Type XIII Collagen Forms Homotrimers with Three Triple Helical Collagenous Domains and Its Association into Disulfide-bonded Trimers Is Enhanced by Prolyl 4-Hydroxylase*

Type XIII collagen is a type II transmembrane protein predicted to consist of a short cytosolic domain, a single transmembrane domain, and three collagenous domains flanked by noncollagenous sequences. Previous studies on mRNAs indicate that the structures of the collagenous domain closest to the cell membrane, COL1, the adjacent noncollagenous domain, NC2, and the C-termi-nal domains COL3 and NC4 are subject to alternative splicing. In order to extend studies of type XIII collagen from cDNAs to the protein level we have produced it in insect cells by means of baculoviruses. Type XIII collagen a chains were found to associate into disulfide-bonded trimers, and hydroxylation of proline residues dramatically enhanced this association. This protein contains altogether eight cysteine residues, and interchain disulfide bonds could be located in the NC1 domain and possibly at the junction of COL1 and NC2, while the two cysteine residues in NC4 are likely to form intrachain bonds. Pepsin and trypsin/chymotrypsin digestions indicated that the type XIII collagen a chains form homotrimers whose three collagenous domains are in triple helical conformation. The thermal stabilities ( T m ) of the COL1, COL2, and COL3 domains were antibody 95D1A and various type XIII collagen-specific polyclonal antibodies. The T m values for the different collagenous domains of type XIII collagen were determined by densitometry of the intensities of the corresponding trypsin/chymotrypsin-resistant fragments using the ImageQuant Software (Molecular Dynamics). Digestion with Trypsin— Recombinant type XIII collagen (del1–38) was produced in High Five cells in the presence of recombinant prolyl 4-hydroxylase as described above. Aliquots of the resulting supernatants were heated at 24 °C for 5 min and treated with trypsin (0.01 or 0.05 mg/ml) for 2 min at room temperature. The trypsin was subsequently inactivated and aliquots of the trypsin-treated samples were electrophoresed on SDS-PAGE, followed by Western blotting using the monoclonal antibody 95D1A and the various type XIII collagen-specific polyclonal antibodies.

The collagens are classically divided into two major groups, fibrillar and nonfibrillar, depending on their structural and functional characteristics, and the heterogeneous group of nonfibrillar collagens can be further divided into several subgroups (1,2). Type XIII collagen belongs to the group of membraneassociated collagens, together with the hemidesmosomal com-ponent type XVII collagen. These two nonfibrillar collagen types are not structurally homologous except that both have been predicted to have a transmembrane domain near their N terminus. Immunoprecipitation of biotinylated type XIII collagen from surface-labeled HT-1080 cells, subcellular fractionation, and immunofluorescence staining have been used to demonstrate that type XIII collagen molecules are indeed located in the plasma membranes of these cells (3). Type XIII collagen molecules are presumed to reside in the plasma membrane in a "type II" orientation with a cDNA-derived predicted structure consisting of a short N-terminal cytosolic domain, a single transmembrane domain, and a large, mainly collagenous ectodomain. The N-terminal noncollagenous domain, NC1, 1 of the human ␣1(XIII) chain encompasses a 38-residue cytosolic domain, a 23-residue transmembrane domain, and the first 60 residues of the noncollagenous extracellular sequences adjacent to the plasma membrane (3). The rest of the ectodomain is predicted to contain three collagenous sequences, COL1-3, with sizes of 104, 172, and 235 residues, respectively, and noncollagenous domains, NC2-4, with sizes of 34, 22, and 18 residues (3)(4)(5)(6). The predicted structure for the mouse type XIII collagen chain is highly homologous (3). A striking feature of type XIII collagen is that its precursor RNAs undergo complex alternative splicing, affecting sequences encoded by 10 exons in both the human and the mouse (5)(6)(7)(8)(9)(10). As a result the sizes of the COL1, NC2, COL3, and NC4 domains of its ␣ chain vary in the ranges 57-104, 12-34, 184 -235, and 13-18 residues, respectively, in human (5,7,9).
The role of type XIII collagen is not known, but it is likely to function either in the adhesion of cells to their surrounding matrix or as a receptor for soluble ligands such as the collagenous proteins scavenger receptors and the receptor MARCO (11)(12)(13)(14). Type XVII collagen is known to be essential for the structural integrity of the skin, as mutations in its primary structure result in blistering skin disease (15,16). The expression of type XVII collagen is restricted to stratified epithelia (17,18), while type XIII collagen appears to occur widely in tissues (9,19). According to Northern and in situ hybridization experiments, type XIII collagen mRNAs are found in all fetal tissues studied, including bone, cartilage, intestine, skin, and striated muscle, and the placenta (9,19).
Until now, type XIII collagen has mainly been characterized on the cDNA and genomic levels, while studies on the protein level have been hampered by its low level of expression and the lack of suitable antibodies. To gain more information on type XIII collagen protein, we expressed it in insect cells using the baculovirus expression system (20). We have now produced two polyclonal antibodies against synthetic peptides corresponding to the NC2 and NC4 domains of human type XIII collagen, and have generated a novel pan-collagen monoclonal antibody that recognizes not only type XIII collagen in Western blotting, but also all the other collagen types studied. The recombinant expression system and the new antibodies were then used to characterize the conditions required for the association of type XIII collagen ␣ chains into disulfide-bonded trimers and the location of the interchain disulfides. Furthermore, pepsin and trypsin/chymotrypsin digestions were used to demonstrate the folding of the ectodomain into three triple helical domains and to assess the T m of these domains.

