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UDP-glucose:glycoprotein glucosyltransferase (UGGT1) promotes substrate solubility in the endoplasmic reticulum.

Ferris SP, Jaber NS, Molinari M, Arvan P, Kaufman RJ - Mol. Biol. Cell (2013)

Bottom Line: Whereas substrate solubility increases directly with the number of N-linked glycosylation sites, our results indicate that additional solubility is conferred by UGGT1 enzymatic activity.Monoglucosylation-dependent solubility decreases both BiP association with NHK and unfolded protein response activation, and the solubility increase is blocked in cells deficient for calreticulin.These results suggest that UGGT1-dependent monoglucosylation of N-linked glycoproteins promotes substrate solubility in the ER.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry and Medical Scientist Training Program, University of Michigan Medical School, Ann Arbor, MI 48109-1621, USA.

ABSTRACT
Protein folding in the endoplasmic reticulum (ER) is error prone, and ER quality control (ERQC) processes ensure that only correctly folded proteins are exported from the ER. Glycoproteins can be retained in the ER by ERQC, and this retention contributes to multiple human diseases, termed ER storage diseases. UDP-glucose:glycoprotein glucosyltransferase (UGGT1) acts as a central component of glycoprotein ERQC, monoglucosylating deglucosylated N-glycans of incompletely folded glycoproteins and promoting subsequent reassociation with the lectin-like chaperones calreticulin and calnexin. The extent to which UGGT1 influences glycoprotein folding, however, has only been investigated for a few selected substrates. Using mouse embryonic fibroblasts lacking UGGT1 or those with UGGT1 complementation, we investigated the effect of monoglucosylation on the soluble/insoluble distribution of two misfolded α1-antitrypsin (AAT) variants responsible for AAT deficiency disease: Hong Kong (NHK) and Z allele. Whereas substrate solubility increases directly with the number of N-linked glycosylation sites, our results indicate that additional solubility is conferred by UGGT1 enzymatic activity. Monoglucosylation-dependent solubility decreases both BiP association with NHK and unfolded protein response activation, and the solubility increase is blocked in cells deficient for calreticulin. These results suggest that UGGT1-dependent monoglucosylation of N-linked glycoproteins promotes substrate solubility in the ER.

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NHK and ATZ, but not wt-AAT, become insoluble over time. (A) Transient transfection of Uggt1−/− MEFs with wt-AAT ± UGGT1 and analysis by pulse chase at the indicated time points (Materials and Methods). No wt-AAT was detected in the insoluble fraction at any time point. (B) Same as A for ATZ. Here ATZ progressively accumulates in the insoluble fraction over time. (C) Same as A for NHK. Here NHK progressively accumulates in the insoluble fraction over time. Soluble NHK was run on a nonreducing gel because under reducing conditions, the reduced IP antibody physically pushes all NHK bands down to the same molecular weight. For A–C, four times the TCA-normalized amount of insoluble fraction was used for IP compared with soluble fraction (actual relative amount of insoluble ATZ and NHK is 25% of what is seen in the gel image). (D) EndoH digest of extracellular, soluble, and insoluble NHK after steady-state labeling. The majority of extracellular NHK is EndoH resistant, evidence of having Golgi-modified N-glycans typically found on secreted glycoproteins. NHK in the soluble and insoluble fractions is completely EndoH sensitive, indicating that these forms are not modified by Golgi-localized N-glycan–modifying enzymes.
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Figure 2: NHK and ATZ, but not wt-AAT, become insoluble over time. (A) Transient transfection of Uggt1−/− MEFs with wt-AAT ± UGGT1 and analysis by pulse chase at the indicated time points (Materials and Methods). No wt-AAT was detected in the insoluble fraction at any time point. (B) Same as A for ATZ. Here ATZ progressively accumulates in the insoluble fraction over time. (C) Same as A for NHK. Here NHK progressively accumulates in the insoluble fraction over time. Soluble NHK was run on a nonreducing gel because under reducing conditions, the reduced IP antibody physically pushes all NHK bands down to the same molecular weight. For A–C, four times the TCA-normalized amount of insoluble fraction was used for IP compared with soluble fraction (actual relative amount of insoluble ATZ and NHK is 25% of what is seen in the gel image). (D) EndoH digest of extracellular, soluble, and insoluble NHK after steady-state labeling. The majority of extracellular NHK is EndoH resistant, evidence of having Golgi-modified N-glycans typically found on secreted glycoproteins. NHK in the soluble and insoluble fractions is completely EndoH sensitive, indicating that these forms are not modified by Golgi-localized N-glycan–modifying enzymes.

