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Analysis of glycoprotein processing in the endoplasmic reticulum using synthetic oligosaccharides.

Ito Y, Takeda Y - Proc. Jpn. Acad., Ser. B, Phys. Biol. Sci. (2012)

Bottom Line: Recent studies have revealed that high-mannose-type glycans play a pivotal role in the QC process.To gain knowledge about the molecular basis of this process with well-defined homogeneous compounds, we achieved a convergent synthesis of high-mannose-type glycans and their functionalized derivatives.We focused on analyses of UDP-Glc: glycoprotein glucosyltransferase (UGGT) and ER Glucosidase II, which play crucial roles in glycoprotein QC; however, their specificities remain unclear.

View Article: PubMed Central - PubMed

Affiliation: RIKEN Advanced Science Institute, Saitama, Japan. yukito@riken.jp

ABSTRACT
Protein quality control (QC) in the endoplasmic reticulum (ER) comprises many steps, including folding and transport of nascent proteins as well as degradation of misfolded proteins. Recent studies have revealed that high-mannose-type glycans play a pivotal role in the QC process. To gain knowledge about the molecular basis of this process with well-defined homogeneous compounds, we achieved a convergent synthesis of high-mannose-type glycans and their functionalized derivatives. We focused on analyses of UDP-Glc: glycoprotein glucosyltransferase (UGGT) and ER Glucosidase II, which play crucial roles in glycoprotein QC; however, their specificities remain unclear. In addition, we established an in vitro assay system mimicking the in vivo condition which is highly crowded because of the presence of various biomacromolecules.

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G-II activity toward G2M9-MTX using the membranous fraction of gene disruptants lacking either the G-II α-subunit or β-subunit. The membranous fraction lacking the β-subunit was inactive against high mannose-type oligosaccharide.
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fig06: G-II activity toward G2M9-MTX using the membranous fraction of gene disruptants lacking either the G-II α-subunit or β-subunit. The membranous fraction lacking the β-subunit was inactive against high mannose-type oligosaccharide.

Mentions: G-II is a heterodimeric protein, consisting of α- and β-subunit. While the α-subunit comprises a catalytic domain, precise function of the other subunit has been obscure.50) To clarify the issue, we prepared strains of Aspergillus oryzae in which either α- or β-subunit was disrupted, and G-II activity of their microsomal fraction was tested, by using G2M9- and G1M9-MTX. Our analysis showed 1) not only the α-subunit disruptant, but the β-subunit disruptant was inactive toward all of these glycans, although the former (but not the latter) was fully active toward a small molecule substrate pNP-Glc, and 2) mixed microsomal fractions of both disruptans exhibited the activity to digest both G2M9 and G1M9.51) Together, these results provide a clear indication that the presence of the β-subunit is essential in order for G-II to exhibit hydrolytic activity in the ER, possibly by virtue of its ability to recognize high-mannose-type glycans. (Fig. 6)


Analysis of glycoprotein processing in the endoplasmic reticulum using synthetic oligosaccharides.

Ito Y, Takeda Y - Proc. Jpn. Acad., Ser. B, Phys. Biol. Sci. (2012)

G-II activity toward G2M9-MTX using the membranous fraction of gene disruptants lacking either the G-II α-subunit or β-subunit. The membranous fraction lacking the β-subunit was inactive against high mannose-type oligosaccharide.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3316936&req=5

fig06: G-II activity toward G2M9-MTX using the membranous fraction of gene disruptants lacking either the G-II α-subunit or β-subunit. The membranous fraction lacking the β-subunit was inactive against high mannose-type oligosaccharide.
Mentions: G-II is a heterodimeric protein, consisting of α- and β-subunit. While the α-subunit comprises a catalytic domain, precise function of the other subunit has been obscure.50) To clarify the issue, we prepared strains of Aspergillus oryzae in which either α- or β-subunit was disrupted, and G-II activity of their microsomal fraction was tested, by using G2M9- and G1M9-MTX. Our analysis showed 1) not only the α-subunit disruptant, but the β-subunit disruptant was inactive toward all of these glycans, although the former (but not the latter) was fully active toward a small molecule substrate pNP-Glc, and 2) mixed microsomal fractions of both disruptans exhibited the activity to digest both G2M9 and G1M9.51) Together, these results provide a clear indication that the presence of the β-subunit is essential in order for G-II to exhibit hydrolytic activity in the ER, possibly by virtue of its ability to recognize high-mannose-type glycans. (Fig. 6)

Bottom Line: Recent studies have revealed that high-mannose-type glycans play a pivotal role in the QC process.To gain knowledge about the molecular basis of this process with well-defined homogeneous compounds, we achieved a convergent synthesis of high-mannose-type glycans and their functionalized derivatives.We focused on analyses of UDP-Glc: glycoprotein glucosyltransferase (UGGT) and ER Glucosidase II, which play crucial roles in glycoprotein QC; however, their specificities remain unclear.

View Article: PubMed Central - PubMed

Affiliation: RIKEN Advanced Science Institute, Saitama, Japan. yukito@riken.jp

ABSTRACT
Protein quality control (QC) in the endoplasmic reticulum (ER) comprises many steps, including folding and transport of nascent proteins as well as degradation of misfolded proteins. Recent studies have revealed that high-mannose-type glycans play a pivotal role in the QC process. To gain knowledge about the molecular basis of this process with well-defined homogeneous compounds, we achieved a convergent synthesis of high-mannose-type glycans and their functionalized derivatives. We focused on analyses of UDP-Glc: glycoprotein glucosyltransferase (UGGT) and ER Glucosidase II, which play crucial roles in glycoprotein QC; however, their specificities remain unclear. In addition, we established an in vitro assay system mimicking the in vivo condition which is highly crowded because of the presence of various biomacromolecules.

Show MeSH
Related in: MedlinePlus