The N-glycan-dependent quality control of glycoprotein folding prevents endoplasmic to Golgi exit of folding intermediates, irreparably misfolded glycoproteins and incompletely assembled multimeric complexes. It also enhances folding efficiency by preventing aggregation and facilitating formation of proper disulfide bonds. The control mechanism essentially involves four components, resident lectin-chaperones that recognize monoglucosylated polymannose glycans, a lectin-associated oxidoreductase acting on monoglucosylated glycoproteins, a glucosyltransferase that creates monoglucosytlated epitopes in protein-linked glycans and a glucosidase that removes the glucose units added by the glucosyltransferase. This last enzyme is the only mechanism component sensing glycoprotein conformations as it creates monoglucosylated glycans exclusively in not properly folded species or in not completely assembled complexes. The glucosidase is a dimeric heterodimer composed of a catalytic subunit and an additional one that is partially responsible for the ER localization of the enzyme and for the enhancement of the deglucosylation rate as its mannose 6-phosphate receptor homologous domain presents the substrate to the catalytic site. This review deals with our present knowledge on the glucosyltransferase and the glucosidase.

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.

The yeast Pichia pastoris is a cost-effective and easily scalable system for recombinant protein production. In this work we compared the conformation of the receptor binding domain (RBD) from severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) Spike protein expressed in P. pastoris and in the well established HEK-293T mammalian cell system. RBD obtained from both yeast and mammalian cells was properly folded, as indicated by UV-absorption, circular dichroism and tryptophan fluorescence. They also had similar stability, as indicated by temperature-induced unfolding (observed Tm were 50 °C and 52 °C for RBD produced in P. pastoris and HEK-293T cells, respectively). Moreover, the stability of both variants was similarly reduced when the ionic strength was increased, in agreement with a computational analysis predicting that a set of ionic interactions may stabilize RBD structure. Further characterization by high-performance liquid chromatography, size-exclusion chromatography and mass spectrometry revealed a higher heterogeneity of RBD expressed in P. pastoris relative to that produced in HEK-293T cells, which disappeared after enzymatic removal of glycans. The production of RBD in P. pastoris was scaled-up in a bioreactor, with yields above 45 mg/L of 90% pure protein, thus potentially allowing large scale immunizations to produce neutralizing antibodies, as well as the large scale production of serological tests for SARS-CoV-2.