Calnexin/Calreticulin and Assays Related to N-Glycoprotein Folding In Vitro.

Calnexin (CNX) and calreticulin (CRT) are ER-resident lectin-like molecular chaperones involved in the quality control of secretory or membrane glycoproteins. They can exert molecular chaperone functions via specific binding to the early processing intermediates of Glc1Man9GlcNAc2 oligosaccharides of N-glycoproteins. CNX and CRT have similar N-terminal luminal domains and share the same jelly roll tertiary structure as legume lectins. In addition to the lectin-like interactions, CNX and CRT also suppress the aggregation of non-glycosylated substrates through interaction with hydrophobic peptide parts, suggesting a general chaperone function in glycan-dependent and glycan-independent manners. This chapter describes the isolation and purification of CRT produced in a bacterial expression system. We also introduce in vitro assays to estimate the molecular chaperone functions of CRT via the interaction with monoglucosylated N-glycans using Jack bean α-mannosidase as a target substrate. These assays are valuable in assessing quality control events related to the CNX/CRT chaperone cycle and lectin functions.

Calreticulin protects insulin against reductive stress in vitro and in MIN6 cells.

Oxidative folding of proinsulin in the endoplasmic reticulum (ER) is critical for the proper sorting and secretion of insulin from pancreatic ß-cells. Here, by using non-cell-based insulin aggregation assays and mouse insulinoma-derived MIN6 cells, we searched for a candidate molecular chaperone for (pro)insulin when its oxidative folding is compromised. We found that interaction between insulin and calreticulin (CRT), a lectin that acts as an ER-resident chaperone, was enhanced by reductive stress in MIN6 cells. Co-incubation of insulin with recombinant CRT prevented reductant-induced aggregation of insulin. Furthermore, lysosomal degradation of proinsulin, which was facilitated by dithiothreitol-induced reductive stress, depended on CRT in MIN6 cells. Together, our results suggest that CRT may be a protective molecule against (pro)insulin aggregation when oxidative folding is defective, e.g. under reductive stress conditions, in vitro and in cultured cells. Because CRT acts as a molecular chaperone for not only glycosylated proteins but also non-glycosylated polypeptides, we also propose that (pro)insulin is a novel candidate client of the chaperone function of CRT.

Top-down chemoenzymatic approach to a high-mannose-type glycan library: synthesis of a common precursor and its enzymatic trimming.

From the stacks: A novel method for construction of a high-mannose-type glycan library by systematic enzymatic trimming of a single synthetic Man9-based precursor was developed. Efficient chemical synthesis of the tetradecasaccharide common precursor and orthogonal enzymatic trimming to obtain all M(8-9) and G(1)M(8-9) derivatives was demonstrated. G = glucose, M = mannose.

Analyses of carbohydrate binding property of lectin-chaperone calreticulin.

Calreticulin (CRT) is a soluble molecular chaperone of the endoplasmic reticulum. It is a lectin that promotes the folding of proteins carrying N-linked glycans. Recent investigations have revealed that glucosylated high-mannose-type glycans are employed as key elements in this process. Here, we performed quantitative analyses of the interaction of CRT with various disaccharides, including fluorine-substituted analogues using a quartz-crystal microbalance (QCM). These experiments revealed the weak affinity of 2- and 3-fluoroglucose derivatives. On the other hand, 6-fluoroglucose derivatives exhibited a significant affinity, indicating that the role of 6-position of OH is less significant for binding to CRT. We also characterized binding epitope of the Glcalpha1-3Man(alpha)Me to CRT by saturation transfer difference (STD) NMR spectroscopy. It is proposed that 2-, 3-, and 4-positions of Glc and 3-, 4-, and 6-positions of Man are in close contact with CRT binding pocket, while 6-position of Glc and 2-position of Man are not. These finding are in excellent agreement with our QCM experiment.

Facile synthesis of oligosaccharide probes for the analysis of protein-carbohydrate interactions.

A strategy for facile oligosaccharide synthesis is described. It obviates chromatographic separation of intermediates and enables the isolation of desired oligomers with capture-release purification and reverse-phase silica-gel cartridge separation. As an example, preparation of monoglucosylated mannotetraose (Glc alpha1-->3Man alpha1-->2Man1alpha-->2Man1alpha-->3Man beta) was conducted. After sequential glycosylation, capture-release purification with Cys-resin, global deprotection, and reverse-phase silica-gel cartridge separation, the target pentasaccharide was isolated, while isolation of shorter oligomers that lack nonreducing-end residues (Man alpha1-->2Man1alpha-->2Man1alpha-->3Man beta, Man1alpha-->2Man1alpha-->3Man beta, Man1alpha-->3Man beta) was also achieved. These products were connected to a thiol-containing linker and immobilized on Au-coated chips. Their affinity to recombinant calreticulin was evaluated by quartz-crystal microbalance.

Synthesis of fluorine substituted oligosaccharide analogues of monoglucosylated glycan chain, a proposed ligand of lectin-chaperone calreticulin and calnexin.

As a part of a exploring the N-glycan-mediated glycoprotein quality control in endoplasmic reticulum, 2-fluorinated derivative Glcalpha1 --> 3Man(F) 1, Glcalpha1 --> 3Man(F)alpha1 --> 2Man2, and Glcalpha1 --> 3Man(F)alpha1 --> 2Manalpha1 --> 2Man 3 were synthesized in a concise manner. These oligosaccharides were subjected to binding studies with calreticulin by using isothermal titration calorimetry. It was revealed that disaccharide 1 was a poor ligand, while tri- (2) and tetrasaccharide (3) had observable affinity.

Pentafluoropropionyl and trifluoroacetyl groups for temporary hydroxyl group protection in oligomannoside synthesis.

Pentafluoropropionyl (PFP) and trifluoroacetyl (TFA) esters were demonstrated to be useful in facile oligosaccharide synthesis. These were well compatible with glycosylation conditions and removable by treatment with pyridine-EtOH, with complete preservation of acetyl groups. Analytically pure products were obtained quantitatively, simply by evaporating the reaction mixtures. Using O-PFP and O-TFA carrying glycosyl halides, trisaccharide (Manalpha1-->2Manalpha1-->2Man) and tetrasaccharide (Glcalpha1-->3Manalpha1-->2Manalpha1-->2Man) portions of monoglucosylated high-mannose type dodecasaccharide (Glc(1)Man(9)GlcNAc(2)), a putative ligand for the ER chaperon, calnexin and calreticulin, were synthesized.