title: Functional analysis of matriptase-2 mutations and domains: insights into the molecular basis of iron-refractory iron deficiency anemia.
abstract: Mutations in the TMPRSS6 gene are associated with severe iron-refractory iron deficiency anemia resulting from an overexpression of hepcidin, the key regulator of iron homeostasis. The matriptase (MT)-2 protein (encoded by the TMPRSS6 gene) regulates hepcidin expression by cleaving hemojuvelin [HJV/hemochromatosis type 2 (HFE2)], a bone morphogenetic protein (BMP) coreceptor in the hepcidin regulatory pathway. We investigated the functional consequences of five clinically associated TMPRSS6 variants and the role of MT-2 protein domains by generating epitope-tagged mutant and domain-swapped MT-2-MT-1 (encoded by the ST14 gene) chimeric constructs and expressing them in HepG2/C3A cells. We developed a novel cell culture immunofluorescence assay to assess the effect of MT-2 on cell surface HJV expression levels, compatible with HJV cleavage. The TMPRSS6 variants Y141C, I212T, G442R, and C510S were retained intracellularly and were unable to inhibit BMP6 induction of hepcidin. The R271Q variant, although it has been associated with iron-refractory iron deficiency anemia, appears to remain functional. Analysis of the chimeric constructs showed that replacement of sperm protein, enterokinase, and agrin (SEA), low-density-lipoprotein receptor class A (LDLRA), and protease (PROT) domains from MT-2 with those from MT-1 resulted in limited cell surface localization, while the complement C1r/C1s, Uegf, Bmp1 (CUB) domain chimera retained localization at the cell surface. The SEA domain chimera was able to reduce cell surface HJV expression, while the CUB, LDLRA, and PROT domain chimeras were not. These studies suggest that the SEA and LDLRA domains of MT-2 are important for trafficking to the cell surface and that the CUB, LDLRA, and PROT domains are required for cleavage of HJV.
authors.affiliation: Membrane Transport Laboratory, QIMR Berghofer Medical Research Institute, Herston, Queensland, Australia;
authors.forename: Cameron J
authors.initials: CJ
authors.lastname: McDonald
authors.affiliation: Membrane Transport Laboratory, QIMR Berghofer Medical Research Institute, Herston, Queensland, Australia;
authors.forename: Lesa
authors.initials: L
authors.lastname: Ostini
authors.affiliation: Membrane Transport Laboratory, QIMR Berghofer Medical Research Institute, Herston, Queensland, Australia;
authors.forename: Nigel
authors.initials: N
authors.lastname: Bennett
authors.affiliation: Membrane Transport Laboratory, QIMR Berghofer Medical Research Institute, Herston, Queensland, Australia;
authors.forename: Nanthakumar
authors.initials: N
authors.lastname: Subramaniam
authors.affiliation: Mater Research Institute-University of Queensland, Translational Research Institute, Brisbane, Queensland, Australia;
authors.forename: John
authors.initials: J
authors.lastname: Hooper
authors.affiliation: Departamento de Bioquímica y Biología Molecular, Instituto Universitario de Oncología, Universidad de Oviedo, Oviedo, Spain; and.
authors.forename: Gloria
authors.initials: G
authors.lastname: Velasco
authors.affiliation: Membrane Transport Laboratory, QIMR Berghofer Medical Research Institute, Herston, Queensland, Australia;
authors.forename: Daniel F
authors.initials: DF
authors.lastname: Wallace
authors.affiliation: Membrane Transport Laboratory, QIMR Berghofer Medical Research Institute, Herston, Queensland, Australia; Faculty of Medicine and Biomedical Sciences, University of Queensland, Brisbane, Queensland, Australia Nathan.Subramaniam@qimrberghofer.edu.au.
authors.forename: V Nathan
authors.initials: VN
authors.lastname: Subramaniam
mesh_terms: D018798:Anemia, Iron-Deficiency / Q000201:enzymology* / Q000235:genetics*; D058851:GPI-Linked Proteins / Q000235:genetics / Q000378:metabolism; D000071020:Hemochromatosis Protein; D056945:Hep G2 Cells; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics* / Q000502:physiology; D009154:Mutation / Q000235:genetics*; D017434:Protein Structure, Tertiary / Q000235:genetics; D021381:Protein Transport / Q000502:physiology; D012697:Serine Endopeptidases / Q000235:genetics* / Q000502:physiology; D016037:Single-Blind Method
chemical_list: D058851:GPI-Linked Proteins; C481006:HJV protein, human; D000071020:Hemochromatosis Protein; D008565:Membrane Proteins; D012697:Serine Endopeptidases; C467801:matriptase 2
keywords: anemia; functional analysis; iron; localization; mutation analysis