Differential effect toward inhibition of papain and cathepsin C by recombinant human salivary cystatin SN and its variants produced by a baculovirus system.
Human salivary cystatin SN (CsnSN) is a member of the cystatin superfamily of cysteine proteinase inhibitors. In this study we used a baculovirus expression system to produce a full-length unaltered CsnSN and its variants. The variants were constructed with the changes in the three predicted proteinase-binding regions: the N-terminus (variant N(12-13), G12A-G13A), beta-hairpin loop I (variant L(56-58), Q56G-T57G-V58G) and beta-hairpin loop II (variant L(106-107), P106G-W107G). The secreted CsnSNs were purified using sequential spiral cartridge ultrafiltration and DE-52 radial flow chromatography. The purified proteins were examined for papain- and cathepsin C-inhibition. The wild-type CsnSN, and variants N(12-13) and L(106-107) bound tightly to papain (K(i) < 10 pM), whereas mutation in the loop I reduced binding affinity 5700-fold (K(i) = 57 nM). On the other hand, the wild-type CsnSN bound to cathepsin C less tightly (K(i) = 100 nM). The mutation in the N-terminus or loop I reduced binding affinity by 16 (K(i) = 1.6 microM)- and 19-fold (K(i) = 1.9 microM), respectively, while mutation in loop II resulted in an ineffective cathepsin C inhibitor (K(i) = 14 microM). Collectively, these results suggest that the N-terminal G12-G13 residues of CsnSN are not essential for papain inhibition but play a role in cathepsin C inhibition; residues Q56-T57-V58 in the loop I are essential for both papain and cathepsin C inhibitions, and residues P106-W107 in the loop II are not important for papain inhibition but essential for cathepsin C inhibition. These results demonstrated that CsnSN variants have different effects toward different cysteine proteinases.
Tseng
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Divisions of Basic Sciences and of Restorative and Prosthodontic Sciences, Room 1027 S, College of Dentistry, New York University, 345 East 24th Street, New York, New York 10010-4086, USA.
Tseng
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Levine
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Bobek
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D000818:Animals; D020789:Cathepsin C / Q000037:antagonists & inhibitors*; D002460:Cell Line; D002845:Chromatography; D002942:Circular Dichroism; D015891:Cystatins / Q000494:pharmacology*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D003636:DEAE-Cellulose / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D015151:Immunoblotting; D007313:Insecta; D007700:Kinetics; D010206:Papain / Q000037:antagonists & inhibitors*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000494:pharmacology*; D012463:Saliva / Q000737:chemistry*; D055333:Salivary Cystatins; D014461:Ultracentrifugation
C525171:CST1 protein, human; D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D018076:DNA, Complementary; D011994:Recombinant Proteins; D055333:Salivary Cystatins; D003636:DEAE-Cellulose; D020789:Cathepsin C; D010206:Papain