THE JOURNAL. OF BIOL.OGXAL CHEMISTRY 0 1990 by The American Society for Biochemistry and Moleculsr Biology, Inc.

Vol. 265, No. 35, Issue of December 15, pp. 2199%21996,lSSO Printed in U.S. A.

Analysis of Site-directed Mutations in Human Pro-&(I) Collagen Which Block Cleavage by the C-proteinase”

(Received for publication, June 26, 1990)

Seung-Taek Lee+, Efrat Kessler& and Daniel S. GreenspanT From the Department of Pathology and Laboratory Medicine and Program in Cell and Molecular Biology, University of Wisconsin, Madison, Wisconsin 53706 and the §Maurice and Gabriela Goldschleger Eye Research Institute, Tel Aviv University

Faculty of Medicine, Sheba Medical Center, Tel Hashomer, 52621, Israel

We have used site-directed mutagenesis to obtain human proa2(1) cDNAs containing novel mutations de- signed to inhibit cleavage at the C-proteinase site. Deletion of six relatively conserved amino acids which surround the cleavage site did not interfere with as- sembly of the triple helix in transfected rat cells, but blocked cleavage of the constituent mutated chains by endogenous C-proteinase. Substitution for a conserved Asp, which forms part of the Ala-Asp bond cleaved by C-proteinase, also blocked cleavage by endogenous C- proteinase. The conserved Asp is, therefore, a neces- sary component of the C-proteinase cleavage site. In- cubation in vitro with a purified mouse C-proteinase, confirmed both mutations to be resistant to cleavage by high concentrations of the physiologically relevant enzyme. Mutant proa2(1) chains, resistant to cleavage by C-proteinase in culture media, were processed in cell layers by a different protease which cleaved telo- peptide domains. Naturally occurring mutations at the C-proteinase site have not been described in human patients. The mutations characterized here, further define the C-proteinase cleavage site and provide re- agents which may be informative when introduced into transgenic mice.

Naturally occurring mutations underlying such inherited collagen disorders as osteogenesis imperfecta, Ehlers-Danlos syndrome, and Marfan syndrome have been mapped to the two genes encoding type I procollagen. Analysis of the mutant nucleic acids and proteins from such cases have partially defined domains within procollagen required for normal syn- thesis, assembly, secretion and fibrillogenesis (for a review, see Ref. 1). A more systematic approach to characterization of functional domains in type I procollagen is to produce mutations at specific sites in the cloned genes, and to utilize tissue culture expression systems for structure/function analysis. Construction of novel mutations, not yet found in human patients, coupled with analysis in cell culture, and in transgenic mice, may also reveal roles for abnormal collagens in diseases of unknown etiology or pathogenesis.

* This work was supported by National Institutes of Health Grant GM-38544 (to D. S. G.) and by a Grant-in-Aid from the American Heart Association (to D. S. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC. Section 1734 solely to indicate this fact.

$Predoctoral fellow of the March of Dimes. llTo whom correspondence should be addressed: Dept. of Pathology

and Laboratory Medicine and Program in Cell and Molecular Biology, 1300 University Ave., University of Wisconsin, Madison, WI 53706. Tel.: 608-262-4676; Fax: 608-262-2327.

We have previously reported that transfection of a full- length human pro-&(I) cDNA into rat cells (W8) which synthesize endogenous pro-al(I), but not pro-cyB(I) chains, results in secretion of a stable rat/human heterotrimer which behaves like normal type I collagen by a number of criteria (2). We report here use of this expression system to analyze two novel site-directed mutations in the pro-cu2(1) cDNA designed to inhibit enzymatic cleavage of the COOH-terminal propeptide.’ We show that heterotrimeric molecules of normal rat/human mutant pro-cu chains are secreted into the media and extracellular space of the cell layers, and that only the constituent mutant human pro-cu2(1) chains are not processed by endogenous C-proteinase activity. In the cell layers normal and mutant chains are subject to digestion by a telopeptidase (3) that cleaves all nonhelical sequences from the amino and carboxyl termini of the procollagen molecules. Implications of these data for normal and abnormal collagen biosynthesis are discussed.