Mentions: Whereas endoglycosidase H (EndoH)-resistant NHK molecules were found in the secreted fraction, both the soluble and detergent-insoluble intracellular fractions were EndoH sensitive, indicating that neither the soluble nor the insoluble intracellular pool had arrived in a Golgi compartment from which they could be further modified by Golgi N-glycan processing enzymes (Figure 2D). We confirmed by several independent approaches that a fraction of mutant NHK molecules forms detergent-insoluble protein aggregates. First, pulse-chase experiments demonstrated that newly synthesized NHK was not initially recovered in the detergent-insoluble fraction but became so as a function of chase, indicating time-dependent specificity (Figure 2C). Second, we compared solubility of NHK using three standard detergent lysis methods that have been used for analysis of AAT and its variants (Schmidt and Perlmutter, 2005; Kroeger et al., 2009; Galli et al., 2011): 1) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS; 2% wt/vol); 2) Nonidet P-40 (1% vol/vol); and 3) Triton X-100 (0.5% wt/vol) and sodium deoxycholate (0.5% wt/vol). In each case, a detergent-insoluble fraction of NHK was recovered (Supplemental Figure S1B). Finally, intracellular wt-AAT was always undetectable in the insoluble fraction (Figures 1C and 2A), indicating that recovery in the detergent-insoluble pool was not caused by failure to disrupt cellular membranes from Uggt1−/− MEFs. These findings, together with other results (Greenblatt et al., 2011), provide strong evidence that NHK is predisposed to aberrant protein aggregation.


UDP-glucose:glycoprotein glucosyltransferase (UGGT1) promotes substrate solubility in the endoplasmic reticulum.

Ferris SP, Jaber NS, Molinari M, Arvan P, Kaufman RJ - Mol. Biol. Cell (2013)

