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Analyses of lysine aldehyde cross-linking in collagen reveal that the mature cross-link histidinohydroxylysinonorleucine is an artifact.

ABSTRACT: Lysyl oxidase-generated intermolecular cross-links are essential for the tensile strength of collagen fibrils. Two cross-linking pathways can be defined, one based on telopeptide lysine aldehydes and another on telopeptide hydroxylysine aldehydes. Since the 1970s it has been accepted that the mature cross-linking structures on the lysine aldehyde pathway, which dominates in skin and cornea, incorporate histidine residues. Here, using a range of MS-based methods, we re-examined this conclusion and found that telopeptide aldol dimerization is the primary mechanism for stable cross-link formation. The C-telopeptide aldol dimers formed labile addition products with glucosylgalactosyl hydroxylysine at ?1(I)K87 in adjacent collagen molecules that resisted borohydride reduction and after acid hydrolysis produced histidinohydroxylysinonorleucine (HHL), but only from species with a histidine in their ?1(I) C-telopeptide sequence. Peptide MS analyses and the lack of HHL formation in rat and mouse skin, species that lack an ?1(I) C-telopeptide histidine, revealed that HHL is a laboratory artifact rather than a natural cross-linking structure. Our experimental results also establish that histidinohydroxymerodesmosine is produced by borohydride reduction of N-telopeptide allysine aldol dimers in aldimine intermolecular linkage to nonglycosylated ?1(I) K930. Borohydride reduction of the aldimine promotes an accompanying base-catalyzed Michael addition of ?1(I) H932 imidazole to the ?,?-unsaturated aldol. These aldehydes are intramolecular at the N terminus but at the C terminus they can be both intramolecular and intermolecular according to present and earlier findings.

SUBMITTER: Eyre DR

PROVIDER: S-EPMC6484114 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Project description:Cross-linked peptides were isolated from chicken bone collagen that had been digested with CNBr or with bacterial collagenase. Analyses of (3)H radioactivity in disc electrophoretic profiles of the CNBr peptides from bone collagens that had been treated with NaB(3)H indicated that a major site of intermolecular cross-linking in chicken bone collagen is located between the carboxy-terminal region of an alpha1 chain and a small CNBr peptide, probably situated near the amino-terminus of an alpha1 or alpha2 chain in an adjacent collagen molecule. A small amount of this cross-linked CNBr peptide was isolated from a CNBr digest of chicken bone collagen by column chromatography. Amino acid analysis showed that the CNBr peptide, alpha1CB6B, the carboxy-terminal peptide of the alpha1 chain, was the major CNBr peptide in the preparation, and the reduced cross-linking components were identified as hydroxylysinohydroxynorleucine (HylOHNle), with a smaller amount of hydroxylysinonorleucine (HylNle). However, the composition and the low recovery of the cross-linking amino acids suggested that the preparation was a mixture of CNBr peptides alpha1CB6B and alpha1CB6B cross-linked to a small CNBr peptide whose identity could not be determined. A small cross-linked peptide was isolated from chicken bone collagen that had been reduced with NaB(3)H(4) and digested with bacterial collagenase. This peptide was the major cross-linked peptide in the digest and contained a stoicheiometric amount of the reduced cross-linking compounds. A peptide which had the same amino acid composition, but contained the cross-linking compounds in their reducible forms, was isolated from a collagenase digest of chicken bone collagen that had not been treated with NaBH(4). The absence of the reduced cross-links from this peptide indicates that, at least for the cross-linking site from which the peptide derives, natural reduction is not a significant pathway for biosynthesis of stable cross-links. However, most of the reducible cross-linking component in the peptide appeared to stabilize in the bone collagen by rearrangement from aldimine to ketoamine form.

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Analyses of lysine aldehyde cross-linking in collagen reveal that the mature cross-link histidinohydroxylysinonorleucine is an artifact.

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