MATERIALS AND METHODS

Plasmid Construction and Site-directed Mutagenesis-An EcoRI- XhoII fragment containing all 012(I) 3’-untranslated sequences and sequences encoding the final 11 amino acids of procy2(1) was removed from a full-length pro-a2(1) cDNA, in the previously described recom- binant plasmid pSTL22 (2), and replaced with the synthetic adapter

5’-AATTCTTTGTGGACATTGGCCCAGTCTGTTTCAAGTAA-3’ 3’-GAAACACCTGTAACCGGGTCAGACAAAGTTCATTCTAG-5’

which reconstitutes the COOH end of pro-a2(1) and links it directly to downstream SV40 sequences (Fig. IA, pSTL29). Sequences in this region of pSTL29 were confirmed by chemical cleavage (4).

A 352-base pair NcoI-XbaI pro-a2(1) cDNA fragment (nucleotides 3246-3598 (5)) (Fig. L4) containing sequences encoding the C-pro- teinase cleavage.&, was blunt-ended ai the NcoI end with Klenow fragment and ligated between the SmaI and XbaI sites of M13mp18. Single-stranded-template, containing the positive strand of the ;2(1) NcoI-XbaI fragment, was annealed to the 29-base oligomer 5’- GGTGCTGAGCGAGGATCTCCATCGTAACC-3’ for deletion of 6 amino acids surrounding the pro-a2(1) C-proteinase cleavage site (recombinant pSTL47), or to the 19-base oligomer 5’- GCGAGGCTGGCCAGCCCTG-3’, for substitution of glycine for as- partate at the cleavage site (recombinant pSTL49). Oligonucleotides were extended with Klenow fragment by the method of Nakamaye and Eckstein (6), using a kit (Amersham Corp.). Mutations were identified by screening for created restriction sites and sequences

’ The nomenclature used is: propeptides, nonhelical amino- and carhoxyl-terminal amino acid sequences unique to unprocessed pro- collagen a chains; C-and N-proteinases, enzymes that excise carboxyl- and amino-terminal propeptides from procollagen, leaving abbrevi- ated nonhelical telopeptides as remnants; telopeptides, nonhelical sequences left at amino and carboxyl termini of triple-helical native collagen; pNa1 and pNa2, processing intermediates of procollagen that contain N- but not C-propeptides; p&l and pCa2, processing intermediates of procollagen that contain C- but not N-propeptides.

21992

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confirmed by dideoxy chain termination (2). Oligonucleotides were synthesized at the University of Wisconsin Biotechnology Center.

Cell Culture and Transfection-K16 rat liver epithelial cells, W8, a transformed variant of K16 cells deficient in production of pro-a2(1) chains, and A2 cells, a clonal line of W8 cells which have been transfected with and express a full-length human pro-cu2(1) cDNA, have been described (2). Cells were cultured in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum. Cotransfection of W8 cells with pSV2neo and with either pSTL47 or pSTL49 was by calcium phosphate precipitation as described (2).

Isolation and Analysis of Radiolabeled Collagen-Confluent cell cultures were supplemented with 75 pg/ml ascorbate for 24 h, depleted in labeling medium (Dulbecco’s modified Eagle’s medium with 5% dialyzed fetal bovine serum and 75 rg/ml ascorbate) without radioi- sotope for 2 h, and radiolabeled in labeling medium plus 20 &i/ml L-[2,3-3H]proline (Du Pont-New England Nuclear) for 20 h. Culture media or labeling media were buffered to pH 7.4 or to pH 7.0 and soybean trypsin inhibitor (SBTI,’ Sigma) was added to 100 pg/ml (3), as indicated in the text. Where noted, labeling medium was made 5% in Dextran T-40 (Pharmacia LKB Biotechnology Inc.).

