Involvement of prolyl 4-hydroxylase in the assembly of trimeric minicollagen XII. Study in a baculovirus expression system.

We have shown previously that hydroxylation played a critical role in the trimer assembly and disulfide bonding of the three constituent α chains of a minicollagen composed of the extreme C-terminal collagenous (COL1) and noncollagenous (NC1) domains of type XII collagen in HeLa cells (Mazzorana, M., Gruffat, H., Sergeant, A., and van der Rest, M. (1993) J. Biol. Chem. 268, 3029-3032). We have further characterized the involvement of prolyl 4-hydroxylase in the assembly of the three α chains to form trimeric disulfide-bonded type XII minicollagen in an insect cell expression system. For this purpose, type XII minicollagen was produced in insect cells from baculovirus vectors, alone or together with wild-type human prolyl 4-hydroxylase or with the human enzyme mutated in the catalytic site of its α or β subunits or with the individual α or β subunits. When type XII minicollagen was produced alone, negligible amounts of disulfide-bonded trimers were found to be produced by the cells. However, coproduction of the collagen with the two subunits of the wild-type human enzyme dramatically increased the amount of disulfide-bonded trimeric type XII minicollagen molecules. In contrast, coproduction of the collagen with α subunits that had a mutation completely inactivating the human enzyme failed to enhance the trimer assembly. These results directly show that an active prolyl 4-hydroxylase is required for the assembly of disulfide-bonded trimers of type XII minicollagen.

The most abundant components of the extracellular matrices are the collagens. Owing to their diverse structural and functional properties, these multidomain proteins contribute significantly to the high diversity of these matrices. The collagens are defined as proteins that contain one or more characteristic triple-helical domains. In these regions, the three constituent polypeptides, termed ␣ chains, are composed of repeated Gly-X-Y amino acid triplets (1)(2)(3). During their biosynthesis, they undergo various post-translational modifications (1)(2)(3). One of these reactions, hydroxylation of many of the proline residues located in the Y positions of the repeating Gly-X-Y triplets into 4-hydroxyprolines, is an absolute requirement for the forma-tion of stable triple helices. It has been suggested that 4-hydroxyprolines contribute to the stability of the collagen triple helix by providing additional interchain water bridges (4). The enzyme catalyzing this modification is prolyl 4-hydroxylase (3,5).
Fibril-Associated Collagens with Interrupted Triple helices (FACITs, 1 types IX, XII, XIV, and XVI) are nonfibrillar collagens characterized by the presence of triple-helical (COL) domains interspersed with non-triple-helical (NC) domains (6,7). They do not form the quarter-staggered fibrils characteristic of the so-called fibril-forming collagens (types I-III, V, and XI), but they are associated with these fibrils (8,9). The precise biological functions of the FACIT collagens are still unknown, but recent data suggest that they participate in bridging processes that allow the binding of the fibrils formed by the fibril-forming collagens to each other (10) and they promote interactions of the fibrils with other components of the extracellular network such as proteoglycans (11,12). Type XII collagen was initially described based on a cDNA clone from chick embryo fibroblasts (13). Type XII collagen molecules are homotrimers consisting of three identical ␣1(XII) chains with two triple-helical (COL1 and COL2) domains and three non-triplehelical (NC1, NC2, and NC3) domains (6,7). It is mainly localized in tissues containing type I collagen, such as tendons, bones, ligaments, dermis, and blood vessel walls (14 -16), but it is also detected in cartilage, which contains type II collagen as its major constituent (17).
In contrast to fibril-forming collagens, FACITs are not synthesized as procollagens and they are devoid of classical Cpropeptides present in fibrillar collagens. In the case of the fibrillar collagens, the C-propeptides play an important role in the selection and correct registration of the three constituent ␣ chains (18 -20). In their absence and assuming that the triple helix of FACITs propagates from the C terminus to the N terminus of these molecules as in fibrillar collagens, we have postulated that their C-terminal domains (COL1 and/or NC1) contain the information required for assembly of the molecules (21). Expression in HeLa cells of a type XII minicollagen containing the sequences coding for these terminal domains has suggested that hydroxylation plays a key role in the assembly of disulfide-bonded trimers of type XII collagen (21).
