|Budget Amount *help
¥2,100,000 (Direct Cost : ¥2,100,000)
Fiscal Year 1991 : ¥900,000 (Direct Cost : ¥900,000)
Fiscal Year 1990 : ¥1,200,000 (Direct Cost : ¥1,200,000)
Human saliva contains a series of cysteine-proteinase inhibitors which are Classified into family II of "Cystatin Superfamily". Up to date, three molecular species of salivary (S-type) cystatins (cystatins S, SA and SN) have been elucidated. The physiological roles of these inhibitors could be the protection of the cells from inappropriate proteolysis and the regulation of cysteine proteinases both of host and bacterial origin. They share about 55% sequence homology with cystatin C, which is abundant in synovial fluid, seminal plasma and cerebrospinal fluid. A variant of cystatin C(Leu^<68> ->Gln) is known to deposit as amyloid fibrils in patients with hereditary cystatin C amyloid angiopathy.
The synthesis of these four inhibitors is controlled by a multigene family leaving 6-7 members which is localized on human chromosome 20 - the cystatin gene family. From this gene family, five genes (named as the CST1, CST2, CST2B, CST3 and CST4) and two pseudogenes CSTP1 and CSTP2 (CST5) were, is
olated and characterized. The three genes (CST1, CST2 and CST4), respectively, code for cystatins SN, SA and S. The CST2B is an allele at the CST2 locus. The CST3 codes for cystatin C. The S-type cystatin genes (CST1, CST2 and CST4) which contain the ATA and CAT boxes in their 5' -flanking regions, are differentially regulated and the expression of the genes is more restricted than of the CST3 gene, for cystatin C. In contrast to the S-type cystatin genes, the cystatin C gene shares some properties with the promoter of house keeping genes : lacking of typical CAT box and the presence of the binding sites of transcription factor Sp 1.
The human cystatin genes sequenced here are composed of three exons encoding 76 or 81 (exon 1), 38 (exon 2) and 27 (exon 3) amino acids. These three-exon genes are closely related to an ancestral three-exon which generated contemporary nine exons coding for the kininogen heavy chain. Further analysis of the homology levels of the cDNA sequences of various proteinase inhibitors revealed that the second and third exons of the family II cystatin genes are significantly homologous with each other, and that DNA sequences of the two exons and exons 2, 3, 5, 6, 8 and 9 in the kininogen (family III cystatin) genes are extremely homologous to the CDNA sequencer encoding inhibitory domains of Bowman-Birk type serine-proteinase inhibitors. The kinetic study with synthetic peptides confirmed that the well conserved sequences in the protein domains encoded by the second and third exons of the family II cystatin gene possess inhibitory activities against the proteinase. Therefore it is evident that the exon-intron organization of the cystatin gene coincides with the structural and/or functional domains of the protein. These findings allowed us to conclude that the cystatin genes of families II and III have evolved by gene duplications from a common ancestral unit-length DNA sequence. Finally, we propose that cysteineproteinase inhibitors belonging to cystatin superfamily and serineproteinase inhibitors of Bowman-Birk family should be classified as the same family.