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A cell-based reglucosylation assay demonstrates the role of GT1 in the quality control of a maturing glycoprotein.

Pearse BR, Gabriel L, Wang N, Hebert DN - J. Cell Biol. (2008)

Bottom Line: GT1 reglucosylated N-linked glycans in the slow-folding stem domain of HA once the nascent chain was released from the ribosome.Maturation mutants that disrupted the oxidation or oligomerization of HA also supported region-specific reglucosylation by GT1.Therefore, GT1 acts as an ER quality control sensor by posttranslationally reglucosylating glycans on slow-folding or nonnative domains to recruit chaperones specifically to critical aberrant regions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Massachusetts, Amherst, MA 01003, USA.

ABSTRACT
The endoplasmic reticulum (ER) protein GT1 (UDP-glucose: glycoprotein glucosyltransferase) is the central enzyme that modifies N-linked carbohydrates based upon the properties of the polypeptide backbone of the maturing substrate. GT1 adds glucose residues to nonglucosylated proteins that fail the quality control test, supporting ER retention through persistent binding to the lectin chaperones calnexin and calreticulin. How GT1 functions in its native environment on a maturing substrate is poorly understood. We analyzed the reglucosylation of a maturing model glycoprotein, influenza hemagglutinin (HA), in the intact mammalian ER. GT1 reglucosylated N-linked glycans in the slow-folding stem domain of HA once the nascent chain was released from the ribosome. Maturation mutants that disrupted the oxidation or oligomerization of HA also supported region-specific reglucosylation by GT1. Therefore, GT1 acts as an ER quality control sensor by posttranslationally reglucosylating glycans on slow-folding or nonnative domains to recruit chaperones specifically to critical aberrant regions.

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Glycan processing in wild-type and MI8-5 cells. (A) Processing of the N-linked glycan in the ER of both wild-type and MI8-5 CHO cells. The glycoform generated by GT1 activity in each cell type is boxed and the point at which trimming is blocked by the presence of the glucosidase inhibitor DNJ is marked. OST, oligosaccharyltransferase; Gls I, glucosidase I; Gls II, glucosidase II; Glc, glucose; Man, Mannose; GlcNAc2, N-acetylglucosamine. (B) Schematic of a 14-member N-linked glycan. Glucoses are depicted as triangles, mannoses as circles, and N-acetylglucosamines as squares. The three mannose branches, A, B, and C, are marked. The three glucoses attached to the A branch are indicated as G3, G2, and G1.
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fig1: Glycan processing in wild-type and MI8-5 cells. (A) Processing of the N-linked glycan in the ER of both wild-type and MI8-5 CHO cells. The glycoform generated by GT1 activity in each cell type is boxed and the point at which trimming is blocked by the presence of the glucosidase inhibitor DNJ is marked. OST, oligosaccharyltransferase; Gls I, glucosidase I; Gls II, glucosidase II; Glc, glucose; Man, Mannose; GlcNAc2, N-acetylglucosamine. (B) Schematic of a 14-member N-linked glycan. Glucoses are depicted as triangles, mannoses as circles, and N-acetylglucosamines as squares. The three mannose branches, A, B, and C, are marked. The three glucoses attached to the A branch are indicated as G3, G2, and G1.

Mentions: Studies of GT1 reglucosylation in the ER are hindered by the presence of two distinct pathways that can create monoglucosylated proteins. Monoglucosylated proteins can be generated by glucose addition to Man9GlcNAc2 glycans via GT1 or through the sequential trimming of newly added glycans by glucosidases I and II (Fig. 1 A, WT). To separate monoglucosylated glycans produced via GT1 activity from those generated by glucosidase activity, the CHO mutant cell line MI8-5 was used. The oligosaccharyltransferase of these cells transfers Man9GlcNAc2 glycans as opposed to triglucosylated high-mannose glycans because of a mutation in the alg6 gene (Fig. 1 A, MI8-5; Quellhorst et al., 1999). Alg6p is responsible for transferring glucose residues to the growing dolichol pyrophosphate precursor (Runge et al., 1984). Therefore, GT1 activity is the sole mechanism for producing monoglucosylated glycoproteins in MI8-5 CHO cells.


