Asparagine-linked oligosaccharides are localized to a luminal hydrophilic loop in human glucose-6-phosphatase

yDeficiency of glucose-6-phosphatase (G6Pase), an endoplasmic reticulum transmembrane glycoprotein, causes glycogen storage disease type la, We have recently shown that human G6Pase contains an odd number of transmembrane segments, supporting a nine-transmembrane helical model for this enzyme. Sequence analysis predicts the presence of three potential asparagine (N)-linked glycosylation sites, (NTS)-T-96, N(203)AS, and (NSS)-S-276, conserved among mammalian G6Pases. According to this model, Asn(96), located in a 37-residue luminal loop, is a potential acceptor for oligosaccharides, whereas Asn(203) and Asn(276), located in a 12-residue cytoplasmic loop and helix 7, respectively, would not be utilized for this purpose. We therefore characterized mutant G6Pases lacking one, two, or all three potential N-linked glycosylation sites. Western blot and in vitro translation studies showed that G6Pase is glycosylated only at Asn(96), further validating the nine-transmembrane topology model. Substituting Asn96 with an Ala (N96A) moderately reduced enzymatic activity and had no effect on G6Pase synthesis or degradation, suggesting that oligosaccharide chains do not play a major role in protecting the enzyme from proteolytic degradation. Ln contrast, mutation of Asn(276) to an Ala (N276A) destabilized the enzyme and markedly reduced enzymatic activity. We present additional evidence suggesting that the integrity of transmembrane helices is essential for C6Pase stability and catalytic activity.