Role of Glucosidase II beta subunit in N-glycan trimming

NIEMEBOKA, B.; STIGLIANO, I.D.; PARODI, A.J.; D'ALESSIO, C.

Phoenix, Arizona,USA.

Congreso; Annual Biomedical Research Conference for Minority Students (ABRCMS); 2009

American Society for Microbiology

The endoplasmic reticulum (ER) of eukaryotic cells discriminates between native and non-native glycoprotein conformations, selectively transporting properly folded ones to their final destinations through the secretory pathway or alternatively, retrotranslocating misfolded ones to the cytosol to be degraded by proteasomes. During N-glycosylation, a glycan (Glc3Man9GlcNAc2) is transferred to proteins that are being translocated into the ER. The outermost Glc from this glycan is then removed by the Glucosidase I (GI) enzyme. The Glucosidase II (GII) enzyme successively removes the middle and innermost Glc. Both of these GII-mediated cleavages play a crucial role in the quality control of glycoprotein folding within the ER. Monoglucosylated glycoproteins generated by GII first cleavage interact with chaperones calnexin or calreticulin to enhance folding efficiency and to prevent the exit of the incompletely folded proteins from the ER. The GII-mediated cleavage of the innermost Glc prevents this interaction and allows glycoproteins to continue through the secretory pathway. However, if the protein is not properly folded, a Glc moiety is added by the UDP-Glc:glycoprotein glucosyltransferase. The glycoproteins cycle between a monoglucosylated and a non-glucosylated conformation until proper folding is achieved. GII has two subunits, alpha and beta (GIIalpha and GIIbeta). We already determined that the GIIalpha has the catalytic site while GIIbeta retains GIIalpha in the ER and modulates the trimming of the oligosaccharides by GII. We hypothesize that GIIbeta is responsible for the presentation of the glycan to be cleaved by GIIalpha. To analyze in vitro if GIIbeta acts as a lectin -i.e. if it directly binds to glycans- we obtained the cDNA coding Schizosaccharomyces pombe GIIbeta. We performed a reverse transcriptase reaction from total RNA of these yeast cells, and cloned the product into a bacterial expression vector fused to a His6 tag, to induce its expression and to affinity purify the protein. Only mutated clones of GIIbeta could be expressed in E. coli, suggesting toxicity due to protein over expression. Since the entire wild type GIIbeta sequence could not be expressed, we expressed and purify an 18 KDa fragment of GIIbeta. We then prepared anti GIIbetab antibodies by injecting mice three times with 15 mg of the 18 KDa protein and obtaining sera. In the future, we will purify endogenous S. pombe GIIbeta using the produced antibodies and analyze its binding to radioactive oligosaccharides.