Nucleus pulposus, the central zone of the intervertebral disc, is gel-like and has a similar collagen phenotype to that of hyaline cartilage. Amino-terminal protein sequence analysis of the ␣1(IX)COL3 domain purified from bovine nucleus pulposus gave a different sequence to that of the long ␣1(IX) transcript expressed in hyaline cartilage and matched the predicted sequence of short ␣1(IX). The findings indicate that the matrix of bovine nucleus pulposus contains only the short form of ␣1(IX)

more »

... t lacks the NC4 domain. The sequence encoded by exon 7, predicted from human COL9A1, is absent from both short and long forms of ␣1(IX) from bovine nucleus pulposus and articular cartilage. A structural analysis of the cross-linking sites occupied in type IX collagen from nucleus pulposus showed that usage of the short ␣1(IX) transcript in disc tissue had no apparent effect on cross-linking behavior. As in cartilage, type IX collagen of nucleus pulposus was heavily cross-linked to type II collagen and to other molecules of type IX collagen with a similar site occupancy. The collagen phenotype of hyaline cartilage is complex; at least seven distinct collagen types have been identified, of which types II, XI, and IX are the cartilage-specific molecules. In addition, significant amounts of collagen types III, VI, XII, and XIV have been identified in cartilage matrix (1-3). Type IX collagen is a structural matrix component that is most abundant in hyaline cartilages of the developing skeleton. It functions as an adhesion protein in the extracellular matrix where it becomes covalently cross-linked to the surface of type II collagen fibrils and most concentrated on thin fibrils of the pericellular domain (4 -6). The molecule is a heterotrimer of genetically distinct ␣1(IX), ␣2(IX), and ␣3(IX) chains, which form three triple-helical segments, COL1, 1 COL2, and COL3 and four non-helical domains, NC1-NC4. The largest, NC4 on ␣1(IX), is basically charged, suggesting that it may serve to interact with proteoglycans (7) . Recent reports have linked genetic polymorphisms to risk of disc degeneration (8, 9) . Specifically, tryptophan substitutions at position 326 of the ␣2(IX) chain, and position 103 of the ␣3(IX) chain, were associated with increased risk of human lumbar disc degeneration and chronic sciatica but not knee osteoarthritis (8, 9) . Nucleus pulposus, the gel-like central zone of the young intervertebral disc, has a similar collagen phenotype to that of hyaline cartilage, with types II, IX, and XI collagens being the principal fibrillar components (10). The association of the tryptophan polymorphisms with clinical conditions involving discs but not synovial joint cartilages implies that the molecular character of collagen IX in intervertebral disc collagen may differ from that in hyaline cartilage. The properties of collagen IX from disc tissue have not been fully characterized. The human ␣1(IX) gene gives rise to two mRNA transcripts, a long and a short form (11). The long form is expressed in hyaline cartilages and the product has a globular NH 2 -terminal domain, NC4. Short ␣1(IX), expressed in embryonic chick cornea (12), is transcribed from an alternative start site and lacks the NC4 domain. A second promoter and an alternative exon 1, located in the intron between exons 6 and 7 of COL9A1, are used to express the short form of ␣1(IX). In the present study, by NH 2 -terminal sequence analysis of COL3(IX) domains isolated from the matrix of bovine nucleus pulposes and articular cartilage, we show that nucleus pulposus contains exclusively the short form of ␣1(IX). The covalent cross-linking properties of nucleus pulposus type IX collagen were also characterized to determine any consequences of having short instead of long ␣1(IX). MATERIALS AND METHODS Preparation of Type IX Collagen-Articular cartilage was dissected from knee joints and nucleus pulposus from lumbar spines of 3-monthold calves. Tissue slices were extracted in 4 M guanidine HCl, 0.05 M Tris-HCl, pH 7.4, at 4°C for 24 h to remove proteoglycans and other matrix proteins then washed thoroughly with water and freeze-dried. Cross-linked collagens were solubilized by digesting the washed residues with pepsin at 4°C. Pepsin digests were fractionated into collagen types II, XI, and IX (COL1 and COL2 trimers) by precipitation at 0.7, 1.2, and 2.0 M NaCl, respectively (13). The COL3 domains of type IX collagen were then isolated from the 4.0 M NaCl precipitated fraction. Column Chromatography-Pepsin-solubilized type IX collagen COL1 and COL2 trimers were resolved on a C8 reverse-phase column (Brownlee Aquapore RP-300; 4.6 mm ϫ 25 cm) with a linear gradient (23-38%) of solvent B in A over 45 min at a flow rate of 1 ml/min collecting 1-ml fractions. Solvent A was 0.1% trifluoroacetic acid (v/v) in water, and solvent B was 0.085% trifluoroacetic acid (v/v) in acetonitrile:n-propyl alcohol (3:1, v/v). Eluent was monitored for 220 nm absorbance, and aliquots of collected fractions (100 l each) were dried and analyzed by SDS-PAGE. The disulfide-bounded COL2 trimer was reduced, and cysteines were carboxymethylated with iodoacetate before rechromatographing the individual chains on the same column with a linear gradient (15-40%) of solvent B in A over 50 min. The COL3 domains of type IX collagen were isolated from the 4.0 M NaCl precipitated fraction by molecular sieve chromatography on a Bio-Gel A5m column (1.5 ϫ 170 cm, 200 -400 mesh, Bio-Rad) eluted with a 0.05 M Tris-HCl buffer, pH 7.5, containing 2 M guanidine HCl, at a flow rate of 4 ml/h, collecting 2.0-ml fractions. Aliquots of collected fractions were desalted and analyzed by SDS-PAGE. Fractions contain-