Addition of beta1-6 GlcNAc branching to the oligosaccharide attached to Asn 772 in the serine protease domain of matriptase plays a pivotal role in its stability and resistance against trypsin.

beta1-6 GlcNAc branching, a product of N-acetylglucosaminyltransferase V (GnT-V), is a key structure that is associated with malignant transformations and cancer metastasis. Although a number of reports concerning tumor metastasis-related glycoproteins that contain beta1-6 GlcNAc branching have appeared, the precise function of beta1-6 GlcNAc branching on glycoproteins remains to be elucidated. We previously reported on the importance of beta1-6 GlcNAc branching on matriptase in terms of proteolytic degradation in tumor metastasis. We report here that matriptase purified from GnT-V transfectant (beta1-6 GlcNAc matriptase) binds strongly to L4-PHA, which preferentially recognizes beta1-6 GlcNAc branches of tri- or tetraantennary sugar chains, indicating that the isolated matriptase contains beta1-6 GlcNAc branching. The beta1-6 GlcNAc matriptase was resistant to autodegradation, as well as trypsin digestion, compared with matriptase purified from mock-transfected cells. Furthermore, N-glycosidase-F treatment of beta1-6 GlcNAc matriptase greatly reduced its resistance to degradation. An analysis of matriptase mutants that do not contain potential N-glycosylation sites clearly shows that the beta1-6 GlcNAc branching on N-glycans attached to Asn 772 in the serine protease domain plays a major role in trypsin resistance. This is the first example of a demonstration of a direct relationship between beta1-6 GlcNAc branching and a biological function at the protein level.

[1]  R. Dickson,et al.  The Activation of Matriptase Requires Its Noncatalytic Domains, Serine Protease Domain, and Its Cognate Inhibitor* , 2003, Journal of Biological Chemistry.

[2]  Baljit Singh,et al.  Tissue microarray analysis of hepatocyte growth factor/Met pathway components reveals a role for Met, matriptase, and hepatocyte growth factor activator inhibitor 1 in the progression of node-negative breast cancer. , 2003, Cancer research.

[3]  S. Wahl,et al.  Matriptase/MT-SP1 is required for postnatal survival, epidermal barrier function, hair follicle development, and thymic homeostasis , 2002, Oncogene.

[4]  S. Nakahara,et al.  Prometastatic Effect ofN-Acetylglucosaminyltransferase V Is Due to Modification and Stabilization of Active Matriptase by Adding β1–6 GlcNAc Branching* , 2002, The Journal of Biological Chemistry.

[5]  Baljit Singh,et al.  Expression of the serine protease matriptase and its inhibitor HAI-1 in epithelial ovarian cancer: correlation with clinical outcome and tumor clinicopathological parameters. , 2002, Clinical cancer research : an official journal of the American Association for Cancer Research.

[6]  Robert Huber,et al.  Catalytic Domain Structures of MT-SP1/Matriptase, a Matrix-degrading Transmembrane Serine Proteinase* , 2002, The Journal of Biological Chemistry.

[7]  R. Schwartz,et al.  N-terminal Processing Is Essential for Release of Epithin, a Mouse Type II Membrane Serine Protease* , 2001, The Journal of Biological Chemistry.

[8]  T. Iwanaga,et al.  A role for membrane-type serine protease (MT-SP1) in intestinal epithelial turnover. , 2001, Biochemical and biophysical research communications.

[9]  M. Monden,et al.  Elevated expression of UDP‐N‐acetylglucosamine: αmannoside β1,6 N‐acetylglucosaminyltransferase is an early event in hepatocarcinogenesis , 2001 .

[10]  R. Dickson,et al.  Activation of Hepatocyte Growth Factor and Urokinase/Plasminogen Activator by Matriptase, an Epithelial Membrane Serine Protease* , 2000, The Journal of Biological Chemistry.

[11]  Jennifer L. Harris,et al.  Cellular Localization of Membrane-type Serine Protease 1 and Identification of Protease-activated Receptor-2 and Single-chain Urokinase-type Plasminogen Activator as Substrates* , 2000, The Journal of Biological Chemistry.

