Sun J, Ooms L, Bird C H, Sutton V R, Trapani J A, Bird P I
Department of Medicine, Monash Medical School, Clive Ward Centre, Box Hill Hospital, Box Hill 3128, Australia.
J Biol Chem. 1997 Jun 13;272(24):15434-41. doi: 10.1074/jbc.272.24.15434.
Serine proteinase inhibitors (serpins) are classically regulators of extracellular proteolysis, however, recent evidence suggests that some function intracellularly. Such "ovalbumin" serpins include the human proteinase inhibitors 6 (PI-6), 8 (PI-8), and 9 (PI-9), plasminogen activator inhibitor 2, and the monocyte/neutrophil elastase inhibitor. PI-9 is a potent granzyme B (graB) inhibitor that has an unusual P1 Glu and is present primarily in lymphocytes. In a search for the murine equivalent of PI-9 we screened cDNA libraries, and performed reverse transcriptase-polymerase chain reaction on RNA isolated from leukocyte cell lines and from lymph nodes and spleens of allo-immunized mice. We identified 10 new ovalbumin serpin sequences: two resemble PI-8, two resemble PI-9, and the remaining six have no obvious human counterparts. By RNA analysis only one of the two sequences resembling PI-9 (designated SPI6) is present in mouse lymphocytes while the other (a partial clone designated mBM2A) is predominantly in testis. SPI6 comprises a 1.8-kilobase cDNA encoding a 374-amino acid polypeptide that is 68% identical to PI-9. mBM2A is 65% identical to PI-9 and over 80% identical to SPI6. Although the reactive loops of SPI6 and mBM2A differ from PI-9, both contain a Glu in a region likely to contain the P1-P1' bond. SPI6 produced in vitro using a coupled transcription/translation system formed an SDS-stable complex with human graB and did not interact with trypsin, chymotrypsin, leukocyte elastase, pancreatic elastase, thrombin, or cathepsin G. Recombinant SPI6 produced in a yeast expression system was used to examine the interaction with human graB in more detail. The second-order rate constant for the interaction was estimated as 8 x 10(4) M-1 s-1, and inhibition depended on the Glu in the SPI6 reactive center. The SPI6 gene was mapped to the same region on mouse chromosome 13 as Spi3, which encodes the murine homolog of PI-6. We conclude that even though their reactive centers are not highly conserved, SPI6 is a functional homolog of PI-9, and that the regulation of graB in the mouse may involve a second serpin encoded by mBM2A. Our identification of multiple sequence homologs of PI-8 and PI-9, and six new ovalbumin serpins, is consonant with the idea that the larger set of granule and other proteinases known to exist in the mouse (compared with human) is balanced by a larger array of serpins.
丝氨酸蛋白酶抑制剂（丝氨酸蛋白酶抑制剂家族）传统上是细胞外蛋白水解的调节因子，然而，最近的证据表明，其中一些在细胞内发挥作用。这类“卵清蛋白”丝氨酸蛋白酶抑制剂包括人类蛋白酶抑制剂6（PI-6）、8（PI-8）和9（PI-9）、纤溶酶原激活物抑制剂2以及单核细胞/中性粒细胞弹性蛋白酶抑制剂。PI-9是一种有效的颗粒酶B（graB）抑制剂，其P1位为谷氨酸，主要存在于淋巴细胞中。为了寻找与PI-9等效的小鼠蛋白，我们筛选了cDNA文库，并对从白细胞系以及同种免疫小鼠的淋巴结和脾脏中分离的RNA进行了逆转录聚合酶链反应。我们鉴定出10个新的卵清蛋白丝氨酸蛋白酶抑制剂序列：两个与PI-8相似，两个与PI-9相似，其余六个在人类中没有明显的对应物。通过RNA分析，两个与PI-9相似的序列中只有一个（命名为SPI6）存在于小鼠淋巴细胞中，而另一个（一个部分克隆命名为mBM2A）主要存在于睾丸中。SPI6包含一个1.8千碱基的cDNA，编码一个374个氨基酸的多肽，与PI-9的同源性为68%。mBM2A与PI-9的同源性为65%，与SPI6的同源性超过80%。尽管SPI6和mBM2A的反应环与PI-9不同，但两者在可能包含P1-P1'键的区域都含有一个谷氨酸。使用偶联转录/翻译系统在体外产生的SPI6与人graB形成了SDS稳定的复合物，并且不与胰蛋白酶、糜蛋白酶、白细胞弹性蛋白酶、胰腺弹性蛋白酶、凝血酶或组织蛋白酶G相互作用。在酵母表达系统中产生的重组SPI6被用于更详细地研究与人graB的相互作用。相互作用的二级速率常数估计为8×10⁴ M⁻¹ s⁻¹，抑制作用取决于SPI6反应中心的谷氨酸。SPI6基因被定位到小鼠13号染色体上与Spi3相同的区域，Spi3编码PI-6的小鼠同源物。我们得出结论，尽管它们的反应中心不是高度保守的，但SPI6是PI-9的功能同源物，并且小鼠中graB的调节可能涉及由mBM2A编码的另一种丝氨酸蛋白酶抑制剂。我们对PI-8和PI-9的多个序列同源物以及六个新的卵清蛋白丝氨酸蛋白酶抑制剂的鉴定，与这样一种观点一致，即已知小鼠（与人类相比）中存在的更大一组颗粒酶和其他蛋白酶是由更大的丝氨酸蛋白酶抑制剂阵列来平衡的。
