A novel 3-hydroxyproline (3Hyp)-rich motif marks the triple-helical C terminus of tendon type I collagen.

Because of its unique physical and chemical properties, rat tail tendon collagen has long been favored for crystallographic and biochemical studies of fibril structure. In studies of the distribution of 3-hydroxyproline in type I collagen of rat bone, skin, and tail tendon by mass spectrometry, the repeating sequences of Gly-Pro-Pro (GPP) triplets at the C terminus of α1(I) and α2(I) chains were shown to be heavily 3-hydroxylated in tendon but not in skin and bone. By isolating the tryptic peptides and subjecting them to Edman sequence analysis, the presence of repeating 3-hydroxyprolines in consecutive GPP triplets adjacent to 4-hydroxyproline was confirmed as a unique feature of the tendon collagen. A 1960s study by Piez et al. (Piez, K. A., Eigner, E. A., and Lewis, M. S. (1963) Biochemistry 2, 58-66) in which they compared the amino acid compositions of rat skin and tail tendon type I collagen chains indeed showed 3-4 residues of 3Hyp in tendon α1(I) and α2(I) chains but only one 3Hyp residue in skin α1(I) and none in α2(I). The present work therefore confirms this difference and localizes the additional 3Hyp to the GPP repeat at the C terminus of the triple-helix. We speculate on the significance in terms of a potential function in contributing to the unique assembly mechanism and molecular packing in tendon collagen fibrils and on mechanisms that could regulate 3-hydroxylation at this novel substrate site in a tissue-specific manner.

Maturation of collagen Ketoimine cross-links by an alternative mechanism to pyridinoline formation in cartilage.

The tensile strength of fibrillar collagens depends on stable intermolecular cross-links formed through the lysyl oxidase mechanism. Such cross-links based on hydroxylysine aldehydes are particularly important in cartilage, bone, and other skeletal tissues. In adult cartilages, the mature cross-linking structures are trivalent pyridinolines, which form spontaneously from the initial divalent ketoimines. We examined whether this was the complete story or whether other ketoimine maturation products also form, as the latter are known to disappear almost completely from mature tissues. Denatured, insoluble, bovine articular cartilage collagen was digested with trypsin, and cross-linked peptides were isolated by copper chelation chromatography, which selects for their histidine-containing sequence motifs. The results showed that in addition to the naturally fluorescent pyridinoline peptides, a second set of cross-linked peptides was recoverable at a high yield from mature articular cartilage. Sequencing and mass spectral analysis identified their origin from the same molecular sites as the initial ketoimine cross-links, but the latter peptides did not fluoresce and were nonreducible with NaBH(4). On the basis of their mass spectra, they were identical to their precursor ketoimine cross-linked peptides, but the cross-linking residue had an M+188 adduct. Considering the properties of an analogous adduct of identical added mass on a glycated lysine-containing peptide from type II collagen, we predicted that similar dihydroxyimidazolidine structures would form from their ketoimine groups by spontaneous oxidation and free arginine addition. We proposed the trivial name arginoline for the ketoimine cross-link derivative. Mature bovine articular cartilage contains about equimolar amounts of arginoline and hydroxylysyl pyridinoline based on peptide yields.

Type III collagen, a fibril network modifier in articular cartilage.

The collagen framework of hyaline cartilages, including articular cartilage, consists largely of type II collagen that matures from a cross-linked heteropolymeric fibril template of types II, IX, and XI collagens. In the articular cartilages of adult joints, type III collagen makes an appearance in varying amounts superimposed on the original collagen fibril network. In a study to understand better the structural role of type III collagen in cartilage, we find that type III collagen molecules with unprocessed N-propeptides are present in the extracellular matrix of adult human and bovine articular cartilages as covalently cross-linked polymers extensively cross-linked to type II collagen. Cross-link analyses revealed that telopeptides from both N and C termini of type III collagen were linked in the tissue to helical cross-linking sites in type II collagen. Reciprocally, telopeptides from type II collagen were recovered cross-linked to helical sites in type III collagen. Cross-linked peptides were also identified in which a trifunctional pyridinoline linked both an alpha1(II) and an alpha1(III) telopeptide to the alpha1(III) helix. This can only have arisen from a cross-link between three different collagen molecules, types II and III in register staggered by 4D from another type III molecule. Type III collagen is known to be prominent at sites of healing and repair in skin and other tissues. The present findings emphasize the role of type III collagen, which is synthesized in mature articular cartilage, as a covalent modifier that may add cohesion to a weakened, existing collagen type II fibril network as part of a chondrocyte healing response to matrix damage.