[12]  S. Ishiguro,et al.  Expression of N-acetylglucosaminyltransferase V in colorectal cancer correlates with metastasis and poor prognosis. , 2000, Clinical cancer research : an official journal of the American Association for Cancer Research.

[13]  J. Dennis,et al.  Suppression of tumor growth and metastasis in Mgat5-deficient mice , 2000, Nature Medicine.

[14]  C. Craik,et al.  Reverse biochemistry: use of macromolecular protease inhibitors to dissect complex biological processes and identify a membrane-type serine protease in epithelial cancer and normal tissue. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[15]  M. Johnson,et al.  Molecular Cloning of cDNA for Matriptase, a Matrix-degrading Serine Protease with Trypsin-like Activity* , 1999, The Journal of Biological Chemistry.

[16]  M. Johnson,et al.  Purification and Characterization of a Complex Containing Matriptase and a Kunitz-type Serine Protease Inhibitor from Human Milk* , 1999, The Journal of Biological Chemistry.

[17]  U. Metzger,et al.  Prognostic Value of β1,6-Branched Oligosaccharides in Human Colorectal Carcinoma , 1998 .

[18]  R. Dickson,et al.  Characterization of a Novel, Membrane-bound, 80-kDa Matrix-degrading Protease from Human Breast Cancer Cells , 1997, The Journal of Biological Chemistry.

[19]  N. Taniguchi,et al.  Suppression of lung metastasis of B16 mouse melanoma by N-acetylglucosaminyltransferase III gene transfection. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[20]  J. Dennis,et al.  Reduced contact-inhibition and substratum adhesion in epithelial cells expressing GlcNAc-transferase V , 1995, The Journal of cell biology.

[21]  N. Niikawa,et al.  cDNA cloning and chromosomal mapping of human N-acetylglucosaminyltransferase V+. , 1994, Biochemical and biophysical research communications.

[22]  N. Hayashi,et al.  N-acetylglucosaminyltransferase III and V messenger RNA levels in LEC rats during hepatocarcinogenesis. , 1993, Cancer research.

[23]  D. Wen,et al.  Isolation, characterization, and expression of a cDNA encoding N-acetylglucosaminyltransferase V. , 1993, The Journal of biological chemistry.

[24]  J. Gu,et al.  Purification and characterization of UDP-N-acetylglucosamine: alpha-6-D-mannoside beta 1-6N-acetylglucosaminyltransferase (N-acetylglucosaminyltransferase V) from a human lung cancer cell line. , 1993, Journal of biochemistry.

[25]  A. Wellstein,et al.  Identification and characterization of a novel matrix-degrading protease from hormone-dependent human breast cancer cells. , 1993, Cancer research.

[26]  M. Pierce,et al.  Purification and characterization of rat kidney UDP-N-acetylglucosamine: alpha-6-D-mannoside beta-1,6-N-acetylglucosaminyltransferase. , 1992, The Journal of biological chemistry.

[27]  R. Kerbel,et al.  Beta 1-6 branching of Asn-linked oligosaccharides is directly associated with metastasis. , 1987, Science.

[28]  P Berg,et al.  Electroporation for the efficient transfection of mammalian cells with DNA. , 1987, Nucleic acids research.

[29]  R. Cummings,et al.  Characterization of the structural determinants required for the high affinity interaction of asparagine-linked oligosaccharides with immobilized Phaseolus vulgaris leukoagglutinating and erythroagglutinating lectins. , 1982, The Journal of biological chemistry.

[30]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[31]  R. Dickson,et al.  Deregulated activation of matriptase in breast cancer cells , 2004, Clinical & Experimental Metastasis.

[32]  M. Monden,et al.  Elevated expression of UDP-N-acetylglucosamine: alphamannoside beta1,6 N-acetylglucosaminyltransferase is an early event in hepatocarcinogenesis. , 2001, International journal of cancer.

[33]  U. Metzger,et al.  Prognostic value of beta1,6-branched oligosaccharides in human colorectal carcinoma. , 1998, Cancer research.

[34]  S. Hakomori,et al.  Aberrant glycosylation in tumors and tumor-associated carbohydrate antigens. , 1989, Advances in cancer research.