A Lysosomal Cysteine Proteinase from Dictyostelium discoideum Contains N-Acetylglucosamine-1-phosphate Bound to Serine but Not Mannose-6-phosphate on N-linked Oligosaccharides

doi:10.1074/jbc.271.18.10897

A Lysosomal Cysteine Proteinase from Dictyostelium discoideum Contains N-Acetylglucosamine-1-phosphate Bound to Serine but Not Mannose-6-phosphate on N-linked Oligosaccharides (*)

From the La Jolla Cancer Research Foundation, La Jolla, California 92037 and
Glycobiology Program, Cancer Center, University of California at San Diego, La Jolla, California 92093

^§ To whom correspondence should be addressed:
La Jolla Cancer Research Foundation, 10901 N. Torrey Pines Rd., La Jolla, CA 92037.
Tel.: 619-455-6480; Fax: 619-450-2101; Hudson{at}ljcrf.edu.

Abstract

Previous studies showed that vegetative Dictyostelium discoideum cells make a lysosomal proteinase, proteinase-1, that contains multiple GlcNAc-α-1-P residues in phosphodiester linkage to serine. We extended these studies and, in contrast to earlier reports, found that proteinase-1 contains 7.5 mol of Fuc, 8 mol of Man, 2 mol of Xyl, and 30 mol of GlcNAc per calculated mol of protein but no Man-6-P residues. The protein binds to concanavalin A and wheat germ agglutinin lectin affinity columns, and PNGase-F digestion released most of the mannose and xylose but little of the GlcNAc. β-Elimination under reducing conditions released only GlcNAc-α-1-P. There was no evidence for the release of disaccharides or of fucitol. A rabbit antiserum and monoclonal antibodies prepared against proteinase-1 recognize GlcNAc-α-1-P residues in immunoblots and are specifically competed by UDP-GlcNAc or GlcNAc-α-1-P. Use of other monoclonal antibodies showed the presence of mannose-6-sulfate on N-linked sugar chains, and α-fucose residues on the protein. Thus, proteinase-1 has at least two types of modifications: GlcNAc-α-1-P-Ser, which we call phosphoglycosylation, and N-linked oligosaccharides. This is the first purified lysosomal enzyme in Dictyostelium that does not contain Man-6-P residues. The GlcNAc-α-1-P-specific antibodies also recognize a group of developmentally regulated proteins, especially enriched in vegetative cells. Some of them are also lysosomal cysteine proteinases, and all bind to the GlcNAc-α-1-P-specific monoclonal antibody but not to the mammalian CI-Man-6-P receptor. Conversely, lysosomal enzymes that have Man-6-P do not bind to the GlcNAc-α-1-P-specific antibody. An exception to this is β-N-acetylglucosaminidase, where 15% of the activity binds to this antibody. Thus, there appear to be two sets of lysosomal enzymes with distinct post-translational modifications.

Footnotes

↵* This work was supported by the National Institute of Environmental Health Sciences Grant 32485. 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:

Man-6-P-OCH

mannose-6-phosphomethyldiester

CI-MPR

bovine cation-independent mannose 6-phosphate receptor

PNGase-F

peptide-N-glycosidase F

LSIMS

liquid secondary ion mass spectrometry

mAb

monoclonal antibody

TBS

Tris-buffered saline

ELISA

enzyme-linked immunosorbent assay

NAG

N-acetyl β-D-glucosaminidase.
- Received December 12, 1995.
- Revision received February 6, 1996.