Inactive matriptase-2 mutants found in IRIDA patients still repress hepcidin in a transfection assay despite having lost their serine protease activity

Université Paris Diderot, Paris, France.
Human Mutation (Impact Factor: 5.14). 09/2012; 33(9):1388-96. DOI: 10.1002/humu.22116
Source: PubMed


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.

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    • "Consistent with previous results [Guillem et al., 2012], R576A and S762A inhibited hepcidin activation even in our assay (Fig. 1A). Using the HuH7 cells, that are hemochromatosis cell lines [Vecchi et al., 2010], a trend toward an impaired inhibitory activity of MT2 mutants in the hepcidin promoter luciferase-based assay was observed (Fig. 1B), although no statistical significance was reached for almost all the MT2 mutants analyzed, with the exception of Y418C and the MASK variant. "

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