propeptides of procollagen v (a,b) in chick embryo crop*

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 256, No. 13, Issue of July 10. pp. 7053-7058, 1981 Printed m U.S.A.

Propeptides of Procollagen V (A,B) in Chick Embryo Crop* (Received for publication, December 15, 1980, and in revised form, March 9, 1981)

Carol A. Kumamoto and John H. Fessler From the Molecular Biology Institute and Biology Department, University of California a t Los Angeles, LOS Angeles, California 90024

The biosynthesis of type V (A,B) collagens was recently found to proceed through the sequential forms proa(A,B), pa(A,B), and fa(A,B). All these chains are larger than the A,B chains extracted from tissues after pepsin digestion. This report shows that all forms contain substantial peptides which are resistant to bacterial collagenase and concludes that type V collagens differ from the interstitial collagens (types I, 11,III) in retaining large noncollagenous peptides in tissues. A peptide becomes transiently attached to the processing intermediate paA by a reducible linkage. The conversion of procollagen V to p-collagen V was inhibited by colchicine and arginine. Previously, the disulfide- linked heterotrimer [(proaB)z (proaA)] was found and additional procollagens containing only B type molecules were inferred. Further investigations reported here agree with these conclusions and also indicate that some trimeric molecules containing more than one chain related to A may exist.

Pulse-chase labeling experiments recently showed that the biosynthesis of type V (A,B) collagens in chick embryo crop proceeds through a set of precursors (1, 2). The largest re- covered precursors were named procollagens v, followed by p-collagens V, which were only slowly converted to the final form, f-collagens V. All these forms gave on pepsin digestion the distinctly smaller A,B chains that have been described after extraction from pepsin-treated tissues (3-14). Here we report additional studies which further delineate the structure of type V collagen precursors and their biosynthesis. Our analysis uses the characteristic electrophoretic mobilities of the corresponding reduced constituent chains: proaA, proaB, paA, paB, faA, and faB established previously.’ These chains had been grouped into homologous A and B types by comparison of the patterns generated with Staphylococcus aureus V8 protease, with those obtained from reference A and B chains. The increasing times needed for labeling gave the biosynthetic sequence within each set of proa, pa, and fa

* This work was supported by the Kroc Foundation and United States Public Health Service Grants AM 13748 and AG 02128. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “adLlertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

’ These chains could also be designated, respectively, proa2(V), proal(V), pa2(V), pal(V), a2(V), and al(V). The terms al(V) and a2(V) have already been used in a review (14) for the B and A chains extracted by pepsin digestion. This is unfortunate, as the naturally occurring chains must be larger, and our results indicate that the faB and faA chains are indeed larger than aB and uA, respectively. Eventually it may be necessary to distinguish between al(V), a2(V), corresponding respectively to faB, faA and al(V) (pepsin), a2(V) (pepsin) corresponding to aB and aA. The relative electrophoretic migrations of the various chains are shown in Fig. 3 of Ref. 2.

chains. Linsenmayer and Little (13) first described chains that probably correspond to some of these and chains associated with A,B biosynthesis, especially by cell cultures, have been reported (8, 12, 15-17).

In order to investigate the propeptides of these chains, we have now removed the collagenous regions with bacterial collagenase. We find that the steps of processing are unlikely to be explained by proteolytic cleavage alone. Probably there are changes in glycosylation and a noncollagenous peptide becomes transiently disulfide-linked to one chain. Further- more, faA and faB chains, the end products of processing, retain significant noncollagenous regions that are lost in the pepsin extraction of A,B chains from tissues.

The composition of individual triple chain V molecules, folded in the collagen helix, is under debate (reviewed in Ref. 14). Although Bentz et al. (18) concluded from reconstitution experiments that three A chains could not form a stable collagen helix, various trimeric combinations of type V A,B and also C (6) could exist. The formulation B*A, proposed by Burgeson et al. (3) and others (18) from the proportions in which they extracted chains from tissues, agrees with the demonstrated disulfide-linked procollagen [ (proaB)~(proaA)] of chick crop (Z), but an excess of precursor B chains in this tissue (2) is also consistent with BB forms reported in cartilage (5) and cell culture (16). We report here further studies on reducible linkages, presumably disulfide bridges, in these type V precursors. Unlike other procollagens there may be triplets which are never interchain-linked, although small electrophoretic changes on reduction suggest that intrachain bridges occur. We have found small amounts of a dimer of chains which on reduction yields only chains related to A. Our results suggest that t-ype V procollagens exist in several different forms.

