by centrifugation and ammonium sulfatewas added to 30% thesized collagens. We observed, however, that alternatefeedof saturation. The precipitate was recovered the following day by ings with low ascorbate medium were necessary to maintain centrifugation a t 10,000 X g for 30 min, redissolved in a phosphate the viability of the cultures. In ascorbate-supplemented mebuffer at 4 "C containing the above protease inhibitors and stored dium, the chondrocytes became more refractile when viewed frozen a t -20 "C. The pH 7.6 phosphate buffer containing 130 mM by phase microscopy, and if maintained constantly in such K,HPO, and 19 mM KH,PO,. The collagen was further purified by a second ammonium sulfate medium they would begin to detach from the tissue culture precipitation as described above. The SCcollagen was separated from dish. Thisdid not occur if cultures were fed on alternate days type I1 collagen by a salt fractionation at acid pH. The collagen, with medium lacking the exogenous ascorbate. The analyses solubilized in phosphate buffer, was dialyzed against two changes of to be described were all performed with SC collagen isolated 0.9 M NaCl in 0.5 M acetic acid. The type I1 collagen precipitated from days of harvest in which the cells had beenin the under these conditions and was removed by centrifugation. The SC presence of ascorbate for the previous 24 h. Inorderto collagen, which required a higher ionicstrength for precipitation, was of the SC collagen from serum subsequently collected by dialyzing the samples against two changes facilitatethepurification proteins, the serum concentrationof the medium from which of 2.0 M NaCl in 0.5 M acetic acid, followed by centrifugation to recover the precipitate.All precipitates were redissolved in phosphate the collagen was to be harvested was lowered to 2%. On buffer containingproteaseinhibitorsandstored frozen. The 45K alternate days itwas raised back to the normal concentration form of SC collagen was prepared by limited pepsin digestion of the (lo%),which was necessary to maintain cell viability. The 59K form at 4 "C for 16 h, as described previously (9). Samples were pooled culture media harvested from twenty 150-mm culture digested with bacterial collagenase (Worthington CLSIII) which had been further purified by the method of Peterkofsky and Diegelmann plates over a 5-week period routinely yielded 40-50 mg of SC (12). The collagenase digestions were performed at 25 "C for 18 h in collagen. SC collagen represents a major collagenousprotein in these a pH 7.5 buffer containing 0.4 M NaCI, 10 mM CaC12, 10 mM N ethylmaleimide, and 50 mM Tris. Under these reaction conditions cultures, and it canbe readily purified from the other collagthispurifiedbacterial collagenaseshowed noproteolyticactivity enous and noncollagenous proteins, as shown by SDS-PAGE toward a high specific activity radiolabeled casein substrate. Samples analysis in Fig. 1. The SDS-PAGE profile of the proteins were electrophoresedin SDS on 7% polyacrylamide gels (13) and precipitated by ammonium sulfate from the medium of a day stained with Coomassie blue (14). Column. Chromatography-Gel filtration chromatography of both 31 harvest is shown in lune 2. For comparison, lune 1 contains native and thermally denatured molecules was performed ona column standards of type I collagen, BSA, and ovalbumin. In lune 2, (0.9 X 98 cm) of Sephacryl S-500 resin (Pharmacia), maintained a t the major band migratingslightly slower than BSA is the 59K

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8 "C. The column was equilibrated and elutedwith the same neutral phosphate buffer used for collagen solubilization or the phosphate buffer containing 25% ethylene glycol to lower hydrophobic interactions between thecolumn resin and the SC collagen (see under "results"). The column was pumped at a flow rate of 3 ml/h and 1ml fractions were collected. The void volume of the column was determined using radiolabeled chondroitin sulfate proteoglycan aggregate isolatedfromchondrocyteculturesafter labeling with H,35S0,. The total columnvolume was determined with 3H20. CM-cellulose chromatography of the denatured chainswas carried out as previouslydescribed (15). A column (1.5 X 5 cm) of CMcellulose (Whatman CM52), water jacketed a t 42 "C, was equilibrated in 0.02 M Na acetate buffer, p H 4.8, containing 1 M urea. Samples dialyzed into this buffer were denatured a t 50 "C for 15 min and applied to the column. The column was eluted with a 300-ml superimposed linear gradient of 0.0-0.16 M NaCl in the above buffer (flow rate 60 ml/h) and 5-min fractionswere collected. Circular Dichroism Measurements-The CD spectra of the two forms of SC collagen were measured ina Cary 61 spectropolarimeter. Samples, a t a concentration of 250 pg/ml, were analyzed a t 23 "C or after denaturation a t 50 "C for 15 min in a p H 7.4 buffer containing 0.4 M NaCl and 20 mM Na phosphate. Amino Acid Analysis-Proteins were hydrolyzed in constant boiling HCI which had been bubbled with N, for 10 min. Samples were sealed in a glass tube under a N, atmosphere and heated to 108 "C for 24 h. Under these conditions, all methionine residues eluted from the ion exchange resin in one peak; no detectable methionine sulfoxide was formed. The hydrolysateswere analyzed ona Beckman 121 amino acid analyzer, using a single column separation (16).

