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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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HA glycans associated with calnexin are preferentially reglucosylated by GT1. (A) HA was translated in the presence of wild-type or MI8-5 SP cells treated with DMJ in the presence or absence of DNJ at 27°C for 60 min. After lysis of the SP cells, radiolabeled HA was immunoprecipitated with polyclonal HA, calnexin (αCNX), or calreticulin (αCRT) antisera. Samples were resolved via 7.5% SDS-PAGE. (B) Quantifications of A (n = 3). Error bars show SD.
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fig3: HA glycans associated with calnexin are preferentially reglucosylated by GT1. (A) HA was translated in the presence of wild-type or MI8-5 SP cells treated with DMJ in the presence or absence of DNJ at 27°C for 60 min. After lysis of the SP cells, radiolabeled HA was immunoprecipitated with polyclonal HA, calnexin (αCNX), or calreticulin (αCRT) antisera. Samples were resolved via 7.5% SDS-PAGE. (B) Quantifications of A (n = 3). Error bars show SD.

Mentions: 35S-labeled HA was coimmunoprecipitated with anti-calnexin or -calreticulin antibodies after 1 h of translation. Both calnexin and calreticulin efficiently bound to HA from wild-type SP cells (Fig. 3, A and B; Hebert et al., 1995, 1996, 1997; Peterson et al., 1995; Molinari et al., 2002). This binding was abolished with the addition of the glucosidase inhibitor DNJ, which supported the accumulation of triglucosylated HA, indicating that the association was glycan-dependent. In contrast, only calnexin bound HA at significant levels in MI8-5 SP cells. DNJ treatment did not inhibit calnexin binding because DNJ promotes the accumulation of monoglucosylated substrates in MI8-5 SP cells (Fig. 3, A [lanes 7, 8, 11, and 12] and B). GT1 appears to recognize the membrane-proximal domain of HA, resulting in the reglucosylation of the glycans that interact with the integral membrane chaperone calnexin. The lack of observable calreticulin binding was likely caused by the different topologies of the lectin chaperones in their native environment rather than by differences in their substrate recognition because GST-calreticulin bound HA from SP cell lysates (Fig. 2 D). Therefore, GT1 specifically reglucosylates glycans on the slow-folding membrane-proximal stem domain of HA.


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)

HA glycans associated with calnexin are preferentially reglucosylated by GT1. (A) HA was translated in the presence of wild-type or MI8-5 SP cells treated with DMJ in the presence or absence of DNJ at 27°C for 60 min. After lysis of the SP cells, radiolabeled HA was immunoprecipitated with polyclonal HA, calnexin (αCNX), or calreticulin (αCRT) antisera. Samples were resolved via 7.5% SDS-PAGE. (B) Quantifications of A (n = 3). Error bars show SD.
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Related In: Results  -  Collection

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fig3: HA glycans associated with calnexin are preferentially reglucosylated by GT1. (A) HA was translated in the presence of wild-type or MI8-5 SP cells treated with DMJ in the presence or absence of DNJ at 27°C for 60 min. After lysis of the SP cells, radiolabeled HA was immunoprecipitated with polyclonal HA, calnexin (αCNX), or calreticulin (αCRT) antisera. Samples were resolved via 7.5% SDS-PAGE. (B) Quantifications of A (n = 3). Error bars show SD.
Mentions: 35S-labeled HA was coimmunoprecipitated with anti-calnexin or -calreticulin antibodies after 1 h of translation. Both calnexin and calreticulin efficiently bound to HA from wild-type SP cells (Fig. 3, A and B; Hebert et al., 1995, 1996, 1997; Peterson et al., 1995; Molinari et al., 2002). This binding was abolished with the addition of the glucosidase inhibitor DNJ, which supported the accumulation of triglucosylated HA, indicating that the association was glycan-dependent. In contrast, only calnexin bound HA at significant levels in MI8-5 SP cells. DNJ treatment did not inhibit calnexin binding because DNJ promotes the accumulation of monoglucosylated substrates in MI8-5 SP cells (Fig. 3, A [lanes 7, 8, 11, and 12] and B). GT1 appears to recognize the membrane-proximal domain of HA, resulting in the reglucosylation of the glycans that interact with the integral membrane chaperone calnexin. The lack of observable calreticulin binding was likely caused by the different topologies of the lectin chaperones in their native environment rather than by differences in their substrate recognition because GST-calreticulin bound HA from SP cell lysates (Fig. 2 D). Therefore, GT1 specifically reglucosylates glycans on the slow-folding membrane-proximal stem domain of HA.

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