MATERIALS AND METHODS

Materials and methods were the same as described previously (2, 19,20) unless stated to the contrary. In outline, crops freshly dissected from 19-day-old chick embryos were incubated with radioactive amino acids in Dulbecco’s modified Eagle’s medium, and homogenized in 1 M NaCl, 50 mM Tris-HC1, pH 7.5, in the presence of protease inhibitors, EDTA, PMSF,* and N-ethylmaleimide (2). All operations were at 0-4 “C. The clarified extract was resolved by DEAE-cellulose ion exchange chromatography in 4 M urea, 50 mM Tris-HC1, pH 7.8, by step elution with 0.15 M NaCl, followed by 1.0 M NaCl except where indicated. When salt gradient elution (2) was used instead, the type V procollagens eluted as a “low salt fraction” at approximately 6.5 X

mho/cm together with some type 111 pN-collagen and a material probably corresponding to precursors of [a1(1)]3. The procollagen V was then further purified by velocity sedimentation (2). The p-collagen V and f-collagen V materials eluted from the DEAE-cellulose column at 12 X IO-” mho/cm and higher salt concentrations and are collectively denoted as “high salt fraction.” These fractions were concentrated by precipitation in the presence of chick tendon carrier collagen by adding NaCl to 1.2 M at acid pH, and all gave A and B chains upon pepsin digestion (2). End products were identified by

’ The abbreviation used is: PMSF, phenylmethylsulfonyl fluoride.

7053

7054 Propeptides of Procollagen V

sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis (2) and fluorograms are shown in all figures. For further analysis after reduction, individual gel bands that had been identified by fluorography were cut out, swelled in 63 mM Tris-HCI, 1% sodium dodecyl sulfate, 1 mM EDTA, pH 7.5, buffer containing 1% (v/v) 2-mercaptoethanol and subjected to re-electrophoresis (19).

To. prepare the high salt fraction material used for the velocity sedimentation experiments of Fig. 2, radioactively labeled extracts were chromatographed on DEAE-cellulose as above, but step-eluted with 0.15 M, 0.2 M, and 1.0 M NaCl in column buffer, and then concentrated by salt precipitation. The 0.2 M NaCl column eluate contained radioactive p-collagen V and a small amount of f-collagen V, and was passed over a small DEAE-cellulose column to remove the carrier collagen that had been added to aid precipitation. After elution from this column with 1.0 M NaCI, the material was dialyzed into sedimentation buffer (2). Samples were layered onto 3.7 ml of 5- 20% (w/v) sucrose gradients in this buffer and sedimented for 29 h a t 56 krpm in a Beckman SW6O rotor a t 4 "C.

For radioactive labeling in the presence of inhibitors, crops were preincubated either for 20 min with 2 p~ colchicine in incubation medium, or for 10 min with 50 mM L-arginine, and then treated as previously described (2).

T o prepare specimens for collagenase digestion in gel pieces, reduced samples were treated with 10 mM dithiothreitol for 1 h a t 35 "C, and then with 40 mM N-ethylmaleimide for 4 h a t 22 "C. Samples were electrophoresed on 4% polyacrylamide gels, which were treated for fluorography. Bands located by fluorography were cut from the gel and reswelled in 20 pl of 50 mM Tris, 0.1 M NaCI, 0.19 Triton X- 100,5 mM CaCIz, pH 7.5, containing bacterial collagenase, followed by 20 pI of buffer alone. Samples were incubated in a humidified atmo- sphere at 26 "C for 2 h. After digestion, 10 pI of a 2% sodium dodecyl sulfate, 2 M urea, 1% 2-mercaptoethanol solution was added, and the samples were electrophoresed on 10 or 128 polyacrylamide gels to resolve the resistant peptides.

