Molecular Genetic Study of the Cystatin Gene Family

人唾液中含有一系列半胱氨酸蛋白酶抑制剂，属于“胱抑素超家族”中的第II家族。迄今为止，已经鉴定了三种唾液（S型）半胱氨酸蛋白酶抑制剂（cystatins S，SA和SN）。这些抑制剂的生理作用可能是保护细胞免受不适当的蛋白水解和调节宿主和细菌来源的半胱氨酸蛋白酶。它们与在滑液、精浆和脑脊液中大量存在的胱抑素C共享约55%的序列同源性。CystatinC的一种变体（Leu^<68>-&gt;Gln）在遗传性CystatinC淀粉样血管病患者中以淀粉样纤维的形式存款，这四种抑制剂的合成受一个位于人类20号染色体上的多基因家族--CystatinC基因家族的控制，该家族共有6-7个成员。在该基因家族中，有5个基因（命名为CST 1、CST 2、CST 2B、CST 3和CST 4）和2个假基因CSTP 1和CSTP 2（CST 5）， ...更多信息 已鉴定和表征。这三个基因（CST 1、CST 2和CST 4）分别编码半胱氨酸蛋白酶抑制剂SN、SA和S。CST 2B是CST 2基因座处的等位基因。CST 3编码胱抑素C。在其5' -侧翼区含有ATA和CAT盒的S-型半胱氨酸蛋白酶抑制剂基因（CST 1、CST 2和CST 4）受到差异调节，并且对于半胱氨酸蛋白酶抑制剂C，所述基因的表达比CST 3基因的表达更受限制。与S型cystatin基因相比，cystatin C基因具有管家基因启动子的一些特点：缺少典型的CAT盒，存在转录因子Sp1的结合位点。这些三外显子基因与产生编码激肽原重链的当代九个外显子的祖先三外显子密切相关。对各种蛋白酶抑制剂的cDNA序列的同源性水平的进一步分析显示，家族II半胱氨酸蛋白酶抑制剂基因的第二和第三外显子彼此显著同源，并且这两个外显子和外显子2、3、5、6的DNA序列，激肽原中的8和9（家族III半胱氨酸蛋白酶抑制剂）基因与编码Bowman-Birk型丝氨酸蛋白酶抑制剂的抑制结构域的cDNA测序仪极其同源。合成肽的动力学研究证实，家族II胱抑素基因的第二和第三外显子编码的蛋白质结构域中的高度保守的序列具有对蛋白酶的抑制活性。因此，很明显，半胱氨酸蛋白酶抑制剂基因的外显子-内含子组织与蛋白质的结构和/或功能域一致。这些发现使我们能够得出结论，家族II和III的胱抑素基因是通过基因复制从共同的祖先单位长度DNA序列进化而来的。最后，我们建议，属于cystatin超家族的半胱氨酸蛋白酶抑制剂和属于Bowman-Birk家族的丝氨酸蛋白酶抑制剂应归为同一家族。少

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 … More 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. Less