title: Purification and properties of the Dictyostelium calpain-like protein, Cpl.
abstract: Calpains are intracellular, cysteine proteases found in plants, animals, and fungi. There is emerging evidence that they are important mediators of cell adhesion and motility in animal cells. Because the cellular slime mold, Dictyostelium discoideum, is a genetically tractable model for cell adhesion and motility, we have investigated whether a calpain-like protein is expressed in this organism. Contig 13130 (Sanger Institute Dictyostelium sequencing project) was identified as a three-exon gene that encodes a calpain-like protein. Using a custom peptide antibody to assay for the presence of this putative protein, we identified Dictyostelium calpain-like protein (Cpl) and purified it to near homogeneity. Cpl is a 72278 Da cytosolic protein. Weak caseinolytic activity inhibitable by cysteine protease inhibitors was copurified with Cpl immunoreactivity, and purified Cpl appeared to undergo autoproteolysis upon transfer to inhibitor-free buffer. The major cleavage, generating a 51291 Da form, occurred after Pro 189. The Cpl domain structure resembles mammalian calpain 10, comprising an N-terminal catalytic domain followed by tandem calpain D-III domains. The putative catalytic domain appears to possess His and Gln active site residues, instead of the canonical His and Asn residues in calpains. The active site Cys has not yet been identified, and definitive proof of a proteolytic function awaits further study. Its phylogenetic distribution in D. discoideum and several protists suggests that the calpain D-III domain evolved early in eukaryotic cells.
authors.affiliation: Department of Pharmacology and Therapeutics, The Medical College of Ohio, Toledo, Ohio 43614-5804, USA.
authors.forename: Xinhua
authors.initials: X
authors.lastname: Huang
authors.forename: Eric
authors.initials: E
authors.lastname: Czerwinski
authors.forename: Ronald L
authors.initials: RL
authors.lastname: Mellgren
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D002154:Calpain / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D002364:Caseins / Q000737:chemistry; D002850:Chromatography, Gel; D004023:Dictyostelium / Q000201:enzymology* / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010802:Phylogeny; D017434:Protein Structure, Tertiary / Q000235:genetics; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D017386:Sequence Homology, Amino Acid
chemical_list: D002364:Caseins; D015800:Protozoan Proteins; D002154:Calpain; C415197:calpain 10