RESULTS

Structure of p-Collagen V-Type V collagens composed of a mixture of pa and fa chains elute from DEAE-cellulose in a high salt fraction (conductivity 12 X lo-" mho/cm and greater) (2). Although the chains resolved electrophoretically after reduction into paB, faB, paA, and faA, in order of increasing mobility' (Fig. l ) , before reduction only three bands, M1, M2, and M3, appeared (Fig. 1 and (2)). The latter three bands were further analyzed after reduction as described under "Materials and Methods." This showed that band M1 contained both paB and paA, M2 contained faB, and M3 contained faA (Fig. 1). The large increase in mobility of paA on reduction was accompanied by release of a peptide, named P (Fig. 1). This peptide migrated approximately with the mobility of the reduced carboxyl propeptides of proal(1) and therefore its apparent molecular weight is estimated to be 35,000. As digestion of a polyacrylamide gel piece containing band M 1 with bacterial collagenase did not change or remove P (Fig. 1, i , j ) , this peptide has a noncollagenous structure. Furthermore, as reduction of the paB contained in M1 caused a shift to slightly slower electrophoretic mobility (Fig. l ) , i t is unlikely that P is attached to paB and we conclude that P is attached to paA.

The components of the high salt fraction were partly resolved by velocity sedimentation under conditions which maintain the collagen triple helix, and Fig. 2 shows duplicate electrophoretic analyses, after reduction, of successive sedimentation fractions. The 4% acrylamide concentration of elec- trophoretogram Fig. 2a gave a resolution of paB. faB, paA, and faA chains while a higher, 7.5-15% concentration was needed to demonstrate peptide P in Fig. 2b. Peptide P was found in the fastest sedimenting fractions which contained paB and paA, again indicating attachment of peptide P to p- collagen V." The faA and faB chains were in the slower

The disulfide-linked paA-I' complex of chick embryo blood vessel p-collagen V sediments ahead of pnB and reduced paA under denaturing conditions which separate the chains:'

a b c d e f g h

- m M! M2 M3

red. - + - f + + + +

i j

*

:II

c

P- .

M1 M!

+ + t

Collagenase

M! pC(1)

+ +

FIG. 1. Effects of reduct ion on p-collagen V and f-collagen V. Crops were labeled for 5 h with [.'H]proline (lanes a-h), for 2.5 h with ["Hlproline, [,"H]leucine, and ['Hltyrosine (lanes k , l ) , or for 5 h with ["Hlproline and ["Hlleucine (lanes i, j ) and high salt fractions from each were prepared by DEAE-cellulose chromatography. Lanes a-d, samples were electrophoresed on a 44, polyacrylamide gel without reduction (lanes a and c, duplicate samples), or after reduction and alkylation (lanes h and d , duplicate samples). Another preparation of the high salt fraction was electrophoresed on a 3-54 gradient gel until a separation about 2-fold better than in lane a was achieved. The bands M1, M2, and M3 were then cut out, reduced, and re- electrophoresed on a 4 4 gel to give lanes f, g, and h, respectively. Lane e shows the original sample after reduction. Samples in lanes i, J, and k were similarly derived from the M1 bands of a 3-5% gel. Lane i, the M1 band was digested with collagenase as described under "Materials and Methods," then reduced. Lane j , control, M1 band not treated with collagenase, then reduced. Lanes i and; are in a 7%- 159 polyacrylamide gradient gel, while lanes h and l are in a 4-157 gradient gel. Lane k , M1 band reduced; lane I , marker pC-collagen I and carboxyl propeptides pC1, pC2.

sedimenting fractions. This, together with their greater electrophoretic mobility, is consistent with a loss of mass during conversion from pa to fa chains. There was considerable overlap of the various chain types in different fractions and paB chains occurred throughout as the major component of each fraction. This suggests several types of trimeric molecules with overlapping sedimentation zones. As the conversion of p- collagen V to f-collagen V proceeds very slowly over a number of hours ((2)4.5)), molecules in which 0, 1, 2, or 3 chains have been cut could readily occur and would sediment differently if the cut-off fragments were lost, or if cleavage caused con- formational changes, or both of these. A larger proportion of the paA chains than of the paB chains appear to have been cleaved.