Location of 3-hydroxyproline residues in collagen types I, II, III, and V/XI implies a role in fibril supramolecular assembly.

Collagen triple helices are stabilized by 4-hydroxyproline residues. No function is known for the much less common 3-hydroxyproline (3Hyp), although genetic defects inhibiting its formation cause recessive osteogenesis imperfecta. To help understand the pathogenesis, we used mass spectrometry to identify the sites and local sequence motifs of 3Hyp residues in fibril-forming collagens from normal human and bovine tissues. The results confirm a single, essentially fully occupied 3Hyp site (A1) at Pro(986) in A-clade chains alpha1(I), alpha1(II), and alpha2(V). Two partially modified sites (A2 and A3) were found at Pro(944) in alpha1(II) and alpha2(V) and Pro(707) in alpha2(I) and alpha2(V), which differed from A1 in sequence motif. Significantly, the distance between sites 2 and 3, 237 residues, is close to the collagen D-period (234 residues). A search for additional D-periodic 3Hyp sites revealed a fourth site (A4) at Pro(470) in alpha2(V), 237 residues N-terminal to site 3. In contrast, human and bovine type III collagen contained no 3Hyp at any site, despite a candidate proline residue and recognizable A1 sequence motif. A conserved histidine in mammalian alpha1(III) at A1 may have prevented 3-hydroxylation because this site in chicken type III was fully hydroxylated, and tyrosine replaced histidine. All three B-clade type V/XI collagen chains revealed the same three sites of 3Hyp but at different loci and sequence contexts from those in A-clade collagen chains. Two of these B-clade sites were spaced apart by 231 residues. From these and other observations we propose a fundamental role for 3Hyp residues in the ordered self-assembly of collagen supramolecular structures.

Matrix deposition of tryptophan-containing allelic variants of type IX collagen in developing human cartilage.

Genetic polymorphisms that encode a tryptophan (Trp) residue in the triple-helical domain of the alpha2 (Trp2) or alpha3 chain (Trp3) of human type IX collagen have been linked to risk of degenerative intervertebral disc disease. To determine whether these two allelic variants express protein that may affect the extracellular matrix of cartilage in vivo, we examined the properties of resident type IX collagen in an anonymous collection of embryonic and fetal human cartilage samples screened for Trp genotypes. No difference was found in the yield and electrophoretic properties of pepsin-solubilized type IX collagen between Trp2, Trp3 and non-Trp cartilage samples. On Western blot analysis, a polyclonal antiserum raised against a synthetic peptide matching the immediate Trp-containing sequence of the Trp3 allele reacted specifically with the alpha3(IX) chain prepared from Trp3 cartilage samples. Two-dimensional peptide mapping of type IX collagen in CNBr-digests of whole tissue gave indistinguishable fingerprints for Trp2, Trp3 and control tissues, including the yield of cross-linked peptides. Analysis of one cartilage sample that was homozygous for the Trp2 allele also gave a normal yield of collagen IX, including its alpha2 chain and a normal profile of cross-linked peptides. Together, the findings indicate that both Trp2 and Trp3 allelic products are incorporated into the cross-linked fibrillar network of developing human cartilage apparently normally. Any pathological consequences are likely, therefore, to be long-term and indirect rather than from overt misassembly of matrix.

Evolutionary origins of C-terminal (GPP)n 3-hydroxyproline formation in vertebrate tendon collagen.

