title: 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.
authors: Metabolism and Genetics Group, Research Institute for Medicines and Pharmaceutical Sciences, Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal.
authors: Marisa I S
authors: MI
authors: Mendes
authors: Henrique G
authors: HG
authors: Colaço
authors: Desirée E C
authors: DE
authors: Smith
authors: Rúben J J F
authors: RJ
authors: Ramos
authors: Ana
authors: A
authors: Pop
authors: Silvy J M
authors: SJ
authors: van Dooren
authors: Isabel
authors: I
authors: Tavares de Almeida
authors: Leo A J
authors: LA
authors: Kluijtmans
authors: Mirian C H
authors: MC
authors: Janssen
authors: Isabel
authors: I
authors: Rivera
authors: Gajja S
authors: GS
authors: Salomons
authors: Paula
authors: P
authors: Leandro
authors: Henk J
authors: HJ
authors: Blom
mesh_terms: D000483:Alleles; D002478:Cells, Cultured; D003541:Cystathionine beta-Synthase / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005347:Fibroblasts / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D005838:Genotype; D006712:Homocystinuria / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D006801:Humans; D009154:Mutation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012189:Retrospective Studies; D012436:S-Adenosylmethionine / Q000235:genetics* / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D012436:S-Adenosylmethionine; D003541:Cystathionine beta-Synthase
references: J Biol Chem. 2008 Dec 12;283(50):34588-95
references: 18849566
references: J Inherit Metab Dis. 1998 Dec;21(8):823-8
references: 9870207
references: Hum Mutat. 2002 Jun;19(6):641-55
references: 12007221
references: Hum Mutat. 2009 Jun;30(6):1021-2
references: 19370759
references: Eur J Pediatr. 1984 Aug;142(3):204-7
references: 6468444
references: N Engl J Med. 1985 Sep 19;313(12):709-15
references: 4033695
references: Hum Mutat. 2011 Jul;32(7):835-42
references: 21520339
references: Anal Biochem. 1985 Oct;150(1):76-85
references: 3843705
references: Eur J Pediatr. 1998 Apr;157 Suppl 2:S84-7
references: 9587032
references: Hum Mutat. 2006 Feb;27(2):211
references: 16429402
references: Biochem J. 2013 Jan 1;449(1):109-21
references: 22985361
references: Arch Biochem Biophys. 2005 Jan 1;433(1):144-56
references: 15581573
references: J Biol Chem. 2004 Jul 16;279(29):29871-4
references: 15087459
references: J Chromatogr B Analyt Technol Biomed Life Sci. 2012 Dec 12;911:186-91
references: 23217323
references: Hum Mol Genet. 2001 Mar 15;10(6):635-43
references: 11230183
references: J Inherit Metab Dis. 2011 Feb;34(1):39-48
references: 20490928
references: EMBO J. 2001 Aug 1;20(15):3910-6
references: 11483494
references: J Inorg Biochem. 2006 Dec;100(12):1988-95
references: 17069888
references: Protein Expr Purif. 2012 Apr;82(2):317-24
references: 22333527
references: Genomics. 1998 Sep 15;52(3):312-24
references: 9790750
references: Eur J Pediatr. 1998 Apr;157 Suppl 2:S67-70
references: 9587029
references: Am J Hum Genet. 1985 Jan;37(1):1-31
references: 3872065
references: FEBS Lett. 1998 Jul 17;431(2):175-9
references: 9708897
references: Methods Enzymol. 1995;252:103-12
references: 7476343
references: Biochemistry. 2002 Oct 1;41(39):11832-7
references: 12269827
references: Am J Physiol Cell Physiol. 2005 Dec;289(6):C1369-78
references: 16275737
references: Biochemistry. 2011 Jan 25;50(3):419-25
references: 21141970
references: Trends Biochem Sci. 1997 Jan;22(1):12-3
references: 9020585
references: J Biol Chem. 2002 Dec 13;277(50):48386-94
references: 12379655
references: Nat Genet. 1998 May;19(1):91-3
references: 9590298
references: Hum Mutat. 1992;1(2):113-23
references: 1301198
references: J Biol Chem. 2010 May 21;285(21):15866-73
references: 20308073
references: J Biol Chem. 1994 Oct 14;269(41):25283-8
references: 7929220
references: J Clin Invest. 1978 Mar;61(3):645-53
references: 641146
references: Biochemistry. 2002 Aug 20;41(33):10454-61
references: 12173932
references: J Clin Invest. 1996 Jul 15;98(2):285-9
references: 8755636
references: Am J Hum Genet. 2001 Jun;68(6):1506-13
references: 11359213
references: Hum Mutat. 2002 Aug;20(2):117-26
references: 12124992
references: Biochemistry. 2012 Jun 12;51(23):4755-63
references: 22612060
references: Hum Mutat. 2010 Jul;31(7):809-19
references: 20506325
references: Protein Expr Purif. 1994 Oct;5(5):442-8
references: 7827502
references: Biochemistry. 2012 Aug 14;51(32):6360-70
references: 22738154
references: PLoS Genet. 2010 Jan;6(1):e1000807
references: 20066033
references: Arch Biochem Biophys. 2008 Feb 1;470(1):64-72
references: 18060852
references: Protein Expr Purif. 2009 Apr;64(2):139-45
references: 19010420
references: Acta Crystallogr D Biol Crystallogr. 2001 Feb;57(Pt 2):289-91
references: 11173483
references: Mol Genet Metab. 2006 Apr;87(4):323-8
references: 16307898
references: J Hum Genet. 2005;50(12):648-54
references: 16205833