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

Cystathionine b-synthase (CBS) deficiency, a genetic disorder in homocysteine (Hcy) metabolism in humans, elevatesplasmaHcy-Thiolactoneand leads to connective tissue abnormalities that affect the cardiovascularand 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 cross-linking, N-homocysteinylation of these lysines should impair cross-linking.Using a Tg-I278T Cbs-/-mousemodel of hyperhomocysteinemia(HHcy)which replicates the connective tissue abnormalities observed in CBS-deficient patients,we found thatN-Hcy-collagenwas elevated in bone, tail, and heartofCbs-/-mice,whereaspyridinoline cross-linksweresignificantly reduced.Plasmadeoxypyridinoline cross-link and cross-linked carboxyterminal telopeptide of type I collagen were also significantly reduced in the Cbs-/-mice. Lysine oxidase activity andmRNAlevelwerenot reducedbythe Cbs-/-genotype.We also showedthat collagen carries S-linked Hcy bound to the thiol of N-linked Hcy. In vitro experiments showed that Hcy-Thiolactone modifies lysine residues in collagen type I a-1 chain. Residue K160, located in the nonhelical N-Telopeptide region and involved in pyridinoline cross-link formation, was also N-homocysteinylated in vivo. Taken together, our findings showed that N-homocysteinylation of collagen in Cbs-/-mice impairs its cross-linking. These findings explain, at least in part, connective tissue abnormalities observed in HHcy.