The cysteine proteinase inhibitors belonging to the cystatin superfamily are subdivided into three subfamilies, stefins, cystatins, and kininogens, based on their structural complexity . An inducible cysteine proteinase inhibitor belonging to cystatin family 2 has been isolated and sequenced from the submandibular glands of adult rats exposed chronically to the ?-adrenergic agonist isoproterenol . The cDNA as well as the gene for rat salivary cystatin have also been cloned and sequenced . We recently described the successful cloning of the coding sequence of the cystatin gene into a pGEX-2T expression vector, and the expression and purification of a functionally and immunologically reactive recombinant cystatin from Escherichia coli .An alignment of the amino acid sequence of the rat salivary cystatin with rat liver and epidermal cysteine proteinase inhibitors (belonging to family-1 cystatin) and kininogen (a family-3 cystatin) revealed very little sequence similarity, but G₅, E₁₉ and Q₄₉ V₅₀ V₅₁ A₅₂ G₅₃ (amino acid numbers refer to the sequence of rat salivary cystatin; ) were highly conserved in members of all three cystatin families. The location of the disulphide bridges is also conserved in family-2 and -3 cystatins. The corresponding residues are also most highly conserved in cystatins from several other sources. Based on the three-dimensional structure of the chicken egg-white cystatin and on information about the conserved residues, proposed an elephant-trunk model for interaction with papain, in which the first 10 amino acid residues in the amino terminus, a ?-hairpin loop containing the conserved QVVAG residues, and a second ?-hairpin loop containing the conserved residues L₁₀₂ and H₁₀₄ in family-1 and Trp₁₀₄ in family-2 cystatins cover the active site cleft of papain or other cysteine proteinases. The X-ray crystallographic structure of recombinant human stefin B in complex with papain further confirms that the conserved residues form a tripartite wedge, which slots into the papain active site . In the present study, to define the binding site of the cystatin to papain, a series of peptides was synthesized corresponding to the entire length of the salivary cystatin. Synthetic peptides have previously been used successfully for epitopic or molecular mapping of proteins .

Recombinant DNA techniques are now being used in studies of the molecular mechanism of inhibition of cysteine proteinases by cystatins . Over the past decade, gene products for human cystatin C , human salivary cystatin , stefin A , rat cystatin , and stefin B , and rat salivary cystatin have been produced by such methods. In an effort to begin to understand the mechanism of inhibition by cystatin S, we now describe the expression, purification, immunological and biochemical characterization of rat salivary cystatin recombinant variants with a truncated amino terminus lacking the N-terminal nine amino acids (cys ?1–9 variant), a first hairpin-loop variant in which QVVAG has been replaced with LVL (variant 49–53) and a first disulphide-loop variant in which amino acid residues TICLKTQGDLTNCP (residues 65–78) have been replaced with amino acid residues PG (variant 65–78).

The synthetic peptides prepared for this study corresponded to the published amino acid sequence of salivary cystatin . The peptides were synthesized in a solid-phase Fmoc procedure with an Applied Biosystems model 431A . Peptides were cleaved from the resin with 95% trifluoroacetic acid–5% thioanisol as a scavenger. Cleaved peptides were precipitated with diethyl ether and lyophilized. Their purity was checked by analytical, high-performance liquid chromatography and >95% appeared as one peak. The composition and sequence of each of the peptides was confirmed by amino acid analysis and sequencing.

To localize the active binding domains of the cystatin to papain, a series of peptides was synthesized, corresponding to the full length of the salivary cystatin . The binding ability of the synthetic peptides was tested by dot-blot analysis. Peptides were immobilized on to a nitrocellulose membrane using a Bio-Dot microfiltration unit. The membrane was washed with PBS for 10 min and blocked with 2% BSA for 2 hr at 37°C. After washing three times with 10 ml PBS at room temperature, the membrane was immersed in 10 ml of ¹²⁵I-labelled papain (5×10⁶ counts/min) in 0.1 M phosphate buffer, pH 6.0, for 1 hr at 37°C. After washes, the nitrocellulose filter was exposed to Kodak XAR film at ?70°C. The autoradiogram was scanned in an LKB ultrascan XL laser densitometer and the data processed using Gelscan XL version 1.2 software.

The papain inhibitory activity of the synthetic peptides corresponding to the full length of the cystatin sequence was tested. The chromogenic substrate BAPNA was used for enzyme inhibition assays. The activity of papain was assayed by a slight modification of the method of Barrett . The enzyme was preincubated for 10 min with an equal volume (50 ?l) of activation buffer (0.1 M phosphate buffer, pH 6.0, containing 0.2 mg/ml dithiothreitol and 0.5 mg/ml Na₂EDTA), with or without cystatin peptides. The reaction was started by adding 25 ?l of a 10 mM solution of BAPNA; it was terminated by the addition of 25 ?l of glacial acetic acid. The amount of liberated p-nitroaniline was determined at 410 nm. The amount of the enzyme used was adjusted to obtain a final optical density between 0.5 and 0.7 in positive controls containing no inhibitors. One nmol of purified rat salivary cystatin completely inhibited BAPNA hydrolytic activity of 1.25 nmol of papain.Amplification was by PCR, carried out for 30 cycles in a DNA thermal cycler (Perkin Elmer Cetus, Norwalk, CN). The cystatin plasmid clone pBSCys2, described previously, was used as a template and with the following procedures: denaturation, 94°C, 1 min; annealing, 55°C, 1 min; and primer extension, 72°C, 1 min. The extension primers used for PCR amplification of coding regions from the pBSCys2 plasmid were synthesized by DNAgency (Collegeville, PA). Primers P1 and P11 are based on the amino acid residues 1–6 (GHFLG) and residues 11–16 (SMEEEG) of the rat salivary cystatin sequence, respectively . These primers incorporated a BamHI restriction site for in-frame cloning into the BamHI site of the pGEX-4T-2 expression vector. The 5? end of these primers also contained a 4-bp overhang to facilitate digestion. The design of primers 5459 and 7985 was based on the nucleotide sequence of the cystatin cDNA corresponding to amino acid residues 54–59 (TLFFFD) and residues 79–85 (DEEADQ), respectively. Primers 4348 and 5964 were designed as reverse (antisense) primers corresponding to amino acid residues 43–48 (VLDVQ) and 59–64 (DVILG), respectively. Primers 4348 and 5459 incorporated a HindIII restriction site, and primers 5964 and 7985 incorporated a SmaI restriction site (underlined) at the 5? ends and also contained 4–6-bp overhangs. Primer T3 is a T3 promotor primer located in the vector, about 60 bp on the outside of pBluescript cloning site.