ABSTRACT
Cystatins are protein inhibitors of papain and related cysteine proteinases. A series of continuous synthetic peptides corresponding to the entire sequence of rat salivary cystatin was used to localize the binding domains of the cystatin to papain. Several synthetic peptides, one from the aminoterminal sequence (peptide 1-24) and others from the carboxylterminal (peptides 66-79, 66-90, 79-90, 79-114), showed binding to papain, but none of the peptides showed inhibition of papain activity. Three recombinant rat salivary cystatin variants (N-terminal truncated protein lacking amino acid residues 1-9; variant 49-53, in which amino acid residues QVVAG of rat salivary cystatin had been replaced with amino acid residues LVL in mutant protein; and variant 65-78, in which amino acid residues 65-78 had been replaced with amino acids PG in mutant protein) were produced using the Escherichia coli expression system pGex-4T. To generate N-terminal truncated protein the desired coding region of the cystatin gene was amplified by polymerase chain reaction (PCR). To produce the variants 49-53 and 65-78, a PCR-based approach of gene splicing by overlap extension was used. Recombinant cystatin proteins were produced as insoluble inclusion bodies as fusion proteins with a glutathione S-transferase (GST) carrier. After solubilization with urea the GST carrier was cleaved from the fusion protein with thrombin and cystatin variants purified by fast liquid chromatography on a MonoQ column. The purified proteins reacted with antibodies to rat salivary cystatin. The N-terminal truncated and variant 49-53 exhibited very little inhibitory activity towards papain, whereas variant 65-78 exhibited papain-inhibitory activity similar to the full-length recombinant cystatin.
Subject(s)
Cystatins/genetics , Cysteine Proteinase Inhibitors/genetics , Escherichia coli/genetics , Salivary Proteins and Peptides/genetics , Alternative Splicing , Amino Acids/genetics , Animals , Antibodies , Carrier Proteins/genetics , Chromatography, Liquid , Gene Amplification , Gene Expression Regulation, Bacterial , Genetic Vectors , Glutathione Transferase/genetics , Inclusion Bodies/genetics , Mutation/genetics , Papain/antagonists & inhibitors , Peptides/genetics , Polymerase Chain Reaction , Rats , Recombinant Fusion Proteins , Recombinant Proteins , Salivary Cystatins , Solubility , Thrombin/chemistry , Urea/chemistryABSTRACT
A unique family of proline-rich proteins (PRPs) is induced in rats following prolonged isoproterenol treatment. PRPs can be divided into glycosylated (GPRP), basic (BPRP) and acidic (APRP) proline-rich proteins based on their physicochemical characteristics. Inducible rat parotid PRPs were isolated from aqueous extracts of parotid glands of isoproterenol-treated animals by sequential chromatography on columns of DEAE-Sepharose CL-6B, Sephadex G-100 and FPLC on Suprose-12 column. The GPRP showed a single homogeneous band on sodium dodecylpolyacrylamide gel electrophoresis with an estimated molecular weight of approximately 220,000. Compositional analysis of GPRP revealed that this protein contained 19.7% glutamic acid/glutamine, 28.2% proline and 9.5% glycine, and 44% carbohydrate, consisting of fucose (2.81g/100g), mannose (9.78g/100g), galactose (9.29g/100g), N-acetylglucosamine (18.03g/100g) and N-acetylgalactosamine (3.90g/100g). Basic PRPs consisted of a family of proteins with estimated molecular masses ranging from 14-45 kDa. These proteins contained 42.6% proline, 20.65% glutamic acid/glutamine and 21.33% glycine. Acidic PRPs also comprised of a family of metachromatically stained ladder of 40-60 kDa containing 29.1% proline, 21.5% glutamic acid/glutamine and 17.8% glycine. APRP were heavily glycosylated containing N-acetylglucosamine (6.34g/100g), N-acetylgalactosamine (19.04g/100g) and glucuronic acid (38.08g/100g).
