ASN49 IS THE UNIQUE GLYCOSYLATION SITE OF THE TROUT RED-BLOOD-CELL NA+ H+ EXCHANGER/

The Na+/H+ exchanger (NHE) is a plasma membrane transport protein foun d in a wide range of biological systems. NHE is involved in various fu nctions including pH homeostasis, volume regulation, cell proliferatio n and transcellular Na+ absorption. This study reports immunodetection results obtained with antibodies generated against the C-terminus of the NHE of trout red blood cells, beta NHE. Immunoblotting of cell mem brane preparation reveals that beta NHE is a protein with an apparent molecular mass of 95 kDa. Moreover enzymatic glycosidase treatment dem onstrates that the antiporter is an N-glycosylated but not O-glycosyla ted protein. The primary structure of beta NHE contains three putative N-glycosylation consensus sites (N-X-S/T) at Asn49, Asn338 and Asn378 . Expression of beta NHE in PS120 fibroblasts, a cell line which lacks an endogenous Na+/H+ exchange, allows to determine the precise sites of glycosylation. The construction of a site-directed mutated beta NHE antiporter, lacking the first predicted motif, shows that beta NHE po ssesses an unique glycosylation site located on the first extracellula r loop of the exchanger (Asn49). Expression of this deglycosylated ant iporter shows that deglycosylation of the protein modifies neither the pH, dependency of the antiporter nor its hormonal stimulation.