The cysteine proteinase gene cprG in Dictyostelium discoideum has a serine-rich domain that contains GlcNAc-1-P.
A lysosomal cysteine proteinase called proteinase-1 is the major proteolytic enzyme in vegetative cells of Dictyostelium 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 to cprG 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 proteinases (CP4 and CP5) that also carry a GlcNAc-1-P-Ser modification. The cprG product, 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 vegetative Dictyostelium cysteine proteinases.
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Burnham Institute, La Jolla, California 92037, USA.
Adessi
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Wang
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Freeze
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D000117:Acetylglucosamine / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics; D004023:Dictyostelium / Q000201:enzymology / Q000235:genetics*; D018507:Gene Expression Regulation, Developmental; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D006031:Glycosylation; D008247:Lysosomes / Q000201:enzymology; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D015800:Protozoan Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry
D018076:DNA, Complementary; D010750:Phosphoproteins; D015800:Protozoan Proteins; D012694:Serine; D003546:Cysteine Endopeptidases; C095028:cprG protein, Dictyostelium; D000117:Acetylglucosamine