title: N-glycosylation is required for matriptase-2 autoactivation and ectodomain shedding.
abstract: Matriptase-2 is a hepatic membrane serine protease that regulates iron homeostasis. Defects in matriptase-2 cause iron deficiency anemia. In cells, matriptase-2 is synthesized as a zymogen. To date, how matriptase-2 expression and activation are regulated remains poorly understood. Here we expressed human matriptase-2 in HEK293 and hepatic BEL-7402, SMMC-7721, and QGY-7703 cells. By labeling cell surface proteins and Western analysis, we examined matriptase-2 cell surface expression, zymogen activation, and ectodomain shedding. Our results show that matriptase-2 was activated on the cell surface but not intracellularly. Activated matriptase-2 underwent ectodomain shedding, producing soluble fragments in the conditioned medium. By testing inactive mutants, R576A and S762A, we found that matriptase-2 activation and shedding were mediated by its own catalytic activity and that the one-chain form of matriptase-2 had little activity in ectodomain shedding. We made additional matriptase-2 mutants, N136Q, N184Q, N216Q, N338Q, N433Q, N453Q, and N518Q, in which each of the predicted N-glycosylation sites was mutated. All of these mutants were expressed on the cell surface. However, mutants N216Q, N453Q, and N518Q, but not the other mutants, had impaired zymogen activation and ectodomain shedding. Our results indicate that N-glycans at specific sites are critical for matriptase-2 activation. Together, these data provide new insights into the cell surface expression, zymogen activation, and ectodomain shedding of matriptase-2.
authors.affiliation: From the Cyrus Tang Hematology Center and Ministry of Education Engineering Center of Hematological Disease, the Department of Clinic Laboratories, the Second Affiliated Hospital.
authors.forename: Jiang
authors.initials: J
authors.lastname: Jiang
authors.affiliation: From the Cyrus Tang Hematology Center and Ministry of Education Engineering Center of Hematological Disease.
authors.forename: Jianfeng
authors.initials: J
authors.lastname: Yang
authors.affiliation: the Department of Clinic Laboratories, the Second Affiliated Hospital.
authors.forename: Ping
authors.initials: P
authors.lastname: Feng
authors.affiliation: From the Cyrus Tang Hematology Center and Ministry of Education Engineering Center of Hematological Disease.
authors.forename: Bin
authors.initials: B
authors.lastname: Zuo
authors.affiliation: From the Cyrus Tang Hematology Center and Ministry of Education Engineering Center of Hematological Disease, the Ministry of Health Key Laboratory of Thrombosis and Hemostasis, Jiangsu Institute of Hematology, the First Affiliated Hospital, and the Collaborative Innovation Center of Hematology, Soochow University, Suzhou 215006, China and.
authors.forename: Ningzheng
authors.initials: N
authors.lastname: Dong
authors.affiliation: From the Cyrus Tang Hematology Center and Ministry of Education Engineering Center of Hematological Disease, the Collaborative Innovation Center of Hematology, Soochow University, Suzhou 215006, China and the Department of Molecular Cardiology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195 wuq@ccf.org.
authors.forename: Qingyu
authors.initials: Q
authors.lastname: Wu
authors.affiliation: From the Cyrus Tang Hematology Center and Ministry of Education Engineering Center of Hematological Disease, the Ministry of Health Key Laboratory of Thrombosis and Hemostasis, Jiangsu Institute of Hematology, the First Affiliated Hospital, and the Collaborative Innovation Center of Hematology, Soochow University, Suzhou 215006, China and heyang1963@163.com.
authors.forename: Yang
authors.initials: Y
authors.lastname: He
mesh_terms: D019943:Amino Acid Substitution; D045744:Cell Line, Tumor; D004789:Enzyme Activation / Q000502:physiology; D004792:Enzyme Precursors / Q000096:biosynthesis* / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D008099:Liver / Q000166:cytology / Q000201:enzymology*; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics; D020125:Mutation, Missense; D012697:Serine Endopeptidases / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D004792:Enzyme Precursors; D008565:Membrane Proteins; D012697:Serine Endopeptidases; C467801:matriptase 2
keywords: Glycosylation; Iron Metabolism; Proteolytic Enzyme; Serine Protease; Shedding