Maturation of Collagen Ketoimine Cross-links by an Alternative Mechanism to Pyridinoline Formation in Cartilage

Abstract

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...

Type III Collagen, a Fibril Network Modifier in Articular Cartilage

“…Such an adduct has been shown to be a maturation product of a pool of ketoimine cross-links in type II collagen of bovine cartilages that do not mature to pyridinolines. It results from ketoimine oxidation and arginine addition (40).…”

Lysine post-translational modifications of collagen

“…In mineralized tissues, the major locus of deH-DHLNL and Pyr involves the 16 C -Hyl ald (α1) and 87-Hyl (α1) [87,92–94]. Apparently not all deH-DHLNL mature into Pyr as the rate of the decrease of deH-DHLNL/ketoamine and an increase of Pyr is often disproportional in vivo as well as during an in vitro incubation ([95], and M. Sricholpech and M. Yamauchi, unpublished work). The fate of all deH-DHLNL is not fully understood but, recently, an alternative mechanism to Pyr formation through its further oxidation and free arginine addition has been suggested [95].…”

Temporal development of near-native functional properties and correlations with qMRI in self-assembling fibrocartilage treated with exogenous lysyl oxidase homolog 2

“…Pyridinoline cross-links represent one type of molecular cross-linking which is associated with the development of collagen fibers natively. Although other types of cross-links exist in collagenous tissues, including pyrroles in mineralized tissues and arginoline in cartilage, pyridinoline cross-links are the most well-characterized and likely the most prevalent of these cross-links in cartilage [16,17]. The formation of pyridinoline cross-links is catalyzed by the lysyl oxidase (LOX) family of enzymes [18].…”

“…However, the temporal characterization of long-term in vitro culture with LOXL2 is unexplored. This is particularly significant because the process of cross-link formation is believed to take weeks to months in vivo [17,20,21]. …”