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 ninetransmembrane helical model for this enzyme. Sequence analysis predicts the presence of three potential asparagine (N) 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 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 G6Pase stability and catalytic activity.-Glucose-6-phosphatase (G6Pase, EC 3.1.3.9), 1 which catalyzes the terminal step in gluconeogenesis and glycogenolysis, is the key enzyme in glucose homeostasis (1). In humans, deficiency in microsomal G6Pase causes glycogen storage disease type 1a (GSD-1a), also known as von Gierke disease (2). It is an autosomal recessive disorder with clinical manifestations of hypoglycemia, growth retardation, hepatomegaly, kidney enlargement, lactic acidemia, hyperlipidemia, and hyperuricemia (2, 3). G6Pase is a transmembrane protein tightly associated with the endoplasmic reticulum (ER) (1). Using N-or C-terminal-tagged G6Pase constructs, we have shown that human G6Pase contains an odd number of transmembrane helices with the N terminus localized in the ER lumen and the C terminus in the cytoplasm (4). Our data support a nine-transmembrane helical model previously proposed for the enzyme based on hydropathy analysis (5).G6Pase is a glycoprotein (6 -8) and three potential asparagine (N)-linked glycosylation sites at N 96 TS, N 203 AS, and N 276 SS are predicted from sequence analysis (7-10). All three sites are conserved in human (7), mouse (8), rat (9), and canine (10) G6Pases. Thus, G6Pase belongs to a class of multispan membrane glycoproteins that are N-glycosylated. Although the presence of a consensus glycosylation site within the amino acid sequence of a membrane protein is suggestive of a glycoprotein, not all consensus sites are necessarily utilized. A survey of mammalian multispan membrane proteins has suggested that, for a potential N-linked glycosylation site to be utilized, it must be situated on the luminal side of the ER, and the size of the hydrophilic loop must be at least 33 amino acids in length (11,12). Analysis of protein glycosylation thus provides useful topological information for membrane proteins.The nine-transmembrane topology model predicts that human G6Pase would contain four short (8 -12 resides) cytoplasmic loops, and two short (7 and 8 residues) and two large (33 and 37 residues) luminal loops (4). According to this model, N 96 TS would be situated in a 37-residue luminal loop, thus it is the only glycosylation site in human G6Pase that satisfies the cri...

The Molecular Basis of Glycogen Storage Disease Type 1a

“…The nucleotide sequence in all constructs was verified by DNA sequencing. D38V-3Ј-FLAG and P178S-3Ј-FLAG mutants have been described previously (19).…”

“…In the presence of a glycosylation inhibitor, tunicamycin (32), only the 37-kDa nonglycosylated G6Pase was detected, confirming their identities. We have previously shown that both forms of G6Pase are enzymatically active and that the nonglycosylated G6Pase retains ϳ40% activity (19). Immunoblot analyses showed that the active site mutants, K76N, R83C, R83H, H119L, and R170Q (Fig.…”

“…As an alternative strategy to monitor biosynthesis of mutated G6Pase, we have constructed all 48 missense and the ⌬F327 G6Pase mutants with the eight-amino acid FLAG marker peptide (DYKDDDDK) at their carboxyl termini. The FLAG tag does not interfere with the expression, stability, or activity of WT G6Pase and has been used successfully to tag human G6Pase (18,19) for topological studies. G6Pase biosynthesis in COS-1 cells was examined by Western blot analysis using a monoclonal antibody against the FLAG epitope.…”

“…Thirty missense mutations (excluding the active site mutation K76N in helix 2) and the ⌬F327 mutation are scattered (18,19). The mutations are indicated and shown in black.…”

“…We have shown that human G6Pase is anchored to the endoplasmic reticulum (ER) by nine transmembrane helices with the amino terminus in the lumen and the carboxyl terminus in the cytoplasm (18,19). Therefore, the large collection of G6Pase mutations can now be studied in the context of their positions with respect to the ER and the cytoplasm.…”