THE CYSTEINE PROTEINASE GENE CPRG IN DICTYOSTELIUM-DISCOIDEUM HAS A SERINE-RICH DOMAIN THAT CONTAINS GLCNAC-1-P

A lysosomal cysteine proteinase called proteinase-l is the major prote olytic enzyme in vegetative cells of Dictyostelium discoideum. This ph osphoglycosylated protein contains multiple residues of GlcNAc-1-P lin ked to peptidyl serines. Here we report the cloning, structure, and ex pression of its cDNA (cprG). Another cDNA (cprF) closely related to cp rG was also cloned and characterized. mRNAs of both genes are present during the vegetative phase and decrease in developing cells. However, the level of cprG mRNA is about 100-fold higher than that of cprF The predicted protein products of both genes contain a unique serine-rich domain that was previously found only in two Dictyostelium proteinase s (CP4 and CP5) that also carry a GlcNAc-1-P-Sermodification . The cpr G product, renamed CP7, was tagged with the FLAG epitope (FLAG-CP7) an d shown to bind to cystatin, a highly specific inhibitor of cysteine p roteinases. The FLAG-CP7 product also contained both N-linked oligosac charides and GlcNAc-1-P. Deletion of the serine-rich domain from FLAG- CP7 yields a product that still binds to cystatin, but no longer carri es GlcNAc-1-P. This finding supports the idea that the GlcNAc-1-P resi dues are normally added to the serine-rich domain, found only in veget ative Dictyostelium cysteine protienases. (C) 1997 Academic Press.