Functional analysis of endoplasmic reticulum glucosyltransferase (UGGT): Synthetic chemistry's initiative in glycobiology.

UGGT1 is called as a folding sensor protein that recognizes misfolded glycoproteins and selectively glucosylates high-mannose-type glycans on the proteins. However, conventional approaches using naturally occurring glycoproteins is not optimum in performing precise analysis of the unique properties of UGGT1. We have demonstrated that high-mannose-type glycans, in which various hydrophobic aglycons were introduced, act as good substrates for UGGT1 and are useful analytical tools for its characterization. Moreover, we found that UGGT2, an isoform UGGT1, is also capable of glucosylating these synthetic substrates. Our strategy stemmed on synthetic chemistry has been further strengthened by total synthesis of homogeneous glycoproteins in correctly folded as well as in intentionally misfolded forms.

The 811 C/T polymorphism in the 3' untranslated region of the selenoprotein 15-kDa (Sep15) gene and breast cancer in Caucasian women.

The 15-kDa selenoprotein (Sep15) is a selenocysteine-containing oxidoreductase in the endoplasmic reticulum that participates in disulfide-bond formation and protein folding control. The 3'-untranslated region (3'-UTR) contains two exclusively linked, polymorphic sites at positions 811 (C/T) and 1125 (G/A), which result in two functional haplotypes: 811C/1125G or 811T/1125A. The 811T/1125A variant occurs significantly more often in African-Americans as compared to Caucasians and has been linked to increased breast cancer risk in black women. We studied the 811C/T (rs5845) Sep15 gene polymorphism in 182 Caucasian women-83 breast cancer cases and 99 healthy controls-by pyrosequencing and polymerase chain reaction. Associations between allelic variants and clinico-pathological variables (e.g., age, stage of disease, tumor type, grading, and receptor status) were investigated. The genotype distribution in breast cancer patients (CC 63.9 %, CT 33.7 %, TT 2.4 %) and controls (69.7 %, CT 28.3 %, TT 2 %) showed no significant difference (OR 0.77, 95 % CI 0.41-1.42, p = 0.4). The overall low prevalence of the T allele was in accordance with that reported for Caucasians in previous studies. There was no significant association between 811C/T Sep15 polymorphism and any of clinico-pathological parameters. In conclusion, we are the first to report on 811C/T SEP 15 polymorphism in white breast cancer patients. Genotype variation within the 3'-UTR of the SEP 15 gene showed no association with breast cancer risk or clinico-pathological parameters in Caucasian women.

Knockdown of 15-kDa selenoprotein (Sep15) increases hLE cells' susceptibility to tunicamycin-induced apoptosis.

In this work, we investigated the effect of Sep15 gene knockdown on apoptosis in human lens epithelial (hLE) cells, trying to understand the relevance of Sep15 to cataract formation in the Sep15 knockout (KO) mice. The results showed that sole knockdown of Sep15 by RNA interference did not result in apoptosis; however, reduction of Sep15 expression aggravated tunicamycin (Tm)-induced cell apoptosis and caspases activation. Furthermore, Tm-induced mitochondrial dysfunction was also exacerbated under the Sep15 knockdown condition by measurement of mitochondrial membrane potential decrease and human cytochrome c release into cytosol. Interestingly, the knockdown of Sep15 exacerbated Tm-induced oxidative stress while endoplasmic reticulum (ER) stress was not correspondingly elevated. These results suggest that the protective role of Sep15 against Tm-induced apoptosis in hLE cells is operated via inhibiting oxidative stress rather than regulating Tm-induced ER stress, and the protective role becomes dependent on Sep15 only in acute stress condition.

The study on polymorphisms of Sep15 and TrxR2 and the expression of AP-1 signaling pathway in Kashin-Beck disease.

