Limits...
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.

Show MeSH

Related in: MedlinePlus

UGGT1 maintains increased total soluble/insoluble ratio for NHK during 6-h chase after 24-h labeling. (A) The 24-h labeling and chase analysis of NHK. After 24-h labeling, cells were chased with unlabeled media for 3 or 6 h, and then extracellular, soluble, and insoluble fractions were subjected to anti-AAT double IP. The amount of newly synthesized NHK recovered from 20-min pulse labeling was used to quantify NHK synthesis for normalization. (B) Total amount of NHK remaining in each fraction after chase, normalized to synthesis. Error bars represent SEM. (C) Plot of total soluble/insoluble NHK at 0-, 3-, and 6-h chase points. Student's t test was used for statistical testing (*p ≤ 0.05). Error bars represent SEM.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3756913&req=5

Figure 4: UGGT1 maintains increased total soluble/insoluble ratio for NHK during 6-h chase after 24-h labeling. (A) The 24-h labeling and chase analysis of NHK. After 24-h labeling, cells were chased with unlabeled media for 3 or 6 h, and then extracellular, soluble, and insoluble fractions were subjected to anti-AAT double IP. The amount of newly synthesized NHK recovered from 20-min pulse labeling was used to quantify NHK synthesis for normalization. (B) Total amount of NHK remaining in each fraction after chase, normalized to synthesis. Error bars represent SEM. (C) Plot of total soluble/insoluble NHK at 0-, 3-, and 6-h chase points. Student's t test was used for statistical testing (*p ≤ 0.05). Error bars represent SEM.

Mentions: As noted, all newly synthesized NHK, ATZ, and wt-AAT molecules begin in the soluble fraction; thus the accumulation of NHK and ATZ molecules takes time. Although steady-state labeling provides useful conditions for determining the sizes of soluble and insoluble intracellular pools, it does not provide insight into the entry or exit of molecules in these fractions. Therefore, after steady-state labeling, we followed pools of NHK for up to 6 h in the absence of further metabolic labeling. As previously observed (Figure 1A), intracellular NHK solubility was enhanced in UGGT1-expressing cells (Figure 4, A and B). At 3 and 6 h after the steady-state labeling period, the pool of soluble NHK decreased in Uggt1−/− MEFs both without and with UGGT1 complementation. Without UGGT1 complementation, however, some of the molecules formerly in the soluble pool entered and expanded the detergent-insoluble pool (Figure 4, A and B), whereas in the presence of UGGT1, expansion of the detergent-insoluble pool was prevented. Indeed, despite a decrease in soluble NHK as a function of chase time, the relative increase in NHK solubility as a consequence of UGGT1 activity was maintained at all times (Figure 4C). Thus the results suggest that UGGT1 activity limits misfolded glycoprotein entry into detergent-insoluble complexes.


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 maintains increased total soluble/insoluble ratio for NHK during 6-h chase after 24-h labeling. (A) The 24-h labeling and chase analysis of NHK. After 24-h labeling, cells were chased with unlabeled media for 3 or 6 h, and then extracellular, soluble, and insoluble fractions were subjected to anti-AAT double IP. The amount of newly synthesized NHK recovered from 20-min pulse labeling was used to quantify NHK synthesis for normalization. (B) Total amount of NHK remaining in each fraction after chase, normalized to synthesis. Error bars represent SEM. (C) Plot of total soluble/insoluble NHK at 0-, 3-, and 6-h chase points. Student's t test was used for statistical testing (*p ≤ 0.05). Error bars represent SEM.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3756913&req=5

Figure 4: UGGT1 maintains increased total soluble/insoluble ratio for NHK during 6-h chase after 24-h labeling. (A) The 24-h labeling and chase analysis of NHK. After 24-h labeling, cells were chased with unlabeled media for 3 or 6 h, and then extracellular, soluble, and insoluble fractions were subjected to anti-AAT double IP. The amount of newly synthesized NHK recovered from 20-min pulse labeling was used to quantify NHK synthesis for normalization. (B) Total amount of NHK remaining in each fraction after chase, normalized to synthesis. Error bars represent SEM. (C) Plot of total soluble/insoluble NHK at 0-, 3-, and 6-h chase points. Student's t test was used for statistical testing (*p ≤ 0.05). Error bars represent SEM.
Mentions: As noted, all newly synthesized NHK, ATZ, and wt-AAT molecules begin in the soluble fraction; thus the accumulation of NHK and ATZ molecules takes time. Although steady-state labeling provides useful conditions for determining the sizes of soluble and insoluble intracellular pools, it does not provide insight into the entry or exit of molecules in these fractions. Therefore, after steady-state labeling, we followed pools of NHK for up to 6 h in the absence of further metabolic labeling. As previously observed (Figure 1A), intracellular NHK solubility was enhanced in UGGT1-expressing cells (Figure 4, A and B). At 3 and 6 h after the steady-state labeling period, the pool of soluble NHK decreased in Uggt1−/− MEFs both without and with UGGT1 complementation. Without UGGT1 complementation, however, some of the molecules formerly in the soluble pool entered and expanded the detergent-insoluble pool (Figure 4, A and B), whereas in the presence of UGGT1, expansion of the detergent-insoluble pool was prevented. Indeed, despite a decrease in soluble NHK as a function of chase time, the relative increase in NHK solubility as a consequence of UGGT1 activity was maintained at all times (Figure 4C). Thus the results suggest that UGGT1 activity limits misfolded glycoprotein entry into detergent-insoluble complexes.

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