MATERIALS AND METHODS
Preparation and Affinity Purification of Antipeptide Antibodies-Two synthetic peptides corresponding to residues 137-149 (ECLSSMPAALRSS) in the NC2 domain and residues 588 -611 (GAPGLDAPCPLGEDGLPVQGCWNK) in the COL3 and NC4 domains of human type XIII collagen (5) were synthesized using an automated Applied Biosystems 433A peptide synthesizer (Department of Biochemistry, University of Oulu, Finland). The peptides were purified by reversed-phase high pressure liquid chromatography and sequenced by peptide sequencing (Applied Biosystems 477A). 5 mg of each purified peptide was conjugated to keyhole limpet hemocyanin (Sigma) by a standard procedure using glutaraldehyde (21). For primary immunization, the coupled peptide solutions were emulsified in complete Freund's adjuvant and injected intradermally into four rabbits. Booster injections with coupled peptide solutions emulsified in incomplete Freund's adjuvant were given at 4-week intervals. The sera were analyzed by enzyme-linked immunosorbent assay (Vectastain, Vector Labs) using the uncoupled peptide as an antigen. Subsequently, the positive antisera were analyzed by Western blotting of lysates from insect cells expressing recombinant human type XIII collagen (wthumanXIII) and, as a negative control, recombinant human prolyl 4-hydroxylase (4PH␣␤, Ref. 22). In accordance with this analysis, two sera were selected and named antibody XIII/NC2-55 and antibody XIII/NC4-SO.
For further studies, antibody XIII/NC4-SO was purified by affinity chromatography by coupling the corresponding peptide to epoxy-activated Sepharose 6B according to the manufacturer's protocol (Amersham Pharmacia Biotech). The antibody was diluted 1:5 with a solution of 0.1 M NaCl and 0.02 M K 2 HPO 4 , pH 7.0, and applied to the column, which was subsequently washed with a solution of 0.5 M NaCl and 0.02 M K 2 HPO 4 , pH 7.0. Specific antibody molecules were sequentially eluted with 0.03 M glycine-HCl, pH 2.9, and 0.1 M triethylamine, pH 11.0. The protein-containing fractions were detected by absorbance at 280 nm, immediately neutralized with 0.2 volume of 2 M Tris-HCl, pH 7.5, pooled, and concentrated to 0.5 mg/ml (Microsep 30, Filtron Technology Corp.).
Construction of Recombinant Baculoviruses-The cDNA clone E-26 (5) covers all of the coding sequences for the human type XIII collagen ␣ chain except for the beginning of the translation. First, a full-length cDNA called huXIII was constructed at the NotI-EcoRI site of pB-S(SKϪ) by linking a polymerase chain reaction-generated 5Ј-fragment covering nucleotides 1-272 of the human type XIII collagen gene (6) with the cDNA E-26 (5). The cDNA huXIII was digested with NotI-EcoRI restriction enzymes and ligated into pVL1392 (Invitrogen), resulting in the construct pVLwthumanXIII. The cDNA del1-38 was generated by polymerase chain reaction from huXIII cDNA so that it lacks the first 124 nucleotides and contains a new initiation methionine replacing the original residue in position 39, at the junction of the cytosolic and transmembrane domains (6). The cDNA del1-38 was digested with NotI-EcoRI and ligated into pVL1392, resulting in the construct pVLdel1-38. Because the cDNA E-26 (5) encodes an in-frame methionine at residue 84, it was also used directly for expression by inserting it into the EcoRI site of pVL1392 resulting in the construct pVLdel1-83. All the pVL constructs generated were separately cotransfected with modified Autographa californica nuclear polyhedrosis virus DNA into Spodoptera frugiperda Sf9 insect cells using the Bacu-loGold transfection Kit (Pharmingen). The recombinant viruses were collected, amplified, plaque-purified, and re-amplified (20). The viruses were named wthumanXIII, del1-38, and del1-83 (Fig. 1).

Analysis of Total 4-Hydroxyproline in Insect Cells Expressing Recombinant Type XIII Collagen with or without Prolyl 4-Hydroxylase-High
Five insect cells were infected with the virus del1-83 alone or together with separate viruses for the ␣ subunit (virus ␣59) and ␤ subunit (virus ␤) of human prolyl 4-hydroxylase. The virus del1-83 was used at m.o.i. 5 and viruses ␣59 and ␤ at m.o.i. 0.1 or 1. The infected cells were grown in TNM-FH medium supplemented with 10% fetal bovine serum, and ascorbate (50 g/ml) was added daily to the culture medium. The cells were harvested 72 h after infection, washed with 1ϫ PBS and homogenized in 0.5 M NaCl, 0.1% Triton, and 0.02 M Tris, pH 8.0. After centrifugation, the sample was hydrolyzed with 6 M HCl at 120°C overnight, and the total amount of 4-hydroxyproline was examined in cell homogenates by the method of Kivirikko et al. (25).
