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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-mediated solubility of NHK requires N-glycan on NHK. (A) Analysis of NHK N-glycosylation mutants in transfected Uggt1−/− MEFs. Uggt1−/− MEFs were transfected with expression vectors encoding NHK N-glycosylation–site mutants with all combinations of the three N-glycosylation sites mutated (Asp(N) to Gln(Q); 1, NHK-N46,83,247QQQ; 2, NHK-N83,247QQ; 3, NHK-N46,247QQ; 4, NHK-N46,83QQ; 5, NHK-N247Q; 6, NHK-N83Q; 7, NHK-N46Q), cotransfected with either empty vector or UGGT1 expression vector, and subjected to steady-state labeling analysis. (B) Quantification of three replicates of experiment in A. A significant portion of the increased solubility of NHK with increasing number of N-glycans added is due to UGGT1 reglucosylation activity. Student's t test was used for statistical testing (*p ≤ 0.05). Asterisks directly above bars indicate a significant difference in total soluble/insoluble ratio between that bar and nonglycosylated NHK (NHKQQQ), either with or without UGGT1 cotransfection. Error bars represent SEM.
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Figure 6: UGGT1-mediated solubility of NHK requires N-glycan on NHK. (A) Analysis of NHK N-glycosylation mutants in transfected Uggt1−/− MEFs. Uggt1−/− MEFs were transfected with expression vectors encoding NHK N-glycosylation–site mutants with all combinations of the three N-glycosylation sites mutated (Asp(N) to Gln(Q); 1, NHK-N46,83,247QQQ; 2, NHK-N83,247QQ; 3, NHK-N46,247QQ; 4, NHK-N46,83QQ; 5, NHK-N247Q; 6, NHK-N83Q; 7, NHK-N46Q), cotransfected with either empty vector or UGGT1 expression vector, and subjected to steady-state labeling analysis. (B) Quantification of three replicates of experiment in A. A significant portion of the increased solubility of NHK with increasing number of N-glycans added is due to UGGT1 reglucosylation activity. Student's t test was used for statistical testing (*p ≤ 0.05). Asterisks directly above bars indicate a significant difference in total soluble/insoluble ratio between that bar and nonglycosylated NHK (NHKQQQ), either with or without UGGT1 cotransfection. Error bars represent SEM.

Mentions: NHK, ATZ, and wt-AAT all have three N-linked glycosylation sites at positions N46, N83, and N247. To investigate the contribution of each of these N-glycans to NHK solubility, we eliminated N-glycosylation at these sites by mutagenizing each Asn acceptor to Gln and prepared all possible combinations of one-site or two-site N-glycosylation mutants. We then measured the total soluble/insoluble ratio for each of the seven constructs with or without UGGT1 complementation (Figure 6, A and B). The majority of unglycosylated NHK (NHK-N46Q_N83Q_N247Q or NHKQQQ) was insoluble, and UGGT1 cotransfection had no effect on the solubility of this construct (Figure 6B, bars 1 and 2). For the NHK constructs with only one N-glycosylation site (NHK-N83Q_N247Q, NHK-N46Q_N247Q, and NHK-N46Q_N83Q), in the absence of UGGT1, solubility did not increase, but solubility was enhanced by expression of UGGT1 (Figure 6B, bars 3–8). The largest UGGT1-mediated solubility increase occurred with the NHK-N46Q_N247Q mutant, perhaps indicating particular importance of the N83 N-glycan (Figure 6B, bars 5 and 6). For the NHK constructs with two N-glycosylation sites (NHK-N247Q, NHK-N83Q, NHK-N46Q), solubility increased both with and without UGGT1, but the increase was greater when UGGT1 was present (Figure 6B, bars 9–14), and each of the double-glycan mutants behaved comparably. Similar to Figure 1A, the fully glycosylated protein was the most soluble NHK construct, but UGGT1 expression conferred additional solubility (Figure 6B, bars 15 and 16). The results indicate that each N-linked glycan contributes to the solubility of NHK, but maximal solubility is achieved only in UGGT1-expressing cells.


