Identification of Two Novel Dictyostelium discoideum Cysteine Proteinases That Carry N-Acetylglucosamine-1-P Modification

doi:10.1074/jbc.270.48.28938

Identification of Two Novel Dictyostelium discoideum Cysteine Proteinases That Carry N-Acetylglucosamine-1-P Modification *

From the La Jolla Cancer Research Foundation, La Jolla, California 92037

^¶ 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

Dictyostelium discoideum makes multiple developmentally regulated lysosomal cysteine proteinases. One of these, a lysosomal enzyme called proteinase I, contains a cluster of GlcNAc-α-1-P-Ser residues. We call this phosphoglycosylation. To study its function, a cDNA library from vegetative cells was screened, and two novel cysteine proteinase clones were characterized (cprD and cprE). Each of them has highly conserved regions expected for cysteine proteinases, but unlike any other, each has a serine-rich domain containing three distinct motifs, poly-S, SGSQ, and SGSG. cprD and cprE cDNAs were overexpressed in Dictyostelium and the active enzymes identified. cprD codes for a protein of approximately 36 kDa (CP4), which is recognized by monoclonal antibodies against GlcNAc-1-P and fucose. cprE corresponds to a 29-kDa protein, which is recognized by antibodies against GlcNAc-1-P. mRNA for both enzymes is present in the vegetative phase and increases during growth on bacteria but decreases throughout development. When the formation of the fruiting body is complete the mRNA for both messages is detected again but in very low levels. Having cloned cDNAs for proteins that carry GlcNAc-1-P should allow us to probe the function of the carbohydrate in these putative lysosomal enzymes.

Footnotes

↵^§ Supported by a postdoctoral fellowship from CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnologico, Brazil).
↵* This work was supported by National Institute of General Medical 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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L36204 [GenBank](CP4) and L36205 [GenBank](CP5).
↵¹G. Souza and H. Freeze, unpublished results.
↵² The abbreviations used are:

p-NA

p-nitroaniline

bp

base pair(s)

kb

kilobase(s)

SSPE

saline/sodium/phosphate/EDTA

PAGE

polyacrylamide gel electrophoresis

N-Cbz

N-benzyloxycarbonyl

ON-p

p-nitrophenyl ester

DTT

dithiothreitol

E-64

trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane

t-Boc

t-butoxycarbonyl

MCA

7-amido-4-methylcoumarin.
↵³C. West and H. Freeze, unpublished results.
- Received September 5, 1995.