Functional Consequences of Homocysteinylation of the Elastic Fiber Proteins Fibrillin-1 and Tropoelastin

Abstract

Homocystinuria caused by cystathionine-␤-synthase deficiency represents a severe form of homocysteinemias, which generally result in various degrees of elevated plasma homocysteine levels. Marfan syndrome is caused by mutations in fibrillin-1, which is one of the major constituents of connective tissue microfibrils. Despite the fundamentally different origins, both diseases share common clinical symptoms in the connective tissue such as long bone overgrowth, scoliosis, and ectopia lentis, whereas they differ in others. Fibrillin-1 contains ϳ13% cysteine residues and can be modified by homocysteine. We report here that homocysteinylation affects functional properties of fibrillin-1 and tropoelastin. We used recombinant fragments spanning the entire fibrillin-1 molecule to demonstrate that homocysteinylation, but not cysteinylation leads to abnormal self-interaction, which was attributed to a reduced amount of multimerization of the fibrillin-1 C terminus. The deposition of the fibrillin-1 network by human dermal fibroblasts was greatly reduced by homocysteine, but not by cysteine. Furthermore, homocysteinylation, but not cysteinylation of elastin-like polypeptides resulted in modified coacervation properties. In summary, the results provide new insights into pathogenetic mechanisms potentially involved in cystathionine-␤-synthasedeficient homocystinuria.

Fibulin-3, -4, and -5 Are Highly Susceptible to Proteolysis, Interact with Cells and Heparin, and Form Multimers

“…Heparin is used as a cost-effective model of heparan sulfate as the two share a similar structure of repeating iduronic/glucuronic acid and N-sulfated glucosamine/N-acetylglucosamine disaccharide units. Heparin was cross-linked to BSA following a protocol established previously (32). Heparin-BSA protein concentrations were determined by BCA assay, and the pools were stored at Ϫ80°C until use.…”

“…In addition, the two glycosaminoglycans share a similar structure, and the clustering of heparan sulfate chains on cell surface proteoglycans is mimicked by coupling three to four heparin molecules per molecule of BSA. This established procedure has been used in many other studies (32,(35)(36)(37)(38).…”

Homocysteine in renovascular complications: Hydrogen sulfide is a modulator and plausible anaerobic ATP generator

“…The sulfhydryl group of Hcy forms stable disulfide bonds with cysteine residues in proteins altering their structure and function [62]. Recently, Hubmacher et al demonstrated that homocysteinylation affected functional properties of fibrillin and tropoelastin [88]. Fibrillin and tropoelastin are essential components for the formation of elastic fibers.…”

“…The N- and C-terminal regions of fibrillin colocalizes to form typical microfibril structure [90], and in this regard disulfide bond-mediated multimerization of the fibrillin-1 C-terminus and N- to C-terminal self-interaction are considered the initial steps for tissue microfibrils formation [91; 92]. Homocysteinylation reduces N- to –C-terminal fibrillin-1 self-interaction properties which is essential for biogenesis of microfibrils [88]. In addition, homocysteinylation on the disulfide bond in tropoelastin changes the self-assembly process which reduces elastic properties of blood vessels [88].…”

Homocysteine and its thiolactone may promote apoptotic events in blood plateletsin vitro

“…NO is a powerful inhibitor of aggregation [13]. Various results indicate that Hcys or its thiolactone cause N-and S-homocysteinylation of human blood platelets, plasma proteins and other cellular or tissue proteins [14][15][16][17]. Moreover, both tested sulphurcontaining compounds reduce concentration of protein carbonyl groups.…”