Prolyl 4-hydroxylase (EC 1.14.11.2) from vertebrates is an ␣ 2 ␤ 2 tetramer built up of two different types of subunits (5,22). The ␣ subunits (ϳ63 kDa) contain most of the catalytic sites but are inactive in the absence of the ␤ subunits (5,22). The ␤ subunits (ϳ55 kDa) were found to be identical to the enzyme protein-disulfide isomerase (PDI, EC 5.3.4.1; see Refs. 23 and 24), which catalyzes thiol/disulfide interchange in protein substrates, leading to the formation of the set of disulfide bonds that permit establishment of the most stable state of the protein (25). The ␤ subunits retain 50% of their protein-disulfide isomerase activity when participating in the prolyl 4-hydroxylase tetramer (26). Recently, a fully active human prolyl 4-hydroxylase tetramer was produced in insect cells by coinfection with two recombinant baculoviruses, one of them coding for the ␣ subunit and the other for the ␤ subunit (27). Furthermore, recombinant human type III collagen with a 4-hydroxyproline content and T m very similar to those of the nonrecombinant collagen was produced in insect cells by coexpressing the type III procollagen chains with the human prolyl 4-hydroxylase subunits (28).
To better understand the contributions of the various cellular post-translational modifications to the assembly of trimeric type XII minicollagen, we have studied the conditions required for the production of disulfide-bonded type XII minicollagen in insect cells using the baculovirus expression system. For this purpose we have infected the cells with recombinant viruses encoding type XII minicollagen together with various combinations of recombinant viruses encoding the wild-type or mutant human prolyl 4-hydroxylase.

Construction of the Baculovirus Transfer Vector and Generation of the Recombinant Virus-
The baculovirus transfer vector coding for the recombinant minigene for type XII collagen was constructed using the transfer vector pVL1392 (29) (PharMingen, San Diego, CA) and our previously described construct pRc/CMV COL XII 23 (30). The eukaryotic expression vector pRc/CMV COL XII 23 contains the sequences coding for the signal peptide of the ␣1 chain of the human type I collagen (COL1A1), a tagging sequence corresponding to a twice-repeated short epitope of the human c-myc protein, and the five last amino acid residues of the NC2 domain and the entire COL1 and NC1 domains of chicken type XII collagen (30). pRc/CMV COL XII 23 and pVL 1392 were digested with HindIII and NotI, respectively. Both linearized DNAs were submitted to filling with Klenow and subsequently digested with XbaI. The HindIII-filled/XbaI fragment of pRc/ CMV COL XII 23 encompassing the type XII collagen-coding sequences was ligated to pVL1392 containing a NotI-filled and a cohesive XbaI extremity. The recombinant pVL construct was cotransfected into Spodoptera frugiperda Sf9 insect cells with a modified Autographa californica nuclear polyhedrosis virus DNA using the BaculoGold transfection kit (PharMingen) and the calcium phosphate method (31,32). The resultant viral pool was collected, amplified, and plaque-purified (33). The ensuing recombinant virus, termed COL XII 23, was checked by a polymerase chain reaction-based method (34).  35 and 36). In the DNA of the ␣-H412S viruses, the codon for His-412 was altered to a codon for Ser by site-directed mutagenesis. The corresponding mutant ␣ subunits are able to form tetramers with the wild type ␤ subunits, but the prolyl 4-hydroxylase activity is completely lost (35). The His is thought to be needed for the iron binding of prolyl 4-hydroxylase (35,37). In the DNA of the PDI/␤NC viruses, the codons of two cysteines (one located at the N-terminal catalytic site of the ␤ subunit and the other at the C-terminal catalytic site) were replaced by two codons for serine (36). The corresponding mutated subunits have no PDI activity but are able to form tetramers with the wild-type ␣ subunits. In these conditions the tetramers retain prolyl 4-hydroxylase activity (36).

