Extracelluar serine protease cascades in animals are often regulated by proteins of approximately 45 kDa known as serpins, which are specific serine protease inhibitors (Silverman et al., 2001). Serpins form covalent complexes with target proteases. The C-terminal region of the serpin is an extended loop that serves as bait for the target protease. Inhibitory selectivity depends on the sequence and conformation of the reactive site loop. The protease binds to the loop and cleaves a specific bond between residues designated P1 and P1'. The P1 residue of the serpin fits into the primary substrate-specificity pocket of the protease and is particularly important in determining the selectivity of a serpin for protease inhibition (Yu et al., 2001). Upon cleavage, the serpin undergoes a major rearrangement, inserting its reactive-site loop into one of its P-sheets and moving the protease with it about 70 A, distorting the protease active site (Whisstock and Bottomley, 2006). The covalent ester linkage between the protease and serpin remains intact, because it is not accessible to water for completion of the hydrolysis reaction (Dementiev et al., 2006).
Serpins from insects were first identified in the lepidopterans, B. mori and M. sexta (Kanost, 1999). They regulate melanization and Toll cascades in D. melanogaster (Reichhart, 2005) and influence activation of melanization in Anopheles gambiae
(Michel et al, 2006). Serpins in M. sexta have been reviewed recently (Kanost, 2007). M. sexta has at least seven serpin genes, but the overall number of functional serpins is higher due to alternative splicing of the ninth exon of serpin-1, yielding 12 serpin-1 isoforms with different reactive-site loops and therefore different protease selectivities (Jiang et al., 1996; Jiang and Kanost, 1997). Serpin-1 is expressed in feeding larvae, but not during larval or pupal moults, and overall protein level is unchanged after immune challenge (Kanost et al., 1995). In response to bacterial challenge, ser-pins-3, -4, -5, and -6 are upregulated in the fat body; serpins-4, -5, and -6 are also upregulated in haemo-cytes (Tong and Kanost, 2005; Zhu et al., 2003b; Zou and Jiang, 2005). Serpin-3 inhibits all three PAPs, while serpin-6 specifically inhibits PAP3 (Zhu et al., 2003b; Wang and Jiang, 2004b). Serpin-1 isoform J also inhibits PAP3 (Jiang and Kanost, 1997). Serpin-protease complexes in plasma have been identified: serpin-4 complexes with HP1, HP6, and HP21, while serpin-5 complexes with HP1 and HP6 (Tong et al., 2005). Addition of recombinant serpins-1J, 3, 4, 5, and 6 to plasma can diminish proPO activation, indicating that a protease they inhibit functions involved in proPO activation. Serpin-4 and serpin-5 inhibit proPO activation but do not inhibit PAPs, consistent with the conclusion that HP6 is involved in a serine protease cascade that leads to proPO activation. Another target of serpin-4, HP21, has a known function in proPO activation through activation of PAP2 and PAP3, as discussed above. HP1 and/or HP6 are apparently part of an additional PAP-activation pathway or a pathway involved in SPH cleavage. Functions for the remaining serpin-1 isoforms and alternative functions for the other extracellular serpins are topics of current investigation.
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