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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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Related in: MedlinePlus

Substrate specificity of G-II. (A) G-II (cleavage-2) activity against various glycoprobes having different oligosaccharide or aglycone structure. (B) Inhibitory activity of various glycoprobes toward Glc trimming from G2M9. (C) Cleavage of various G1M9 analogues by G-II. Gal, Galactose; GlcA, Glucuronic acid; G3d, 3-Deoxyglucose; G4d, 4-Deoxyglucose; G6d, 6-Deoxyglucose.
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fig05: Substrate specificity of G-II. (A) G-II (cleavage-2) activity against various glycoprobes having different oligosaccharide or aglycone structure. (B) Inhibitory activity of various glycoprobes toward Glc trimming from G2M9. (C) Cleavage of various G1M9 analogues by G-II. Gal, Galactose; GlcA, Glucuronic acid; G3d, 3-Deoxyglucose; G4d, 4-Deoxyglucose; G6d, 6-Deoxyglucose.

Mentions: In the ER, combined action of UGGT and G-II (cleavage-2) interconverts M9 and G1M9 glycoforms. Simultaneously, trimming by mannosidase(s) occurs, giving G0 and G1 glycans that have less than 9 Man residues. Studies in 1980s concluded that G-II cleaves G1M9 exceedingly faster than other monoglucosylated glycoforms.21,46,47) However, our study using structurally defined substrates (CHO-MTX; CHO = G1M9-7) revealed that the difference of reactivity was not as large as has been believed. Most noticeably, the reactivity of G1M8B-MTX which lacks a Man residue of the B-arm, was nearly identical with G1M9-MTX, while reaction rate of its regioisomer (G1M8C-MTX) was reduced to ca. 50%. (Fig. 5A) Interestingly, when conjugated to DHFR, the activity of G1M8B was markedly higher than G1M9. These results implicate that the specificity of G-II is sensitive to nature of aglycon, and possibly, to folding state of proteins.


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

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

Substrate specificity of G-II. (A) G-II (cleavage-2) activity against various glycoprobes having different oligosaccharide or aglycone structure. (B) Inhibitory activity of various glycoprobes toward Glc trimming from G2M9. (C) Cleavage of various G1M9 analogues by G-II. Gal, Galactose; GlcA, Glucuronic acid; G3d, 3-Deoxyglucose; G4d, 4-Deoxyglucose; G6d, 6-Deoxyglucose.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig05: Substrate specificity of G-II. (A) G-II (cleavage-2) activity against various glycoprobes having different oligosaccharide or aglycone structure. (B) Inhibitory activity of various glycoprobes toward Glc trimming from G2M9. (C) Cleavage of various G1M9 analogues by G-II. Gal, Galactose; GlcA, Glucuronic acid; G3d, 3-Deoxyglucose; G4d, 4-Deoxyglucose; G6d, 6-Deoxyglucose.
Mentions: In the ER, combined action of UGGT and G-II (cleavage-2) interconverts M9 and G1M9 glycoforms. Simultaneously, trimming by mannosidase(s) occurs, giving G0 and G1 glycans that have less than 9 Man residues. Studies in 1980s concluded that G-II cleaves G1M9 exceedingly faster than other monoglucosylated glycoforms.21,46,47) However, our study using structurally defined substrates (CHO-MTX; CHO = G1M9-7) revealed that the difference of reactivity was not as large as has been believed. Most noticeably, the reactivity of G1M8B-MTX which lacks a Man residue of the B-arm, was nearly identical with G1M9-MTX, while reaction rate of its regioisomer (G1M8C-MTX) was reduced to ca. 50%. (Fig. 5A) Interestingly, when conjugated to DHFR, the activity of G1M8B was markedly higher than G1M9. These results implicate that the specificity of G-II is sensitive to nature of aglycon, and possibly, to folding state of proteins.

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