Purification and N-terminal Sequence Analysis of Recombinant Type XIII Collagen ␣ Chains-Del1-83 protein for purification was expressed in Sf9 cells, since these are easier to grow in suspension than High Five cells. The cells were infected with the del1-83 virus at m.o.i. 5 as described above, harvested 48 h after infection, washed with PBS, homogenized in a 0.013 M CHAPS, 1 mM EDTA-Na 2 , 1 mM EGTA, 1 mM N-ethylmaleimide, 0.01% phenylmethylsulfonyl fluoride, and 0.05 M Hepes buffer, pH 8.0, and ultracentrifuged at 100,000 ϫ g for 60 min. The resulting supernatant in 10% glycerol was loaded onto a phosphocellulose column (P-11, Whatman) which had been equilibrated with buffer A (10% glycerol, 0.5 mM EDTA-Na 2 , 0.5 mM EGTA, 1 mM Nethylmaleimide, 0.02% sodium azide, and 0.05 M Hepes buffer, pH 8.0). The column was washed with buffer A and eluted with a salt gradient from buffer B (buffer A including 0.3 M betaine) to buffer C (buffer B including 1 M NaCl). Fractions containing type XIII collagen were pooled and loaded onto a Resource RPC column (Amersham Pharmacia Biotech) which had been equilibrated in a 0.1% trifluoroacetic acid and 10% acetonitrile buffer. The column was eluted with a 10 -80% acetonitrile gradient in the presence of 0.1% trifluoroacetic acid. Finally, the type XIII collagen fraction was evaporated to dryness and re-suspended in 25% acetonitrile and 0.1 M ammonium acetate buffer, pH 5.0. This sample was chromatographed on a Sephadex 75 column (Amersham Pharmacia Biotech) equilibrated and eluted with 25% acetonitrile and 0.1 M ammonium acetate buffer, pH 5.0.
In order to analyze the N-terminal sequence of purified del1-83 ␣ chains, an aliquot of the fraction containing the Resource RPC-purified type XIII collagen was applied to SDS-PAGE and then electroblotted onto a ProBlott membrane (Perkin-Elmer). The band, which was visu-alized by Coomassie Blue staining and matched antibody detection with antibody XIII/NC3-1, was cut down and loaded directly into a Procise 492 Protein Sequencer (Applied Biosystems Inc.).
Production of Monoclonal Antibodies against Purified Recombinant Type XIII Collagen-Monoclonal antibodies were produced and prescreened by enzyme-linked immunosorbent assay commercially (Dia-Bor OY, Oulu, Finland). Five mice were immunized with Resource RPC-purified type XIII collagen. The sera of all these immunized mice were positive in enzyme-linked immunosorbent assay and Western blotting (data not shown). One mouse was chosen for final boosting with gel filtration-purified del1-83 protein. Six of the culture media of the fused cells (95D1-6) were positive in enzyme-linked immunosorbent assay, and the fusion 95D1 was cloned further. Three clones, 95D1A-C, were obtained, and the IgG of clone 95D1A was purified.
For preliminary epitope mapping of the resulting monoclonal antibodies, five recombinant fragments covering the indicated regions of human type XIII collagen ( Fig. 1) were produced in bacteria using the QIAexpressionist Kit (Qiagen) and analyzed by denaturing SDS-PAGE, followed by staining with Coomassie Brilliant Blue or Western blotting with the resulting monoclonal antibodies. Recombinant type I, II, and III procollagens produced in insect cells (22,26,27) (a kind gift of Johanna Myllyharju and Minna Nokelainen, Department of Medical Biochemistry, University of Oulu) were also analyzed by Western blotting using the monoclonal antibody 95D1A.
A (Pro-Pro-Gly) 10 peptide (Peptide Institute Inc., Osaka, Japan) and seven synthetic peptides, GFPGFPGPIG and GPQGQKGEKG, corresponding to residues 178 -187 and 211-220 in the COL1 domain of human type XIII collagen (3), respectively, APPGPKGEAG, corresponding to residues 403-412 in the COL2 domain, GPPGVKGENG and GPAGPKGERG, corresponding to residues 530 -539 and 646 -655 in the COL3 domain, respectively (Innovagen, Lund, Sweden), ECLSSMPAALRSS, corresponding to residues 229 -241 in the NC2 domain, and DYNGNINEALQEIRTL, corresponding to residues 454 -469 in the NC3 domain (Department of Biochemistry, University of Oulu, Finland), were used to block the antibody 95D1A reaction with recombinant type XIII collagen. The antigen competition experiments were performed by incubating 95D1A with an excess of purified peptide or with PBS as a control overnight at 4°C, followed by use in Western blotting.
Digestion with Pepsin and a Mixture of Trypsin and Chymotrypsin-Recombinant type XIII collagen (del1-38) was produced in High Five cells in the presence of exogenous prolyl 4-hydroxylase as described above. Aliquots of the resulting supernatants were digested with 0.15 mg/ml pepsin for 2 min, 5 min, or 1 h at room temperature (28). The pepsin was inactivated and the pepsin-resistant fragments were analyzed by SDS-PAGE, followed by Western blotting using the monoclonal antibody 95D1A and various type XIII collagen-specific polyclonal antibodies (Fig. 6C). The thermal stability of the pepsin-resistant recombinant type XIII collagen was studied by digestion with a mixture of trypsin and chymotrypsin (28). The samples pepsinized for 2 min were heated at 24 -70°C for 5 min and subsequently treated with the mixture of trypsin and chymotrypsin (0.02/0.05 mg/ml) for 2 min at room temperature (28). After inactivation of trypsin and chymotrypsin, aliquots of the pepsinized and trypsin/chymotrypsin-treated samples were electrophoresed on SDS-PAGE and analyzed by Western blotting using the monoclonal antibody 95D1A and various type XIII collagen-specific polyclonal antibodies. The T m values for the different collagenous domains of type XIII collagen were determined by densitometry of the intensities of the corresponding trypsin/chymotrypsin-resistant fragments using the ImageQuant Software (Molecular Dynamics).
Digestion with Trypsin-Recombinant type XIII collagen (del1-38) was produced in High Five cells in the presence of recombinant prolyl 4-hydroxylase as described above. Aliquots of the resulting supernatants were heated at 24°C for 5 min and treated with trypsin (0.01 or 0.05 mg/ml) for 2 min at room temperature. The trypsin was subsequently inactivated and aliquots of the trypsin-treated samples were electrophoresed on SDS-PAGE, followed by Western blotting using the monoclonal antibody 95D1A and the various type XIII collagen-specific polyclonal antibodies.