Infection of Insect Cells with Recombinant Viruses-Sf9 cells or
A recently described baculovirus expression system for efficient expression of triple-helical collagen was used (28). The insect cells were cultured in a mixture (50:50) of two culture media, IPL 41 (Sigma) and Sf900 (Life Technologies, Inc.) containing 0.05% pluronic acid (Life Technologies, Inc.) and 5% heat-inactivated fetal calf serum at 27°C in culture dishes (diameter 10 cm). To produce recombinant proteins, 10 7 cells/dish were seeded and infected at a multiplicity of infection of 5 when the cells were infected with COL XII 23 viruses alone, 5:1 when cells were infected with COL XII 23 and ␣ or ␤ viruses, and 5:1:1 when cells were infected with the three viruses (COL XII 23, ␣, and ␤). The same ratios of multiplicity were used when infection was performed with the mutant viruses ␣-H412S and PDI/␤NC in the different combinations described above. Sodium ascorbate, when present, was added at 50 g/ml immediately after the replacement of the viral inocula with fresh media (7 ml/dish). Cells and media were collected 65 h after infection.
Analysis of the Produced Recombinant Minicollagen XII-The minicollagen XII produced by Sf9 cells was recovered from the culture media by immunoprecipitation using protein G-Sepharose. The protocol essentially followed that described by Sugrue (38) except that 0.5 mM phenylmethylsulfonyl fluoride and 10 mM N-ethylmaleimide were added to the samples before dialysis. We have used a mouse monoclonal antibody produced by the hybridoma cells Myc-9E10.2 (ATCC CRL 1729) designated in this report as 9E10. This antibody is directed against the tagging sequences derived from the human c-myc protein (39) present at the N terminus of the minicollagen XII construct (30). Ascite fluid was used for immunoprecipitation at 1:500 dilution. The protein G-Sepharose-antibody-antigen complexes were dissolved in sample buffer containing 1% SDS in presence or absence of 1% 2-mercaptoethanol and analyzed by a 7.5-15% gradient SDS-PAGE, followed by Western blotting with antibody 9E10 (hybridoma supernatant 1:100 dilution).
To recover the minicollagen from cell homogenates, Sf9 cells were gently suspended in 7 ml of phosphate-buffered saline (PBS) or in a 25 mM Tris/HCl, pH 7.4 buffer, containing 140 mM NaCl, 5 mM KCl, 0.7 mM Na 2 HPO 4 , 5.5 mM glucose, and 0.5 mM EDTA). They were centrifuged at 200 ϫ g for 5 min at room temperature. The cell pellet was resuspended in 160 l of 20 mM Tris/HCl buffer, pH 7.2, containing 2 mM CaCl 2 in ice and transferred in 1.5-ml Eppendorf tubes. Protease inhibitors were added at the concentrations described for the media. Samples were treated with 0.3% SDS in the absence of a reducing agent and boiled for 5 min in a water bath. After cooling in ice, they were digested with 3.3 g of DNase (stock solution, 1 mg/1.5 ml of 500 mM Tris/HCl buffer, pH 7, containing 50 mM MgCl 2 ) in order to decrease their viscosity (40). Finally, the samples were frozen in liquid nitrogen. When utilized, they were thawed and 50 mM Tris/HCl buffer, pH 7.6, containing 0.2 M NaCl, 0.5% Triton X-100, and 1 mM EDTA were added to complete the volume to 1 ml. After centrifugation (11,200 ϫ g for 5 min at 4°C) to eliminate cell debris, supernatants were treated for immunoprecipitation under conditions described above.
Trypsin Digestion of Type XII Minicollagen Present in the Culture Medium of Sf9 Cells-The conditions and the products used for the digestion are the same as those described in a previous paper (21). The monoclonal antibody 9E10 was used for the immunoprecipitation of the tagged minicollagen XII with protein-G-Sepharose before elution with glycine and enzymatic digestion. SDS-PAGE and analysis of the digested samples by Western blotting were carried out under conditions described above. However, the blotted protein was revealed with the mouse monoclonal antibody 75d7 kindly provided by Dr. S. P. Sugrue (Harvard Medical School, Boston, MA) (15), since trypsin eliminates the epitopes of the antibody 9E10 but not the epitope of 75d7 (a short peptide of the NC1 domain) (30).