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-mediated solubility of NHK requires N-glycan on NHK. (A) Analysis of NHK N-glycosylation mutants in transfected Uggt1−/− MEFs. Uggt1−/− MEFs were transfected with expression vectors encoding NHK N-glycosylation–site mutants with all combinations of the three N-glycosylation sites mutated (Asp(N) to Gln(Q); 1, NHK-N46,83,247QQQ; 2, NHK-N83,247QQ; 3, NHK-N46,247QQ; 4, NHK-N46,83QQ; 5, NHK-N247Q; 6, NHK-N83Q; 7, NHK-N46Q), cotransfected with either empty vector or UGGT1 expression vector, and subjected to steady-state labeling analysis. (B) Quantification of three replicates of experiment in A. A significant portion of the increased solubility of NHK with increasing number of N-glycans added is due to UGGT1 reglucosylation activity. Student's t test was used for statistical testing (*p ≤ 0.05). Asterisks directly above bars indicate a significant difference in total soluble/insoluble ratio between that bar and nonglycosylated NHK (NHKQQQ), either with or without UGGT1 cotransfection. Error bars represent SEM.
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Figure 6: UGGT1-mediated solubility of NHK requires N-glycan on NHK. (A) Analysis of NHK N-glycosylation mutants in transfected Uggt1−/− MEFs. Uggt1−/− MEFs were transfected with expression vectors encoding NHK N-glycosylation–site mutants with all combinations of the three N-glycosylation sites mutated (Asp(N) to Gln(Q); 1, NHK-N46,83,247QQQ; 2, NHK-N83,247QQ; 3, NHK-N46,247QQ; 4, NHK-N46,83QQ; 5, NHK-N247Q; 6, NHK-N83Q; 7, NHK-N46Q), cotransfected with either empty vector or UGGT1 expression vector, and subjected to steady-state labeling analysis. (B) Quantification of three replicates of experiment in A. A significant portion of the increased solubility of NHK with increasing number of N-glycans added is due to UGGT1 reglucosylation activity. Student's t test was used for statistical testing (*p ≤ 0.05). Asterisks directly above bars indicate a significant difference in total soluble/insoluble ratio between that bar and nonglycosylated NHK (NHKQQQ), either with or without UGGT1 cotransfection. Error bars represent SEM.
Mentions: NHK, ATZ, and wt-AAT all have three N-linked glycosylation sites at positions N46, N83, and N247. To investigate the contribution of each of these N-glycans to NHK solubility, we eliminated N-glycosylation at these sites by mutagenizing each Asn acceptor to Gln and prepared all possible combinations of one-site or two-site N-glycosylation mutants. We then measured the total soluble/insoluble ratio for each of the seven constructs with or without UGGT1 complementation (Figure 6, A and B). The majority of unglycosylated NHK (NHK-N46Q_N83Q_N247Q or NHKQQQ) was insoluble, and UGGT1 cotransfection had no effect on the solubility of this construct (Figure 6B, bars 1 and 2). For the NHK constructs with only one N-glycosylation site (NHK-N83Q_N247Q, NHK-N46Q_N247Q, and NHK-N46Q_N83Q), in the absence of UGGT1, solubility did not increase, but solubility was enhanced by expression of UGGT1 (Figure 6B, bars 3–8). The largest UGGT1-mediated solubility increase occurred with the NHK-N46Q_N247Q mutant, perhaps indicating particular importance of the N83 N-glycan (Figure 6B, bars 5 and 6). For the NHK constructs with two N-glycosylation sites (NHK-N247Q, NHK-N83Q, NHK-N46Q), solubility increased both with and without UGGT1, but the increase was greater when UGGT1 was present (Figure 6B, bars 9–14), and each of the double-glycan mutants behaved comparably. Similar to Figure 1A, the fully glycosylated protein was the most soluble NHK construct, but UGGT1 expression conferred additional solubility (Figure 6B, bars 15 and 16). The results indicate that each N-linked glycan contributes to the solubility of NHK, but maximal solubility is achieved only in UGGT1-expressing cells.

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