NORMAL GENOMIC

• Margis R, Reis EM, Villeret V
• Structural and phylogenetic relationships among plant and animal cystatins.
• Arch Biochem Biophys. 1998; 359: 24-30
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• The plant cystatins or phytocystatins (PhyCys) are cysteine proteinase inhibitors containing the QxVxG motif and have been placed in the cystatin superfamily of proteins. The primary sequences of PhyCys have a high degree of homology with the members of the cystatin family, but they resemble stefins by the absence of disulfide bonds and cysteine residues. A multialignment and a phylogenetic analysis of 63 cystatins, 32 of which are PhyCys, demonstrate that all PhyCys cluster in a major evolutionary tree branch and support the classification of PhyCys as a new cystatin family. The PhyCys also possess a specific consensus sequence [LVI]-[AGT]-[RKE]-[FY]-[AS]-[VI]-x-[EDQV]-[HYFQ] -N placed on the region corresponding to a predictable amino-terminal alpha-helix. This sequence can be used to specifically identify PhyCys on protein data banks and to differentiate them from the other members of the superfamily.

• Abe K
• [Oryzacystatin-oryzain relation: from biological dissection to applications]
• Tanpakushitsu Kakusan Koso. 1997; 42: 2348-54

• Abrahamson M
• Cystatins.
• Methods Enzymol. 1994; 244: 685-700

• Murzin AG
• Sweet-tasting protein monellin is related to the cystatin family of thiol proteinase inhibitors.
• J Mol Biol. 1993; 230: 689-94
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• The structure of the intensely sweet protein monellin, isolated from an African berry, and the structures of two thiol proteinase inhibitors, cystatin and stefin B, are found to be very similar. An alignment of sequences of monellin and the inhibitors, deduced from the structural comparisons, has been extended to include other members of the cystatin superfamily. There is a significant homology (up to 23% identical residues) with oryzacystatins, the only well defined plant cystatins. These results clearly indicate that monellin is a close relative of cystatins. Monellin and cystatins do not have the same sequence in the regions homologous to the cystatin active site. It is suggested here, however, that this region in monellin may be essential for a function in situ, because one of the loops comprising this part of the structure is found to be cleaved.

• Lalmanach G, Hoebeke J, Moreau T, Ferrer-Di Martino M, Gauthier F
• An immunochemical approach to investigating the mechanism of inhibition of cysteine proteinases by members of the cystatin superfamily.
• J Immunol Methods. 1992; 149: 197-205
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• Antibodies were raised against a synthetic dodecameric peptide KGAGQVVAGPWK (K12K), encompassing sequences thought to be important for the function of the cysteine proteinase inhibitors of the cystatin superfamily. These antibodies specifically recognized molecules of family 3, i.e., kininogens, in the serum of seven mammalian species tested in this study. The only notable exception was that of rat thiostatin (T kininogen) which is structurally related to the kininogen family. The antibodies also discriminated between family 2 (cystatins) and family 3 (kininogens) of the cystatin superfamily, since neither chicken cystatin nor human and rat cystatins C and S, which all belong to family 2 were recognized. The cystatin-like inhibitory domains resulting from fragmentation of human low molecular weight kininogen by bovine trypsin, were still recognized by antibodies, indicating that discrimination does not require two neighbouring inhibitory sites on the kininogen heavy chain. The antibodies blocked the capacity of kininogens to inhibit papain, suggesting that they recognize a conformational epitope at or near the kininogen inhibitory sites. The inhibitory properties of family 2 cystatins remained unchanged, confirming that members of this family do not interact with anti K12K antibodies. These antibodies are thus a new tool able to discriminate functionally and structurally between the members of the cystatin superfamily.

• Abe K, Kondo H, Watanabe H, Emori Y, Arai S
• Oryzacystatins as the first well-defined cystatins of plant origin and their target proteinases in rice seeds.
• Biomed Biochim Acta. 1991; 50: 637-41
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• Two cystatins occur in mature seeds of the rice, Oryza sativa L. japonica, which are named oryzacystatin I (OC-I) and oryzacystatin II (OC-II). These are highly homologous to each other and are significantly homologous to cystatin superfamily members of animal origin, especially to family-2 cystatins. However, both lack disulfide bonds as in the case of family-1 cystatins (stefins). Each of OC-I and OC-II thus seems to be chimerical of family-1 and family-2 cystatins, and we propose that a new category such as "phytocystatin" be opened for these cystatins of plant origin. For specificity it was observed that OC-I inhibits papain 100 times more efficiently than cathepsin H, whereas, OC-II inhibits cathepsin H 100 times more efficiently than papain. A cysteine proteinase, named oryzain alpha, exists in germinating rice seeds. cDNA cloning studies have disclosed that two other related species, named oryzain beta and gamma, are also present. In respect to the amino acid sequence, oryzain alpha and beta are homologous to papain and oryzain gamma is homologous to cathepsin H. These observations suggest the possibility that either or both of oryzain alpha and beta are target enzymes of OC-I and oryzain gamma is a target enzyme of OC-II.

• Saitoh E, Isemura S, Sanada K, Ohnishi K
• Cystatins of family II are harboring two domains which retain inhibitory activities against the proteinases.
• Biochem Biophys Res Commun. 1991; 175: 1070-5
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• Two cyclic peptides, Ac-CTKSQPNLDTC-NH2 (SA-LOOP1) and Ac-CSFQIYEVPWE DRMSLVNSRC-NH2 (SA-LOOP2) were prepared. These sequences are respectively found in the second and third exons of cystatin SA and are well conserved among the cystatins of family II. In addition, these sequences are extremely homologous to the inhibitory regions of several serine-proteinase inhibitors. The peptides were assayed for their inhibiting properties towards serine- and cysteine-proteinases. SA-LOOP1 inhibited porcine pancreatic trypsin (Ki = 370 microM), but did not inhibit cysteine-proteinases. SA-LOOP2 inhibited not only porcine pancreatic alpha-chymotrypsin (Ki = 23 microM) but also papain (Ki = 24 microM) and ficin (Ki = 52 microM). These data indicate that the exon-intron organization of the cystatin genes coinside with the structural and/or functional domains of the protein, and may have significant implications for understanding the active sites of cystatins.