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 th e Na+/H+ exchanger isoform, NHE2. By Western blotting, Ab597 recognize d proteins of 75 and 85 kDa in PS120/NHE2 membranes (PS120 cells stabl y transfected with NHE2), and this antibody did not cross-react with N HE1 and NHE3. When Ab597 was used to immunocytochemically stain PS120/ NHE2 cells, permeabilization of the cells was required for staining, c onfirming the putative membrane topology of NHE2 that the C-terminus i s cytoplasmic. NHE1 is N-glycosylated. NHE2 was predicted to be N-glyc osylated as it contains one potential N-linked glycosylation site ((NV S)-V-350), which is conserved among NHE1, NHE3, and NHE4. However, NHE 2 was resistant to peptide:N-glycosidase F (PNGase F) and endoglycosid ase H (Endo H) digestion, suggesting that NHE2 is not N-glycosylated. In contrast, neuraminidase shifted the mobility of the 85 kDa NHE2 pro tein in PS120/NHE2 membranes into an 81 kDa band, and O-glycanase furt her shifted the mobility of the neuraminidase-treated 81 kDa protein t o 75 kDa. Incubation of PS120/NHE2 cells with benzyl N-acetyl-alpha-D- galactosaminide (Bz alpha GalNAc), an O-glycosylation inhibitor, decre ased the size of the 85 kDa protein to 81 kDa. This treatment had no e ffect 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. T hese results suggest that the 85 kDa protein is an O-glycosylated form of NHE2, while the 75 kDa protein is an unglycosylated form. Thus, un like NHE1, which is N- and O-glycosylated, NHE2 has only O-linked glyc osylation.