IDENTIFICATION OF 2 NOVEL DICTYOSTELIUM-DISCOIDEUM CYSTEINE PROTEINASES THAT CARRY N-ACETYLGLUCOSAMINE-1-P MODIFICATION

Dictyostelium discoideum makes multiple developmentally regulated lyso somal cysteine proteinases. One of these, a lysosomal enzyme called pr oteinase I, contains a cluster of GlcNAc-alpha-1-P-Ser residues. We ca ll this phosphoglycosylation. To study its function, a cDNA library fr om vegetative cells was screened, and two novel cysteine proteinase cl ones were characterized (cprD and cprE). Each of them has highly conse rved 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 Dictyostel ium and the active enzymes identified, cprD codes for a protein of app roximately 36 kDa (CP4), which is recognized by monoclonal antibodies against GlcNAc-1-P and fucose, cprE corresponds to a 29-kDa protein, w hich is recognized by antibodies against GlcNAc-1-P. mRNA for both enz ymes 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 a gain but in very low levels. Having cloned cDNAs for proteins that car ry GlcNAc-1-P should allow us to probe the function of the carbohydrat e in these putative lysosomal enzymes.