Purification of Type XII Minicollagen on Affinity Column Myc-9E10.2-Insect cells (Sf9 or High Five) were infected as described above, and 60 ml of the resulting culture medium were used. The affinity column was prepared by immobilizing the monoclonal antibody 9E10 on Avidgel (Avidchrom, Hydrazide Avidgel, Unisyn Technologies) according to the manufacturer's recommendations. The beads of the gel were first equilibrated in PBS and then batch-incubated (1 h at 4°C) with the medium of infected Sf9 or High Five cells. The column was packed and washed first with PBS alone (two volumes), this being followed by 20 volumes of PBS containing 0.5 M NaCl at 4°C. The antigen was eluted from the column with 0.1 M glycine, pH 2.3, and the corresponding fractions immediately neutralized with 1 M Tris/HCl, pH 8. Then, 300 l of these fractions were precipitated with 8% trichloracetic acid, dried, washed with acetone, dissolved in nonreducing sample buffer containing 1% SDS, and loaded onto a 10% SDS-PAGE according to Laemmli (41).
Collagenase Digestion of the Type XII Minicollagen Purified on Affinity Column-The fractions containing the minicollagen XII were pooled, and 500 l were used for the enzymatic digestion. The dry trichloracetic acid-precipitate was dissolved in collagenase buffer (50 mM Tris/HCl buffer, pH 7.5, containing 0.5 M NaCl, 6 mM CaCl 2 , and 10 mM N-ethylmaleimide). The suspension was digested with 0.6 unit of collagenase (clostridiopeptidase A, form III, Advance Biofactures Corp.) for 90 min at 33°C, and then treated with five volumes of acetone for 1 h at Ϫ20°C to stop the reaction. After centrifugation at 15,000 ϫ g for 10 min at 4°C, the samples were analyzed by SDS-PAGE as described above.

Involvement of Prolyl 4-Hydroxylase in the Formation of
Disulfide-bonded Trimeric Type XII Minicollagen-In HeLa cells, we have shown that the assembly of disulfide-bonded trimers of minicollagen XII did not occur under conditions preventing hydroxylation of proline residues (absence of ascorbate or presence of ␣,␣Ј-dipyridyl, see Ref. 21). In contrast to HeLa cells, insect cells contain a low prolyl 4-hydroxylase activity, which can be markedly increased by infecting the cells with recombinant baculoviruses directing the synthesis of prolyl 4-hydroxylase (28,42). The insect cells thus represent a useful tool to determine the essential parameters required for the synthesis of this protein by comparison with the data obtained in HeLa cells (21). For this purpose, Sf9 cells were infected with COL XII 23 viruses alone (under these conditions recombinant human type III collagen has been shown to be underhydroxylated; see Ref. 28) or with COL XII 23 viruses together with ␣ and ␤ viruses encoding the two types of subunit of human prolyl 4-hydroxylase (co-expression of recombinant human type III collagen and the human prolyl 4-hydroxylase subunits has been shown to result in full hydroxylation; see Ref. 28). In the culture medium, at 65 h post-infection, negligible amounts of trimeric molecules of minicollagen XII were observed when cells were infected with the COL XII 23 viruses alone (Fig. 1A, lanes 2 and 3), whereas trimers were clearly visible when the cells were simultaneously infected with the three types of viruses, as shown after Western blot analysis of the immunoprecipitated minicollagen XII (Fig. 1A, lanes 4 and  5; T, 105 kDa). These results show that hydroxylation of the COL XII 23 chains, arising from the presence of an active human prolyl hydroxylase, is essential for the assembly of disulfide-bonded trimeric type XII minicollagen molecules.
At least three bands corresponding to individual COL XII 23 ␣ chains (based on their apparent molecular masses) were visible ( Fig. 1A; M1, 44 kDa; M2, 42.5 kDa; M3, 37 kDa). The presence of the exogenous human enzyme modified the migration of the polypeptides corresponding to M2 and M3 promoting their shift toward the higher molecular mass M1 form. This suggests that the M1 chains contained more hydroxylated proline residues. However, the shift was too marked to be due only to the change in molecular mass rendered by a more extensive hydroxylation, and it may reflect in addition a change in conformation. Dimeric assemblies of the previously described isolated chains were also visible in variable amounts, particularly in the absence of hydroxylation ( Fig. 1A; D1, 91 kDa; D2, 83 kDa; D3, 74 kDa).