Propeptides of Type V Collagen-To investigate the propeptides, all the type V chains were obtained after treatment with dithiothreitol and N-ethylmaleimide as individual electrophoretic bands by the above-mentioned and the previously described methods (2). After fluorographic location, the bands were cut from the gels, digested with bacterial collagenase, and re-electrophoresed as described under "Materials and Methods." The results are shown in Figs. 3 and 4. Table I lists the principal, reproducibly obtained peptides which remained after collagenase digestion and indicates their potential rela- tionships." Apparent, rounded-off molecular weights relative

Fessler, L. I., Kumamoto, C. A.. Meis, M. E.. and Fessler. .J. H. (1981) J. Biol. Chem. 256, in press.

"Fessler. L. I., Robinson. W. J., and Fessler, J . H. (1981) J. B i d . Chem. 256, in press.

li Each collagenase-resistant peptide was assigned a letter to denote whether it was derived from an A or a B type chain and a number. The sequence of numbers for the principal peptides is in order of decreasing apparent size and successive appearance in the series proa, pa, and fa chain. A minor peptide of pronB was denoted as Bla to

Propeptides of Procollagen V 7055

0 s-

18 16 14 12 IO 8

b

diL cx2’

I/

PC{ PC2

.P

FIG. 2. Analysis of p-collagen V and f-collagen V by velocity sedimentation. Crops were labeled for 2.5 h with [“Hlproline, [“Hlleucine, and [“Hltyrosine. The DEAN-cellulose high salt fraction was prepared for velocity sedimentation and centrifuged as described under “Materials and Methods.” 50 ~1 of the sample was used in a and 125 ~1 in b. 609~1 fractions were collected from the bottom, reduced, and electrophoresed on a 4”r polyacrylamide gel (a) or on a 7%15% gradient gel (b). Significant amounts of radioactivity were found only in fractions 8 through 18. Partially processed pC-collagen 1 was also electrophoresed on gel h and (I chains and carboxyl propeptides are indicated.

to globular protein markers are given as a measure of relative electrophoretic mobility but actual molecular weights could be different. Some minor bands of Fig. 3 could be due to incomplete separation of reduced chains before digestion. Controls incubated under identical conditions but without enzyme did not show any evidence of degradation and pepsin- derived A and B chains digested with collagenase under these conditions failed to yield any resistant peptides. Collagenase digestion of materials contained in gel pieces gave the same products as digestion of solutions of the starting materials before slab gel electrophoretic separation. The evidence for this is shown in Fig. 4 in which the electrophoretic migrations of collagenase-resistant peptides Bl and B3 are independent of digestion conditions.

Chain proaB contains two principal collagenase-resistant peptides Bl and B2 (Fig. 3~). On conversion to paB, the peptide B2 was lost and the migration of Bl was possibly slightly retarded (Fig. 36). When only proline label was used, the further conversion of paB to faB caused loss of Bl and appearance of a new peptide, B3 (Fig. 4, a and b). This suggests cleavage of Bl to give B3.

Adequate labeling of the collagenase-resistant portions of

indicate that it behaved like Bl in being found in collagenase digests of both proaB and puB, but not of faB chains. Bla could be a variant of Bl. A very small change in the electrophoretic behavior of peptide Al suggested the notation Al’, as the two peptides were not found together in the same material.

m U m 2 u a 0

a b

B3

m u Lc

C

-A3

FIG. 3. Collagenase-resistant peptides from type V collagen chains. Substrates labeled with the indicated amino acids were prepared as described under “Materials and Methods.” Procollagen V, [“Hlproline, [“Hlleucine, [“Hjtyrosine, p-collagen V and f-collagen V, [:‘H]proline, and [“Hlleucine. After digestion with bacterial collagenase, as described under “Materials and Methods,” resistant peptides were analyzed by electrophoresis on a IO? polyacrylamide gel, Lane a, prooB; lane b, puB; lane c, f&B; lane rl, prouA; lane e, paA; lane A faA. A set of globular proteins was electrophoresed on the same slab gel and detected by Coomassie blue staining (not shown). Corresponding approximate, nominal molecular weights are given in Table I.

