title: Inactive matriptase-2 mutants found in IRIDA patients still repress hepcidin in a transfection assay despite having lost their serine protease activity.
abstract: Mutations of the TMPRSS6 gene, which encodes Matriptase-2, are responsible for iron-refractory iron-deficiency anemia. Matriptase-2 is a transmembrane protease that downregulates hepcidin expression. We report one frameshift (p.Ala605ProfsX8) and four novel missense mutations (p.Glu114Lys, p.Leu235Pro, p.Tyr418Cys, p.Pro765Ala) found in IRIDA patients. These mutations lead to changes in both the catalytic and noncatalytic domains of Matriptase-2. Analyses of the mutant proteins revealed a reduction of autoactivating cleavage and the loss of N-Boc-Gln-Ala-Arg-p-nitroanilide hydrolysis. This resulted either from a direct modification of the active site or from the lack of the autocatalytic cleavage that transforms the zymogen into an active protease. In a previously described transfection assay measuring the ability of Matriptase-2 to repress the hepcidin gene (HAMP) promoter, all mutants retained some, if not all, of their transcriptional repression activity. This suggests that caution is called for in interpreting the repression assay in assessing the functional relevance of Matriptase-2 substitutions. We propose that Matriptase-2 activity should be measured directly in the cell medium of transfected cells using the chromogenic substrate. This simple test can be used to determine whether a sequence variation leading to an amino acid substitution is functionally relevant or not.
authors.affiliation: Université Paris Diderot, Paris, France.
authors.forename: Flavia
authors.initials: F
authors.lastname: Guillem
authors.forename: Caroline
authors.initials: C
authors.lastname: Kannengiesser
authors.forename: Claire
authors.initials: C
authors.lastname: Oudin
authors.forename: Anne
authors.initials: A
authors.lastname: Lenoir
authors.forename: Pavle
authors.initials: P
authors.lastname: Matak
authors.forename: Jean
authors.initials: J
authors.lastname: Donadieu
authors.forename: Bertrand
authors.initials: B
authors.lastname: Isidor
authors.forename: Francoise
authors.initials: F
authors.lastname: Méchinaud
authors.forename: Patricia
authors.initials: P
authors.lastname: Aguilar-Martinez
authors.forename: Carole
authors.initials: C
authors.lastname: Beaumont
authors.forename: Sophie
authors.initials: S
authors.lastname: Vaulont
authors.forename: Bernard
authors.initials: B
authors.lastname: Grandchamp
authors.forename: Gael
authors.initials: G
authors.lastname: Nicolas
mesh_terms: D000293:Adolescent; D000328:Adult; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D018798:Anemia, Iron-Deficiency / Q000201:enzymology* / Q000235:genetics; D023181:Antimicrobial Cationic Peptides / Q000097:blood / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D002648:Child; D002675:Child, Preschool; D002863:Chromogenic Compounds / Q000378:metabolism; D003470:Culture Media / Q000378:metabolism; D004789:Enzyme Activation; D057075:Enzyme Assays; D004792:Enzyme Precursors / Q000378:metabolism; D016368:Frameshift Mutation; D020868:Gene Silencing; D005820:Genetic Testing; D006367:HeLa Cells; D064451:Hepcidins; D006801:Humans; D007223:Infant; D008297:Male; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D010375:Pedigree; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D057057:Serine Proteases / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection / Q000379:methods*
chemical_list: D023181:Antimicrobial Cationic Peptides; D002863:Chromogenic Compounds; D003470:Culture Media; D004792:Enzyme Precursors; C578702:HAMP protein, human; D064451:Hepcidins; D008565:Membrane Proteins; D012097:Repressor Proteins; D057057:Serine Proteases; D012697:Serine Endopeptidases; C504318:TMPRSS6 protein, human