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at 222 nm and a negative rotation beginning a t 215 nm. After denaturation of the molecule by heating at 50 "C for 15 min (dashed line), the positive rotation a t 222 nm is completely lost. Measurements performed on native and denatured samples of the 45K form (not shown)give profiles similar to those of the 59K form. We next asked whether the native 59K and 45K forms of SC collagen exist as individual molecules or whether their minimum molecular arrangement is somehigher order structure or aggregate. For this we examined their mobility when chromatographed on columns of Sephacryl S-500 gel filtration resin run under nondenaturing conditions in neutral phosphate buffer a t 8 "C (Fig. 3). As chromatographic standards, we used cartilage proteoglycan aggregate (8) to mark thevoid BSAvolume, intact type I1 collagen to determine the elutionposition of a native helical collagen molecule composed of three CY chains, and denatured nl(I1) chains to mark position the of individual 95,000 molecular weightn chains. Bothof the latter two standards eluted within the included volume of the col0 VAumn (Fig. 3A). The native type I1 standard eluted as a large peak a t fraction 37, plus a smaller one between fractions 28 and 30, whichprobably contains highermolecular weight aggregates. The major peak of the denatured al(I1) chains FIG. 1. SDS-PAGE of SC collagen isolated from mass chon- was recovered in fraction 53. drocyte cultures. Lune 1 , standards of chick type I collagen, RSA, The results obtainedwith the two different forms of native and ovalbumin (OVA). Lane 2, ammonium sulfate-precipitated proteins from culture medium collected on day 31. Samples similar to SC collagen are shown inFig. 3B. In the standard phosphate that shown in lane 2 were digested with either pepsin (lone 3 ) or buffer, the 45K form eluted a t fraction 45, a position interbacterial collagenase (lane 4 ) . Lane 5 showsthe 59K form of SC mediate between the native and denatured typeI1 standards. collagen after purification by two precipitations with 30% ammonium Although no definitivemolecular weight can be obtained from sulfate followed by an acid-salt fractionation. Lane 6 showsthat this data, it seems consistent with a homogeneous population limited pepsinization of the native 59K form generates a form with of individual native molecules about half the lengthof type I1 45K molecular weight chains. All the samples were heated to 100 "C collagen. and reduced with @-mercaptoethanolprior to electrophoresis. The 59K form eluted slightly later than the 45K form in

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chain of SC collagen (see below). The bands, with mobilities I I I I slightly slower than then l chain, show the precursorsof type I1 collagen. When samples of native material are subjected to limited pepsinization before SDS-PAGE, the 59K molecular weight chains of the SC collagen are cleaved to 45K, and those of the type I1 procollagen are converted to an CY chain size band(see lane 3). Digestion of sampleswith highly purified bacterial collagenase (12) completelyremoved the bands of both SC and type I1 collagens (lane 4 ) . These data intheculture show SC collagen to be amajorcollagen medium, representing greater than 90% of those produced. The remainder of the culture medium collagen is predominantly type 11. Further purification of the SC collagen and its separation from the type I1 procollagens can be achieved by a second ammonium sulfate precipitation, followed by fractional salt precipitation performed a t acid pH. The additional ammoniumsulfateprecipitation removes any residual BSA and chondroitin sulfate proteoglycan. If fractional salt separation is performed in 0.5 M acetic acid, type I1 collagen and its precursors precipitate when the salt concentration is raised to 0.9 M, whereas the 59K and 45K forms of SC collagen remain in solution. Both SC collagen forms precipitate when the salt concentration raised is to 2.0 M. Lanes 5 and 6 of Fig. 1 show the results of such a separation. The pepsin resistance, collagenase susceptibility, and amino acid composition (presented later) of the SC collagen suggest I I I the major molecular domain of the molecule is in the confor220 240 280 mation of a collagen triple helix. Additional evidence to supWavelength fnml port this claim was obtained from optical rotation measureFIG. 2. CD spectra of the 5 9 K form of SC collagen. The ments of the molecules (Fig. 2), which show the spectrum spectrum of the native 59K form of SC collagen is represented by a characteristic of a collagenous molecule. The native59K form solid line. When the 59K form was heated to 50 "C for 15 min, the of the molecule (solid line) shows a maximum positive rotation spectrum represented by the dashed lines was obtained.

