Function of glycoprotein glycans

Olden, Kenneth; Bernard, Bruno; Humphries, Martin J.; Yeo, Tet-Kin; Yeo, Kiang-Teck; White, Sandra L.; Newton, Sheila A.; Bauer, H.; Parent, J B

doi:10.1016/0968-0004(85)90238-5

Cited by 186 publications

(83 citation statements)

References 25 publications

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“…The glycan moieties of glycoproteins have been shown to play important roles, including the direction of protein folding, regulation of cell-surface expression, maintenance of polypeptide conformation and solubility, and increase in the half-life of proteins (21,22). The N96A mutant directed the synthesis of a 32-kDa nonglycosylated G6Pase, whereas the WT construct supported the synthesis of a glycoprotein of 36 kDa.…”

Section: Characterization Of G6pase Mutants Lacking One or More Glycomentioning

confidence: 99%

Asparagine-linked Oligosaccharides Are Localized to a Luminal Hydrophilic Loop in Human Glucose-6-Phosphatase

Pan¹,

Lei²,

Chou³

1998

Journal of Biological Chemistry

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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...

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Section: Characterization Of G6pase Mutants Lacking One or More Glycomentioning

confidence: 99%

Asparagine-linked Oligosaccharides Are Localized to a Luminal Hydrophilic Loop in Human Glucose-6-Phosphatase

Pan¹,

Lei²,

Chou³

1998

Journal of Biological Chemistry

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“…The fact that the position of the single N-glycosylation site in the plant GnTI sequences is only conserved among the two solanaceous species, but not in Arabidopsis, argues against their actual use in planta, or at least indicates that they do not play a role other than modulation of the physicochemical properties of the polypeptide (e.g. stabilization of protein conformation; Olden et al, 1985). …”

Section: Gnti-cdna Sequencesmentioning

confidence: 99%

Isolation and Characterization of Plant N-Acetyl Glucosaminyltransferase I (GntI) cDNA Sequences. Functional Analyses in the Arabidopsis cgl Mutant and in Antisense Plants

Wenderoth

Schaewen

2000

Plant Physiology

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We report on the isolation and characterization of full-length cDNA sequences coding for N-acetylglucosaminyltransferase I (GnTI) from potato (Solanum tuberosum L.), tobacco (Nicotiana tabacum L.), and Arabidopsis. The deduced polypeptide sequences show highest homology among the solanaceous species (93% identity between potato and tobacco compared with about 75% with Arabidopsis) but share only weak homology with human GnTI (35% identity). In contrast to the corresponding enzymes from animals, all plant GnTI sequences identified are characterized by a much shorter hydrophobic membrane anchor and contain one putative N-glycosylation site that is conserved in potato and tobacco, but differs in Arabidopsis. Southern-blot analyses revealed that GntI behaves as a single-copy gene. Northern-blot analyses showed that GntI-mRNA expression is largely constitutive. Arabidopsis cgl mutants deficient in GnTI activity also possess GntI mRNA, indicating that they result from point mutations. GntI-expression constructs were tested for the ability to relieve the GnTI block in protoplasts of the Arabidopsis cgl mutant and used to obtain transgenic potato and tobacco plants that display a substantial reduction of complex glycan patterns. The latter observation indicates that production of heterologous glycoproteins with little or no antigenic glycans can be achieved in whole plants, and not in just Arabidopsis, using antisense technology.

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“…Among the approaches used to accomplish this are (i) introduction of disulphide bonds, (ii) substitution of specific residues in order to increase ahelical stability, and (iii) substitution of residues involved in non-covalent interactions and folding patterns. Although there is circumstantial evidence that glycan moieties contribute to enhanced protein stability and may protect some proteins against proteolytic attack (Olden et al, 1985;Gu et al, 1989), the effect on thermostability of adding glycan groups to normally unglycosylated enzymes has not been systematically investigated. In yeast, similar to the situation in higher eukaryotes, N-linked glycosylation may occur at the Asn residue of the sequence Asn-Xxx-Ser/Thr while O-linked glycosylation occurs at either Ser or Thr (Innis, 1989).…”

Section: Introductionmentioning

confidence: 99%