Collagen, for pepsinization, was prepared from media as described (2) or from cell layers by scraping cells into 0.5 M acetic acid, shaking overnight at 4 “C, and removal of cell debris by low speed centrifu- gation. Digestion was with 100 Kg/ml pepsin in 0.5 M acetic acid (pH 2.0) for 6 h at 4 “C.

Procollagen, which was not pepsinized, was isolated by (NH&SO, fractionation (2) of media which contained protease inhibitors (0.1 mM phenylmethylsulfonyl fluoride, 25 mM EDTA, 10 mM N-ethyl- maleimide, and 1 mM p-aminobenzoic acid). Collagen forms from ECM were prepared by seauential extraction of cell lavers with 1% deoxycholate followed by 2% SDS in a modification of the procedure of McKeown-Longo and Mosher (7). Cells were scraped into buffer A (1% deoxycholate, 50 mM Tris-HCl (pH 8.3), 2 mM EDTA, 2 rg/ ml leupeptin, 2 rg/ml aprotinin, 1 pg/ml pepstatin, and 1 mM phenyl- methylsulfonyl fluoride), vortexed, and centrifuged at 17,000 x g for 20 min. Deoxycholate-insoluble material was washed twice with buffer A and solubilized by boiling in 2% SDS-gel buffer prior to electrophoresis.

To prepare substrate for in vitro C-proteinase cleavage assavs. cells were double-labeled with 2.8 &i/ml -[i4C]proline (Am&sham Corp.) and 11 uCilm1 13Hltrvotonhan (Amersham Corn.) for 6 h. Dulbecco’s modified Eagle’s me&urn (pH 7.0) used for doublelabeling was devoid of serine and tryptophan, contained 100 pg/mI SBTI, and did not contain serum. Procollagen was isolated, from the medium, by (NH&SO, fractionation, 75% ethanol precipitation (2), and lyophi- lization. The lyophilate was dissolved in and dialyzed against 50 mM Tris-HCI (pH 7.5), 150 mM NaCl, 1 mM EDTA, and stored at -70 “C. Double-labeled human procollagen (DL7) was prepared from the culture medium of normal human skin fibroblasts (CRL 1121, Amer- ican Type Culture Collection) as described (8).

Collagen samples were reduced with ,&mercaptoethanol, unless otherwise noted, and analyzed by electrophoresis in 5% SDS-poIy- acrylamide gels.

Incubation with Purified C-proteinase-C-proteinase was purified from the medium of cultured mouse 3T6 fibroblasts approximately 18,500-fold, as described previously (9). Substrate was incubated with 0.15 unit of purified C-proteinase for 3 h at 37 “C in 50 ~1 of assay buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM CaCl,), with or without added enhancer (0.2 rg). Reactions were stopped by heat denaturation in SDS (9).

RESULTS

Mutagenesis and Plasmid Construction-As shown in Fig. lB, there is relative conservation of amino acid residues around and including the C-proteinase cleavage site of a number of pro-a chains thought to be cleaved by the same enzyme (10, 11). In an attempt to render pro-& chains resistant to cleavage by the C-proteinase, sequences encoding six amino acids were deleted from a full-length pro-a2(1) cDNA in plasmid pSTL29, to give mutant pSTL47 (Fig. 1C). Because the initial association of procollagen chains is in the

*The abbreviations used are: SBTI, soybean trypsin inhibitor; ECM, extracellular matrix; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis.

