Cystatins proteases

The N-terminal sequence of one peptide from the 35 kDa zone of H-gal-GP showed some homology to cathepsin B-like cysteine proteases. Molecular cloning has also identified a thrombospondin homologue associated with the diffusely staining region between zones A and B, a galectin associated with zone D (Newlands et al., 1999) and a low molecular weight (approximately 13 kDa) cysteine protease inhibitor, cystatin. 

Stefin or cystatin (cysteine protease inhibitor) Homo sapiens 95 (dimer form) 358-363... 

A straightforward approach is to hunt for short polypeptides that meet the specificity requirement of an enzyme but which, because of peculiarities of the sequence, are acted upon very slowly. Such a peptide may contain unusual or chemically modified amino acids. For example, the peptide Thr-Pro-nVal-NMeLeu-Tyr-Thr (nVal=norvaline NMeLeu = N-methylleucine) is a very slow elastase substrate whose binding can be studied by X-ray diffraction and NMR spectroscopy.6 Thiol proteases are inhibited by succinyl-Gln-Val-Val-Ala-Ala-p-nitroanilide, which includes a sequence common to a number of naturally occurring peptide inhibitors called cystatins.f They are found in various animal tissues where they inhibit cysteine proteases. 

Arabidopsis clialiana (mouse-ear cress) (Brassicaceae) PRL1 PRL2 (proprotein 23 kDa) Cysteine protease inhibitor protein (cystatin) homologues [147]... 

Saccharum officinarum (sugar cane) (Poaceae) Sugar cane cystatins Scl- Sc25 (Phytocystatins) Cysteine proteases [184]... 

The Cystatin Superfamily of Inhibitors of Papain-like Cysteine Proteases. . 64... 

Fig. 1. Amino acid sequence and schematic structure of human cystatin C. The shaded area marks the inhibitory site for papain-like cysteine proteases, which does not overlap with the inhibitory site for mammalian legumains comprising, inter alia, the Asn39 residue. The arrow indicates the Leu68 residue, which is replaced with a Gin residue in the cerebral hemorrhage producing cystatin C variant. The asterisk marks the Pro3 residue, which is partly hydroxylated.

All human cystatins are assumed to have major biological roles as inhibitors of one or more target proteases of human and/or nonhuman origin. Identification of target proteases of biomedical relevance is, however, difficult. Only few examples of clear-cut identifications of target proteases for inhibitors are known, and these identifications usually rely on experiments by Nature and not by Man. Two examples are the identifications of granulocyte elastase as a target enzyme... 

The distribution in body fluids of the different cystatins is remarkably different (Fig. 3). For example, while cystatin C is present in appreciable amounts in all investigated body fluids, cystatins S, SN, and SA are virtually confined to saliva, tears, and seminal plasma (Al). Cystatin D is present only in saliva and tear fluid (Al, F3). In some body compartments, e.g., spinal fluid, cystatin C represents more than 90% of the total molar concentration of cysteine protease... 

Equilibrium Constants for Dissociation of Complexes between Human Cystatins and Cysteine Proteases Ki (nM)... 

In addition to being an inhibitor of papain-like cysteine proteases, cystatin C has recently been shown be an efficient inhibitor of some of the cysteine proteases of another family of cysteine proteases, called the peptidase family C13, with human legumain as a typical enzyme (C6). Human legumain has, like cathepsin S, been proposed to be involved in the class n MHC presentation of antigens (M3). It has also been shown that the cystatin C inhibitory site for mammalian legumain does not overlap with the cystatin C inhibitory site for papain-like cysteine proteases (Fig. 1) and that the same cystatin C molecule therefore is able to simultaneously inhibit one cysteine protease of each type (A 10). 

When HCCAA is suspected because of clinical observations and/or family history, the diagnosis can easily be verified by demonstrating a low cerebrospinal fluid level of cystatin C (G10, J3) or the presence of the mutated cystatin C allele producing the Leu 68 Gin cystatin C variant (A6, PI). The first-mentioned procedure requires lumbar puncture and, to secure a stable level of cystatin C in the sample, the addition of a serine protease inhibitor, e.g., benzamidinium chloride, directly when the sample is drawn (Grubb, A., unpublished results). The PCR-based procedure is more robust, does not require lumbar puncture, and can be used for prenatal diagnosis and is therefore presently the preferable diagnostic method. 

CIO. Colle, A., Tavera, C., Laurent, P., Leung-Tack, J., and Girolami, J. P., Direct radioimmunoassay of rat cystatin C Increased urinary excretion of this cysteine proteases inhibitor during chromate nephropathy. J. Immunoassay 11(2), 199-214 (1990). 

K7. Korant, B., Towatari, T., Kelley, M., Brzin, J., Lenarcic, B., and Turk V., Interactions between a viral protease and cystatins. Biol. Chem. Hoppe Seyler 369(Suppl.), 281—286 (1988). 

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