Abstract
Calreticulin was identified by immunochemical and sequence analyses to be the higher molecular mass (60 kDa) component of the polypeptide doublet previously observed in a rat liver Golgi endomannosidase preparation obtained by chromatography on a Glc alpha 1 --> 3Man-containing matrix. The affinity for this saccharide ligand, which paralleled that of endomannosidase and was also observed with purified rat liver calreticulin, suggested that this chaperone has lectin-like binding properties. Studies carried out with immobilized calreticulin and a series of radiolabeled oligosaccharides derived from N-linked carbohydrate units revealed that interactions with this protein were limited to monoglucosylated polymannose components. Although optimal binding occurred with Glc1Man9GlcNAc, substantial interaction with calreticulin was retained after sequential trimming of the polymannose portion down to the Glc1Man5GlcNAc stage. The alpha 1 --> 6-mannose branch point of the oligosaccharide core, however, appeared to be essential for recognition as Glc1Man4GlcNAc did not interact with the calreticulin. The carbohydrate-peptide linkage region had no discernible influence on binding as monoglucosylated oligosaccharides in N-glycosidic linkage interacted with the chaperone to the same extent as in their unconjugated state. The immobilized calreticulin proved to be a highly effective tool for sorting out monoglucosylated polymannose oligosaccharides or glycopeptides from complex mixtures of processing intermediates. The copurification of calreticulin and endomannosidase from a Golgi fraction in comparable amounts and the strikingly similar saccharide specificities of the chaperone and the processing enzyme have suggested a tentative model for the dissociation through glucose removal of calreticulin-glycoprotein complexes in a post-endoplasmic reticulum locale; in this scheme, deglucosylation would be brought about by the action of endomannosidase rather than glucosidase II.
Highlights
Tant role in the quality control of the secretory pathway by influencing the folding and assembly of the proteins to which they are attached [1,2,3]
Immobilized Calreticulin Resolves Glc1Man9GlcNAc from Unglucosylated Man9GlcNAc—In view of our previously demonstrated understanding that endomannosidase has a high degree of specificity for monoglucosylated polymannose oligosaccharides, which is responsible for its retention on Glc-ManAffi-Gel [12], we explored the possibility that the binding of calreticulin to this matrix has a similar basis
When an incompletely resolved mixture of radiolabeled Man9GlcNAc and Glc1Man9GlcNAc was chromatographed on a calreticulinSepharose column, thin layer chromatographic examination of the resulting two peaks indicated that the glucosylated oligosaccharide was bound, while the unglucosylated polymannose component was unretained (Fig. 3)
Summary
Tant role in the quality control of the secretory pathway by influencing the folding and assembly of the proteins to which they are attached [1,2,3]. Studies carried out with immobilized calreticulin and a series of radiolabeled oligosaccharides derived from N-linked carbohydrate units revealed that interactions with this protein were limited to monoglucosylated polymannose components.
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