Production of Polyclonal Antibodies against Human Type XIII Collagen and Analysis of Their Specificity-Antibodies
have previously been produced against the NC1 and NC3 domains of type XIII collagen (3). 2 Here polyclonal antibodies were produced against two synthetic peptides, one covering part of the noncollagenous NC2 domain and another covering two Gly-X-Y triplets of the COL3 domain and all of the NC4 domain of human type XIII collagen. The amino acid sequence of the NC2 peptide represents the exon 12 alternative splice variant and has low homology with the corresponding mouse sequence, whereas the sequence of the COL3/NC4 peptide is completely conserved between these two species (3). Three recombinant baculoviruses were generated, wthumanXIII, del1-38, and del1-83, and used to study various properties of human type XIII collagen (see below) and to test the specificity of the ensuing antibodies XIII/NC2-55 and XIII/NC4-SO by Western blotting of the recombinant type XIII collagen variants produced in insect cells (Fig. 1). The wthumanXIII virus codes for full-length type XIII collagen ␣ chains, del1-38 for N-terminally truncated ␣ chains, where the cytosolic portion has been deleted and the N terminus starts from the transmembrane domain, and del1-83 for N-terminally truncated ␣ chains, where the N terminus starts from a methionine at residue 84, thus lacking the cytosolic and transmembrane domains and part of the ectodomain of the NC1 domain. These three viruses were used separately to infect High Five cells, which were also solely infected with a virus coding for the two subunits, ␣ and ␤, of human prolyl 4-hydroxylase (see Ref. 22). Forty-eight hours post-infection, insect cell supernatants were analyzed by SDS-PAGE under reducing conditions, followed by Western blotting with antibody XIII/NC2-55 (Fig. 2, lanes 1 to 4) or antibody XIII/NC4-SO (Fig. 2, lanes 5 to 8). The two antibodies gave virtually identical results, and the observed molecular masses for the wthumanXIII, del1-38, and del1-83 ␣ chains were 103, 83, and 81 kDa, respectively, while the corresponding calculated molecular masses are 65, 61.4, and 56.5 kDa, respectively. It should be noted that retarded migration is a common feature of collagens analyzed by SDS-PAGE (29). In the case of antibody XIII/NC2-55 there was no staining against cells expressing solely prolyl 4-hydroxylase, and in the case of XIII/ NC4-SO only very faint background staining was observed (Fig. 2, lanes 4 and 8), confirming their specificity for type XIII collagen.
The Del1-83 ␣ Chain Variant Is Subject to 4-Hydroxylation of Prolines-Previous studies have shown that expression of the fibrillar collagen types I, II, and III in insect cells results in translocation and post-translational modification of the ␣ chains in the lumen of the endoplasmic reticulum (22,26,27). Due to the presence of the N-terminal transmembrane domain, the wthumanXIII and del1-38 ␣ chains were presumed to be inserted into the membrane of the endoplasmic reticulum when expressed in insect cells and consequently their ectodomains were expected to be subject to the modifications that occur to collagen chains in the lumen of the endoplasmic reticulum. In fact, this assumption was confirmed by the slower SDS-PAGE mobility of the del1-38 ␣ chains when expressed in combination with prolyl 4-hydroxylase (see below). The del1-83 ␣ chains have a somewhat hydrophobic sequence at their N terminus, and thus we wanted to test whether these chains are translocated to the endoplasmic reticulum by assaying the 4-hydroxyproline content in insect cells expressing this collagen. Since the endogenous prolyl 4-hydroxylase level is not sufficient to hydroxylate recombinant collagen chains being expressed at high levels, viruses encoding the ␣ (virus ␣59) and ␤ (virus ␤) subunits of human prolyl 4-hydroxylase were used in co-infections (26) (Table I). These results indicate that the del1-83 ␣ chains are subject to prolyl 4-hydroxylation, a modification occurring only in the endoplasmic reticulum, and thus although they lack a typical signal sequence, they nevertheless appear to behave like secreted proteins.
Purification of Denatured Type XIII Collagen Ectodomain Portion-In order to study the fragments of type XIII collagen that resist digestion with proteolytic enzymes, antibodies were required against the collagenous sequences. Thus we set out to produce monoclonal antibodies against the del1-83 ␣ chains, since these chains mainly consist of collagenous sequences and were expected to be more easily purified than longer forms due to lack of the transmembrane domain (Fig. 1). Insect cells expressing del1-83 ␣ chains were harvested 48 h post-infection, washed, homogenized, and centrifuged at 100,000 ϫ g. Since the pI of the del1-83 polypeptide was calculated to be 9.33 (EMBL WWW Gateway to Isoelectric Point Service), the supernatant was fractionated by cation exchange chromatography followed by reverse phase chromatography (data not shown). Subsequently the fractions containing the del1-83 ␣ chains were pooled and fractionated on a gel filtration column. SDS-PAGE analysis followed by Coomassie staining and Western blotting (Fig. 3) suggests that the purified protein is free from contaminants. Furthermore, N-terminal sequencing of the purified protein revealed it to be a mixture of two polypeptides differing with respect to the N-terminal end. The longer form had the sequence XVNQLLDEKX, which agrees with the sequence RVNQLLDEKW beginning at residue 91 in the cDNA-deduced full-length polypeptide, and the slightly shorter form had the sequence SPGXNXPPGPP, which agrees with the sequence SPGCNCPPGPP beginning at residue 114.