Reduction of the minicollagen XII produced in the presence of the active human enzyme showed that the trimers obtained in these conditions were indeed reducible. As reduction did not promote a real increase in the amount of a particular monomer, it could not be determined in our experimental conditions from which monomers the trimers were constituted. Furthermore, reduction induced a shift toward higher apparent molecular masses for the three bands of monomers, showing that intrachain oxidation occurred. However, reduction did not promote the transformation of one monomer to an other, indicating that cyclization due to the formation of intrachain disulfide bridges did not account for the different apparent molecular masses observed between the monomers (data not shown).
The presence of sodium ascorbate in the culture medium did not improve the formation of trimers (Fig. 1A, lanes 4 and 5). These results are different from those observed in HeLa cells. In these cells, in the absence of sodium ascorbate, the amounts of disulfide-bonded trimers present in the culture medium were very low (21). These data suggest that the amount of the overproduced exogenous enzyme present in the insect cells was sufficient to hydroxylate the available substrate (␣ minicollagen XII chains), even in the absence of sodium ascorbate. The high amount of the enzyme probably compensated for the loss of its activity, and the substrate concentration was the limiting factor with respect to the enzymatic reaction. However, in insect cells, the effect of ascorbate on the formation of disulfidebonded trimers may be masked by its partial degradation due to its instability in solution (sodium ascorbate was added only once at the beginning of the experiments, and media were collected 65 h after infection of the insect cells and only 24 h after transfection of HeLa cells).
The results were almost identical for the corresponding cell homogenates (Fig. 1B). Only minor amounts of trimeric molecules were observable in cells infected with COL XII 23 viruses alone (Fig. 1B, lanes 2 and 3). Dimeric assemblies are not visible in the cell extract shown in Fig. 1B, but they were observed in the cell extracts of other experiments, indicating that these associations were not artifactual assemblies restricted to the culture medium. In the cells infected with the three types of viruses, the amount of the trimeric protein present in the cell homogenates was similar to the amount of the protein secreted in the culture medium (Fig. 1B, lanes 4 and 5). Previously, we had never observed trimers in HeLa cell homogenates (21), suggesting that the rate of secretion of the recombinant minicollagen XII is slower in insect cells than in HeLa cells. The low rate of secretion of secretory proteins is considered a characteristic of insect cells, and we have previously observed that also recombinant type III collagen molecules are secreted at a lower rate from insect cells than from mammalian cells (28). All in all, the data reported here clearly show that the addition of the exogenous human enzyme promotes the formation of trimers of minicollagen XII. The data thus provide direct evidence for the involvement of prolyl 4-hydroxylase in the formation of trimeric molecules of type XII minicollagen. Fig.  2. The digestion of the immunoprecipitated minicollagen XII with trypsin (Fig. 2, lane 2) resulted in the disappearance of the bands corresponding to the previously described monomers, which, in contrast to COL1, are not protected from the trypsin by the triple-helical conformation. As also expected, the band corresponding to the trimers disappeared and gave rise to the new bands located between the two arrows in Fig. 2 (lane 2). Their molecular masses range from 56 to 68 kDa. Since the NC1 domain contains several arginine and lysine residues, these bands correspond to the undigested COL1 domain with various fragments of the partially digested NC1 domain.

Conformation of the Trimeric Molecules Synthesized in Insect Cells-The triple-helical conformation of the COL1 domain of the type XII minicollagen produced and secreted by insect cells infected with the three types of viruses is demonstrated in
Respective Roles of the ␣ and ␤ Subunits of Prolyl 4-Hydroxylase in the Formation of Trimeric Molecules of Minicollagen XII-Sf9 cells were infected with COL XII 23 viruses in combination with wild-type or mutant ␣ or/and ␤ viruses. As previously mentioned, the mutations in the catalytic sites of the ␣ and ␤ subunits result in the loss of hydroxylase and proteindisulfide isomerase activities, respectively, but do not prevent the tetrameric association of mutant ␣ subunits with wild-type ␤ subunits and vice versa (35,36). As expected, trimeric type XII minicollagen molecules were observed in the culture medium of Sf9 cells infected with the three types of viruses (COL XII 23, ␣, and ␤) (Fig. 3, T, lane 3). Furthermore, when the cells were infected in the same conditions, but with COL XII 23 and ␣ viruses, trimeric molecules of minicollagen XII were again observed (Fig. 3, lane 5). The presence of trimers of minicollagen XII under these conditions shows that sufficient amounts of an active prolyl 4-hydroxylase was formed by complementation of the human ␣ subunits with the insect cell PDI (␤ subunits). In contrast, trimers of type XII minicollagen were not present when the cells were infected with COL XII 23 and ␤ viruses (Fig. 3, lane 4).