NHK and ATZ, but not wt-AAT, become insoluble over time. (A) Transient transfection of Uggt1−/− MEFs with wt-AAT ± UGGT1 and analysis by pulse chase at the indicated time points (Materials and Methods). No wt-AAT was detected in the insoluble fraction at any time point. (B) Same as A for ATZ. Here ATZ progressively accumulates in the insoluble fraction over time. (C) Same as A for NHK. Here NHK progressively accumulates in the insoluble fraction over time. Soluble NHK was run on a nonreducing gel because under reducing conditions, the reduced IP antibody physically pushes all NHK bands down to the same molecular weight. For A–C, four times the TCA-normalized amount of insoluble fraction was used for IP compared with soluble fraction (actual relative amount of insoluble ATZ and NHK is 25% of what is seen in the gel image). (D) EndoH digest of extracellular, soluble, and insoluble NHK after steady-state labeling. The majority of extracellular NHK is EndoH resistant, evidence of having Golgi-modified N-glycans typically found on secreted glycoproteins. NHK in the soluble and insoluble fractions is completely EndoH sensitive, indicating that these forms are not modified by Golgi-localized N-glycan–modifying enzymes.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 2: NHK and ATZ, but not wt-AAT, become insoluble over time. (A) Transient transfection of Uggt1−/− MEFs with wt-AAT ± UGGT1 and analysis by pulse chase at the indicated time points (Materials and Methods). No wt-AAT was detected in the insoluble fraction at any time point. (B) Same as A for ATZ. Here ATZ progressively accumulates in the insoluble fraction over time. (C) Same as A for NHK. Here NHK progressively accumulates in the insoluble fraction over time. Soluble NHK was run on a nonreducing gel because under reducing conditions, the reduced IP antibody physically pushes all NHK bands down to the same molecular weight. For A–C, four times the TCA-normalized amount of insoluble fraction was used for IP compared with soluble fraction (actual relative amount of insoluble ATZ and NHK is 25% of what is seen in the gel image). (D) EndoH digest of extracellular, soluble, and insoluble NHK after steady-state labeling. The majority of extracellular NHK is EndoH resistant, evidence of having Golgi-modified N-glycans typically found on secreted glycoproteins. NHK in the soluble and insoluble fractions is completely EndoH sensitive, indicating that these forms are not modified by Golgi-localized N-glycan–modifying enzymes.
Mentions: Whereas endoglycosidase H (EndoH)-resistant NHK molecules were found in the secreted fraction, both the soluble and detergent-insoluble intracellular fractions were EndoH sensitive, indicating that neither the soluble nor the insoluble intracellular pool had arrived in a Golgi compartment from which they could be further modified by Golgi N-glycan processing enzymes (Figure 2D). We confirmed by several independent approaches that a fraction of mutant NHK molecules forms detergent-insoluble protein aggregates. First, pulse-chase experiments demonstrated that newly synthesized NHK was not initially recovered in the detergent-insoluble fraction but became so as a function of chase, indicating time-dependent specificity (Figure 2C). Second, we compared solubility of NHK using three standard detergent lysis methods that have been used for analysis of AAT and its variants (Schmidt and Perlmutter, 2005; Kroeger et al., 2009; Galli et al., 2011): 1) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS; 2% wt/vol); 2) Nonidet P-40 (1% vol/vol); and 3) Triton X-100 (0.5% wt/vol) and sodium deoxycholate (0.5% wt/vol). In each case, a detergent-insoluble fraction of NHK was recovered (Supplemental Figure S1B). Finally, intracellular wt-AAT was always undetectable in the insoluble fraction (Figures 1C and 2A), indicating that recovery in the detergent-insoluble pool was not caused by failure to disrupt cellular membranes from Uggt1−/− MEFs. These findings, together with other results (Greenblatt et al., 2011), provide strong evidence that NHK is predisposed to aberrant protein aggregation.

Bottom Line: Whereas substrate solubility increases directly with the number of N-linked glycosylation sites, our results indicate that additional solubility is conferred by UGGT1 enzymatic activity.Monoglucosylation-dependent solubility decreases both BiP association with NHK and unfolded protein response activation, and the solubility increase is blocked in cells deficient for calreticulin.These results suggest that UGGT1-dependent monoglucosylation of N-linked glycoproteins promotes substrate solubility in the ER.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry and Medical Scientist Training Program, University of Michigan Medical School, Ann Arbor, MI 48109-1621, USA.

ABSTRACT
Protein folding in the endoplasmic reticulum (ER) is error prone, and ER quality control (ERQC) processes ensure that only correctly folded proteins are exported from the ER. Glycoproteins can be retained in the ER by ERQC, and this retention contributes to multiple human diseases, termed ER storage diseases. UDP-glucose:glycoprotein glucosyltransferase (UGGT1) acts as a central component of glycoprotein ERQC, monoglucosylating deglucosylated N-glycans of incompletely folded glycoproteins and promoting subsequent reassociation with the lectin-like chaperones calreticulin and calnexin. The extent to which UGGT1 influences glycoprotein folding, however, has only been investigated for a few selected substrates. Using mouse embryonic fibroblasts lacking UGGT1 or those with UGGT1 complementation, we investigated the effect of monoglucosylation on the soluble/insoluble distribution of two misfolded α1-antitrypsin (AAT) variants responsible for AAT deficiency disease: Hong Kong (NHK) and Z allele. Whereas substrate solubility increases directly with the number of N-linked glycosylation sites, our results indicate that additional solubility is conferred by UGGT1 enzymatic activity. Monoglucosylation-dependent solubility decreases both BiP association with NHK and unfolded protein response activation, and the solubility increase is blocked in cells deficient for calreticulin. These results suggest that UGGT1-dependent monoglucosylation of N-linked glycoproteins promotes substrate solubility in the ER.

Show MeSH
Related in: MedlinePlus