y i , Adaptor

-

a + pro-a2(1) Human Phe Tyr Arg Ala Asp Gin

Chick TP

pro-al(I) Human Tyr Tyr Arg Ala Asp Asp Chick

pro-cll(II) HUllXl Tyr Met Arg Ala Asp Gin Chick Glu

pro-Ul(III) Human Tyr . . . Tyr Gly Asp Glu Chick Glu Arg

C + pS~L29 Gly Asp Phe Tyr Arg Ala Asp Gin Pro Arg Ser Ala

GGA GAC TTC TAC AGG GCT GAC CAG CCT CGC TCA GCA

pSTI.47

pSTL49

GGA GAT '** *'* l ** l ** *** ***ET CGC TCA GCA

GGA GAC TTC TAC AGG GCT G& CAG CCT CGC TCA GCA Gly

FIG. 1. Construction of cDNA expression vector and C-pro- teinase cleavage mutants. A, schematic representation of the pSTL29 expression vector. Stippled and white boxes represent RSV- LTR and pro-a2(1) cDNA sequences, respectively. The horizontal black bar represents SV40 sequences downstream of the pro-&(I) cDNA and the dashed angle represents the small t splice site. The horizontal arrow indicates the transcriptional start site, and the vertical arrow represents the SV40 polyadenylation site. H, HindIII; E, EcoRI; BP, BgIII; N. NcoI: X. XbaI. Black and hatched boxes represent pa& of the cDNA clone encoding the main triple-helical region and terminal propeptides, respectively, of pro-cu2(1). B, con- servation of amino acids which bracket the C-proteinase cleavage site in various pro-n2(1) (12), pro-al(I) (I3), pro-al(II) (14), and pro- cul(II1) (15) chains. The vertical arrow shows the site of physiologic cleavage. C, nucleotide sequences of C-proteinase cleavage mutants. *, deleted nucleotides in pSTL47. Bold underlined nucleotides repre- sent substitutions. Overlined sequences represent XhoII and Bali sites created in pSTL47 and pSTL49, respectively.

C-propeptide, followed by assembly of the triple helix in a COOH-terminal to amino-terminal direction (16), it was pos- sible that the deletion in pSTL47 might interfere with the assembly of pro-o2 and pro-al chains into heterotrimers. Accordingly, a second less extensively modified mutation was constructed in which only the conserved Asp residue of the C-proteinase cleavage site was replaced by Gly to yield con- struct pSTL49 (Fig. 1C).

Expression of Mutagenized Pro-a2(I) Collagen in Trans- fected W8 Cells-Recombinant plasmids, pSTL47 and pSTL49 were transfected, with the selectable marker pSV2neo (17), into WS cells which secrete cul(I) homotrimers but do not produce detectable a2(1) chains (18). Clonal lines of transfected cells, resistant to the neomycin analogue G418, were isolated and analyzed by Sl nuclease mapping for the presence of human a2(1)-specific transcripts (data not shown). Those clonal lines found to contain high levels of human a2(1)-specific RNA for each mutant were metaboli- cally labeled with [3H]proline, and the cell lines found to secrete the highest levels of o2(1) chains, lines S47 and S49 for mutants pSTL47 and mutant pSTL49, respectively, were selected for further analysis.

The A2 cell line, which was created in a previous study by transfection of W8 cells with a construct (pSTL22) containing a wild type human a2(1) cDNA, produces both rat al(I) homotrimers and chimeric type I heterotrimers comprised of normal rat al(I) and human cy2(1) chains (2). Like the het- erotrimers from A2 cells, which contain normal human CUB(I) chains, heterotrimers containing mutant a2(1) chains secreted into culture media by either S47 or S49 cells were found to be pepsin-resistant (Fig. 2). The ratios of ~yl to a2 chains in

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21994 Pro-a2(I) Collagen Mutations Resistant to C-proteinase

A2 s47 s49 m c m c m cmcmcmc

Dextran - _ + + _ _ + + _ _ + +

FIG. 2. Incorporation of cuZ(I) and al(I) chains into A2, S47, and S49 cell layers. Pepsin-resistant collagen from A2, S47, and S49 media (m) or cell layers (c) radiolabeled with [3H]proline in the presence (+) or absence (-) of 5% dextran, was analyzed by SDS- PAGE and autofluorography.

