The Human Cystatins

A5. Abrahamson, M., Islam, M. Q., Szpirer, J., Szpirer, C., and Levan G., The human cystatin C gene (CST3), mutated in hereditary cystatin C amyloid angiopathy, is located on chromosome 20. Hum. Genet. 82(3), 223-226 (1989). 

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... 

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). 

The complete amino acid sequence of the single polypeptide chain of human cystatin C was determined in 1981 (Gil) and later corroborated by identification and sequencing of the corresponding cDNA (Fig. 4) (A3) and gene (A5, A7). 

The three-dimensional structure of cystatin C is not yet determined, although some crystallographic data are available (K8), but it can be presumed that it is similar to that described for the homologous protein chicken cystatin (B19) and this has, at least partially, been confirmed by NMR studies (El). A schematic structure for human cystatin C is given in Fig. 1. 

DNA sequence The nucleotide sequence data are available from the EMBL, GenBank, and DDBJ Nucleotide Sequence Databases under accession number X52255 Half-life About 20 min (experimentally determined for human cystatin C in rat plasma. The similarity in distribution volume and renal clearance between human cystatin C and acknowledged markers of human glomerular filtration, i.e., iohexol and 51 Cr-EDTA, suggests that the substances are eliminated at the same rate in humans with a half-life of approximately 2 h in individuals with normal renal function)... 

Establishment of reference values of general use requires general availability of a well-defined calibrator. The availability of such a calibrator also facilitates accreditation of procedures for quantitative determination of the corresponding analyte. Recombinant human cystatin C can easily be produced and isolated and used for establishing reliable calibrators (A2, Dl). A first step toward an international calibrator for cystatin C has been taken by the production of a solution of recombinant human cystatin C of high purity, determining the concentration of this... 

A3. Abrahamson, M., Grubb, A., Olafsson, I., and Lundwall, A., Molecular cloning and sequence analysis of cDNA coding for the precursor of the human cysteine proteinase inhibitor cystatin... 

A8. Abrahamson, M., Ritonja, A., Brown, M. A., Grubb, A., Machleidt, W., and Barrett, A. I., Identification of the probable inhibitory reactive sites of the cysteine proteinase inhibitors human cystatin C and chicken cystatin. J. Biol. Chem. 262(20), 9688—9694 (1987). 

F3. Freije, J. P., Balbin, M., Abrahamson, M., Velasco, G., Dalb0ge, H., et al., Human cystatin D. cDNA cloning, characterization of the Escherichia coli expressed inhibitor, and identification of the native protein in saliva. J. Biol. Chem, 268(21), 15737-15744 (1993). 

H4. Hall, A., Dalbpge, H., Grubb, A., and Abrahamson, M., Importance of the evolutionarily conserved glycine residue in the N-terminal region of human cystatin C (Gly-11) for cysteine en-dopeptidase inhibition. Biochem. J. 291 (Pt 1), 123-129 (1993). 

L6. Leung-Tack, J., Tavera, C., Gensac, M. C., Martinez, J., and Colle, A., Modulation of phagocytosis-associated respiratory burst by human cystatin C Role of the N-terminal tetrapep-tide Lys-Pro-Pro-Arg. Exp. Cell. Res. 188(1), 16-22 (1990). 

L9. Lignelid, H., Collins, V. P., and Jacobsson, B., Cystatin C and transthyretin expression in normal and neoplastic tissues of the human brain and pituitary. Acta Neuropathol. (Berl.) 93(5), 494-500 (1997). 

M10. Mussap, M., Ruzzante, N., Varagnolo, M., and Plebani, M., Quantitative automated particle-enhanced immunonephelometric assay for the routinary measurement of human cystatin C. Clin. Chem. Lab. Med. 36(11), 859-865 (1998). 

Tenstad O, Roald AB, Grubb A, Aukland K. Renal handling of radiolabelied human cystatin C in the rat. Scand J Clin Lab Invest 1996 56 409-14. 

Figure B3.1.3 An isoelectric focusing (IEF) gel, pH 3 to 10. Lane 1, 4 pg purified egg white cystatin. Lane M, broad-range pi standards trypsinogen (pi 9.3), lentil lectin-basic band (pi 8.65), lentil lectin-middle band (pi 8.45), lentil lectin-acidic band (pi 8.15), myoglobin-basic band (pi 7.35 visible as a broad band), myoglobin-acidic band (pi 6.85), human carbonic anhydrase B (pi 6.55), bovine carbonic anhydrase (pi 5.85), a-lactoglobulin A (pi 5.20), soybean trypsin inhibitor (pi 4.55), and amyloglucosidase (pi 3.50) in order shown from top of gel. The pi values of the two purified egg white cystatin isomers were determined to be 6.6 (upper band) and 5.8 (lower band). Adapted from Akpinar (1998) with permission from author.

Chin K. O. A., Johnsson M., Bergey E. J., Levine M. J., and Nancollas G. H. (1993) A constant composition kinetics study of the influence of salivary cystatins, statherin, amylase, and human serum albumin on hydroxyapatite dissolution. Coll. Surf. A Physicochem. Eng. Aspects 78, 229-234. 

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