A liquid chromatography mass spectrometry method for the measurement of cystathionine β-synthase activity in cell extracts

Smith, Desirée E.C.; Mendes, Marisa I.; Kluijtmans, Leo A. J.; Janssen, Mirian C. H.; Smulders, Yvo M.; Blom, Henk J.

doi:10.1016/j.jchromb.2012.10.041

Cited by 8 publications

(8 citation statements)

References 34 publications

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“…The availability of a highly sensitive and rigorous method of Cth quantitation [Smith et al., ] enabled us to determine the kinetic parameters of the WT and variant CBS for both substrates (Ser and Hcy), which are presented in Table . At a fixed Hcy concentration of 2.7 mM, the WT CBS displayed for Ser a typical Michaelis–Menten kinetics (Supp.…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2014

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Cystathionine beta-synthase (CBS) catalyzes the formation of cystathionine from homocysteine and serine. CBS is allosterically activated by S-adenosylmethionine (SAM), which binds to its C-terminal regulatory domain. Mutations in this domain lead to variants with high residual activity but lacking SAM activation. We characterized six C-terminal CBS variants (p.P427L, p.D444N, p.V449G, p.S500L, p.K523Sfs*18, and p.L540Q). To understand the effect of C-terminal mutations on the functional/structural properties of CBS, we performed dynamic light scattering, differential scanning fluorimetry, limited proteolysis, enzymatic characterization, and determination of SAM-binding affinity. Kinetic data confirm that the enzymatic function of these variants is not impaired. Although lacking SAM activation, the p.P427L and p.S500L were able to bind SAM at a lower extent than the wild type (WT), confirming that SAM binding and activation can be two independent events. At the structural level, the C-terminal variants presented various effects, either showing catalytic core instability and increased susceptibility toward aggregation or presenting with similar or higher stability than the WT. Our study highlights as the common feature to the C-terminal variants an impaired binding of SAM and no increase in enzymatic activity with physiological concentrations of the activator, suggesting the loss of regulation by SAM as a potential pathogenic mechanism.

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Section: Resultsmentioning

confidence: 99%

“…The enzyme activity of recombinant CBS proteins was determined in a reaction volume of 110 μL, as previously described [Smith et al., ], using 0.165 μg of protein and 30 min incubation. Specific enzyme activities were expressed as μmol Cth formed per hour per milligram CBS protein, at 37°C (μmol Cth h −1 mg −1 ).…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2014

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“…The gold standard for confirming CBS deficiency is generally considered to be the determination of cystathionine production from Hcy and serine in cultured fibroblasts using radioactive or deuterium labelled substrates (Kraus 1987; Smith et al 2012). The fibroblast CBS activity may, however, be normal in mild forms of the disease, despite biochemical and clinical abnormalities and mutations in the CBS gene.…”

Section: Recommendationsmentioning

confidence: 99%

Guidelines for the diagnosis and management of cystathionine beta‐synthase deficiency

Morris

Kožich

Santra

et al. 2016

J of Inher Metab Disea

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Cystathionine beta-synthase (CBS) deficiency is a rare inherited disorder in the methionine catabolic pathway, in which the impaired synthesis of cystathionine leads to accumulation of homocysteine. Patients can present to many different specialists and diagnosis is often delayed. Severely affected patients usually present in childhood with ectopia lentis, learning difficulties and skeletal abnormalities. These patients generally require treatment with a low-methionine diet and/or betaine. In contrast, mildly affected patients are likely to present as adults with thromboembolism and to respond to treatment with pyridoxine. In this article, we present recommendations for the diagnosis and management of CBS deficiency, based on a systematic review of the literature. Unfortunately, the quality of the evidence is poor, as it often is for rare diseases. We strongly recommend measuring the plasma total homocysteine concentrations in any patient whose clinical features suggest the diagnosis. Our recommendations may help to standardise testing for pyridoxine responsiveness. Current evidence suggests that patients are unlikely to develop complications if the plasma total homocysteine concentration is maintained below 120 μmol/L. Nevertheless, we recommend keeping the concentration below 100 μmol/L because levels fluctuate and the complications associated with high levels are so serious.

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“…Because a high through-put method that required low levels of protein was required, in the soluble fraction of E.coli lysates the catalytic activity of recombinant CBS proteins was determined by following Cth production using a LC-MS/MS method, according to Smith et al (2012b) with some modifications. Briefly, the enzymatic assay conditions were firstly optimized regarding substrates concentration (0.5-50 mmol/L Ser, 0.27-27 mmol/L Hcy), total protein amount (20 μg and 40 μg) and reaction times (5 min-4 h).…”

Section: Assay Of Cbs Activity In Fibroblasts and E Coli Lysatesmentioning

confidence: 99%

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

Mendes

Colaço

Smith

et al. 2013

J of Inher Metab Disea

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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.

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A liquid chromatography mass spectrometry method for the measurement of cystathionine β-synthase activity in cell extracts

Cited by 8 publications

References 34 publications

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

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

Guidelines for the diagnosis and management of cystathionine beta‐synthase deficiency

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

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