- FN -

pNnl- al- a2-

-pmal- -proal

al, -pNaP,

a2-

FIG. 3. Processing of procollagen in culture media. A, pro- collagens were isolated from culture media of cells radiolabeled with [“Hlproline for 20 h (see “Materials and Methods”); or B, media prelabeled with [“HJproline were made 20 mM in nonradiolabeled proline and overlaid on unlabeled confluent Kl6 monolayers for 20 h. Samples were reduced with fl-mercaptoethanol and analyzed by SDS-PAGE and autofluorography. FN, fibronectin.

pepsin-treated material was also somewhat greater than the 2:l expected from a pure population of heterotrimers. There- fore, S47 and S49 cells, like A2 cells, secrete a mixture of rat al(I) homotrimers and chimeric rat cYl(I)/human mutant o2(1) heterotrimers. As with many cell types, procollagens secreted by S47, S49, or A2 cells accumulate predominantly in the culture medium with little enzymatic processing or incorporation into the ECM of the cell layer. Addition of neutral polymers, which increase effective macromolecule concentrations by a volume exclusion mechanism, has been shown to speed both procollagen processing and incorporation into ECM in human fibroblasts in culture (19). Addition of 5% dextran to the culture medium of A2 cells resulted in incorporation of the majority of secreted collagen into cell layer matrix. In the presence of dextran, heterotrimers con- taining mutant a2 chains, from either the S47 and S49 line, were incorporated into cell layers as efficiently as wild type heterotrimers from the A2 line (Fig. 2).

Resistance of Mutant Chains to C-proteinme-The above data from pepsinized samples showed that chimeric mutant molecules were assembled and secreted into the media and cell layers, but offered no indication of whether the constitu- ent mutant pro-a2 chains were resistant to endogenous C- proteinase activity. Gel analysis of unpepsinized media from A2, S47, and S49 cultures was relatively uninformative, be- cause little procollagen processing, aside from some conver- sion of wild type procollagen chains to pN forms, occurred in the media of these cultures over the 20-h labeling period (Fig. 3A). K16 is a rat epithelial line that more rapidly processes procollagens accumulating in the culture media than lines A2, S47, and S49 (Fig. 3A). To take advantage of the presumed greater activity of N- and C-proteinases secreted by Kl6 cells, media from A2, S47, and S49 cultures radiolabeled with [3H] proline, were overlaid on K16 monolayers for 20 h. Prior to overlay, media samples were made 20 mM with proline to prevent radiolabeling of endogenous K16 collagens. After

overlay, unpepsinized media samples were analyzed in gels (Fig. 3B). Mutant procollagen chains in S47 and S49 media were now found predominantly as forms which retained the C-propeptide (pro-a2 and p&2). By contrast, wild type pro- (~2 chains in A2 media were almost entirely converted to pNa2 or a2 chains. Wild type pro-al chains were converted to pNa1 or al chains in all three cases.

As an additional test of the resistance of the mutant chains, radiolabeled procollagens from A2, S47, and S49 medium, and from the medium of a control human fibroblast culture (sub- strate designated DL7), were concentrated and incubated in uitro with a highly purified mouse C-proteinase (9) (see “Ma- terials and Methods”). After a 3-h incubation, electrophoresis in nonreducing gels showed that pro-al chains had been cleaved to pNa1 for all samples (Fig. 4A). In contrast, mutant pro-a2 chains were not cleaved by C-proteinase (Fig. 4A, S47 and S49) although wild type pro+2 chains were cleaved to produce pNa2 (Fig. 4A, DL7, A2). A 36-kDa glycoprotein, which has been shown to enhance C-proteinase activity by up to an order of magnitude (9, 20), was added to some incuba- tions (Fig. 4, Enh.), but did not increase levels of cleavage of the control and wild type pro-a chains because C-proteinase was already in excess. The mutant pro-a:! chains were not processed even in the presence of enhancer, further substan- tiating their resistance to cleavage by the C-proteinase. Ra- diolabeling with [3H]tryptophan allowed identification of cleaved C-propeptides. Cleavage of human type I procollagen with C-proteinase produced a disulfide-linked C-propeptide fragment comprised of two C1 subunits (cleaved from pro-al chains) and one CZ subunit (cleaved from pro-cu2) (Fig. 4A, DL7, C&. Incubation of procollagen from A2 cells produced two species of C-propeptides; one which co-migrated with the