Approximately half the proline residues in fibrillar collagen are hydroxylated. The predominant form is 4-hydroxyproline, which helps fold and stabilize the triple helix. A minor form, 3-hydroxyproline, still has no clear function. Using peptide mass spectrometry, we recently revealed several previously unknown molecular sites of 3-hydroxyproline in fibrillar collagen chains. In fibril-forming A-clade collagen chains, four new partially occupied 3-hydroxyproline sites were found (A2, A3, A4 and (GPP)n) in addition to the fully occupied A1 site at Pro986. The C-terminal (GPP)n motif has five consecutive GPP triplets in α1(I), four in α2(I) and three in α1(II), all subject to 3-hydroxylation. The evolutionary origins of this substrate sequence were investigated by surveying the pattern of its 3-hydroxyproline occupancy from early chordates through amphibians, birds and mammals. Different tissue sources of type I collagen (tendon, bone and skin) and type II collagen (cartilage and notochord) were examined by mass spectrometry. The (GPP)n domain was found to be a major substrate for 3-hydroxylation only in vertebrate fibrillar collagens. In higher vertebrates (mouse, bovine and human), up to five 3-hydroxyproline residues per (GPP)n motif were found in α1(I) and four in α2(I), with an average of two residues per chain. In vertebrate type I collagen the modification exhibited clear tissue specificity, with 3-hydroxyproline prominent only in tendon. The occupancy also showed developmental changes in Achilles tendon, with increasing 3-hydroxyproline levels with age. The biological significance is unclear but the level of 3-hydroxylation at the (GPP)n site appears to have increased as tendons evolved and shows both tendon type and developmental variations within a species.

Type IX collagen neo-deposition in degenerative discs of surgical patients whether genotyped plus or minus for COL9 risk alleles.

STUDY DESIGN: Immunohistochemical analysis of type IX collagen in disc tissue from spinal fusion patients. OBJECTIVE: To determine if collagen IX can be detected in adult disc tissue removed at spinal fusion surgery from patients either with or without degeneration-associated tryptophan single nucleotide polymorphisms (SNPs) and whether the distribution is associated either with severity of degeneration or incidence of a collagen IX SNP genotype. SUMMARY OF BACKGROUND DATA: Genetic factors are strongly associated with risk of development and/or progression of disc degeneration. Two SNPs that introduce tryptophan polymorphisms in COL9A2 and COL9A3 are independently linked to an increased risk of lumbar disc disease. Although tryptophan variants are associated with accelerated degeneration, it is not known if collagen IX can be detected in adult disc tissue. METHODS: We selected age-matched disc samples from five clinical groups: fracture with Trp(-) (six cases), herniation (six cases), degeneration (five cases), spondylolisthesis with Trp(-) (eight cases), and spondylolisthesis/herniation/fracture with Trp(+) (six cases of Trp3 allele and one case of Trp2 allele). Using hematoxylin and eosin staining and immunohistochemical staining (collagens IX and IIA), 78 sections from 32 patients were analyzed. Selected disc tissues were assayed biochemically for collagen IX. RESULTS: Focal deposition of collagen IX was observed in regions of adult human disc tissue from spines showing degenerative changes in patients whether or not they were positive for a tryptophan SNP. However, in nondegenerative control disc tissue from fracture cases, little or no collagen IX was detected. The latter finding was confirmed by direct biochemical analyses for collagen IX in pooled samples of normal adult human annulus fibrosus or nucleus pulposus. CONCLUSION: During growth and maturation of the disc, collagen IX is presumably removed completely during matrix remodeling so that the protein is absent from normal adult annulus and nucleus but can reappear at sites of degeneration presumably as part of a repair response to mechanical injury.

Differences in chain usage and cross-linking specificities of cartilage type V/XI collagen isoforms with age and tissue.

