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 found in a wide range of biological systems. NHE is involved in various functions including pH homeostasis, volume regulation, cell proliferation 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, betaNHE. Immunoblotting of cell membrane preparation reveals that betaNHE is a protein with an apparent molecular mass of 95 kDa. Moreover enzymatic glycosidase treatment demonstrates that the antiporter is an N-glycosylated but not O-glycosylated protein. The primary structure of betaNHE contains three putative N-glycosylation consensus sites (N-X-S/T) at Asn49, Asn338 and Asn378. Expression of betaNHE 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 betaNHE antiporter, lacking the first predicted motif, shows that betaNHE possesses an unique glycosylation site located on the first extracellular loop of the exchanger (Asn49). Expression of this deglycosylated antiporter shows that deglycosylation of the protein modifies neither the pH(i) dependency of the antiporter nor its hormonal stimulation.
Malapert
M
M
Laboratoire Jean Maetz, ERS 1253, Département de Biologie Cellulaire et Moléculaire CEA, Villefranche-sur-mer, France.
Pellissier
B
B
Borgese
F
F
D000818:Animals; D001216:Asparagine / Q000737:chemistry*; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D017931:DNA Primers; D004912:Erythrocytes / Q000737:chemistry*; D006031:Glycosylation; D017136:Ion Transport; D016296:Mutagenesis; D017923:Sodium-Hydrogen Exchangers / Q000097:blood*; D014337:Trout / Q000097:blood*
D017931:DNA Primers; D017923:Sodium-Hydrogen Exchangers; D001216:Asparagine