Reduced response of Cystathionine Beta‐Synthase (CBS) to S‐Adenosylmethionine (SAM): Identification and functional analysis of CBS gene mutations in Homocystinuria patients

Abstract

A reduced response of cystathionine beta-synthase (CBS) to its allosteric activator S-adenosylmethionine (SAM) has been reported to be a cause of CBS dysfunction in homocystinuria patients. In this work we performed a retrospective analysis of fibroblast data from 62 homocystinuria patients and found that 13 of them presented a disturbed SAM activation. Their genotypic background was identified and the corresponding CBS mutant proteins were produced in E. coli. Nine distinct mutations were detected in 22 independent alleles: the novel mutations p.K269del, p.P427L, p.S500L and p.L540Q; and the previously described mutations p.P49L, p.C165Rfs*2, p.I278T, p.R336H and p.D444N. Expression levels and residual enzyme activities, determined in the soluble fraction of E. coli lysates, strongly correlated with the localization of the affected amino acid residue. C-terminal mutations lead to activities in the range of the wild-type CBS and to oligomeric forms migrating faster than tetramers, suggesting an abnormal conformation that might be responsible for the lack of SAM activation. Mutations in the catalytic core were associated with low protein expression levels, decreased enzyme activities and a higher content of high molecular mass forms. Furthermore, the absence of SAM activation found in the patients' fibroblasts was confirmed for all but one of the characterized recombinant proteins (p.P49L). Our study experimentally supports a deficient regulation of CBS by SAM as a frequently found mechanism in CBS deficiency, which should be considered not only as a valuable diagnostic tool but also as a potential target for the development of new therapeutic approaches in classical homocystinuria.

Insights into the Regulatory Domain of Cystathionine Beta-Synthase: Characterization of Six Variant Proteins

“…Wild‐type (WT) CBS cDNA was cloned into the Nde I and Xho I sites of the pET28b expression vector, as previously reported [Mendes et al., ]. The CBS mutations c.1280C>T (p.P427L), c.1330G>A (p.D444N), c.1499C>T (p.S500L), and c.1619T>A (p.L540Q) were obtained as previously described [Mendes et al., ].…”

“…In this article, we characterize six C‐terminal CBS variants, including one novel variant (c.1346T>G, p.V449G), four previously reported but not characterized (c.1280C>T, p.P427L; c.1499C>T, p.S500L; c.1566delG, p.K523Sfs*18; and c.1619T>A, p.L540Q) [Castro et al., ; Mendes et al., ], and one partially characterized at the functional level (c.1330G>A, p.D444N) [Evande et al., ]. Since all these variants presented high residual enzymatic activities and limited response to SAM, we performed further functional and biophysical studies to understand the molecular basis of the associated CBS‐deficient phenotypes.…”

S-Adenosyl-l-methionine Modulates CO and NO• Binding to the Human H2S-generating Enzyme Cystathionine β-Synthase

“…CBS regulation by AdoMet has been recognized historically as physiologically relevant (24). Defects in this regulatory mechanism are currently considered as playing a role in the pathogenesis of classic homocystinuria (27,28), the inherited error of metabolism associated with CBS deficiency. More recently, AdoMet has been shown to markedly modulate the proliferation of colorectal cancer cells as compared with non-tumorigenic cells (29).…”

CBS mutations are good predictors for B6‐responsiveness: A study based on the analysis of 35 Brazilian Classical Homocystinuria patients

“…The novel mutations c.284T>C and c.329A>T were expressed in E. coli using a protocol adapted after Mendes et al., (), as described below.…”

“…The novel mutations c.284T>C and c.329A>T were expressed in E. coli using a protocol adapted after Mendes et al, (2014), as described below. For expression of wild-type (WT) and mutant CbS, WT cDNA was first cloned in pOTB7 vector (Thermo Scientific, Lafayette, CO, USA), between restriction sites EcoRI and XhoI.…”