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.

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.

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.

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.

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.

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.

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.

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.

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.

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.