Calreticulin Functions as a Molecular Chaperone in the Biosynthesis of Myeloperoxidase

doi:10.1074/jbc.270.9.4741

Calreticulin Functions as a Molecular Chaperone in the Biosynthesis of Myeloperoxidase (*)

From the Department of Medicine, Veterans Administration Medical Center and the University of Iowa, College of Medicine, Iowa City, Iowa 52242

^§ A Clinical Investigator in the Dept. of Veterans Affairs. To whom correspondence should be addressed:
Dept. of Medicine, University of Iowa, 200 Hawkins Dr., Iowa City, IA 52242;
Tel.: 319-356-1739; Fax: 319-356-4600.

Abstract

Myeloperoxidase (MPO), a lysosomal heme protein found exclusively in neutrophils and monocytes, is necessary for efficient oxygen-dependent microbicidal activity. Acquisition of heme by the heme-free MPO precursor apopro-MPO appears to be a prerequisite for its subsequent proteolytic processing and advancement along the biosynthetic pathway to mature MPO. We present data indicating that calreticulin (CRT), a high capacity calcium-binding protein residing in the lumen of the endoplasmic reticulum of a wide variety of cells, interacts specifically with fully glycosylated apopro-MPO. Biosynthetically radiolabeled CRT (60 kDa) and apopro-MPO (90 kDa) were coprecipitated from PLB 985 cells by monospecific antiserum against CRT when the immunoprecipitations were performed either under nondenaturing conditions or following reversible cross-linking. Nonglycosylated MPO precursors synthesized in the presence of tunicamycin did not interact with CRT. The CRT-apopro-MPO interaction was restricted to an early phase of MPO biosynthesis, and CRT did not interact with the later appearing, heme-containing species of MPO, i.e. pro-MPO or the heavy subunit of mature MPO. These data show that CRT participates in the posttranslational processing of MPO, perhaps by maintaining apopro-MPO in a conformation competent to accommodate insertion of the heme group. In this general way, CRT shares certain functional properties with the structurally homologous transmembrane calcium-binding endoplasmic reticulum protein calnexin. Both interact with glycosylated biosynthetic precursors of proteins selectively expressed in specialized cells.

Footnotes

↵* This work was supported in part by merit review grants from the Dept. of Veterans Affairs (to W. M. N. and R. A. C.). 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:

PMNs

polymorphonuclear neutrophils

-ALA

-aminolevulinic acid

CRT

calreticulin

DTSSP

3,3′-dithiobis(sulfosuccinimidyl proprionate)

DTT

dithiothreitol

GRP94

glucose-regulated protein, 94 kDa

hsp90

heat shock protein, 90 kDa

IEF

isoelectric focusing

MPO

myeloperoxidase

NEPHGE

non-equilibrium pH gel electrophoresis

PAGE

polyacrylamide gel electrophoresis

TBS

tris-buffered saline

TM

tunicamycin

ER

endoplasmic reticulum.
↵²W. M. Nauseef, unpublished data.
↵³W. M. Nauseef and S. J. McCormick, unpublished data.
- Received July 18, 1994.
- Revision received December 9, 1994.