Na+/H+ exchanger-2 is an O-linked but not an N-linked sialoglycoprotein

Biochemistry. 1994 Nov 8;33(44):12954-61. doi: 10.1021/bi00248a003.


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)

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Acetylgalactosamine / analogs & derivatives*
  • Acetylgalactosamine / pharmacology
  • Animals
  • Benzyl Compounds / pharmacology*
  • Blotting, Western
  • Cell Line
  • Cell Membrane Permeability
  • Cells, Cultured
  • Cross Reactions
  • DNA, Complementary / chemistry
  • Electrophoresis, Polyacrylamide Gel
  • Enzyme Induction
  • Fibroblasts / cytology
  • Fibroblasts / drug effects*
  • Glutathione Transferase / chemistry
  • Glutathione Transferase / immunology
  • Glutathione Transferase / metabolism*
  • Glycosylation / drug effects
  • Immune Sera / immunology
  • Immunohistochemistry
  • Molecular Weight
  • Rabbits
  • Sialoglycoproteins / chemistry*
  • Sialoglycoproteins / metabolism
  • Skin / cytology
  • Sodium-Hydrogen Exchangers / chemistry*
  • Sodium-Hydrogen Exchangers / immunology
  • Sodium-Hydrogen Exchangers / metabolism
  • Staining and Labeling
  • Transfection


  • Benzyl Compounds
  • DNA, Complementary
  • Immune Sera
  • Sialoglycoproteins
  • Sodium-Hydrogen Exchangers
  • benzyl-alpha-N-acetylgalactosamine
  • Glutathione Transferase
  • Acetylgalactosamine