Na+/H+ exchanger-2 is an O-linked but not an N-linked sialoglycoprotein.
A polyclonal antibody (Ab597) was produced in rabbit against a fusion protein of glutathione-S-transferase and the last 87 amino acids of the Na+/H+ exchanger isoform, NHE2. By Western blotting, Ab597 recognized proteins of 75 and 85 kDa in PS120/NHE2 membranes (PS120 cells stably transfected with NHE2), and this antibody did not cross-react with NHE1 and NHE3. When Ab597 was used to immunocytochemically stain PS120/NHE2 cells, permeabilization of the cells was required for staining, confirming the putative membrane topology of NHE2 that the C-terminus is cytoplasmic. NHE1 is N-glycosylated. NHE2 was predicted to be N-glycosylated as it contains one potential N-linked glycosylation site (N350VS), which is conserved among NHE1, NHE3, and NHE4. However, NHE2 was resistant to peptide:N-glycosidase F (PNGase F) and endoglycosidase H (Endo H) digestion, suggesting that NHE2 is not N-glycosylated. In contrast, neuraminidase shifted the mobility of the 85 kDa NHE2 protein in PS120/NHE2 membranes into an 81 kDa band, and O-glycanase further shifted the mobility of the neuraminidase-treated 81 kDa protein to 75 kDa. Incubation of PS120/NHE2 cells with benzyl N-acetyl-alpha-D-galactosaminide (Bz alpha GalNAc), an O-glycosylation inhibitor, decreased the size of the 85 kDa protein to 81 kDa. This treatment had no effect on the initial rate of Na+/H+ exchange of PS120/NHE2 cells. The 75 kDa protein was not affected by the glycosidase treatment of PS120/NHE2 membranes or the Bz alpha GalNAc treatment of PS120/NHE2 cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Levine
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Yun
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Khurana
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Donowitz
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D000116:Acetylgalactosamine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D001593:Benzyl Compounds / Q000494:pharmacology*; D015153:Blotting, Western; D002460:Cell Line; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D003429:Cross Reactions; D018076:DNA, Complementary / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects*; D005982:Glutathione Transferase / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D006031:Glycosylation / Q000187:drug effects; D007106:Immune Sera / Q000276:immunology; D007150:Immunohistochemistry; D008970:Molecular Weight; D011817:Rabbits; D012795:Sialoglycoproteins / Q000737:chemistry* / Q000378:metabolism; D012867:Skin / Q000166:cytology; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D013194:Staining and Labeling; D014162:Transfection
D001593:Benzyl Compounds; D018076:DNA, Complementary; D007106:Immune Sera; D012795:Sialoglycoproteins; D017923:Sodium-Hydrogen Exchangers; C080739:benzyl-alpha-N-acetylgalactosamine; D005982:Glutathione Transferase; D000116:Acetylgalactosamine