Metabolic syndrome perturbs deglucosylation and reglucosylation in the glycoprotein folding cycle.

Deglucosylation and reglucosylation of glycoproteins by glucosidase II and uridine diphosphate-glucose: glycoprotein glucosyltransferase 1 (UGGT1), respectively, are important steps in glycoprotein quality control. Misfolded glycoprotein accumulation is associated with endoplasmic reticulum stress and can lead to protein misfolding diseases such as metabolic syndrome. Here, we analyzed the expression and activities of glucosidase II and UGGT1 in rat models of obesity and obese type 2 diabetes, and phenotypes associated with moderate and severe metabolic syndrome, respectively. In obesity, the mRNA and protein levels of glucosidase II and UGGT1 are decreased and their activities are reduced. In obese type 2 diabetes, the mRNA and protein levels of these enzymes are increased, and glucosidase II activity is slightly recovered, although UGGT1 activity is reduced. Our findings suggest that metabolic syndrome affects deglucosylation/reglucosylation enzymes according to disease severity.

Stratified analysis of lectin-like chaperones in the folding disease-related metabolic syndrome rat model.

The metabolic syndrome including obesity and diabetes mellitus is known to be a major health problem worldwide. A recent study reported that obesity causes endoplasmic reticulum (ER) stress and subsequently leads to insulin resistance and type 2 diabetes. However, little is known about the alterations in the components of the calnexin/calreticulin (CNX/CRT) cycle, which promote glycoprotein folding in obese and diabetic conditions. To understand the operating status of the lectin-like chaperones related to the CNX/CRT cycle in the metabolic syndrome, we analyzed the chaperones for the activity, protein expression, and mRNA expression levels using Zucker fatty (ZF) and Zucker diabetic fatty (ZDF) rat models for obesity and diabetes, respectively. We demonstrated that misfolded proteins were gradually increased with progression of the syndrome, obesity to diabetes. The individual chaperone activities of CNX and CRT were both decreased in the ZF rat ER and, in contrast, were increased in the ZDF rat ER. The protein quantities and mRNA expressions of CNX and CRT were decreased in the ZF rats, but increased in the ZDF rats compared with those of the healthy model. Therefore, these results indicate that obesity down-regulates CNX and CRT expressions and their activities and diabetes up-regulates the expressions and activities of CNX and CRT. Our findings clearly suggest that metabolic syndrome affects the lectin-like chaperones in the CNX/CRT cycle at both the activity and expression levels.

Analytical method for determining relative chaperone activity using an ovalbumin-conjugated column.

Investigating the relative efficiencies of molecular chaperones is important for understanding protein biosynthesis inside a cell. We developed an analytical method for estimating relative chaperone activity under physiological, multi-chaperone conditions using a protein-conjugated column. A chaperone mixture was subjected to chromatography on a column conjugated with denatured ovalbumin, and the elution positions of target chaperones were compared using western blotting to determine the relative affinity of each chaperone for the denatured protein. Because molecular chaperones should be eluted according to their strength of association with the denatured ovalbumin in the column, the elution position must accord with the chaperone activity and can be used as an indicator of relative chaperone activity. We found that the column procedure was effective in an assay of a mixture of calreticulin and BiP, the molecular chaperones in the endoplasmic reticulum; the assay showed that calreticulin associated with denatured ovalbumin more strongly than BiP.