Full-length heparin did significantly accelerate SPN48 inhibition of SPE, though lower weight heparin molecules did not. detailed overview of the process. [8,9], and amino acid sequences confirmed that the and inhibitors were serpins [8,10]. Serpin sequences have now been identified in many arthropod transcriptomes and genomes, with 30C40 serpin genes in many species, including 34 in [11], 32 in (M. Kanost, unpubished data), 31 in a beetle, [12], 29 in species [13]. Other species have significantly fewer serpin genes, including just 7 in the honeybee, [14] and 10 in the tsetse [15]. Mosquito species vary from 18 serpin genes in [16]. Ticks and mites also have considerable variation in the serpin gene content of their genomes, with 45 serpin genes in the blacklegged tick [17], 22 in the cattle tick [18], and only 10 in the scabies mite, [19]. Besides gene duplication, the number of unique serpins encoded by a given arthropod genome can also be increased post-transcriptionally. Some insect serpin genes have a unique structure, in which mutually AQ-13 dihydrochloride exclusive alternate splicing of an exon that encodes the RCL results in production of several inhibitors with different inhibitory activities. This phenomenon was first observed in the gene for serpin-1, which contains 14 copies of its 9th exon [20] (M. Kanost, unpublished data). Each version of exon 9 encodes a different sequence for the carboxyl-terminal 39C46 residues, including the RCL (Fig. 2), and the resulting serpin variants inhibit a different spectrum of proteinases [21,22]. Orthologous serpin-1 genes from other lepidopteran species, with alternate exons in the same position as in serpin-1, Ncam1 have been identified [23C25]. The serpin-1 gene of SRPN10 [27] and in spn4 orthologs in multiple species [28] (discussed more in Section 2.3). Open in a separate window Fig. 2 Outline of alternative splicing in insect serpins. (A) Structure of serpin1K showing the alternatively spliced RCL region (red) with the P1-P1 residue (yellow). (B) Simplified splice variant diagram in serpin-1. Exons that are always expressed are shown in black and alternatively spliced exon 9 variants are colored. Depicted is the splicing diagram of serpin1A, wherein the A isoform of exon 9 is expressed. Expression AQ-13 dihydrochloride of BCD, results in expression of serpin1B, ?1C, ?1D, etc. (C) Simplified splice variant diagram in serpin-1. The solid line indicates expression of the isoform of exon 9, resulting in isoform 1. Expression of b and c exons results in isoforms 2 and 3, respectively. The dotted line depicts expression of both b and c exons, resulting in isoform 4 expression. (D) Simplified splice variant diagram in SRPN10. The solid line shows expression of the K isoform of exon 9 (KRAL isoform). Expression of R, F, and C exons results in RCM, FCM, and CAM isoforms, respectively. (E) Simplified splice variant diagram in Spn4. The expression of exon 1 results in Spn4B, D-F, and I isoforms, and expression of exon 2 results in Spn4A, G, H, J, and K isoforms. The solid line depicts the expression of Spn4B and the dotted line depicts expression of Spn4A. Expression of additional Spn4 isoforms arises from alternative splicing of exons 6, 7, and 8. 2. Biological functions of arthropod serpins in insect immunity Arthropods produce and secrete serpins into their hemolymph to regulate proteinase cascade pathways that amplify signals resulting from detection of pathogens, eliciting innate immune responses. Regulation of such pathways by serpins is an ancient aspect of immune system evolution, occurring in the hemolymph coagulation pathway of horseshoe crabs [29]. The following section will provide specific examples on how insect innate immunity is regulated by serpins. 2.1. Regulation of Toll pathway in hemolymph The Toll pathway for stimulation of gene expression, particularly of antimicrobial peptides, has been best characterized in and other species is a member of the clip domain serine proteinase family, which are serine proteinases with an amino-terminal clip domain, common as immune factors in arthropods [31,32]. Serpins that regulate Sp?tzle-processing proteinases and their upstream activating proteinases have been identified through biochemical studies in and a beetle HP6. Genetic experiments also implicate Spn1 as a regulator of an upstream proteinase in the Toll pathway [38]. 