DEFINITION OF THE LECTIN-LIKE PROPERTIES OF THE MOLECULAR CHAPERONE, CALRETICULIN, AND DEMONSTRATION OF ITS COPURIFICATION WITH ENDOMANNOSIDASE FROM RAT-LIVER GOLGI

Calreticulin was identified by immunochemical and sequence analyses to be the higher molecular mass (60 kDa) component of the polypeptide do ublet previously observed in a rat liver Golgi endomannosidase prepara tion obtained by chromatography on a Glc alpha 1-->3Man-containing mat rix. The affinity for this saccharide ligand, which paralleled that of endomannosidase and was also observed with purified rat liver calreti culin, suggested that this chaperone has lectin-like binding propertie s. Studies carried out with immobilized calreticulin and a series of r adiolabeled oligosaccharides derived from N-linked carbohydrate units revealed that interactions with this protein were limited to monogluco sylated polymannose components. Although optimal binding occurred with Glc(1)Man(9)GlcNAc, substantial interaction with calreticulin was ret ained after sequential trimming of the polymannose portion down to the Glc(1)Man(5)GlcNAc stage. The alpha 1-->6-mannose branch point of the oligosaccharide core, however, appeared to be essential for recogniti on as Glc(1)Man(4)GlcNAc did not interact with the calreticulin. The c arbohydrate-peptide linkage region had no discernible influence on bin ding as monoglucosylated oligosaccharides in N-glycosidic linkage inte racted with the chaperone to the same extent as in their unconjugated state. The immobilized calreticulin proved to be a highly effective to ol for sorting out monoglucosylated polymannose oligosaccharides or gl ycopeptides from complex mixtures of processing intermediates. The cop urification of calreticulin and endomannosidase from a Golgi fraction in comparable amounts and the strikingly similar saccharide specificit ies of the chaperone and the processing enzyme have suggested a tentat ive model for the dissociation through glucose removal of calreticulin -glycoprotein complexes in a post-endoplasmic reticulum locale; in thi s scheme, deglucosylation would be brought about by the action of endo mannosidase rather than glucosidase II.