It is well documented that in insect cells the expression of the ␣ subunit alone results in formation of mainly insoluble aggregates, but some of the ␣ subunits remain soluble and associate with the insect cell PDI/␤ subunits to form small amounts of active prolyl 4-hydroxylase (27,42). Consequently, minor amounts of prolyl 4-hydroxylase activity can be detected in insect cells only infected with the human ␣ subunit compared with those coinfected with both the human ␣ and ␤ subunits (27,42). Moreover, the complementation of Caenorhabditis elegans ␣ subunits with human ␤ subunits has recently been demonstrated in insect cells (42). The type XII minicollagen production level using the Sf9 cells coexpression system was lower than that recently observed for human type III collagen (28). Thus, in these cells infected with the COL XII 23 and ␣ viruses, the contribution of the endogenous insect cell PDI/␤ subunits could be sufficient to allow the formation of enough active human/insect hybrid enzyme to hydroxylate the available minicollagen chains followed by their assembly into disulfide-bonded trimeric molecules.
Infection of the cells with mutant ␣ viruses (␣-H412S viruses) with a mutation abolishing hydroxylase activity of the enzyme without affecting enzyme tetramer assembly failed to produce the band corresponding to the trimers (Fig. 3, lane 2). In contrast, a mutation in the catalytic site of the human ␤ subunits did not impair the formation of trimers of minicollagen XII as indicated by the data obtained in the cells infected with COL XII 23, ␣ and PDI/␤NC viruses (Fig. 3, lane 1). The results demonstrate that prolyl 4-hydroxylase is an absolute requirement for the formation of disulfide-bonded trimers of minicollagen XII through its ␣ subunits and thus through its hydroxylase activity.
Affinity Chromatography of Type XII Minicollagen Produced in the Culture Medium of Sf9 and High Five Cells-Cells were infected with the three types of viruses (COL XII 23, ␣, and ␤). After 65 h of infection, 60 ml of the culture medium containing sodium ascorbate were applied to the affinity column Myc-9E10.2. The minicollagen XII was mainly recovered in the glycine-eluted fractions 4 -6. The major bands corresponded to the disulfide-bonded trimeric molecules of minicollagen XII (Fig. 4A, T) and, to the isolated ␣ minicollagen XII chains (Fig.  4A, M) present as multiple bands as previously observed in the Western blots in Figs. 1-3. In addition, a 69-kDa band was observed, which was likely to represent bovine serum albumin based on its apparent molecular mass (Fig. 4A, BSA) and resistance to collagenase digestion (Fig. 4B, BSA). The recovery of secreted type XII minicollagen using the affinity chromatography was roughly estimated to be 0.5 mg/liter of cell cultures based on Coomassie Blue staining.
In order to clearly identify the bands relevant to minicollagen XII, fractions 4 -6 were pooled and an aliquot was treated with bacterial collagenase. This enzyme is specific for the sequence repeats (Gly-X-Y) n characteristic of the helical part of the collagens. As shown in Fig. 4B, the bands corresponding to the trimers (T) and monomers (M) of the minicollagen XII disappeared. The intact disulfide-bonded noncollagenous NC1 domain was clearly visible (Fig. 4B, lane 2, NC1, 22 kDa). As expected, the band presumed to be bovine serum albumin was not digested by the enzyme.
