Molecular Site Specificity of Pyridinoline and Pyrrole Cross-links in Type I Collagen of Human Bone

Abstract

Compared with soft tissue collagens, bone type I collagen displays a distinctive pattern of covalent cross-linking, with evidence of preferred sites of molecular interaction and a prominence of both immature, divalent crosslinks and mature, trivalent cross-links in the adult tissue. In this study the site-specificity of the mature cross-links in human bone collagen was examined. Peptides containing fluorescent pyridinoline cross-links and Ehrlich's-reactive pyrrole cross-links were isolated from a bacterial collagenase digest of demineralized bone matrix. The digest was fractionated by molecular sieve chromatography, monitoring for peptide absorbance, pyridinoline fluorescence, pyrroles by Ehrlich's reagent, and immunoassay for crosslinked N-telopeptides. Individual cross-linked peptides were resolved by ion-exchange and reverse-phase HPLC. Structures were established by NH 2 -terminal microsequencing, cross-link analysis, electrospray mass spectrometry, and immunoassay. Two, about equally occupied, sites of pyridinoline cross-linking were identified, N-telopeptide to helix and C-telopeptide to helix. Pyrroles were alternative cross-linking products at the same sites, but concen- The chemistry of lysine-mediated intermolecular cross-linking in the fibrillar collagens is understood in some detail (1-4). The three principal collagens, types I, II, and III, have four cross-linking sites at equivalent locations in their molecules, one in each telopeptide and two others at sites in the triplehelical domains at or close to residues 87 and 930. The initial intermolecular cross-links in new fibrils are borohydride-reducible residues that are condensation products between aldehyde side chains formed on the telopeptides by lysyl oxidase, and hydroxylysine or lysine residues in helical domains of adjacent molecules. Both N-telopeptide-to-helix and C-telopeptide-tohelix cross-links occur (5-12). However, as young connective tissues mature, borohydride-reducible cross-links disappear, progressing to more stable, non-reducible compounds (13). The best characterized and most widely distributed mature crosslinks are the fluorescent compound, hydroxylysylpyridinoline (HP) 1 (14) and its deoxy analog, lysylpyridinoline (LP; Ref. 15), also referred to as pyridinoline (Pyr) and deoxypyridinoline (16), respectively. HP is widely distributed in the collagens of most internal, vertebrate connective tissues, whereas LP, although widely distributed, features most prominently in bone and dentin. The pattern of cross-linking in bone collagen is also strikingly different from that of soft tissue collagens in other respects, including retention of a significant pool of the borohydride-reducible, initial cross-links throughout adult life (4). The distinctive pattern of cross-linking interactions in bone collagen may be a result of or, as proposed, a necessary requirement for its mineralization (11,12).In recent years, the pyridinoline residues HP and LP have become widely adopted in clinical studies as urinary markers of bone resorption. The...

Structural Characterization of Pyrrolic Cross-links in Collagen Using a Biotinylated Ehrlich's Reagent

“…Enzyme digests of decalcified bone matrix have been used in attempts to isolate pyrrole-containing peptides, but because these peptides are reduced in size and enriched, the pyrrole tends to oxidize or polymerize. 1 In a recent study, Ehrlich chromogen-containing peptides were enriched and analyzed by mass spectrometry, revealing peaks that were interpreted to be derived from peptides containing oxidized pyrrolic cross-link (11). These experiments supported previous preliminary indications that the pyrroles were concentrated at the N-telopeptide site.…”

“…A similar affinity chromatography approach was used to demonstrate that Ehrlich chromogen cross-links were present at the same loci as the pyridinium cross-links in bovine tendon (10). This work culminated in a proposed structure and mechanism of formation for pyrroles analogous to that for pyridinium cross-link formation: this mechanism involves reaction of a difunctional, keto-imine cross-link with a lysyl aldehyde-derived component rather than a hydroxylysyl aldehyde-derived component (10), where the latter may be a second difunctional cross-link (11). Taken together, these studies suggest that Ehrlich chromogens exist in many different tissues and probably have different chemistries based on their stability and the different spectra observed upon reaction with p-dimethylaminobenzaldehyde (9).…”

Consortium for osteogenesis imperfecta mutations in the helical domain of type I collagen: regions rich in lethal mutations align with collagen binding sites for integrins and proteoglycans

“…MLBR 3 extends from helical residue 920 to the end of the helical region. The gly910-964 stretch of lethal mutations includes one of the two major residues for intermolecular collagen cross-linking (the lysyl residue at helical position 930) [Hanson and Eyre, 1996], as well as sites important for the binding of the decorin proteoglycan core protein , the glycosaminoglycan heparin [San Antonio et al, 1994;Sweeney et al, 1998], and possibly the DDR2 receptor [Schlessinger, 1997;Shrivastava et al, 1997;Vogel et al, 1997]. MLBR 1 is located toward the amino end of the type I collagen helix, where most glycine substitutions are nonlethal.…”

An overview on bone protein extract as the new generation of demineralized bone matrix

“…Collagen in DBM has also been confirmed to have osteoconductive functions [39]. Hanson et al [40] found that the stereospecific features of bone collagen at the molecular level, such as the pattern of cross-linking and the interchain placement of bonds, were markedly different from other tissue collagens, in particular type I collagen, which may alter collagen mineralization. As BPE is a lyophilisate in collagenous matrix form, consisting of collagen type I chains and other insoluble proteins, the osteoconductive capacity is obvious.…”

“…In the early 1970s, Bachra [39] verified that bone collagen was a good nucleation catalyst for mineral deposition compared with rat tail collagen. Hanson et al [40] studied the stereospecific features of type I collagen in human bone, which is convenient for collagen mineralization. Although there has been no scientific paper on type I collagen in BPE, it has been documented by the FDA.…”