Generation of a Pan-collagen Antibody Recognizing a KGEcontaining Epitope-Mouse monoclonal antibodies were generated using the purified del1-83 protein, and one of them, termed 95D1A, was used for further studies. In order to map the epitope of 95D1A, five recombinant fragments covering various portions of the human type XIII ␣ chain were produced in bacteria (Fig. 1). The antibody detected all four fragments 2-5 containing collagenous sequences, but not fragment 1 corresponding to the NC1 domain (data not shown). Furthermore, the affinity of 95D1A for fragment 2, covering the COL1 and NC2 sequences, was much weaker when compared than its action against fragments 3-5 (data not shown). In the light of these results the epitope recognized by 95D1A appeared to be collagenous in nature and probably not restricted to type XIII collagen. As shown in the Coomassie-stained gel (Fig. 4A) and the Western blot (Fig. 4B), 95D1A recognizes not only type XIII collagen but clearly also collagen types I, II, and III. Cells expressing solely the recombinant prolyl 4-hydroxylase show that there is negligible background staining with 95D1A (Fig.  4B, lane 5). Thus the monoclonal antibody 95D1A is not type XIII collagen-specific, but recognizes several types of collagens.
Further epitope mapping was performed by antigen competition experiments using six peptides encoding collagenous sequences and two encoding the NC2 and NC3 domains. We considered first the possibility that the sequence Pro-Pro-Gly occurring in all collagens would be included in the epitope, but a (Pro-Pro-Gly) 10 peptide did not block the 95D1A antibody reaction with type XIII collagen in Western blotting (Table II). Comparison of the amino acid sequences of the three collagenous domains of type XIII collagen revealed the sequence KGE at least once in all of them. Several peptides derived from the type XIII collagen sequence were tested, and the results suggested that the KGE sequence is included in the epitope, but adjacent sequences affect the antibody reaction (Table II). More specifically, the synthetic peptides APPGPKGEAG and GP-PGVKGENG derived from the COL2 and COL3 domains, respectively, blocked the 95D1A antibody reaction, whereas the  peptides GPQGQKGEKG and GPAGPKGERG did not. The most notable difference between the four peptides is the occurrence of a basic residue following the conserved KGE triplet in the nonfunctional ones, which appears to adversely affect the affinity of these peptides for the antibody. All in all, polyclonal antibodies against the NC2 and NC4 domains and a monoclonal antibody recognizing the COL2 and 3 domains strongly and the COL1 domain weakly were generated to facilitate the further characterization of the type XIII collagen protein.

Effect of Prolyl 4-Hydroxylation on Trimer Association in Recombinant
Type XIII Collagen-Type XIII collagen ␣ chains have eight cysteine residues in the NC1, COL1, NC2, and NC4 domains (see Fig. 6C) and thus may form disulfide-bonded trimers. To investigate the possible occurrence of interchain disulfide bonds, High Five cells were infected and 48 h postinfection aliquots of the cell pellets, the cell supernatants, and the culture media were analyzed on SDS-PAGE under reducing or nonreducing conditions, followed by Western blotting with the antibody XIII/NC3-1. Since pilot studies indicated that the del1-38 variant and the full-length chains gave identical results, we subsequently used the del1-38 variant to test this possibility, because of its more than 20-fold higher expression level. When insect cells were infected with this virus and the corresponding cell pellet was analyzed under reducing conditions, an immunoreactive monomer of size 83 kDa was seen (Fig. 5A, lane 1). This band could be verified as originated from the del1-38 virus, since it was not observed when cells were infected only with a virus encoding the two subunits of human prolyl 4-hydroxylase (Fig. 5A, lane 3). Analysis of nonreduced samples nevertheless revealed only minute amounts of trimeric disulfide-bonded type XIII collagen molecules (Fig. 5B, lane 1).
In the case of fibrillar collagens, the C-propeptides play an important role in the selection and correct registration of the three constituent ␣ chains (30 -32). Many of the nonfibrillar collagens are devoid of large C-propeptides, and thus additional factors may be needed for the assembly of trimeric structures. This is evident from previous studies with type XII collagen, a member of the subgroup of fibril-associated collagens with interrupted triple helices, which have indicated that hydroxylation of proline residues enhances association of the ␣ chains into disulfide-bonded trimers (33)(34)(35). To investigate the possible effect of proline hydroxylation on the assembly of type XIII collagen trimers, insect cells were co-infected with the del1-38 virus and the 4PH␣␤ virus, which encodes both human prolyl 4-hydroxylase subunits (Fig. 5A, lane 2). Somewhat more retarded migration and broadening of the del1-38 monomer band could be observed, suggesting that the ␣ chains were hydroxylated. Interestingly, trimer formation was enhanced dramatically in the nonreduced gels (Fig. 5B, lane 2). This positive effect of prolyl 4-hydroxylation on trimer formation in type XIII collagen was most clearly observed in the cell pellet fractions, but it was also detectable in the cell supernatant and culture media fractions (data not shown). Use of separate viruses for the ␣ (virus ␣59) and ␤ subunits (virus ␤) of human prolyl 4-hydroxylase together with virus del1-38 also led to enhanced trimer association of ␣1(XIII) chains (Fig. 5B, lane 3), but in a less effective fashion than with a single virus coding for both subunits (compare lanes 2 and 3 in Fig. 5B). This may be due to the fact that in the case of a single virus encoding both subunits all the infected insect cells start to express both types of subunit at the same time, whereas in the case of the two separate viruses some cells can begin to express one type of subunit before the other. In fact, when insect cells were infected with a single virus encoding both subunits the level of prolyl 4-hydroxylase activity was about 30% higher than that obtained with a similar amount of two separate viruses (22). Previous studies with an D414A mutant ␣ subunit have indi-   Ϫ indicates an identical immunosignal compared with 95D1A preincubated with PBS and ϩ indicates a markedly reduced immunosignal. In ϩ(ϩ) the immunosignal is even more strongly blocked. cated that a tetrameric enzyme is formed between two D414A mutant ␣ subunits and two ␤ subunits but that this enzyme lacks any prolyl 4-hydroxylase activity (24). Use of the virus encoding the D414A mutant ␣ subunit instead of the one encoding the wild-type ␣ subunit together with that encoding the ␤ subunit reduced the amount of trimeric type XIII collagen observed to the same level as in infections where no viruses encoding prolyl 4-hydroxylase were used (Fig. 5B, lane 4). Thus efficient association of the type XIII collagen ␣ chains into disulfide-bonded trimers requires hydroxylation of proline residues.