A(Pro)collagen Chain ShortChondrocytes Aged from

fraction 49. This result was inconsistent with the molecular weights of the component chains of the two forms as determined by SDS-PAGE. From the SDS-PAGE data, we would have predicted that the59K form should eluteslightly earlier than the 45K form or that the differences between the two would be small enough that they would co-elute from this resin which separates molecules with veryhighmolecular weights or extended structures. We reasoned that onepossible explanation for this discrepancy could be that the pepsin-sensitive domain, present only in the 59K form, had some affinity for the Sephacryl resin. Hydrophobic interactions seemedalikelypossibility, since the amino acid analyses of the two forms of SC collagen showed the 59K form to have a considerably higher level of aromatic residues (presentedlater).Inordertotestthis possibility the two forms of SC collagen were chromato-

graphed in phosphate buffer containing ethylene glycol, an agent used to elute proteinsfrom hydrophobic resins (17, 18). Fig. 3C shows that in thepresence of 25% ethylene glycol the 59K form elutes four fractions earlier, whereas the elution position of the 45K form remains unchanged. Thus, the two forms now coelute from the column asexpected. The elution positions of the native and denatured typeI1 collagen do not change with the inclusion of the ethylene glycol in the phosphate buffer (data not shown). The chains of the two different forms of SC collagen were further characterized and purified by CM-cellulose chromatography run under denaturing conditions. As shown in Fig. 4, the chains of the 45K form elute as a single peak in a position slightly before the a2 chain of type I collagen (conductivity = 9 mmho). The chains of the 59K form also elute as a single peak, but at a considerably higher salt concentration (conductivity = 12.5 mmho). The amino acid compositions of the CM-cellulose-purified 45K and 59K forms further confirm the identity of SC collagen as a new and different collagen type. These analyses are presented in Table I along with those of several other chick collagens for comparison. As can be seen in this table, the chains of both SC forms contain high amounts of hydroxyproline and hydroxylysine, the two amino acids indicative of collagenous molecules. The 45K form contains approximately one-third glycine suggesting that its chains are arranged predominantly, if not exclusively, in the triplehelical conformation. Its content of imino acids (25%) is slightly higher than most other collagens. The arginine content is about half of that in most collagens, and the methionineis about twice the typical value. Besides its unusual methionine and arginine content, SC collagen differs from the pepsin-extracted high molecular weight and low molecular weight cartilage collagens recently characterized by Reese and Mayne(19), chiefly in its lack of cysteine. The amino acid composition of the 59K form is consistent with itshaving both collagenous and noncollagenous domains, the latter containinga high concentration of aromatic amino acids. Compared to the 45K form, the 59K form contains reduced levels of imino acids and of glycine as would be expected for a collagenous molecule containing a nonhelical domain (i.e. a procollagen or a procollagen-like molecule such as typeIV). Most procollagen-like molecules contain cysteine; the 59K form of SC collagen, however, does not. Since N -

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FIG. 4. CM-cellulose chromatograms of the two forms of SC collagen run under denaturing conditions. The elution profile of the 45K chains are shown by the dashed line; the 59K chains are shown by the solid line. The two arrows mark the elution positionsof the a1 and a 2 chains of chick type I collagen. The dottedline represents the conductivity of the fractions collected from the NaCl elution gradient.

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Fraction FIG. 3. Gel filtration chromatography of the native59K and 45K forms of SC collagen on a Sephacryl 5-500column. A shows the elution profile of 250 pg of native chick type I1 collagen (solid line) in neutral phosphate buffer and a similar sample chromatographed under identical conditions after heating to50 "C for 15 min (dashed line).B shows the elution profileof the two forms of SC collagen in the phosphate buffer. The59K form is represented by the dashed line, and the 45K form by the solid line. C shows the elution profile of the two forms of SC collagen when 25% ethylene glycol has been added to the phosphate buffer. The 45K form is represented by the solid line and the 59K form by the dashed line. The void volume of the column (V,) and the total column volume ( Vt) were determined with radiolabeledchondroitinsulfateproteoglycanaggregateand 3H20,respectively.

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A Chain (Pro)collagen ShortChondrocytes Aged from

TABLE I Amino acid composition of cartilage collagens The 59K and 45K chains of SC collagen were purified by chromatographyon CM-cellulose,hydrolyzed, andanalyzedas described under "Materials and Methods." No corrections have been made for loss or incomplete release of the amino acids. For comparison, the composition of other chick cartilage collagens is listed.