Collagen type V/XI is a minor but essential component of collagen fibrils in vertebrates. We here report on age- and tissue-related variations in isoform usage in cartilages. With maturation of articular cartilage, the alpha1(V) chain progressively replaced the alpha2(XI) chain. A mix of the molecular isoforms, alpha1(XI)alpha1(V)alpha3(XI) and alpha1(XI)alpha2(XI)alpha3(XI), best explained this finding. A prominence of alpha1(V) chains is therefore characteristic and a potential biomarker of mature mammalian articular cartilage. Analysis of cross-linked peptides showed that the alpha1(V) chains were primarily cross-linked to alpha1(XI) chains in the tissue and hence an integral component of the V/XI polymer. From nucleus pulposus of the intervertebral disc (in which the bulk collagen monomer is type II as in articular cartilage), type V/XI collagen consisted of a mix of five genetically distinct chains, alpha1(XI), alpha2(XI), alpha3(XI), alpha1(V), and alpha2(V). These presumably were derived from several different molecular isoforms, including alpha1(XI)alpha2(XI)alpha3(XI), (alpha1(XI))(2)alpha2(V), and others. Meniscal fibrocartilage shows yet another V/XI phenotype. The findings support and extend the concept that the clade B subfamily of COL5 and COL11 gene products should be considered members of the same collagen subfamily, from which, in combination with clade A gene products (COL2A1 or COL5A2), a range of molecular isoforms has evolved into tissue-dependent usage. We propose an evolving role for collagen V/XI isoforms as an adaptable polymeric template of fibril macro-architecture.

Advances in collagen cross-link analysis.

The combined application of ion-trap mass spectrometry and peptide-specific antibodies for the isolation and structural analysis of collagen cross-linking domains is illustrated with examples of results from various types of collagen with the emphasis on bone and cartilage. We highlight the potential of such methods to advance knowledge on the importance of post-translational modifications (e.g., degrees of lysine hydroxylation and glycosylation) and preferred intermolecular binding partners for telopeptide and helical cross-linking domains in regulating cross-link type and placement.

Intervertebral disc collagen. Usage of the short form of the alpha1(IX) chain in bovine nucleus pulposus.

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 alpha1(IX)COL3 domain purified from bovine nucleus pulposus gave a different sequence to that of the long alpha1(IX) transcript expressed in hyaline cartilage and matched the predicted sequence of short alpha1(IX). The findings indicate that the matrix of bovine nucleus pulposus contains only the short form of alpha1(IX) that lacks the NC4 domain. The sequence encoded by exon 7, predicted from human COL9A1, is absent from both short and long forms of alpha1(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 alpha1(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.

Covalent cross-linking of the NC1 domain of collagen type IX to collagen type II in cartilage.

From a study to understand the mechanism of covalent interaction between collagen types II and IX, we present experimental evidence for a previously unrecognized molecular site of cross-linking. The location relative to previously defined cross-linking sites predicts a specific manner of interaction and folding of collagen IX on the surface of nascent collagen II fibrils. The initial evidence came from Western blot analysis of type IX collagen extracted by pepsin from fetal human cartilage, which showed a molecular species that had properties indicating an adduct between the alpha1(II) chain and the C-terminal domain (COL1) of type IX collagen. A similar component was isolated from bovine cartilage in sufficient quantity to confirm this identity by N-terminal sequence analysis. Using an antibody that recognized the putative cross-linking sequence at the C terminus of the alpha1(IX) chain, cross-linked peptides were isolated by immunoaffinity chromatography from proteolytic digests of human cartilage collagen. They were characterized by immunochemistry, N-terminal sequence analysis, and mass spectrometry. The results establish a link between a lysine near the C terminus (in the NC1 domain) of alpha1(IX) and the known cross-linking lysine at residue 930 of the alpha1(II) triple helix. This cross-link is speculated to form early in the process of interaction between collagen IX molecules and collagen II polymers. A model of molecular folding and further cross-linking is predicted that can spatially accommodate the formation of all six known cross-linking interactions to the collagen IX molecule on a fibril surface. Of particular biological significance, this model can accommodate potential interfibrillar as well as intrafibrillar links between the collagen IX molecules themselves, so providing a mechanism whereby collagen IX could stabilize a collagen fibril network.