Pepsin Resistance of Recombinant Type XIII Collagen-We have predicted based on the primary structure of type XIII collagen that its structure is largely triple helical and has three collagenous domains, but this has never been investigated at the protein level. We therefore co-infected High Five cells with the del1-38 and 4PH␣␤ viruses, and harvested, homogenized, and centrifuged them 48 h later. The Triton X-100-soluble proteins in the cell supernatant were digested with pepsin for 2, 5, or 60 min at room temperature and the samples analyzed by SDS-PAGE under reducing condition, followed by Western blotting with various antibodies. After 2 min of pepsin digestion the monoclonal antibody 95D1A revealed five pepsin-resistant fragments, their observed molecular masses being 26, 29, 30, 32, and 36 kDa (Fig. 6A, lane 1). Since pepsin digests non-triple helical collagen ␣ chains while triple helical sequences are resistant to it, the above fragments are likely to be derived from the various triple helical domains of this collagen. To study their origin, various type XIII collagen-specific antibodies were used in Western blotting. Antibody XIII/NC4-SO reacted only with the 36-kDa fragment, which was thus considered to represent the COL3 and NC4 domains (Fig. 6A, lane  2, and Fig. 7C, lane P). Western blotting with antibody XIII/ NC3-1, produced against a synthetic peptide corresponding to the whole NC3 domain of human type XIII collagen (3), detected the 30-and 32-kDa fragments, which were thus considered to represent the COL2 and NC3 domains (Fig. 6A, lane 3,  and Fig. 7B, lane P). Since only the 30-kDa fragment of these two remained after 5 min of pepsin digestion, it was considered to originate from the 32-kDa fragment after digestion of part of the NC3 domain (data not shown). The 30-and 32-kDa fragments were also faintly visible after 2 min pepsinization with antibody XIII/NC2-55 (Fig. 6A, lane 4), confirming that these fragments represent domains COL2 and NC3 and also include part of the NC2 domain. The 26-kDa fragment, and also the 29-kDa fragment to a faint extent (Fig. 6A, lane 4, and Fig. 7A, lane P), were recognized with antibody XIII/NC2-55. These fragments were also detected with antibody XIII/NC1-Q610, produced against a bacterial fragment corresponding to the NC1 domain of mouse type XIII collagen that excluded the transmembrane domain 2 (Fig. 6B, lane 2, ϩ Red.). Collectively, these results indicate that the 26-and 29-kDa fragments represent the COL1 domain of type XIII collagen and include parts of the NC1 and NC2 domains. The antibody XIII/NC1-Q610 also detected two additional fragments of 19 and 22 kDa that were not detected with 95D1A (the 19-kDa fragment is seen at the bottom in Fig. 6B, lane 2, ϩ Red., while the minor 22-kDa fragment is not visible). These fragments are likely to represent mainly the NC1 domain and possibly part of the COL1 domain. After 60 min pepsinization only the COL3 and NC4derived 36-kDa fragment was detected with 95D1A (Fig. 6A,  lane 6). This fragment was also detected with the NC4-specific antibody XIII/NC4-SO (data not shown).
The reduction sensitivity of the pepsin-resistant fragments following digestion for 2 min was tested by comparing their mobilities by SDS-PAGE under reducing and nonreducing conditions, followed by Western blotting. When antibody 95D1A was used only the NC1-COL1-NC2-derived 26-and 29-kDa fragments were found to be reduction-sensitive (compare the ϩ and ϪRed. samples in lane 1, Fig. 6B). The 26-and 29-kDa fragments disappeared and additional, more slowly migrating bands were also detected with antibodies XIII/NC1-Q610 (Fig.  6B, lane 2) and XIII/NC2-55 (Fig. 6B, lane 3) in the nonreduced samples. The other three pepsin-resistant fragments had the same mobility as the reduced and nonreduced samples even when detected using antibodies XIII/NC3-1 and XIII/NC4-SO (data not shown). The 19-and 22-kDa fragments identified with antibody XIII/NC1-Q610 were also sensitive to reduction (Fig. 6B, lane 2).