A DL7 ,, A2 ,,S47,,%9, ,

C-pmt. -+ + -I- + + -I- + +I- + + Enh. - _ + +,- - + +, - - +, - _ +

9

prod,

pNal-

pNaP-

A2 s47 s49

--prom2

C-pmt. - ++ - - ++ - + + Enh. - -+ +I- -+f- - +

FIG. 4. Processing of procollagen from CRL 1121 fibro- blast, A2, S47, and 549 media incubated in vitro, with puri- fied C-proteinase. Collagenous proteins from media (see “Materials and Methods”) of CRL 1121 (X7), A2, S47, or S49 cells, radiolabeled with [“‘Clproline and [3H]tryptophan, were incubated in the presence (+) or absence (-) of purified C-proteinase (C-prot.) and/or enhancer protein (Enh.) (9, 20), for 3 h at 37 “C. Samples were analyzed by SDS-PAGE without prior reduction (A) or after reduction with @- mercaptoethanol (B). al”, pNd’, and pNa1’ refer to the human and rat forms, respectively, which have different electrophoretic mobili- ties.

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Pro-a2(I) Collagen Mutation-s Resistant to C-proteinase 21995

C1,2 propeptide from normal type I procollagen, and another, more slowly migrating form, comprised of three C, subunits derived from pro-al homotrimers. In contrast, incubation of C-proteinase with S47 or S49 procollagens, produced only the slower migrating species (C,), consistent with cleavage of C1 from pro-al homotrimers. The demonstrated cleavage of pro- (~1 chains in the mutant heterotrimers, should generate an intermediate composed of an uncleaved mutant pro+2 chain with two disulfide-attached C1 propeptides. Such an inter- mediate appears only in enzyme-digested samples of S47 and S49 (arrow, Fig. 4A).

Reduced gel samples again revealed complete digestion of wild type pro-a2 chains (A2) to pNa2 forms, but no detectable cleavage of mutant pro-a2 chains (S47 or S49) after a 3-h incubation (Fig. 4B). Both types of mutant pro-a:! chains were completely resistant to C-proteinase even after a 6-hr incubation (data not shown).

We conclude from all of these data that the transfected mutant pro-a2 chains were resistant to endogenous or exog- enous C-proteinase activity.

Processing in Cell Layers-Although mutant pro-a2 chains were resistant to cleavage by C-proteinase they were incor- porated into cell layers as efficiently as wild type pro-a2 chains (Fig. 2). Since published studies of procollagen polym- erization in vitro had suggested that retention of C-propep- tides interferes with fibril formation (22), it was important to determine the extent of processing of mutant pro-a2 chains, in the ECM of cell culture systems.

Radiolabeled media from A2, S47, and S49 cultures were supplemented with proline and dextran and overlaid on K16 cell layers for 20 h. Radiolabeled collagens incorporated into K16 ECM from the added media were then analyzed (Fig. 5). The ECMs overlaid with mutant media contained small amounts of pro-a2 and pCa2 rather than pNa2, the processing intermediate present in the A2 matrix. This result again supports the resistance of the mutant pro-a2 chains to endog- enous C-proteinase activity, however, the mutant lanes also displayed strong signals from (u2* chains moving with a slightly faster mobility than a2 chains from the A2 overlay. Additionally the mutant lanes have faster moving al* chains, and they also lack p forms as compared to the A2 lane. Collagen p components are formed by nonreducible interchain cross-links between telopeptide residues (21), and so the data indicate that telopeptides are missing from the cx chains in the mutant samples.

