Characterization by spectroscopic, kinetic and equilibrium methods of the interaction between recombinant human cystatin A (stefin A) and cysteine proteinases

Abstract

The near-UV spectroscopic changes induced by the binding of recombinant human cystatin A to papain were appreciably different from those induced by cystatin C, reflecting mainly interactions involving the single tryptophan of cystatin C, Trp-106. Cystatin A bound tightly and rapidly to papain and cathepsin L, with dissociation equilibrium constants of approximately 10(-11)-10(-13) M and association rate constants of 3 x 10(6)-5 x 10(6) M-1.s-1. These affinities are at least 50-100-fold higher than previously reported values. The kinetics of binding to papain were consistent with a simple reversible bimolecular reaction mechanism, indicating that cystatin A, like chicken cystatin and cystatin C, binds to papain with no appreciable conformational adaptation of either reacting protein. Cystatin A bound more weakly to actinidin and cathepsins B, C and H, with dissociation equilibrium constants of 10(-8)-10(-9) M. The weaker binding to cathepsin B was largely due to a considerably reduced association rate constant (approximately 4 x 10(4) M-1.s-1), consistent with the 'occluding loop' of cathepsin B markedly restricting the access of cystatin A to the active site. The lower affinities for actinidin and cathepsins C and H were due partly to lower association rate constants (2 x 10(5)-6 x 10(5) M-1.s-1) but primarily to higher dissociation rate constants. The mode of binding of cystatin A to inactivated papains indicated that there is appreciably less space around the active-site cysteine of papain in the complex with cystatin A than in the complexes with chicken cystatin and cystatin C. An N-terminally truncated form of cystatin A, lacking the first six residues, had considerably lower affinity for papain than the full-length inhibitor, consistent with an intact N-terminal region being of importance for proteinase binding.

Inhibition of cruzipain, the major cysteine proteinase of the protozoan parasite, Trypanosoma cruzi, by proteinase inhibitors of the cystatin superfamily

“…Protein concentration was determined spectrophotometrically at 280 nm with the use of published [21,23] or calculated absorbance coefficients and molecular masses obtained from amino acid sequences [27].…”

“…Human stefin A [20], recombinant human stefin A [21], recombinant human stefin B [22], chicken cystatin [23], recombinant human cystatin C [24] and human low molecular weight kininogen [25] were purified by published procedures.…”

Interaction of human cathepsin C with chicken cystatin

“…Cathepsin C is inhibited by stefins A and B [13][14][15] and chicken cystatin [13,16], three protein inhibitors of cysteine proteinases from the cystatin superfamily [17]. All inhibitors were shown to be of the competitive, reversible type, with stefin A being the weakest by 2-3 orders of magnitude [13][14][15][16].…”

“…All inhibitors were shown to be of the competitive, reversible type, with stefin A being the weakest by 2-3 orders of magnitude [13][14][15][16]. However, the mechanism of interaction between cathepsin C and cystatins is not known.…”

Interaction between Human Cathepsins K, L, and S and Elastins

“…44, and the cystatin-like domain 3 of human kininogen (kininogen domain 3) was isolated as described previously (45) and recombinant thyroglobulin type 1 (Tg1) domain of human testican-1 as described previously (46). The recombinant human p41 fragment of the major histocompatibility complex class II-associated invariant chain, recombinant Tg1 domain of human nidogen-1, and recombinant Tg1 domain 1 of human nidogen-2 were produced in-house.…”