A cell-based reglucosylation assay demonstrates the role of GT1 in the quality control of a maturing glycoprotein.

Pearse BR, Gabriel L, Wang N, Hebert DN - J. Cell Biol. (2008)

Glycan processing in wild-type and MI8-5 cells. (A) Processing of the N-linked glycan in the ER of both wild-type and MI8-5 CHO cells. The glycoform generated by GT1 activity in each cell type is boxed and the point at which trimming is blocked by the presence of the glucosidase inhibitor DNJ is marked. OST, oligosaccharyltransferase; Gls I, glucosidase I; Gls II, glucosidase II; Glc, glucose; Man, Mannose; GlcNAc2, N-acetylglucosamine. (B) Schematic of a 14-member N-linked glycan. Glucoses are depicted as triangles, mannoses as circles, and N-acetylglucosamines as squares. The three mannose branches, A, B, and C, are marked. The three glucoses attached to the A branch are indicated as G3, G2, and G1.
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Related In: Results  -  Collection

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fig1: Glycan processing in wild-type and MI8-5 cells. (A) Processing of the N-linked glycan in the ER of both wild-type and MI8-5 CHO cells. The glycoform generated by GT1 activity in each cell type is boxed and the point at which trimming is blocked by the presence of the glucosidase inhibitor DNJ is marked. OST, oligosaccharyltransferase; Gls I, glucosidase I; Gls II, glucosidase II; Glc, glucose; Man, Mannose; GlcNAc2, N-acetylglucosamine. (B) Schematic of a 14-member N-linked glycan. Glucoses are depicted as triangles, mannoses as circles, and N-acetylglucosamines as squares. The three mannose branches, A, B, and C, are marked. The three glucoses attached to the A branch are indicated as G3, G2, and G1.
Mentions: Studies of GT1 reglucosylation in the ER are hindered by the presence of two distinct pathways that can create monoglucosylated proteins. Monoglucosylated proteins can be generated by glucose addition to Man9GlcNAc2 glycans via GT1 or through the sequential trimming of newly added glycans by glucosidases I and II (Fig. 1 A, WT). To separate monoglucosylated glycans produced via GT1 activity from those generated by glucosidase activity, the CHO mutant cell line MI8-5 was used. The oligosaccharyltransferase of these cells transfers Man9GlcNAc2 glycans as opposed to triglucosylated high-mannose glycans because of a mutation in the alg6 gene (Fig. 1 A, MI8-5; Quellhorst et al., 1999). Alg6p is responsible for transferring glucose residues to the growing dolichol pyrophosphate precursor (Runge et al., 1984). Therefore, GT1 activity is the sole mechanism for producing monoglucosylated glycoproteins in MI8-5 CHO cells.

Bottom Line: GT1 reglucosylated N-linked glycans in the slow-folding stem domain of HA once the nascent chain was released from the ribosome.Maturation mutants that disrupted the oxidation or oligomerization of HA also supported region-specific reglucosylation by GT1.Therefore, GT1 acts as an ER quality control sensor by posttranslationally reglucosylating glycans on slow-folding or nonnative domains to recruit chaperones specifically to critical aberrant regions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Massachusetts, Amherst, MA 01003, USA.

ABSTRACT
The endoplasmic reticulum (ER) protein GT1 (UDP-glucose: glycoprotein glucosyltransferase) is the central enzyme that modifies N-linked carbohydrates based upon the properties of the polypeptide backbone of the maturing substrate. GT1 adds glucose residues to nonglucosylated proteins that fail the quality control test, supporting ER retention through persistent binding to the lectin chaperones calnexin and calreticulin. How GT1 functions in its native environment on a maturing substrate is poorly understood. We analyzed the reglucosylation of a maturing model glycoprotein, influenza hemagglutinin (HA), in the intact mammalian ER. GT1 reglucosylated N-linked glycans in the slow-folding stem domain of HA once the nascent chain was released from the ribosome. Maturation mutants that disrupted the oxidation or oligomerization of HA also supported region-specific reglucosylation by GT1. Therefore, GT1 acts as an ER quality control sensor by posttranslationally reglucosylating glycans on slow-folding or nonnative domains to recruit chaperones specifically to critical aberrant regions.

Show MeSH
Related in: MedlinePlus