Bateman et al. (3) have reported that pro-a chains in tissue culture cell layers may be shortened by a proteolytic activity that cleaves telopeptides, yielding LY chains shorter than those generated by the N- and C-proteinases. The telopeptidase

P=

anal,

rn’\

pNru27 a2

,proa2 -pCaP ‘IX,* 'cY2"

FIG. 5. Processing of procollagen from A2, 547 and 549 media overlaid on K16 monolayers. Media from A2, S47, or S49 cells, radiolabeled with [3H]proline, were made 20 mM in nonradio- labeled proline, 5% in dextran, and overlaid on unlabeled confluent K16 monolayers for 20 h. ECM was isolated by deoxycholate extrac- tion and unpepsinized, reduced samples analyzed by SDS-PAGE and autofluorography.

activity was reported to be restricted to the cell layer, poten- tiated by the addition of dextran to the culture, and to be inhibited by SBTI and by maintaining the cultures in the pH range 6.5-7.0. Such a telopeptidase activity in K16 cultures is demonstrated in Fig. 6. For comparison, lane 1 shows the position in gels of (~1 and CY~ chains processed by N- and C- proteinases in the media, while lane 2 shows the position of pepsin-treated media cul and a2 chains, which lack telopep- tides. Lane 4 is a sample of ECM from a Kl6 culture treated with dextran and maintained at pH 7.4. The (Y chains move as doublets, the faster moving chains (al*, cr2*) representing the chains lacking telopeptide residues. The a2* chain is more apparent in a longer exposure of lane 4 (lane 4’). Unlike the results obtained by Bateman et al. (3) with cultured human fibroblasts, the K16 telopeptidase activity was only partially inhibited at pH 7.0 in the presence of SBTI (Fig. 6, he 3). ECMs of A2, as well as S47 and S49 cultures, labeled for 20 h in the presence of dextran contained al* and a2* chains (data not shown). Mobilities of (Y chains in these ECM were not affected by radiolabeling at pH 7.0 in the presence of SBTI, suggesting that the WS-derived mutant lines may contain higher levels of telopeptidase activity than the paren- tal K16 line.

We conclude from all these data that the transfected mutant pro-cY2(1) chains in both media and cell layers are resistant to C-proteinase activity, but that in the cell layers they are subject to a different enzyme that cleaves through the telo- peptide domain.

DISCUSSION

We have constructed and characterized two mutations at the site where human pro-a2(1) chains are cleaved by C- proteinase during normal processing of type I procollagen in vivo. One construction deleted six amino acids, -Phe-Tyr-Arg- Ala-Asp-Gln-, which bracket the cleavage site, and the other replaced Asp with Gly at the -Ala-Asp- cleavage bond. Neither mutation blocked assembly or secretion of chimeric rodent/ mutant human triple helical heterotrimers in transfected cells, but both mutations blocked excision of the pro-a2(1) C- propeptide by C-proteinase. The mutant chains were resistant to endogenous C-proteinase activity in the media and cell layers of the culture systems, and resisted cleavage in vitro by a highly purified mouse C-proteinase under conditions that caused complete cleavage of the wild type pro-al(I) chains. The Asp residue, which is conserved at the C-propeptide cleavage site of type I, II, and III procollagens of different species is, thus, shown to be required for recognition and/or cleavage by both rat and mouse C-proteinase. The pro-cu2(1)

pepsm - + - _ uG _ pH ZO 7.0 7.0 7.4 7.4

SBTI + + + - FIG. 6. Effect of pH and SBTI on electrophoretic mobilities

of al(I) and a2(1) chains from K16 ECM. K16 cells were radio- labeled with [3H]proline at pH 7.0 in the presence of SBTI, in the absence of dextran, and collagen forms isolated from media without (lane I) or with (lane 2) treatment with pepsin. K16 cells were radiolabeled in the presence of dextran at pH 7.0 in the presence of SBTI (lane 3) or at pH 7.4 in the absence of SBTI (lane 4) for analysis of ECM collagen forms. Lane 4’ is a longer exposure of lane 4. Samples were run on long 5% SDS-polyacrylamide gels and ana- lyzed by autofluorography. Med, media samples. ECM, samples iso- lated from ECM by deoxycholate extraction.