Since High Five cells have been shown to express significantly higher levels of recombinant proteins than other insect cells (28,43), we compared the amounts of minicollagen XII produced in Sf9 and in High Five cells. Affinity chromatography of High Five culture medium was conducted in the same conditions on the Myc-9E10.2 column (Fig. 5). The results show that High Five cells infected with the three types of viruses synthesized larger amounts of minicollagen XII than Sf9, the recovery of the affinity-purified collagen being approximately 4 mg/liter of cell culture medium. Furthermore, the trimeric assembly seemed more efficient in High Five cells than in Sf9 cells, since the ratio between trimers and monomers was clearly higher in High Five cells than in Sf9 cells. DISCUSSION The data reported here show that in insect cells, which have a low level of endogenous prolyl 4-hydroxylase activity (28,42), the introduction of an exogenous active human enzyme clearly promotes the trimer assembly of type XII minicollagen molecules. Thus these experiments directly demonstrate the involvement of prolyl 4-hydroxylase in the formation of trimers. A previous work on whole collagen XII synthesized by WISH cells showed that proper formation of interchain disulfide--bonded molecules involves prolyl hydroxylation (16). In this report we further characterize the way by which the enzyme participates in the trimeric assembly of collagen XII, showing that the ␤ subunits of the enzyme (expressing the proteindisulfide isomerase activity) are not sufficient by themselves to promote the formation of disulfide-bonded trimers of collagen XII. In contrast, the data demonstrate that the enzyme is involved in the trimeric assembly of type XII minicollagen through its ␣ subunits and thus through its hydroxylase activity. These results suggest that the molecular assembly of collagen XII (and more generally of the FACITs) is distinct from that described for fibrillar collagen molecules in which preventing hydroxylation and helix formation does not impair the formation of disulfide bonds (45), which happens before the triple helix propagation and stabilization (46,47).
The way in which hydroxylation is involved in the trimeric assembly of the minicollagen XII is still unclear. It is interesting to note that the amounts of disulfide-bonded dimers were lower in the culture medium of cells producing high exogenous human hydroxylase activity (Fig. 3, lanes 1, 3, and 5) than in the media of cells that did not produce exogenous enzymatic activity (cells were infected with COL XII 23 viruses and were not simultaneously infected with ␣ and ␤ viruses or were infected with ␤ viruses without ␣ viruses or infected with ␤ and ␣-H412S viruses; Fig. 3, lanes 6, 4, and 2, respectively). These dimers, also observed in Fig. 1, may represent abortive associations formed only when monomers cannot be properly incorporated into trimers. Alternatively, they would be true intermediates in the assembly process of the minicollagen and would become significantly present when hydroxylation of the third chain does not take place. The isolation of each individual molecular species (monomers, dimers, and trimers) and their biochemical characterization will help us to determine if hydroxylation of specific proline residues governs the initial events of the trimeric assembly of FACIT collagens. The amounts of recombinant minicollagen XII produced by insect cells will allow us to perform these characterizations. In addition, these biochemical studies will undoubtedly explain the existence of the multiple forms simultaneously observed with monomers, such as dimers and trimers. Indeed, migrating in SDS-PAGE slower than the major trimeric form (noted as T in the figures), collagenase-sensitive bands are present in various amounts both after immunoprecipitation (see, for example, Fig.  3, lane 5) or immunopurification (Fig. 5) of insect cell media. In the future, experiments will be necessary to further characterize these minor bands, which show the same dependence toward hydroxylation as their counterpart, T (see Fig. 3).
Parameters other than hydroxylation are also involved in the trimeric assembly of FACITs. In vitro reassociation studies on collagen XIV indicate that the treatment of natural chains purified from a mixture of COL1 and COL2 domains with the alkylating agent N-ethylmaleimide prevents the folding of the COL1 domain (44), suggesting the importance of the disulfide bridges in the folding process. Our recent in vitro reassociation studies of synthetic peptides corresponding to the junction of the COL1/NC1 domains of the three chains of collagen IX show that these peptides form reducible trimers and associate specifically with the native chains. These data demonstrate that they contain part of the information required for chain recognition and chain assembly. 2 On the basis of all these data, we postulate the following sequence of events governing the trimeric assembly of FACITs: 1) recognition of the three chains occurring through the information (primary sequence) contained in the COL1/NC1 junction, 2) folding in a triple-helical conformation of the last triplets of the COL1 domain and stabilization of their conformation by hydroxylation of prolyl residues contributing to the formation of the disulfide linkages which further locks the registered chains, and 3) triple-helical folding of the remaining part of the COL1 domain, which propagates from the C terminus to the N terminus of the molecule. It is possible that this special mechanism of folding for the collagen FACITs is a consequence of the absence of the large C-propeptides present in fibrillar collagens. Folding and stabilization of the C-terminal part of the COL1 domain may be interpreted as a way to mitigate the consequence of the decrease of the number of interactions in the C-terminal part of the molecule.