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 ␣ subunit (GII␣) bears the glycosyl hydrolase active site, whereas its ␤ subunit (GII␤) role is controversial and has been reported to be involved in GII␣ ER retention and folding. Here, we report that in the absence of GII␤, the catalytic subunit GII␣ 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 ␣-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 GII␤ and mannoses in the B and/or C arms of the glycans mediates glycan hydrolysis enhancement. We present evidence that also in mammalian cells GII␤ modulates G2M9 and G1M9 trimming.

Glypican-3 (GPC3) is a proteoglycan involved in migration, proliferation and cell survival modulation in several tissues. There are many reports demonstrating a downregulation of GPC3 expression in some human tumors, including mesothelioma, ovarian and breast cancer. Previously, we determined that GPC3 reexpression in the murine mammary adenocarcinoma LM3 cells induced an impairment of their in vivo invasive and metastatic capacities together with a higher susceptibility to in vitro apoptosis. Currently, the signaling mechanism of GPC3 is not clear. First, it was speculated that GPC3 regulates the insulin-like growth factor (IGF) signaling system. This hypothesis, however, has been strongly challenged. Recently, several reports indicated that at least in some cell types GPC3 serves as a selective regulator of Wnt signaling. Here we provide new data demonstrating that GPC3 regulates Wnt pathway in the metastatic adenocarcinoma mammary LM3 cell line. We found that GPC3 is able to inhibit canonical Wnt signals involved in cell proliferation and survival, as well as it is able to activate non canonical pathway, which directs cell morphology and migration. This is the first report indicating that breast tumor cell malignant properties can be reverted, at least in part, by GPC3 modulation of Wnt signaling. Our results are consistent with the potential role of GPC3 as a metastasis suppressor.

Background: Glucosidase II is an endoplasmic reticulum enzyme involved in quality control of glycoprotein folding. Results: The structure of the lectin domain of GII was determined by NMR spectroscopy. Conclusion: GII lectin domain structure contains a unique Trp residue critical for GII activity. Significance: GII␤ MRH domain structure is the first determined of an MRH domain present in a protein with enzymatic activity.

A decrease in N-glycan mannose content significantly diminishes in vivo glucosidase II–mediated deglucosylation rates but does not affect in vivo UDP-glucose:glycoprotein glucosyltransferase–mediated glucosylation, thus increasing the possibility of displaying monoglucosylated structures able to interact with calnexin/calreticulin for longer time periods.