0omQ.q 8 u ‘d 75 Mixture a- ---

Bio- Bi ..

l33-

Collagenose: + + + + + - abcde f

FIG. 4. Comparison of collagenase-resistant peptides pro- duced by digestion in solution and in gel pieces. Crops were labeled with [“HJproline and the high salt fraction, containing p- collagen V and f-collagen V, was obtained. As described under “Ma- terials and Methods,” the individual chains were separated after reduction, digested with collagenase while contained in polyacrylamide gel bands, and then analyzed by electrophoresis on a 12% polyacrylamide gel (lanes a-d). Aliquots of the original high salt fraction, containing the mixed type V collagen chains, as well as some other proteins, were treated with dithiothreitol and N-ethylmaleimide, and then incubated in solution with or without collagenase at 35 “C for 2 h, then electrophoresed on the same slab gel (lanes e and f). Lane a, p&B; lane b, faB; lane c, paA; lane d, faA; lane e, mixture with collagenase; lane f, mixture without collagenase.


TABLE I Collagenase-resistant peptides

Apparent, nominal molecular weights X 10 ~" are given, after round- ing off to the nearest 5 units, relative to globular protein markers electrophoresed on the same slab gels. These values are intended as approximate references of relative electrophoretic mobility. The actual molecular weights could be substantially different due to anomalous migration. The following markers were used: 8-galactosidase, phosphorylase A, bovine serum albumin, ovalbumin, and lysozyme. The molecular weight of P is estimated from its migration relative to carboxyl propeptides of type I procollagen. See text for anomalous nature of peptides Al, Al', indicated by parentheses.

Chain proa(V) pN(V) f n W )

B B1 85 B1 85 B3 45

A (A1 85) (Al' 90) (AI' 90) B2 35

A2 40 A2 40 A3 20 I' 35

the A chains required ["Hlleucine and ["Hltyrosine (Fig. 3, d- f , and Fig. 4, c and d). The proaA chain yields two peptides, A1 and A2. Conversion to paA is accompanied only by a slight slowing of electrophoretic migration of peptide A1 to Al', but A2 is retained. This is consistent with the very similar electrophoretic mobilities of proaA and paA (2). Peptide A2 is subsequently lost in the conversion of paA to faA and may have given rise to a new peptide A3. Peptide Al' seems to be retained in faA.

A minor band, B l a (Figs. 3 and 4) , is probably a variant of B1, perhaps differing in extent of glycosylation or other post- translational modification. I t is not removed by extending collagenase digestion from 2-5 h and is present in both proaB and paB. The DEAE-cellulose "high salt fraction" from which the p-collagen V and f-collagen V chains are obtained also contains other, unrelated, and smaller noncollagenous peptides. Some of these are seen in Fig. 4, e and f and in Fig. 26.

Chain Composition of Procollagen V-An excess of B type over A type chains in all fractions of Fig. 2a supports our previous conclusion (2) that trimers with greater B content than B2A must exist, i.e. B:l. An excess of B chains relative to the formula B2A was also found in pepsin digests of individual fractions of Fig. 2a.

Conclusive proof for a Bn trimer would be isolation of a corresponding interchain disulfide-linked precursor but this was not found (2). A renewed search failed to demonstrate disulfide-linked (proaB)3 or (proaB)2, but unexpectedly indicated a very small proportion of pairwise disulfide-linked proaA chains. Crops were labeled for 2.5 h with [,"H]proline, ["Hlleucine, and ["'SS]methionine, and extracts were chromatographed on DEAE-cellulose using a linear 75-275 mM NaCl gradient (2). Nine successive column fractions in the "low salt" region expected to contain type V procollagen were pooled into three groups of three consecutive fractions. Each group was concentrated by salt precipitation and then sepa- rately sedimented as described (2). Successive sedimentation fractions were analyzed by electrophoresis without reduction. The first pooled group of chromatographic fractions gave results (not shown) as described before (2) and also exempli- fied in Fig. 6. The second pooled group of chromatographic fractions gave the results of Fig. 5 and the third group contained insufficient radioactive materials for analysis. Fig. 5, lanes (a) and (b) show electrophoretograms of successive sedimentation fractions of the procollagens V sedimentation peak. As described before (2) (see also Fig. 6), there were unlinked proaA and proaB chains and disulfide-linked combinations of these: band L2, [(proaA)(proaB)] and band L1 [ (proaA) (pr~cuB)~]. However, there was an additional band L2a. Individual bands were cut out of similar 3.7R preparative

