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 ␣1(I) and ␣2(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 ␣1(I) and ␣2(I) chains but only one 3Hyp residue in skin ␣1(I) and none in ␣2(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 tissuespecific manner.Prolyl 3-hydroxylation, a long recognized quantitatively minor post-translational modification of collagen (1), has received much attention in the last few years after gene mutations affecting its formation were found to cause recessive forms of osteogenesis imperfecta (2-5). A single primary site of 3-hydroxyproline (3Hyp) 2 is present in normal collagen ␣1(I) and ␣1(II) chains at Pro-986 of the triple-helix (6, 7) but is not hydroxylated in the tissues of mice and humans with recessive osteogenesis imperfecta caused by mutations in CRTAP or LEPRE1 (the gene encoding P3H1) (2-5). The LEPRE1 gene encodes P3H1, which is one of three prolyl 3-hydroxylases (P3H1, P3H2, and P3H3) in the mammalian genome. CRTAP encodes a protein that is homologous to the N-terminal half of P3H1, which it associates with P3H1 together with cyclophilin B to form the functional enzyme complex required for Pro-986 3-hydroxylation of unfolded collagen chains in the endoplasmic reticulum (8).We recently identified several other sites of prolyl 3-hydroxylation in fibrillar collagen chains including Pro-707 in ␣2(I) and ␣2(V), Pro-944 in ␣1(II) and ␣2(V), Pro-470 in ␣2(V), and Pro-434, Pro-665, and Pro-692 in ␣1(V), ␣1(XI), and ␣2(XI) but a lack of any 3-hydroxyproline in the mammalian ␣1(III) chain (7). The D-periodic spacing between three of these additional sites in clade A collagen chains (␣1(I), ␣2(I), ␣1(II), and ␣2(V)) and between two in clade B (␣1(V), ␣1(XI), and ␣2(XI)) suggested a role in fibril formation. In pursuing this concept further, we investigated the potential for differences in 3-hydrox...
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 crosslinked 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 crosslinked 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.All fibril-forming collagens, types I, II, III, V, and XI, of vertebrates are cross-linked by the lysyl oxidase mechanism (1, 2). Two reaction pathways can be defined, one based on lysine aldehydes and the other on hydroxylysine aldehydes, which operate tissue-dependently. A combination of both pathways is evident in some tissues, for example in the unique crosslinking profile of bone collagen (3). Cartilage collagens use the hydroxylysine aldehyde route exclusively (4, 5). Newly made type II collagen in hyaline cartilages and other tissues becomes cross-linked almost exclusively by divalent ketoimines, each formed by the addition of a telopeptide, hydroxylysine aldehyde, to a hydroxylysine at either residue 87 or 930 of the triple helix. As fibrils mature, divalent ketoimines interact spontaneously to form a trivalent cross-link, hydroxylysyl pyridinoline (HP) 2 (6, 7). The stoichiometry is such that 1 mol of HP is formed from 2 mol of hydroxylysino-ketonorleucine (HLKNL; the ketoimine). This was established by direct amino acid analysis after borohydride reduction of tissue to stabilize the ketoimines to acid hydrolysis (7, 8) and also by radiolabeling of cartilage with [14...
There is growing evidence that a spectrum of chondrodysplasias are caused by mutations in the gene coding for type II collagen. The basic molecular defect in diastrophic dysplasia has not been defined, but it appears not to be in collagen type II. Cartilage contains other tissue-specific collagens, types IX, X, and XI, but no mutations have yet been found in their genes in clinical disease. Type IX collagen is hypothesized to play a role in the regulation of type II collagen fibril organization and structure in cartilage extracellular matrix. In this study, we have examined iliac crest growth cartilage from a patient with diastrophic dysplasia. Although collagen fibrils were markedly increased in diameter on transmission electron microscopy, type II collagen appeared to be normal biochemically. Type XI collagen was also normal. However, type IX collagen appeared abnormal on sodium dodecyl sulfate polyacrylamide gel electrophoresis with a pronounced excess of the COL1 domain of the molecule in pepsin extracts. The findings point to an abnormality in structure or metabolism of type IX collagen in diastrophic dysplasia.
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