According to these pepsinization results, recombinant type XIII collagen produced in insect cells folds into a triple-helical conformation with pepsin-sensitive regions in the non-collagenous domains. Some of the cysteines in the NC1 domain, and possibly the cysteines at the junction of the COL1 and NC2 domains seem to be interchain-linked. The two cysteine residues in NC4 are likely to form intrachain disulfides, since the 36-kDa pepsin-resistant fragment detected by the antibody XIII/NC4-SO was not reduction-sensitive. It was considered possible, however, that pepsin could have digested part of the NC4 domain, including the two cysteine residues within it. Thus in additional experiments, the cell supernatant containing the del1-38 variant was digested only with trypsin and the products were analyzed as above (data not shown). These studies provided the same results with respect to reduction sensitivity as the pepsin digestions (data not shown). Since there are no predicted cleavage sites for trypsin at the NC4 domain, these results confirm that the two cysteine residues within this domain do not participate in interchain disulfide formation. The analyses of reduced and nonreduced pepsinized recombinant type XIII collagen are summarized in Fig. 6C. Trypsin/Chymotrypsin Resistance of Type XIII Collagen-Trypsin/chymotrypsin digestion was used to confirm further the triple-helical nature of the type XIII collagen produced by the insect cells and to assess its thermal stability. Cell supernatant containing the del1-38 protein was first treated with pepsin for 2 min, after which the pepsin was inactivated and the samples were heated at 24 -70°C and subsequently digested with a mixture of trypsin and chymotrypsin. Aliquots of the pepsinized and trypsin/chymotrypsin-treated samples were analyzed by SDS-PAGE under reducing conditions, and antibody 95D1A revealed seven resistant fragments, with apparent molecular masses of 23,24,25,29,31,33, and 37 kDa after preheating the sample at 24°C (data not shown). When antibody XIII/NC2-55 was employed, one fragment of size 26 kDa could be observed (Fig. 7A, lanes 24 -55). Since the 26-kDa fragment could also be detected with antibody XIII/NC1-Q610 (data not shown), it is likely to represent part of the NC1 domain, all of COL1 and part of NC2. The T m value for the COL1 domain was evaluated to be 38°C by densitometering the intensity of the 26-kDa fragment after preheating the sample at elevated temperatures (Fig. 7D). Antibody XIII/NC3-1 detected 23-, 24-, and 25-kDa resistant fragments (Fig. 7B,  lanes 24 -55), which thus appear to represent parts of the COL2 and NC3 domains. There is a potential trypsin cleavage site in the COL2 domain, at a short imperfection in the repeating GXY sequences. This site appears to be utilized, since the pepsinresistant 30 -32-kDa fragments are degraded to 23-25-kDa fragments even at the lowest temperatures of trypsin-chymotrypsin treatment. The total intensity of the 23-25-kDa fragments was measured and a T m value of about 49°C was obtained for COL2 (FIG. 7D). Antibody XIII/NC4-SO detected a major 37-kDa fragment and minor 31-and 33-kDa fragments (Fig. 7C, lanes 24 -55), which thus appear to represent the COL3 and NC4 domains. The T m value for COL3 was calculated to be about 40°C by measuring the intensity of the 37-kDa fragment (Fig. 7D). DISCUSSION There is very little protein level data on the transmembrane collagen type XIII, since it is expressed at low levels in various cultured cells and in tissues, but we have overcome the problem here by producing this collagen in insect cells. Full-length type XIII collagen ␣ chains were found to be expressed only at low levels, while markedly better expression levels were observed when the chains lacked the first 38 residues, encompassing the cytosolic domain, or the first 83 residues which cover in addition the transmembrane domain and part of the extracellular noncollagenous domain. Since the lack of the cytosolic domain did not affect the formation of disulfide-bonded trimers, we therefore mainly concentrated on studying the characteristics of the del1-38 ␣ chains.
Previous studies had indicated that insect cells contain low endogenous activity levels of the key enzyme in the biosynthesis of collagens, prolyl 4-hydroxylase (26,36). When the pro-␣1 chains of human type III collagen were produced in insect cells, they formed triple helical molecules with a T m of only about 32-34°C, and since coexpression with constructs encoding human prolyl 4-hydroxylase resulted in an increase in the T m to about 40°C, it became clear that the recombinant expression of collagens in insect cells requires coexpression with prolyl 4-hydroxylase in order to generate correctly hydroxylated recombinant collagen molecules (26). The fact that coexpression of the del1-38 ␣ chains with the ␣ and ␤ subunits of human prolyl 4-hydroxylase increased the apparent molecular mass of the former is indicative of prolyl 4-hydroxylation of the type XIII collagen synthesized in insect cells by the recombinant enzyme. Since the collagenous portion of the del1-38 ␣ chains was accessible to prolyl 4-hydroxylation occurring in the lumen of the endoplasmic reticulum, the ␣ chains had a type II transmembrane orientation despite the lack of the N-terminal cytosolic domain. Surprisingly, when the del1-83 ␣ chains were expressed in insect cells with or without the human prolyl 4-hydroxylase, the 4-hydroxyproline content of the crude cell homogenate was shown to increase in relation to the expression of recombinant prolyl 4-hydroxylase. Thus it was of interest to note that the del1-83 ␣ chains also appeared to be translocated to the lumen of the endoplasmic membrane, presumably via the use of an internal methionine at residue 84. N-terminal sequencing of the del1-83 ␣ chains produced in insect cells re- vealed two polypeptide forms, one with the arginine at position 91 and the other one with the serine at position 114 to be the extreme residue. We do not know whether the shorter form is derived from the longer one or whether they represent del1-83 ␣ chains present in the endoplasmic reticulum and in the cytosolic compartment, and thus being subject to different proteolytic processing. Nevertheless, the amino acid residues adjacent to methionine 84 contain some hydrophobic residues, and the occurrence of 4-hydroxylation of prolines suggest that this internal sequence, although not fulfilling the criteria for a classic signal peptide, may act as such at least in situations where the synthesis of full-length ␣ chains is prohibited.