trimers

dimers

monomers

I L2 I L 2 a

" p r o d B - p r o d B

- p r o u A -prodA

a b c d e f non-reduced reduced

FIG. 5. Analysis of dimers formed from procollagen V chains. Radioactively labeled procollagen V was prepared and frac- tionated by DEAE-cellulose chromatography and velocity sedimentation, as described in text and under "Materials and Methods." Lanes a and b show aliquots of successive sedimentation fractions electrophoresed on 4% polyacrylamide gels without reduction. L1 denotes disulfide-linked trimer [(proaA) (proaB)Z] (2). Bands proaA, proaB, L2a, and L2 were cut from similar preparative 3.7% polyacrylamide gel electrophoretograms, reduced, and re-electrophoresed on one 4% polyacrylamide gel to give, respectively, lanes c, d , e, and f.

.. l i

/"- " trimers - - . L1 \

t c ---- -- -prouB- .I

monomers B i

"- - -prootA /

4"

Colchicine a b c d

pulse ,45 90 135, min. chose

FIG. 6. Lack of further conversion of monomeric procollagen V chains to disulfide-linked oligomers in pulse-chase experiments. Lanes a-d, crops were first labeled for 45 min with [ 'Hlproline and ['Hlleucine, and then chased for an additional 45,90, or 135 min with nonradioactive amino acids present. Lane e, colchicine, 2 VM, was present throughout a 20-min preincubation, 30-min labeling with [:'H]proline and [ 'Hlleucine, and 90-min chase of crops. Lane f , control, incubated as in ( e ) but without colchicine. Procolla- gens V were isolated as described by DEAE-cellulose chromatography (75-275 mM NaCl gradient elution) followed by velocity sedimentation. Sedimentation peak materials were electrophoresed without reduction on 4% polyacrylamide gels. Lane a, 45-min pulse; lane 6, 45-min chase; lane c, 90-min chase; lane d , 135-min chase. Lane e, colchicine-treated; lane f , control.

Propeptides of Procollagen V 7057

control + arg - u2(1)" 0

"A - a b c d e f

FIG. 7. Inhibition of conversion of low salt fraction to high salt fraction materials by arginine. Extracts from crops incubated with ["Hlproline for 1.75 h in the presence or absence of 50 mM arginine were separated by DEAE-cellulose step elution chromatography into an unbound fraction, a low salt fraction (0.15 M NaCI), and a high salt fraction (1.0 M NaCI). Equal aliquots were digested with pepsin, precipitated, and redissolved, and samples of an indicated volume were electrophoresed on a 4 9 polyacrylamide gel. Control, a- c; arginine-treated, d-f. Unbound fraction, 10 pl, a and d; low salt fraction, 40 pl, b and e; high salt fraction, 40 pl, c and f. The A chain bands of the low salt fraction are not fully resolved from contaminat- ing intense a1 chain bands (see text and Ref. 2).

gels and after reduction and electrophoresis gave lanes c-f of Fig. 5. Band L2a essentially only gave proaA chains (Fig. 5e) while band L2 gave about equal amounts of proaA and proaB chains (Fig. Sf) as before (2).

