N-Homocysteinylation impairs collagen cross-linking in cystathionine β-synthase-deficient mice: a novel mechanism of connective tissue abnormalities

Cystathionine β-synthase (CBS) deficiency, a genetic disorder in homocysteine (Hcy) metabolism in humans, elevates plasma Hcy-thiolactone and leads to connective tissue abnormalities affecting cardiovascular and skeletal systems. However, the underlying mechanism of these abnormalities is not understood. Hcy-thiolactone has the ability to form isopeptide bonds with protein lysine residues, which generates N-homocysteinylated protein. Because lysine residues are involved in collagen crosslinking, N-homocysteinylation of these lysines should impair crosslinking. Using a Tg-I278T Cbs -/- mouse model of hyperhomocysteinamia (HHcy) that recapitulates connective tissue abnormalities observed in CBS-deficient patients, we show that N-Hcy-collagen was elevated in bone, tail, and heart of Cbs -/- mice, while pyridinoline crosslinks were significantly reduced. Plasma deoxypyridinoline crosslink and crosslinked carboxyterminal telopeptide of type I collagen were also significantly reduced in Cbs -/- mice. Lysine oxidase activity and mRNA level were not reduced by the Cbs -/- genotype. We also show that collagen carries S-linked Hcy bound to the thiol of N-linked Hcy. In vitro experiments show that Hcy-thiolactone modifies lysine residues in collagen type I alpha-1 chain. Residue K160, located in the non-helical N-telopeptide region and involved in pyridinoline crosslink formation, was also N-homocysteinylated in vivo. Taken together, our findings show that N- homocysteinylation of collagen in Cbs -/- mice impairs its crosslinking. These findings explain at least in part connective tissue abnormalities observed in HHcy