The aim of the study was to investigate the association between rs5859 in Sep15, rs1139793 in TrxR2 polymorphisms with the risks of KBD and to detect the expression of AP-1 pathway in KBD subjects and in vitro. 208 KBD and 206 control subjects were included. PCR-Restriction Fragment Length Polymorphism (RFLP), Amplification Refractory Mutation Specific-PCR (ARMS-PCR) and Western Blotting were conducted. The results showed the minor A-allele frequency of rs5859 in KBD was statistically significantly higher than that in the control group (P < 0.05). The cases carrying A-allele had a 2-fold (95%CI: 1.064-3.956) increased risk of developing KBD compared with the G-allele carriers. There was no significant difference in genotype and allele distribution of rs1139793 between KBD patients and controls (P > 0.05). The frequency of the minor A allele of rs5859 was significantly different in Chinese healthy population compared with European, African and American. The frequency of the minor A allele of rs1139793 showed significant difference when compared with African and American. The levels of JunB, JunD, P65 proteins in KBD group were higher than those in control group (P < 0.0001). The expression of JunB, JunD, P65 proteins all increased in tBHP-induced C28/I2 oxidative damage model compared with control group (P < 0.05) and decreased after Se supplementation. Our finding indicated Sep15 is a possible candidate susceptibility gene for KBD. Combined with the in vitro study, our studies reveal novel insights into the mechanism of Se supplementation as an antioxidant via inhibiting the AP-1 signaling pathway in patients with KBD.

Role of Selenof as a Gatekeeper of Secreted Disulfide-Rich Glycoproteins.

Selenof (15-kDa selenoprotein; Sep15) is an endoplasmic reticulum (ER)-resident thioredoxin-like oxidoreductase that occurs in a complex with UDP-glucose:glycoprotein glucosyltransferase. We found that Selenof deficiency in mice leads to elevated levels of non-functional circulating plasma immunoglobulins and increased secretion of IgM during in vitro splenic B cell differentiation. However, Selenof knockout animals show neither enhanced bacterial killing capacity nor antigen-induced systemic IgM activity, suggesting that excess immunoglobulins are not functional. In addition, ER-to-Golgi transport of a target glycoprotein was delayed in Selenof knockout embryonic fibroblasts, and proteomic analyses revealed that Selenof deficiency is primarily associated with antigen presentation and ER-to-Golgi transport. Together, the data suggest that Selenof functions as a gatekeeper of immunoglobulins and, likely, other client proteins that exit the ER, thereby supporting redox quality control of these proteins.

SLC30A3 and SEP15 gene polymorphisms influence the serum concentrations of zinc and selenium in mature adults.

Because of their numerous roles in several biological processes, zinc and selenium are the most commonly studied micronutrients in the elderly. Therefore, we hypothesized that the polymorphisms in the genes that are responsible for the transport of zinc and selenium may have a genotype-dependent effect on the serum concentration of these micronutrients. The objective of this study was to determine the effects of solute carrier family 30 member 3 (SLC30A3) and 15-kd selenoprotein (SEP15) polymorphisms on zinc and selenium concentrations, respectively, in the serum. This cross-sectional study included 110 individuals who were aged 50 years or older. Serum micronutrient concentrations were determined by flame atomic absorption spectrophotometry (for zinc) and by atomic absorption spectrophotometry with a graphite furnace (for selenium). The single-nucleotide polymorphisms, rs73924411 and rs11126936 of the SLC30A3 gene and rs5859, rs5854, and rs561104 of the SEP15 gene, were examined by real-time polymerase chain reaction. Regarding rs11126936, the serum zinc concentration was lower in CC homozygotes (0.75 ± 0.31 mg/L) than in A carriers (0.89 ± 0.28 mg/L, P = .016). Concerning rs561104, the serum selenium concentration was higher in CC homozygotes (5.65 ± 1.11 µg/dL) compared with T carriers (4.88 ± 1.25 µg/dL, P = .044). Our results demonstrate the influence of SLC30A3 and SEP15 gene polymorphisms on the serum concentrations of zinc and selenium, respectively. The effects of these associations should be further investigated to help elucidate the modes of action of trace elements and to identify biomarkers, which could ultimately define the optimal intake of these micronutrients at the molecular level. More research must be performed before the roles of these polymorphisms in the serum concentrations of zinc and selenium can be fully understood.