To see whether the relatively large pool of unlinked proaB chains acted as a source for subsequently disulfide-linked proaB chains, crops were pulse-labeled for 45 min with [''HI- proline and ["Hlleucine, then chased for 45, 90, and 135 min with nonradioactive proline and leucine and analyzed as before. Comparison of the relative amounts of monomers and oligomers in the low salt, procollagen-containing fraction showed no significant differences a t different times (Fig. 6, a- d). The same conclusions were reached when these experiments were repeated in the presence of compounds which, we show later in this paper, inhibit the conversion of procollagen V to p-collagen V: colchicine and arginine. In the presence of 2 p~ colchicine, radioactive procollagens V were synthesized for 30 min, then chased for 90 min, and isolated as before (Fig. 6e). Comparison with the control (Fig. 6f) shows that the relative intensities of the oligomeric bands L2 and L1 were not increased and a large proaB band remained. Therefore, these monomeric proaB chains do not behave as precursors to L1 and L2, in contrast to monomeric proal(1) and proa2(I) chains which can be chased into disulfide-linked [(proal)2 (proa2)l in similar experiments (21). Thus, there are proaB chains which never become disulfide-linked to other chains, and this is consistent with the postulate of nondisulfide-linked (proaB)3 molecules.

Inhibition of Procollagen V Processing-Arginine can inhibit normal proteolytic removal of the carboxyl propeptides of procollagens I, 11, and I11 in cell-free systems and tissues (22,23),' and causes some accumulation of procollagens I and 111. Crops were labeled for 105 min with ["Hlproline either with or without 50 mM arginine. The extracts were separated by DEAE-cellulose chromatography into unbound, high salt, and low salt fractions, and converted to A,B chains by pepsin digestion (see "Materials and Methods," and Ref. 2). Fig. 7

' D. J. dofuku, L. I. Fessler. and J . H. Fessler. manuscript in preparation.

shows that arginine substantially inhibited conversion of procollagen V, found in the low salt fraction, and also caused some general decrease of protein labeling. As in this experi- ment, the velocity sedimentation purification step was not used and the low salt fractions (Fig. 7 , h and e) contain additional [al(II1)]:1 and a1 chains; the latter overlap with the pepsin-derived A chains. When 2 p~ colchicine or 50 mM arginine were present during a 65-min pulse label followed by a 30-min chase, similar inhibition was observed (not shown).

DISCUSSION

The procollagens of types I, 11, and I11 all contain substantial amino and carboxyl propeptides with interchain disulfide linkages between the carboxyl propeptides. While our results for type V procollagens show collagenase-resistant portions which we interpret as propeptides located at the ends of the established, pepsin-resistant collagen chains aA and aR, we do not know the arrangement of these propeptides. There is a significant deficiency of the expected disulfide links between any propeptides of adjacent chains and there must be more than one trimeric arrangement of A and R chains. Although a definitive model of the structure of type V procollagens cannot yet be stated, the subsequent discussion is relative to the known arrangements of procollagens I, 11, and 111.

An unusual feature of procollagen V processing is the attachment of a noncollagenous peptide P to the intermediate paA by a reducible linkage, which is presumably a disulfide bond. This peptide was not detected when proaA or bands M2 and M3 of Fig. 1 were reduced (not shown) and must be removed during the further conversion of paA to faA. We neither know where peptide P comes from, nor how it becomes associated with the paA chain. ProaB chains contain a collagenase-resistant portion B2, approximately the same size as P, which is lost during the conversion of proaB to paB (Fig. 3 and Table I). Since disulfide links between proaB and proaA occur through the propeptides, one possibility is that peptide P originates as a propeptide of a proaB chain. When the proaB chain is proteolytically processed to paB, the propeptide could remain attached to paA by disulfide links. Contrary to this is the finding that while essentially all paA chains are found in the disulfide-linked form as band M1 (Fig. l ) , and therefore are coupled to P peptides, only some of the proaA chains are found disulfide-linked to proaB chains (bands L2 and L1 of Figs. 5 and 6 and Ref. 2). Thus, although peptide P could be derived from proaB, we consider its origin as un- known a t this time, and this is under further study:'