2.2. Regulation of proPO activation A prominent and broad spectrum arthropod innate immune response that is stimulated by serine proteinase cascade pathways is the activation of prophenoloxidase (proPO) in hemolymph. ProPO activation leads to synthesis of melanin,.Two insect serpin structures were determined containing a partial insertion of the hinge region: SPN48 (pdb code: 3OZQ) [35] and SRPN2 (pdb code: 3PZF) [125]. 30C40 serpin genes in many species, including 34 in [11], 32 in (M. Kanost, unpubished data), 31 in a beetle, [12], 29 in species [13]. Other species have significantly fewer serpin genes, including just 7 in the honeybee, [14] and 10 in the tsetse [15]. Mosquito species vary from 18 serpin genes in [16]. Ticks and mites also have considerable variation in the serpin gene content of their genomes, with 45 serpin genes in the blacklegged tick [17], 22 in the cattle tick [18], and only 10 in the scabies mite, [19]. Besides gene duplication, the number of unique serpins encoded by a given arthropod genome can also be increased post-transcriptionally. Some insect serpin genes have a unique structure, in which mutually exclusive alternate splicing of an exon that encodes the RCL results in production of several inhibitors with different inhibitory activities. This phenomenon was first observed in the gene for serpin-1, which contains 14 copies of its 9th exon [20] (M. Kanost, unpublished data). Each version of exon 9 encodes a different sequence for the carboxyl-terminal 39C46 residues, including the RCL (Fig. 2), and the resulting serpin variants inhibit a different spectrum of proteinases [21,22]. Orthologous serpin-1 genes from other lepidopteran species, with alternate exons in the same AQ-13 dihydrochloride position as in serpin-1, have been identified [23C25]. The serpin-1 gene of SRPN10 [27] and in spn4 orthologs in multiple species [28] (discussed more in Section 2.3). Open in a separate window Fig. 2 Outline of alternative splicing in insect serpins. (A) Structure of serpin1K showing the alternatively spliced RCL region (red) with the P1-P1 residue (yellow). (B) Simplified splice variant diagram in serpin-1. Exons that are always expressed are shown in black and alternatively spliced exon 9 variants are colored. Depicted is the splicing diagram of serpin1A, wherein the A isoform of exon 9 is expressed. Expression of BCD, results in expression of serpin1B, ?1C, ?1D, etc. (C) Simplified splice variant diagram in serpin-1. The solid line indicates expression of the isoform of exon 9, resulting in isoform 1. Appearance of b and c exons leads to isoforms 2 and 3, respectively. The dotted series depicts appearance of both b and c exons, leading to isoform 4 appearance. (D) Simplified splice version diagram in SRPN10. The solid series shows expression from the K isoform of exon 9 (KRAL isoform). Appearance of R, F, and C exons leads to RCM, FCM, and CAM isoforms, respectively. (E) Simplified splice version diagram in Spn4. The appearance of exon 1 leads to Spn4B, D-F, and I isoforms, and appearance of exon 2 leads to Spn4A, G, H, J, and K isoforms. The solid series depicts the appearance of Spn4B as well as the dotted series depicts appearance of Spn4A. Appearance of extra Spn4 isoforms comes from choice splicing of exons 6, 7, and 8. 2. Biological features of arthropod serpins in insect immunity Arthropods generate and secrete serpins to their hemolymph to modify proteinase cascade pathways that amplify indicators resulting from recognition of pathogens, eliciting innate immune system responses. Legislation of such pathways by serpins can be an ancient facet of immune system progression, taking place in the hemolymph coagulation pathway of horseshoe crabs [29]. The next section provides specific examples on what insect innate immunity is normally governed by serpins. 2.1. Legislation of Toll pathway in hemolymph The Toll pathway for arousal of gene appearance, especially of antimicrobial peptides, continues to be greatest characterized in and various other types is normally a member from the clip domains serine proteinase family members, that are serine proteinases with an amino-terminal clip domains, common as immune system elements in arthropods [31,32]. Serpins that regulate Sp?tzle-processing proteinases and their upstream activating proteinases have already been identified through biochemical research in and a beetle HP6. Genetic experiments implicate also.