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of more mature chondrocytes. Recently, a plethora of collagenous molecules with short chains has been isolated from various sources of fetal and adult cartilages (19, 23-30). These, however, have allbeen obtained by proteolytic extraction, aprocedurewhich has been shown tosignificantly alter the structure of some collagenous molecules from that which is normally found in tissues Residue 59K 45K HMW" LMW" la" 20;" 3a0 11" (31). Such extraction procedures can even produce differences residues/1000 when the same collagen type is extracted from two different 4-Hw 80 118 103 95 98 105 96 112 species. This is evidenced by the different sizes of type IV 46 44 42 49 35 53 48 50 ASP collagen fragments obtained from the proteolytic extraction 14 19 26 17 20 11 16 13 Thr of chick tissue (32) compared to those from mouse (33).Thus, 22 37 34 26 26 38 16 38 Ser the relationship(s) among the differentlow molecular weight 91 81 105 90 87 90 85 97 Glu 110 Pro 118 136 90 11792 111 115 cartilage molecules, isolated by proteolytic extraction, still 321 310 259 323 316 329 322 336 GlY remains to be resolved. At present, none of them seems tobe 57 53 53 11257 68 63104 Ala related in any obvious way to SC collagen. This is evidenced 0 0 2 0 0 0 9 0 CYS most readily by the presence of cysteine and the resulting 31 25 22 18 16 36 25 23 Val disulfide bonding in almost all of the pepsin-extracted mole7 5 11 24 23 9 Met 11 6 cules. There is a complete absence of this aminoacid in both 24 17 13 8 8 31 28 27 Ile 38 53 5748 56 33 26 26 Leu forms of SC collagen. The higher number of methionine 6 4 4 2 2 25 3 5 TYr residues and the low arginine content of SC collagen also 26 11 8 8 12 1315 14 Phe distinguishitfromother collagens. This high methionine 20 33 35 19 23 30 35 34 HYl content is consistent with our previous observation (9) that 20 13 13 17 25 20 21 18 LYS cyanogen bromide digestion produces onlypeptides with very 214 2 12 7 8 10 7 His low molecular weights (go%) type be responsible for the unexpected retention of the 59K form present in the culture medium. Previously, we also observed on the SephacrylS-500 column. In addition, suchhydrophobic the synthesis of additional collagens which by SDS-PAGE interactions may also participate in the rather strong interanalysis appeared to be la, 2a, and 3a (20). The molecule(s) chain associations we have observed to be present within the containing these latter chains were found predominantly, if nonhelical domainof SC collagen (preliminary observations). We do not know whether the presence of the nonhelical not exclusively, in the cell layer (9). They were not pursued in this present study which deals exclusively with material domain indicates that 59K is the procollagen form of SC collagen. We also cannot conclude with complete certainly derived from the culturemedium. Others workers have reported that some sternal chondro- that the 59K is the largest form secreted by the cells. Early cyte cultures synthesize a molecule with molecular properties work on procollagens suggested that the presence of one or similar, if not identical, to SC collagen (21, 22). Gibson et al. more relatively large pepsin-sensitive domains within a col(21) reported that therelative proportion of their short chain lagenous molecule identified it as a precursor of a smaller molecule (termed G-collagen) increased with the duration of form which would ultimately become deposited withinan time the chondrocyteswere in culture. This is consistent withextracellularmatrix, Morerecently, it has become evident our observations and hypothesis that SC collagen is a product that this is not always the case. For example, the molecular

A Short Chain(Pro)collagc!nfrom Aged Chondrocytes form of type IV collagen which becomes incorporated into 14. basement membranes contains several large nonhelical domains (34, 35) and to a lesser extent the same may be true 15. for type V collagen (36, 37). Experiments on the synthesisof 16. type V also raise thepossibility that its largest precursor form may be veryshort lived, possibly undergoing an initial proteo- 17. lytic conversion near the cell surface. If so, then the predom- 18. inant molecular form which accumulates in culture medium mayhave alreadyundergonecertain proteolytic modifica19. tions. Is a short-lived form of SC collagen, larger than 59K 20. form, synthesizedby the chondrocytes? What molecular form of SC collagen becomes deposited within the cartilage matrix,21. and precisely where within the tissue does this occur? What role, if any, does SC collagen play in theprocess of chondro- 22. cyte aging and endochondral bone formation? As probes for approaching these questions, we are currentlyproducing monoclonal antibodies specific for the different domains of SC 23. collagen, an approach we have recently shown tobe effective 24. in studies of type IV collagen (38, 39). Acknowledgments-We would like to thank Dr. Bart Holmquist for the use of his spectropolarimeter, Eileen Gibney for her technical assistance, and Dr. Jerome Gross for his helpful discussions.

25. 26.

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