N-Glycosylation Is Required for Matriptase-2 Autoactivation and Ectodomain Shedding

Jiang, Jiang; Yang, Jianfeng; Feng, Ping; Zuo, Bin; Dong, Ningzheng; Wu, Qingyu; He, Yao

doi:10.1074/jbc.m114.555110

Cited by 35 publications

(32 citation statements)

References 47 publications

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“…1B). A previous study reports two additional cleaved products (28). In this study, we indeed detected a robust activity for soluble MT2 ectodomain and a minor activity for soluble S/P domain ( Figs.…”

Section: Mechanistic Studies Of Matriptase-2 Activitysupporting

confidence: 84%

“…MT2 is synthesized as a zymogen in the endoplasmic reticulum. Its activation is predicted to occur on the cell surface by autocleavage (1,28). MT2 also undergoes ectodomain shedding, and multiple sizes of soluble MT2 are detected in the conditioned medium (CM) of transfected cells (25,28,29).…”

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confidence: 99%

“…Its activation is predicted to occur on the cell surface by autocleavage (1,28). MT2 also undergoes ectodomain shedding, and multiple sizes of soluble MT2 are detected in the conditioned medium (CM) of transfected cells (25,28,29). At least four functional forms of MT2 have been suggested, including an uncleaved full-length form, an activated form with the cleaved S/P domain attached to membrane-tethered stem region through disulfide bonds, the shed ectodomain where the entire ectodomain is released into the medium and the shed S/P domain (1,2,16,25,28,30).…”

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confidence: 99%

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The catalytic, stem, and transmembrane portions of matriptase-2 are required for suppressing the expression of the iron-regulatory hormone hepcidin

Mao

Wortham

Enns

et al. 2019

Journal of Biological Chemistry

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Edited by F. Peter Guengerich Matriptase-2 (MT2) is a type-II transmembrane, trypsin-like serine protease that is predominantly expressed in the liver. It is a key suppressor for the expression of hepatic hepcidin, an ironregulatory hormone that is induced via the bone morphogenetic protein signaling pathway. A current model predicts that MT2 suppresses hepcidin expression by cleaving multiple components of the induction pathway. MT2 is synthesized as a zymogen that undergoes autocleavage for activation and shedding. However, the biologically active form of MT2 and, importantly, the contributions of different MT2 domains to its function are largely unknown. Here we examined the activities of truncated MT2 that were generated by site-directed mutagenesis or Gibson assembly master mix, and found that the stem region of MT2 determines the specificity and efficacy for substrate cleavage. The transmembrane domain allowed MT2 activation after reaching the plasma membrane, and the cytoplasmic domain facilitated these processes. Further in vivo rescue studies indicated that the entire extracellular and transmembrane domains of MT2 are required to correct the low-hemoglobin, low-serum iron, and high-hepcidin status in MT2 ؊/؊ mice. Unlike in cell lines, no autocleavage of MT2 was detected in vivo in the liver, implying that MT2 may also function independently of its proteolytic activity. In conjunction with our previous studies implicating the cytoplasmic domain as an intracellular iron sensor, these observations reveal the importance of each MT2 domain for MT2-mediated substrate cleavage and for its biological function.

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Section: Mechanistic Studies Of Matriptase-2 Activitysupporting

confidence: 84%

mentioning

confidence: 99%

mentioning

confidence: 99%

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The catalytic, stem, and transmembrane portions of matriptase-2 are required for suppressing the expression of the iron-regulatory hormone hepcidin

Mao

Wortham

Enns

et al. 2019

Journal of Biological Chemistry

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“…In addition, wild hepsin overexpression enhanced cell migration and invasion (Supplemental Figure 4), whereas hepsin mutant N112Q attenuated the invasion and metastatic potential compared to wild hepsin in MGC80-3 cells (Supplemental Figure 4). Similar roles of N-glycosylation in regulating cell surface expression and protease activity have been reported in other type II transmembrane serine proteases, such as corin24, enteropeptidase25, matriptase26, and matriptase-227, which are involved in blood pressure regulation, food digestion, epithelial function, and iron metabolism, respectively242829.…”

Section: Resultssupporting

confidence: 64%

High Hepsin expression predicts poor prognosis in Gastric Cancer

Zhang

Zhao

Tang

et al. 2016

Sci Rep

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Hepsin, a membrane-associated serine protease, is frequently upregulated in epithelial cancers and involved in cancer progression. Our study aims to describe the expression pattern and evaluate the clinical implication of hepsin in gastric cancer patients. The mRNA expression of hepsin was analyzed in 50 gastric cancer and matched non-tumor tissues, which was downregulated in 78% (39/50) of gastric cancer. By searching and analyzing four independent datasets from Oncomine, we obtained the similar results. Furthermore, we evaluated the hepsin expression by IHC in tissue microarray (TMA) containing 220 Gastric Cancer specimens. More importantly, Kaplan-Meier survival and Cox regression analyses were taken to access the prognosis of gastric cancer and predicted that hepsin protein expression was one of the significant and independent prognostic factors for overall survival of Gastric Cancer.

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“…Our data demonstrate that mutations in MT-2 can cause intracellular retention of the protein or an impaired ability to decrease HJV expression at the cell surface, a function compatible with proteolytic cleavage. Recently, Jiang et al (16) reported that N-linked glycosylation played an important role in MT-2 activation. We have shown that, at lower levels of expression, the Y141C, I212T, G442R, and C510S mutants are predominantly retained intracellularly, probably in the endoplasmic reticulum (ER), and are not transported to the cell surface, whereas the R271Q mutant shows WT-like cell surface localization.…”

Section: Discussionmentioning

confidence: 99%

Functional analysis of matriptase-2 mutations and domains: insights into the molecular basis of iron-refractory iron deficiency anemia

McDonald

Ostini

Bennett

et al. 2015

American Journal of Physiology-Cell Physiology

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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.

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N-Glycosylation Is Required for Matriptase-2 Autoactivation and Ectodomain Shedding

Cited by 35 publications

References 47 publications

The catalytic, stem, and transmembrane portions of matriptase-2 are required for suppressing the expression of the iron-regulatory hormone hepcidin

The catalytic, stem, and transmembrane portions of matriptase-2 are required for suppressing the expression of the iron-regulatory hormone hepcidin

High Hepsin expression predicts poor prognosis in Gastric Cancer

Functional analysis of matriptase-2 mutations and domains: insights into the molecular basis of iron-refractory iron deficiency anemia

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