Definition of the Lectin-like Properties of the Molecular Chaperone, Calreticulin, and Demonstration of Its Copurification with Endomannosidase from Rat Liver Golgi (*)
- From the (1)Departments of Biological Chemistry and Medicine, Harvard Medical School and the Joslin Diabetes Center, Boston, Massachusetts 02215 and the
- (2)Abteilung Biochemie, Zentrum Innere Medizin, Universität Göttingen, D-3400 Göttingen, Germany
- § To whom correspondence and reprint requests should be addressed: Elliott P. Joslin Research Laboratory, Joslin Diabetes Center, One Joslin Place, Boston, MA 02215. Tel.: 617-732-2568; Fax: 617-732-2569.
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 Glcα1
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 Glc
Man
GlcNAc, substantial interaction with calreticulin was retained after sequential trimming of the polymannose portion down to
the Glc
Man
GlcNAc stage. The α1
6-mannose branch point of the oligosaccharide core, however, appeared to be essential for recognition as Glc
Man
GlcNAc 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.
Footnotes
-
↵* This work was supported by Grants DK 17325 and DK 17477 from the National Institutes of Health (to R. G. S), as well as European Community Contract CIPA-CT92-3014 and Grant So 43/47-3 from the Deutsche Forschungsgemeinschaft (to H. S. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
-
↵1 The abbreviations used are:
- ER
-
endoplasmic reticulum
- Glc-Man-Affi-Gel
-
Glcα1
3Man-O-(CH
)
CONH-Affi-Gel
- CAPS
-
3-(cyclohexylamino)propanesulfonic acid
- MES
-
2-(N-morpholine)ethanesulfonic acid
- endo H
-
endoglycosidase H. All sugars mentioned in the text are in the D-configuration.
-
- Received December 19, 1995.
- Revision received February 26, 1996.
- © 1996 by The American Society for Biochemistry and Molecular Biology, Inc.
