Title : The Na+/H+ exchanger
NHE-1 possesses N- and O-linked glycosylation restricted to the first N-terminal extracellular
domain
Abstract :
- The ubiquitously-expressed human Na+H+ exchanger ( NHE-1 ) contains three consensus sites ( Asn-X-Ser/Thr) for N-linked glycosylation at asparagines 75, 370, and 410
- The first extracellular loop is rich in serine and threonine residues which may contain O-linked carbohydrate
- In order to determine unambiguously the sites of glycosylation and their role in biosynthesis and cation transport, site-directed mutagenesis at the individual potential N-glycosylation sites ( Asn to Asp ) was performed and all possible double and triple mutants were constructed
- The mutated DNAs were expressed in PS120 hamster fibroblasts lacking endogenous exchanger, and the transfected cells were selected by their ability to survive acute intracellular acidification
- All constructs produced 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 showed that only the first N-glycosylation site ( Asn 75 ) is utilized
- In addition, treatment of NHE-1 with neuraminidase and O-glycosidase demonstrated that NHE-1 also contains O-linked oligosaccharide
- Two forms of NHE-1 was consistently observed, a mature form with a molecular mass of 110,000 Da which contains N-linked and O-linked oligosaccharide and is expressed at the cell surface, and a lower molecular mass form (85,000 Da) present in the endoplasmic reticulum which only contains N-linked high-mannose oligosaccharide
- NHE-3 , an apically-expressed epithelial isoform which does not possess the N75 N-linked putative glycosylation site and any extracellular loops enriched in serine and threonine residues , does not exhibit any detectable glycosylation
Output (sent_index, trigger,
protein,
sugar,
site):
- 1. glycosylation, , -, -, asparagines 75, 370, and 410
- 3. N-glycosylation, , -, -, Asp
- 3. N-glycosylation, , -, -, sites
- 3. glycosylation, , -, -, sites
- 6. N-glycosylation, , -, -, Asn 75
- 6. N-glycosylation, , -, -, site
- 6. utilized, , -, -, Asn 75
- 6. utilized, , -, -, site
- 7. contains, , NHE-1, O-linked oligosaccharide, -
- 9. glycosylation, , -, -, site
- 9. serine, , -, -, serine and threonine residues
Output(Part-Of) (sent_index,
protein,
site):
- 1. NHE-1, sites
- 9. NHE-3, site
*Output_Site_Fusion* (sent_index,
protein,
sugar,
site):
- 1. NHE-1, -, asparagines 75, 370, and 410
- 6. NHE-1, -, Asn 75