Title :
Asparagine Asparagine-linked oligosaccharides are localized to a luminal hydrophilic loop in human
glucose-6-phosphatase
Abstract :
- Deficiency of glucose-6-phosphatase ( G6Pase ), an endoplasmic reticulum transmembrane glycoprotein , causes glycogen storage disease type 1a
- 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 , N96TS, N203AS, and N276SS, conserved among mammalian G6Pases
- According to this model, Asn96 , located in a 37-residue luminal loop, is a potential acceptor for oligosaccharides , whereas Asn203 and Asn276 , 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 Asn96 , 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
- In contrast, mutation of Asn276 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 G6Pase stability and catalytic activity