The type XIII collagen ␣ chains expressed in insect cells were found to form disulfide-bonded trimers, but only at low efficiency, whereas coexpression with human prolyl 4-hydroxylase enhanced this trimer formation dramatically. The fact that some disulfide-bonded trimers of type XIII collagen were observed without simultaneous expression of recombinant prolyl 4-hydroxylase may be due to the presence of endogenous prolyl 4-hydroxylase. When this collagen was expressed together with a mutant ␣ subunit and a wild-type ␤ subunit, the enhancement of trimer formation was abolished. These mutant ␣ subunits are known to be able to form only a catalytically inactive tetramer with wild-type ␤ subunits (24), demonstrating that the enhanced trimer formation of type XIII collagen is dependent on active recombinant prolyl 4-hydroxylase. The positive effect of prolyl hydroxylation on the formation of interchain disulfide-bonded molecules was first shown for type XII collagen, one of the FACIT group (fibril-associated collagens with interrupted triple helices) (33)(34)(35). Expression of type XII minicollagen in insect cells has demonstrated that prolyl 4-hydroxylase is involved in the trimeric assembly of this collagen type through its ␣ subunit, and thus through its hydroxylase activity (35). With respect to the fibrillar collagens, it is known that the C-propeptides of type I procollagen, pro-␣ chains can associate and form interchain disulfide bonds independent of the hydroxylation state of the respective pro-␣ chains, and this is thought to apply to all fibrillar collagens (37). Recent studies on type III procollagen by Bulleid et al. (38) indicate, however, that hydroxylation and nucleation of the triple helix are necessary for the formation of the interchain disulfide bonds at its C-telopeptides. Thus hydroxylation enhances the formation of disulfide-bonded trimeric collagen molecules in the case of the nonfibrillar FACIT collagens and type XIII collagen, and also in the case of fibrillar collagens.
In order to be able to study type XIII collagen in more detail at the protein level, we have generated two novel polyclonal antibodies, one against part of the NC2 domain and one against the C-terminal end of COL3 and all of the NC4 domain. Since we have already produced antibodies that recognize the NC1 domain 2 and the NC3 domain (3), we now have a palette of polyclonal antibodies against all four noncollagenous domains FIG. 7. Analysis of the thermal stability of the recombinant type XIII collagen by trypsin/chymotrypsin digestion. High Five cells were infected and treated in the same way as for the pepsin digestions (Fig. 6). The cell supernatant was treated with pepsin for 2 min at room temperature, after which samples were digested with a mixture of trypsin and chymotrypsin (0.02/0.05 mg/ml) at temperatures between 24 to 55°C (or to 70°C in the case of COL2) and the digestion reactions were terminated by adding soybean trypsin inhibitor. of human type XIII collagen that can be used for Western blot analysis of the protein. In addition to these antibodies, an antibody against the triple-helical region of type XIII collagen was needed to facilitate studies of the pepsin and trypsin/ chymotrypsin resistance of the collagenous domains of the recombinant collagen. Thus monoclonal antibodies were produced against recombinant N-terminally truncated ␣ chains of human type XIII collagen, which were mainly composed of collagenous sequences. The ensuing monoclonal antibody, 95D1A, was found to detect several other collagen types in addition to type XIII. Epitope mapping indicated that collagenous peptides with the sequence KGE can block the antibody reaction. Subsequently, this monoclonal pancollagen antibody has proven useful for detecting various types of recombinant collagens and their fragments in Western blotting.
Analyses of the pepsin and trypsin/chymotrypsin-resistant fragments using the monoclonal antibody 95D1A and the type XIII collagen-specific antibodies indicated that the three collagenous domains of the del1-38 protein are in triple-helical conformation. Further examination of the pepsin-resistant fragments under nonreducing conditions produced information on the disulfide bonding of the cysteine residues of the del1-38 protein. This protein contains altogether eight cysteine residues, two in the transmembrane domain, two in the C-terminal end of the NC1 domain, two at the junction of the COL1 and NC2 domains, and two in the NC4 domain (Fig. 6C). Interchain disulfide bonds could be located in the NC1 domain and possibly at the junction of COL1 and NC2, while the cysteine residues in NC4 are likely to form intrachain bonds.
Studies on type XIII collagen mRNAs derived from many tissues and a number of cultured cells have shown that they are products of complex alternative splicing affecting sequences encoding the COL1, NC2, COL3, and NC4 domains (5-10). As a result, it is predicted that these domains vary markedly in size, and the same cell appears to synthesize several splice variants. Consequently, it is possible that both homotrimeric and heterotrimeric type XIII collagen molecules are formed. We have demonstrated here that the splice variant represented by the del1-38 ␣ chains can form homotrimeric molecules with stable triple helices and that the T m of the shortest collagenous domain in these molecules, COL1, is 38°C, that of the COL2 domain is 49°C and that of the C-terminal COL3 domain 40°C. The T m values of the collagenous triple helices are usually slightly above body temperature, as in the cases of the fibrillar collagen types I-III, with T m of about 40 -42°C (2). A T m well above body temperature has been observed for one of the three collagenous domains of type IX collagen, namely 49.0°C for the COL1 domain (39,40). Our results also indicate that the central collagenous domain of type XIII collagen has an unusually high T m . Since this domain is invariant in terms of alternative splicing, it could be suggested that the central portion has an important role in the stability of the molecule.
All in all, most of the type XIII collagen ectodomain appears to occur in a triple helical conformation and thus it is predicted that the ectodomain is rodlike, with only one or two flexible hinges. The longest noncollagenous sequence in the type XIII collagen ectodomain is the portion of about 60 residues in the NC1 domain immediately adjacent to the plasma membrane of the cells, while the NC2-NC4 domains, located in the central part and at the free end of the ectodomain vary between 9 and 34 residues, and the three collagenous domains COL1-3 encompass altogether about 500 residues. This is in sharp contrast to the other known collagenous transmembrane proteins, which have ectodomains with substantial noncollagenous portions and only short collagenous portions, ranging from about 60 to 270 residues, namely the EDA gene product, C1q, the macrophage scavenger receptors, MARCO (41)(42)(43)(44), or highly interrupted collagenous portions, as is the case for type XVII collagen (45). Thus type XIII collagen, which has only recently been identified as a member of the collagenous transmembrane proteins, presents a structurally separate entity among them.