title: N-Glycan Branching Affects the Subcellular Distribution of and Inhibition of Matriptase by HAI-2/Placental Bikunin.
abstract: The gene product of SPINT 2, that encodes a transmembrane, Kunitz-type serine protease inhibitor independently designated as HAI-2 or placenta bikunin (PB), is involved in regulation of sodium absorption in human gastrointestinal track. Here, we show that SPINT 2 is expressed as two species of different size (30-40- versus 25-kDa) due to different N-glycans on Asn-57. The N-glycan on 25-kDa HAI-2 appears to be of the oligomannose type and that on 30-40-kDa HAI-2 to be of complex type with extensive terminal N-acetylglucosamine branching. The two different types of N-glycan differentially mask two epitopes on HAI-2 polypeptide, recognized by two different HAI-2 mAbs. The 30-40-kDa form may be mature HAI-2, and is primarily localized in vesicles/granules. The 25-kDa form is likely immature HAI-2, that remains in the endoplasmic reticulum (ER) in the perinuclear regions of mammary epithelial cells. The two different N-glycans could, therefore, represent different maturation stages of N-glycosylation with the 25-kDa likely a precursor of the 30-40-kDa HAI-2, with the ratio of their levels roughly similar among a variety of cells. In breast cancer cells, a significant amount of the 30-40-kDa HAI-2 can translocate to and inhibit matriptase on the cell surface, followed by shedding of the matriptase-HAI-2 complex. The 25-kDa HAI-2 appears to have also exited the ER/Golgi, being localized at the cytoplasmic face of the plasma membrane of breast cancer cells. While the 25-kDa HAI-2 was also detected at the extracellular face of plasma membrane at very low levels it appears to have no role in matriptase inhibition probably due to its paucity on the cell surface. Our study reveals that N-glycan branching regulates HAI-2 through different subcellular distribution and subsequently access to different target proteases.
authors.affiliation: Lombardi Comprehensive Cancer Center, Department of Oncology, Georgetown University, Washington DC, United States of America.
authors.forename: Ying-Jung J
authors.initials: YJ
authors.lastname: Lai
authors.affiliation: Lombardi Comprehensive Cancer Center, Department of Oncology, Georgetown University, Washington DC, United States of America; Department of Biochemistry, National Defense Medical Center, Taipei City, Taiwan.
authors.forename: Hsiang-Hua D
authors.initials: HH
authors.lastname: Chang
authors.affiliation: Lombardi Comprehensive Cancer Center, Department of Oncology, Georgetown University, Washington DC, United States of America.
authors.forename: Hongyu
authors.initials: H
authors.lastname: Lai
authors.affiliation: Lombardi Comprehensive Cancer Center, Department of Oncology, Georgetown University, Washington DC, United States of America.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Xu
authors.affiliation: Lombardi Comprehensive Cancer Center, Department of Oncology, Georgetown University, Washington DC, United States of America.
authors.forename: Frank
authors.initials: F
authors.lastname: Shiao
authors.affiliation: Lombardi Comprehensive Cancer Center, Department of Oncology, Georgetown University, Washington DC, United States of America.
authors.forename: Nanxi
authors.initials: N
authors.lastname: Huang
authors.affiliation: Lombardi Comprehensive Cancer Center, Department of Oncology, Georgetown University, Washington DC, United States of America; Affiliated Hospital of Hunan Traditional Chinese Medicine Research Institute, Changsha, Hunan, China,s.
authors.forename: Linpei
authors.initials: L
authors.lastname: Li
authors.affiliation: Graduate Institute of Biochemistry and Molecular Biology, College of Medicine National Taiwan University, Taipei City, Taiwan.
authors.forename: Ming-Shyue
authors.initials: MS
authors.lastname: Lee
authors.affiliation: Lombardi Comprehensive Cancer Center, Department of Oncology, Georgetown University, Washington DC, United States of America.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Johnson
authors.affiliation: Department of Biochemistry, National Defense Medical Center, Taipei City, Taiwan.
authors.forename: Jehng-Kang
authors.initials: JK
authors.lastname: Wang
authors.affiliation: Lombardi Comprehensive Cancer Center, Department of Oncology, Georgetown University, Washington DC, United States of America.
authors.forename: Chen-Yong
authors.initials: CY
authors.lastname: Lin
mesh_terms: D000595:Amino Acid Sequence; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation; D006031:Glycosylation; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011134:Polysaccharides / Q000737:chemistry*; D011247:Pregnancy; D021381:Protein Transport; D012697:Serine Endopeptidases / Q000378:metabolism*
chemical_list: D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D011134:Polysaccharides; C109148:SPINT2 protein, human; D012697:Serine Endopeptidases; C467801:matriptase 2