The Cysteine Proteinase GenecprGinDictyostelium discoideumHas a Serine-Rich Domain That Contains GlcNAc-1-P

A lysosomal cysteine proteinase called proteinase-1 is the major proteolytic enzyme in vegetative cells ofDictyostelium discoideum.This phosphoglycosylated protein contains multiple residues of GlcNAc-1-P linked to peptidyl serines. Here we report the cloning, structure, and expression of its cDNA (cprG). Another cDNA (cprF) closely related tocprGwas also cloned and characterized. mRNAs of both genes are present during the vegetative phase and decrease in developing cells. However, the level ofcprGmRNA is about 100-fold higher than that ofcprF.The predicted protein products of both genes contain a unique serine-rich domain that was previously found only in twoDictyosteliumproteinases (CP4 and CP5) that also carry a GlcNAc-1-P-Ser modification. ThecprGproduct, renamed CP7, was tagged with the FLAG-epitope (FLAG-CP7) and shown to bind to cystatin, a highly specific inhibitor of cysteine proteinases. The FLAG-CP7 product also contained both N-linked oligosaccharides and GlcNAc-1-P. Deletion of the serine-rich domain from FLAG-CP7 yields a product that still binds to cystatin, but no longer carries GlcNAc-1-P. This finding supports the idea that the GlcNAc-1-P residues are normally added to the serine-rich domain, found only in vegetativeDictyosteliumcysteine protienases.