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21996 Pro-cyB(I,, Collagen Mutations Resistant to C-proteinme

mutations did not block cleavage by C-proteinase of the wild type pro-al(I) chains in the heterotrimer. This observation is consistent with previous findings that cleavages of pro-al(I) chains can occur in the absence of cleavages of the pro-a2(1) chain (22, 23).

Processing of both mutant and wild type pro-cr chains in cell layers appeared to also occur by cleavage within telopep- tide domains, as collagens were identified that lacked ,f3- components and that contained al(I) and a2(1) chains which migrated slightly faster than al(I) and (u2(1) chains cleaved by N- and C-proteinases. Like a previously reported proteo- lytic activity shown to cleave type I collagen telopeptides in cell layers of cultured human dermal fibroblasts (3), the activity reported here was restricted to the cell layer and was partially inhibited, in K16 monolayers, by lowered pH and the presence of SBTI. In experiments, in which prelabeled media from A2, S47, and S49 cells were overlaid on K16 monolayers, relatively small amounts of exogenous collagen chains were incorporated into K16 extracellular matrix. In- terestingly, al(I) and (u2(1) chains processed and incorporated into K16 extracellular matrix from procollagen in A2 media, appeared to retain telopeptides. Thus, efficient cleavage of wild type heterotrimers from A2 media by C-proteinase seems to preclude further processing by telopeptide-cleaving activ- ity. In contrast, the majority of al(I) and (u2(1) chains incor- porated into matrix of K16 cells from overlaid S47 and 549 media appear to have been cleaved within telopeptides. This suggests that retention of C-propeptides on pro-a chains selects for cleavage by a telopeptidase, and the resultant (Y chains might be better incorporated into assembling fibrils than intact pro-a chains or pC forms thereof (24). But (Y chains lacking telopeptides would not be efficiently cross- linked to other collagen molecules and mechanically unstable fibrils could result. Whether telopeptidase activity is ex- pressed in uiuo and whether it is an important element in normal procollagen processing and fibrillogenesis, is presently unknown. Telopeptidase activity might be induced and im- portant in the settings of organogenesis and wound healing, wherein collagen fibrils must be rapidly laid down and remain susceptible to turnover.

of pro-cYB(I) collagen clones bearing either of the C-proteinase cleavage mutations described here, may also yield phenotypes arising from defects in fibril integrity. Even if the mutant chains are cleaved by telopeptidases in certain tissues, the resultant molecules would not be properly cross-linked and connective tissue defects might be expected.

Acknowbdgments-We thank Dr. Burton Goldberg for critical reading of the manuscript and Guy G. Hoffman and Ellen Pluim for excellent technical assistance.

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Further studies, employing the systems described here, should further elucidate the principles of C-proteinase action. Substitutions for other amino acid residues which bracket the cleavage site, for example, could reveal roles for these residues in C-proteinase-procollagen interactions. In addition, substi- tution of the conserved Asp with Asn and Glu may demon- strate which aspects of the Asp side chain are necessary for cleavage.

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The demonstrated absence of cleavage by the physiologi- cally relevant C-proteinase suggests that pro-a2(1) chains bearing either of the two mutations would not be cleaved by this enzyme in uiuo. Naturally occurring mutations which block cleavage of the N-propeptide, in either pro-cy2(1) (25, 26) or pro-al(I) (27) collagen chains, are expressed in a dominant fashion in human patients with the heritable col- lagen disease Ehlers-Danlos syndrome type VIIB. In these cases, retention of N-propeptides appear to interfere with fibril assembly and cross-linking, and profound connective

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S T Lee, E Kessler and D S Greenspancleavage by the C-proteinase.

Analysis of site-directed mutations in human pro-alpha 2(I) collagen which block

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