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1.
J Biol Chem ; 288(23): 16460-16475, 2013 Jun 07.
Article in English | MEDLINE | ID: mdl-23609449

ABSTRACT

Here we report for the first time the three-dimensional structure of a mannose 6-phosphate receptor homology (MRH) domain present in a protein with enzymatic activity, glucosidase II (GII). GII is involved in glycoprotein folding in the endoplasmic reticulum. GII removes the two innermost glucose residues from the Glc3Man9GlcNAc2 transferred to nascent proteins and the glucose added by UDP-Glc:glycoprotein glucosyltransferase. GII is composed of a catalytic GIIα subunit and a regulatory GIIß subunit. GIIß participates in the endoplasmic reticulum localization of GIIα and mediates in vivo enhancement of N-glycan trimming by GII through its C-terminal MRH domain. We determined the structure of a functional GIIß MRH domain by NMR spectroscopy. It adopts a ß-barrel fold similar to that of other MRH domains, but its binding pocket is the most shallow known to date as it accommodates a single mannose residue. In addition, we identified a conserved residue outside the binding pocket (Trp-409) present in GIIß but not in other MRHs that influences GII glucose trimming activity.


Subject(s)
Endoplasmic Reticulum , Glycoproteins , Protein Folding , Schizosaccharomyces pombe Proteins , Schizosaccharomyces/enzymology , alpha-Glucosidases , Crystallography, X-Ray , Endoplasmic Reticulum/chemistry , Endoplasmic Reticulum/genetics , Endoplasmic Reticulum/metabolism , Glycoproteins/chemistry , Glycoproteins/genetics , Glycoproteins/metabolism , Mannose/chemistry , Mannose/genetics , Mannose/metabolism , Protein Structure, Secondary , Protein Structure, Tertiary , Schizosaccharomyces/genetics , Schizosaccharomyces pombe Proteins/chemistry , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces pombe Proteins/metabolism , alpha-Glucosidases/chemistry , alpha-Glucosidases/genetics , alpha-Glucosidases/metabolism
2.
Mol Biol Cell ; 22(11): 1810-23, 2011 Jun 01.
Article in English | MEDLINE | ID: mdl-21471007

ABSTRACT

Glucosidase II (GII) sequentially removes the two innermost glucose residues from the glycan (Glc(3)Man(9)GlcNAc(2)) transferred to proteins. GII also participates in cycles involving the lectin/chaperones calnexin (CNX) and calreticulin (CRT) as it removes the single glucose unit added to folding intermediates and misfolded glycoproteins by the UDP-Glc:glycoprotein glucosyltransferase (UGGT). GII is a heterodimer in which the α subunit (GIIα) bears the active site, and the ß subunit (GIIß) modulates GIIα activity through its C-terminal mannose 6-phosphate receptor homologous (MRH) domain. Here we report that, as already described in cell-free assays, in live Schizosaccharomyces pombe cells a decrease in the number of mannoses in the glycan results in decreased GII activity. Contrary to previously reported cell-free experiments, however, no such effect was observed in vivo for UGGT. We propose that endoplasmic reticulum α-mannosidase-mediated N-glycan demannosylation of misfolded/slow-folding glycoproteins may favor their interaction with the lectin/chaperone CNX present in S. pombe by prolonging the half-lives of the monoglucosylated glycans (S. pombe lacks CRT). Moreover, we show that even N-glycans bearing five mannoses may interact in vivo with the GIIß MRH domain and that the N-terminal GIIß G2B domain is involved in the GIIα-GIIß interaction. Finally, we report that protists that transfer glycans with low mannose content to proteins have nevertheless conserved the possibility of displaying relatively long-lived monoglucosylated glycans by expressing GIIß MRH domains with a higher specificity for glycans with high mannose content.


Subject(s)
Glycoproteins/metabolism , Mannose/metabolism , Schizosaccharomyces/enzymology , alpha-Glucosidases/metabolism , Carbohydrate Sequence , Endoplasmic Reticulum/metabolism , Gene Knockout Techniques , Glucosyltransferases/genetics , Glucosyltransferases/metabolism , Half-Life , Hexosyltransferases/metabolism , Molecular Sequence Data , Polysaccharides/chemistry , Polysaccharides/metabolism , Protein Folding , Protein Interaction Domains and Motifs , Protein Stability , Protein Structure, Tertiary , Schizosaccharomyces/genetics , alpha-Glucosidases/genetics , alpha-Mannosidase
3.
Mol Biol Cell ; 20(17): 3974-84, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19605557

ABSTRACT

Glucosidase II (GII) plays a key role in glycoprotein biogenesis in the endoplasmic reticulum (ER). It is responsible for the sequential removal of the two innermost glucose residues from the glycan (Glc(3)Man(9)GlcNAc(2)) transferred to Asn residues in proteins. GII participates in the calnexin/calreticulin cycle; it removes the single glucose unit added to folding intermediates and misfolded glycoproteins by the UDP-Glc:glycoprotein glucosyltransferase. GII is a heterodimer whose alpha subunit (GIIalpha) bears the glycosyl hydrolase active site, whereas its beta subunit (GIIbeta) role is controversial and has been reported to be involved in GIIalpha ER retention and folding. Here, we report that in the absence of GIIbeta, the catalytic subunit GIIalpha of the fission yeast Schizosaccharomyces pombe (an organism displaying a glycoprotein folding quality control mechanism similar to that occurring in mammalian cells) folds to an active conformation able to hydrolyze p-nitrophenyl alpha-d-glucopyranoside. However, the heterodimer is required to efficiently deglucosylate the physiological substrates Glc(2)Man(9)GlcNAc(2) (G2M9) and Glc(1)Man(9)GlcNAc(2) (G1M9). The interaction of the mannose 6-phosphate receptor homologous domain present in GIIbeta and mannoses in the B and/or C arms of the glycans mediates glycan hydrolysis enhancement. We present evidence that also in mammalian cells GIIbeta modulates G2M9 and G1M9 trimming.


Subject(s)
Polysaccharides/metabolism , Protein Structure, Tertiary , Protein Subunits/chemistry , Protein Subunits/metabolism , Schizosaccharomyces pombe Proteins/chemistry , Schizosaccharomyces pombe Proteins/metabolism , alpha-Glucosidases/chemistry , alpha-Glucosidases/metabolism , Amino Acid Sequence , Animals , Carbohydrate Conformation , Carbohydrate Sequence , Catalytic Domain , Glucosides/metabolism , Isoenzymes/chemistry , Isoenzymes/genetics , Isoenzymes/metabolism , Molecular Sequence Data , Mutagenesis , Protein Folding , Protein Multimerization , Protein Structure, Quaternary , Protein Subunits/genetics , Rats , Receptor, IGF Type 2/genetics , Receptor, IGF Type 2/metabolism , Schizosaccharomyces/enzymology , Schizosaccharomyces pombe Proteins/genetics , Sequence Alignment , alpha-Glucosidases/genetics
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