The proportion of monomeric proaA chains varies with incubation conditions and preparation, and may be low after extensive chase or inhibition of processing, but a substantial portion of the proaB chains does not form interchain disulfide bridges at any time. In spite of our precautions during isolation, interchain disulfide links could have been lost by possi- ble, unusual rearrangements with intrachain S-S bridges. We conclude that there are unlinked proaA and proaB chains which are in pepsin-resistant triple helical conformation (2), but we cannot exclude that eventually, before cleavage to p- collagen V, most of the proaA chains might become disulfide- linked to fellow components of triplet molecules. There is an excess of B type chains relative to the formulation B2A in procollagen V of chick crop and in the subsequently processed materials. While estimates of the proportions of B and A type chains depend on assumptions of approximately equal mole contents of the radioactive amino acids in the labeled precursor chains, this excess was also found in pepsin digests of proline-labeled type V p-collagens, and the resulting aA and a B chains are known to have very similar proline contents (3). Overall, a B:A ratio of approximately 3:l was found. Therefore


it seems that some proaB chains must associate to and are processed as such, but do not form interchain disulfide bridges.

We interpret our data only in terms of A- and €3-type chains because pepsin digests of chick embryo crop and analysis of CNBr peptides (2) have not shown evidence for aC(V) or other chains (24). There are, however, some weak, unidenti- fied, and collagenase-sensitive bands in analyses of some precursor fractions before pepsin digestion, e.g. some weak bands in Fig. 1, b, d, and e. Various possibilities therefore imply caution in interpretation of our findings of a small amount of proaA chains in mutual disulfide linkage (Fig. 5). There is too little L2a band material for further identification of these apparent proaA chains. We would have expected a greater difference of electrophoretic mobilities of the dimers [ (proaA)(proaB)] and [(pr~aA)~]. Band L2a (Fig. 5) could also have contained proaA chains combined with other material which was subsequently lost or was inadequately labeled. Unusual changes in disulfide bonding could have occurred during preparation in spite of precautions, but the state of disulfide bridging of procollagens I and 111, and their deriva- tives, in the same tissue did not show any unexpected features. As AB trimers are considered unstable (18), the finding of (proaA)z may indicate some molecules of form A2B.

Conversion of procollagen V to p-collagen V is likely to occur extracellularly, as indicated by the inhibition by arginine and colchicine. The subsequent conversion of p-collagen is slow and probably proceeds through intermediates in which only some component chains of a triplet molecule are cut.

Reduced proaA and paA chains are so similar, as judged by electrophoretic mobility, sedimentation under denaturing con- d i t i on~~ and collagenase-resistant peptides that the change, if any, between them is small. The difference lies in the conversion of collagenase-resistant peptide A1 to the slightly slower moving peptide Al'. While glycosylation changes could cause this, the existence of peptides A1 and Al' seems anomalous. The relative electrophoretic mobilities of faA and pepsin- derived aA chains seem not sufficiently different to allow for a collagenase-resistant peptide of the mobility of Al'. Fur- thermore, while the apparent molecular weights of Table I could of course be greatly influenced by anomalous electrophoretic behavior, they are not unreasonable, with the excep- tion of peptides A1 and Al'. These peptides could result from unrelated contaminants, but they arose when materials from three different sources, proaA, paA, and faA, were digested. Although collagenase digestion in gel bands can sometimes be incomplete, the peptides were obtained in separate digestions, and extending the incubation time had no effect. Faint bands similar to Al, Al' have been seen occasionally in digestions of other collagen precursors."

The conversion of proaB to paB chains differs from that of proaA to paA in loss of a noncollagenous peptide B2. This change could be related to the marked increase in binding to DEAE-cellulose of the p-collagen V. Although the paB chains

C. A. Kumamoto and J . H. Fessler, unpublished observations.

do not participate in interchain disulfide bridging, the slight, reproducible slowing of electrophoretic migration of paB on reduction (Fig. 1) indicates intrachain looping through inter- nal disulfide links. Similar behavior occurs with the amino propeptides of procollagens I and I11 (19,20). Small, collagenase-resistant peptides could exist in addition to those listed in Table I, as labeling and resolution would not have been adequate to detect peptides in the 10,000 molecular weight range. The retention of noncollagenous peptides in the fa(V) chains may be important for their participation in an insoluble connective tissue matrix from which A and B chains are derived by pepsin cleavage.

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propeptides of procollagen v (a,b) in chick embryo crop*

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