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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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UGGT1 increases the solubility of α1-antitrypsin mutants NHK and ATZ. (A–C) The 24-h metabolic labeling of Uggt1−/− MEFs transfected with (A) NHK, (B) ATZ, or (C) wt-AAT ± UGGT1 cotransfection. Uggt1−/− MEFs were transfected with expression vectors encoding the misfolded α1-antitrypsin variants NHK or ATZ or wild-type AAT (wt-AAT) and cotransfected with either empty vector or UGGT1 expression vector. Cells were radiolabeled from 24 to 48 h posttransfection in complete medium plus 10 μCi/ml [35S]methionine/cysteine. Extracellular, soluble, and insoluble fractions were produced (Materials and Methods), and TCA-precipitation normalized volumes of each fraction were subjected to double–anti-AAT quantitative IP and PNGaseF digestion and analyzed by reducing SDS–PAGE and autoradiography. Substrate solubility was calculated by dividing total soluble (extracellular + intracellular soluble) substrate amount by insoluble substrate amount. Middle, Student's t test used for statistical testing; right, paired Student's t test used for statistical testing (*p ≤ 0.05). Error bars represent SEM. For ATZ, all UGGT1+ bands were darkened with imaging software to make synthesis (data not shown) ± UGGT1 appear equal.
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Figure 1: UGGT1 increases the solubility of α1-antitrypsin mutants NHK and ATZ. (A–C) The 24-h metabolic labeling of Uggt1−/− MEFs transfected with (A) NHK, (B) ATZ, or (C) wt-AAT ± UGGT1 cotransfection. Uggt1−/− MEFs were transfected with expression vectors encoding the misfolded α1-antitrypsin variants NHK or ATZ or wild-type AAT (wt-AAT) and cotransfected with either empty vector or UGGT1 expression vector. Cells were radiolabeled from 24 to 48 h posttransfection in complete medium plus 10 μCi/ml [35S]methionine/cysteine. Extracellular, soluble, and insoluble fractions were produced (Materials and Methods), and TCA-precipitation normalized volumes of each fraction were subjected to double–anti-AAT quantitative IP and PNGaseF digestion and analyzed by reducing SDS–PAGE and autoradiography. Substrate solubility was calculated by dividing total soluble (extracellular + intracellular soluble) substrate amount by insoluble substrate amount. Middle, Student's t test used for statistical testing; right, paired Student's t test used for statistical testing (*p ≤ 0.05). Error bars represent SEM. For ATZ, all UGGT1+ bands were darkened with imaging software to make synthesis (data not shown) ± UGGT1 appear equal.

Mentions: To directly investigate the effect of UGGT1 on NHK, ATZ, or wild-type AAT (wt-AAT) solubility, we metabolically labeled Uggt1−/− MEFs (or those complemented with plasmid-encoded UGGT1) expressing these substrates with [35S]methionine/cysteine to approach steady state. By quantitative immunoprecipitation (IP; Supplemental Figure S1A), we determined that over the course of the metabolic labeling period (24 h), a fraction of mutant NHK molecules was secreted; a second portion was recovered in the intracellular detergent-soluble supernatant fraction; and a third was recovered as aggregates that failed to be solubilized from Uggt1−/− MEFs lysed in nonionic detergents (Figure 1A). This “detergent-insoluble fraction” was indeed solubilized in SDS-containing detergent extracts.


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)

UGGT1 increases the solubility of α1-antitrypsin mutants NHK and ATZ. (A–C) The 24-h metabolic labeling of Uggt1−/− MEFs transfected with (A) NHK, (B) ATZ, or (C) wt-AAT ± UGGT1 cotransfection. Uggt1−/− MEFs were transfected with expression vectors encoding the misfolded α1-antitrypsin variants NHK or ATZ or wild-type AAT (wt-AAT) and cotransfected with either empty vector or UGGT1 expression vector. Cells were radiolabeled from 24 to 48 h posttransfection in complete medium plus 10 μCi/ml [35S]methionine/cysteine. Extracellular, soluble, and insoluble fractions were produced (Materials and Methods), and TCA-precipitation normalized volumes of each fraction were subjected to double–anti-AAT quantitative IP and PNGaseF digestion and analyzed by reducing SDS–PAGE and autoradiography. Substrate solubility was calculated by dividing total soluble (extracellular + intracellular soluble) substrate amount by insoluble substrate amount. Middle, Student's t test used for statistical testing; right, paired Student's t test used for statistical testing (*p ≤ 0.05). Error bars represent SEM. For ATZ, all UGGT1+ bands were darkened with imaging software to make synthesis (data not shown) ± UGGT1 appear equal.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 1: UGGT1 increases the solubility of α1-antitrypsin mutants NHK and ATZ. (A–C) The 24-h metabolic labeling of Uggt1−/− MEFs transfected with (A) NHK, (B) ATZ, or (C) wt-AAT ± UGGT1 cotransfection. Uggt1−/− MEFs were transfected with expression vectors encoding the misfolded α1-antitrypsin variants NHK or ATZ or wild-type AAT (wt-AAT) and cotransfected with either empty vector or UGGT1 expression vector. Cells were radiolabeled from 24 to 48 h posttransfection in complete medium plus 10 μCi/ml [35S]methionine/cysteine. Extracellular, soluble, and insoluble fractions were produced (Materials and Methods), and TCA-precipitation normalized volumes of each fraction were subjected to double–anti-AAT quantitative IP and PNGaseF digestion and analyzed by reducing SDS–PAGE and autoradiography. Substrate solubility was calculated by dividing total soluble (extracellular + intracellular soluble) substrate amount by insoluble substrate amount. Middle, Student's t test used for statistical testing; right, paired Student's t test used for statistical testing (*p ≤ 0.05). Error bars represent SEM. For ATZ, all UGGT1+ bands were darkened with imaging software to make synthesis (data not shown) ± UGGT1 appear equal.
Mentions: To directly investigate the effect of UGGT1 on NHK, ATZ, or wild-type AAT (wt-AAT) solubility, we metabolically labeled Uggt1−/− MEFs (or those complemented with plasmid-encoded UGGT1) expressing these substrates with [35S]methionine/cysteine to approach steady state. By quantitative immunoprecipitation (IP; Supplemental Figure S1A), we determined that over the course of the metabolic labeling period (24 h), a fraction of mutant NHK molecules was secreted; a second portion was recovered in the intracellular detergent-soluble supernatant fraction; and a third was recovered as aggregates that failed to be solubilized from Uggt1−/− MEFs lysed in nonionic detergents (Figure 1A). This “detergent-insoluble fraction” was indeed solubilized in SDS-containing detergent extracts.

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