THE NA+ H+ EXCHANGER NHE-1 POSSESSES N-LINKED AND O-LINKED GLYCOSYLATION RESTRICTED TO THE FIRST N-TERMINAL EXTRACELLULAR DOMAIN/

The ubiquitously-expressed human Na+/H+ exchanger (NHE-1) contains thr ee consensus sites (Asn-X-Ser/Thr) for N-linked glycosylation at aspar agines 75, 370, and 410. The first extracellular loop is rich in serin e and threonine residues which may contain O-linked carbohydrate. In o rder to determine unambiguously the sites of glycosylation and their r ole in biosynthesis and cation transport, site-directed mutagenesis at the individual potential N-glycosylation sites (Asn to Asp) was perfo rmed and all possible double and triple mutants were constructed. The mutated DNAs were expressed in PS120 hamster fibroblasts lacking endog enous exchanger, and the transfected cells were selected by their abil ity to survive acute intracellular acidification. All constructs produ ced functional exchangers that had transport rates and pharmacological profiles that were similar to that of wild-type. Immunoblot analysis of the expressed proteins with and without N-glycosidase F treatment s howed that only the first N-glycosylation site (Asn 75) is utilized. I n addition, treatment of NHE-1 with neuraminidase and O-glycosidase de monstrated that NHE-1 also contains O-linked oligosaccharide. Two form s of NHE-1 were consistently observed, a mature form with a molecular mass of 110 000 Da which contains N-linked and O-linked oligosaccharid e and is expressed at the cell surface, and a lower molecular mass for m (85 000 Da) present in the endoplasmic reticulum which only contains N-linked high-mannose oligosaccharide. NHE-3, an apically-expressed e pithelial isoform which does not possess the N75 N-linked putative gly cosylation site and any extracellular loops enriched in serine and thr eonine residues, does not exhibit any detectable glycosylation.