Characterization of Recombinant Human Type IX Collagen

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-terminal 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 ␣ chains were found to associate into disulfidebonded 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 ␣ 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 38, 49 and 40°C, respectively. The T m of the central collagenous domain is unusually high, which in the light of this domain being invariant in terms of alternative splicing suggests that the central portion of the molecule may have an important role in the stability of the molecule. All in all, most of the type XIII collagen ectodomain appears to be present in triple helical conformation, which is in clear contrast to the short or highly interrupted triple helical domains of the other known collagenous transmembrane proteins.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 component 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 ...

Section: Discussionsupporting

confidence: 65%

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

Snellman¹,

Keränen²,

Hägg³

et al. 2000

“…Another conclusion was that the COL2-NC2 region alone is not sufficient for trimerization (16), which directly contradicts our present finding of the role of the NC2 domain. It is worth mentioning that even for the full-length chains, the yield of heterotrimeric collagen in the baculovirus system was no more than 10% of total chain production, with most of material trapped in monomers and dimers as analyzed on a denaturing gel under non-reducing conditions (16,21). This indicates protein misfolding that leads to aggregation.…”

Section: Discussionmentioning

confidence: 99%

The NC2 Domain of Collagen IX Provides Chain Selection and Heterotrimerization

Boudko

Zientek

Vance

et al. 2010

Self Cite

The mechanism of chain selection and trimerization of fibrilassociated collagens with interrupted triple helices (FACITs) differs from that of fibrillar collagens that have special C-propeptides. We recently showed that the second carboxylterminal non-collagenous domain (NC2) of homotrimeric collagen XIX forms a stable trimer and substantially stabilizes a collagen triple helix attached to either end. We then hypothesized a general trimerizing role for the NC2 domain in other FACITs. Here we analyzed the NC2 domain of human heterotrimeric collagen IX, the only member of FACITs with all three chains encoded by distinct genes. Upon oxidative folding of equimolar amounts of the ␣1, ␣2, and ␣3 chains of NC2, a stable heterotrimer with a disulfide bridge between ␣1 and ␣3 chains is formed. Our experiments show that this heterotrimerization domain can stabilize a short triple helix attached at the carboxylterminal end and allows for the proper oxidation of the cystine knot of type III collagen after the short triple helix.The fibril-associated collagens with interrupted triple helices (FACITs) 2 include types IX, XII, XIV, XVI, XIX, XX, XXI, and XXII. Collagen IX is a heterotrimer composed of three distinct ␣ chains, and all others are homotrimers whose ␣ chains are characterized by short collagenous domains (COL) interrupted by several non-collagenous domains (NC) (1, 2). Unlike the fibril-forming collagens, the FACITs have significantly shorter NC1 domains (the carboxyl-terminal NC domains) (75 residues for human collagen XII, fewer than 30 residues for human collagen IX, and even fewer than 20 residues for human collagen XIX), whereas those of fibrillar collagens are of a different type and comprise about 260 residues. The FACITs share a remarkable sequence homology at their COL1/NC1 junctions by having two strictly conserved cysteine residues separated by four residues in the NC1 domain. These cysteines form interchain disulfide bonds, a so-called cystine knot, but only after the triple helix is formed (3-6). In other words, the NC1 domain cannot trimerize itself and requires exogenous alignment of three chains. It has been suggested that the NC2 domain in all FACITs is able to form an ␣-helical coiled coil, thus bearing an ability to trimerize those collagens (7). Experimental evidence for that was recently reported by us for the NC2 domain of collagen XIX (6).Collagen IX contains three collagenous domains (COL1-COL3) and four noncollagenous domains (NC1-NC4) (Fig. 1). The molecule is covalently associated with the surface of interstitial collagen fibrils in cartilage (8), where it plays a role in maintaining the long term structural integrity of this tissue (9). Loss of collagen IX is associated with such diseases as osteoarthritis, rheumatoid arthritis, intervertebrate disk degeneration, ocular defects, loss of hearing, and others (9 -13).Collagen IX is the most intriguing FACIT collagen in terms of its chain selection and heterotrimerization properties. Several attempts were made to decipher its code. Reasso...

“…Recombinant human collagen IX was produced as described previously (41). Briefly Trichoplusia ni insect cells (High Five, Invitrogen) grown in suspension at 27°C were seeded at 1.0 -1.5 ϫ 10 6 cells/ml in Sf900 II SFM medium (Invitrogen) and supplemented with 5% fetal bovine serum.…”

Section: Methodsmentioning

confidence: 99%

The Fibril-associated Collagen IX Provides a Novel Mechanism for Cell Adhesion to Cartilaginous Matrix

Käpylä

Jäälinoja

Tulla

et al. 2004

Self Cite

Collagen IX is the prototype fibril-associated collagen with interruptions in triple helix. In human cartilage it covers collagen fibrils, but its putative cellular receptors have been unknown. The reverse transcription-PCR analysis of human fetal tissues suggested that based on their distribution all four collagen receptor integrins, namely ␣ 1 ␤ 1 , ␣ 2 ␤ 1 , ␣ 10 ␤ 1 , and ␣ 11 ␤ 1 , are possible receptors for collagen IX. Furthermore primary chondrocytes and chondrosarcoma cells express the four integrins simultaneously. Chondrosarcoma cells, as well as Chinese hamster ovary cells transfected to express ␣ 1 ␤ 1 , ␣ 2 ␤ 1 , or ␣ 10 ␤ 1 integrin as their only collagen receptor, showed fast attachment and spreading on human recombinant collagen IX indicating that it is an effective cell adhesion protein. To further study the recognition of collagen IX we produced recombinant ␣I domains in Escherichia coli. For each of the four ␣I domains, collagen IX was among the best collagenous ligands, making collagen IX exceptional compared with all other collagen subtypes tested so far. Rotary shadowing electron microscopy images of both ␣ 1 I-and ␣ 2 Icollagen IX complexes unveiled only one binding site located in the COL3 domain close to the kink between it and the COL2 domain. The recognition of collagen IX by ␣ 2 I was considered to represent a novel mechanism for two reasons. First, collagen IX has no GFOGER motif, and the identified binding region lacks any similar sequences. Second, the ␣ 2 I domain mutations D219R and H258V, which both decreased binding to collagen I and GFOGER, had very different effects on its binding to collagen IX. D219R had no effect, and H258V prevented type IX binding. Thus, our results indicate that collagen IX has unique cell adhesion properties when compared with other collagens, and it provides a novel mechanism for cell adhesion to cartilaginous matrix.Collagen IX was the first member to be discovered of a subgroup of collagens, now known as the fibril-associated collagens with interrupted triple helix (FACITs) 1 (1). At the present, FACITs also include collagens XII, XIV, XVI, XIX, XX, XXI, and XXII. Collagen IX is composed of three different ␣ chains, termed ␣1(IX), ␣2(IX), and ␣3(IX). Structurally the collagen IX molecule can be divided into three triple helical domains (COL1, COL2, and COL3) separated and flanked by non-triple helical (NC) domains. Collagen IX is expressed in cartilage and in a limited number of other locations, including developing eye. Collagen IX can be covalently cross-linked to collagen II, and typically it covers the large fibrils formed by collagens II and XI (2-5). There is a kink between COL2 and COL3 domains making the COL3 domain project into the perifibrillar space, whereas the COL1 and COL2 domains are arranged on the surface of the fibril. Many but not all collagen IX molecules are proteoglycans because a glycosaminoglycan (GAG) chain may be attached to NC3 domain via a serine residue in the ␣2(IX) chain (6). Collagen IX is essential for the normal st...