Serpins in arthropod biology

Abstract

Serpins are the largest known family of serine proteinase inhibitors and perform a variety of physiological functions in arthropods. Herein, we review the field of serpins in arthropod biology, providing an overview of current knowledge and topics of interest. Serpins regulate insect innate immunity via inhibition of serine proteinase cascades that initiate immune responses such as melanization and antimicrobial peptide production. In addition, several serpins with anti-pathogen activity are expressed as acute-phase serpins in insects upon infection. Parasitoid wasps can downregulate host serpin expression to modulate the host immune system. In addition, examples of serpin activity in development and reproduction in Drosophila have also been discovered. Serpins also function in host-pathogen interactions beyond immunity as constituents of venom in parasitoid wasps and saliva of blood-feeding ticks and mosquitoes. These serpins have distinct effects on immunosuppression and anticoagulation and are of interest for vaccine development. Lastly, the known structures of arthropod serpins are discussed, which represent the serpin inhibitory mechanism and provide a detailed overview of the process.

Keywords: Development; Host-pathogen interactions; Innate immunity; Insect; Tick.

Fig. 1

The known physiological functions of serpins in arthropod physiology. The majority of insect serpins are produced in fat body and hemocytes and are then secreted into the hemocoel. In addition, serpins are expressed in a number of additional tissues, including tick and mosquito salivary glands (orange), midgut (green), trachea (blue), and in male accessory glands (MAGs), as well as the venom glands of parasitoid wasps (pink). Major known functions are listed and discussed in detail in the different sections of the manuscript.

Fig. 2

Outline of alternative splicing in insect serpins. (A) Structure of M. sexta serpin1K showing the alternatively spliced RCL region (red) with the P1-P1′ residue (yellow). (B) Simplified splice variant diagram in M. sexta 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 B–D, etc. results in expression of serpin1B, −1C, −1D, etc. (C) Simplified splice variant diagram in Bombyx mori 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 An. gambiae 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 Drosophila melanogaster 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.

Fig. 3

Saliva Serpins of Mosquitoes and Ticks Facilitate Blood Feeding on Mammals. (A) Mosquitoes insert their stylets into subcutaneous blood vessels, and obtain a blood meal within minutes. In contrast, ticks use their hypostome to rupture blood vessels causing blood to pool at the bite site, and then take a blood meal over the course of several days. Both ticks and some species of mosquitoes inject serpins as part of their saliva (shown in yellow) to counterbalance the mammalian host response to injury. These saliva serpins are inhibitors of several mammalian proteases required for coagulation, platelet aggregation, and inflammation. (B) Several factors of the intrinsic coagulation pathway (gray ovals) can be inhibited by either mosquito or tick serpins (yellow boxes, see Section 4.2). The mammalian serpins that target the same pathway are shown in comparison (blue boxes) [–158]. (C) The evolutionary history of tick and mammalian thrombin-inhibiting serpins does not indicate any orthologies between individual tick and human serpin pairs. The ability for serpins to inhibit thrombin has therefore evolved independently in the tick and human lineage. Phylogenetic analysis was performed using the Maximum Likelihood method based on the JTT matrix-based model [159]. The percentage of trees in which the associated taxa clustered together is shown next to the branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. All positions containing gaps and missing data were eliminated. All evolutionary analyses were conducted in MEGA7 [160]. (D) Alignment of thrombin-inhibiting arthropod and human serpins, reveals significant protein sequence similarity of their reactive center loops (RCL). Based the results of the phyogenetic analysis, this sequence similarity is likely the product of convergent sequence evolution. Alignment was executed using MUSCLE [161], and visualized in Jalview2[162]. Alboserpin, Ae. aegypti (Genbank accession number AAC31158); AT, human antithrombin(NP_000479.1); HCII, human heparin cofactor II (NP_000176.2); IRS-2, Ixodes ricinus (ABI94056); IxscS-1E1, Ixodes scapularis (KF990169); Rms-15, Rhipicephalus microplus (AHC98666); RmS-17, R. microplus (AHC98658); PCI, human Protein C Inhibitor (NP_000615.3); PN1, human Protease nexin 1 (NP_006207.1); ZPI, human Protein Z-dependent protease inhibitor.

Fig. 4

Structures of Insect Serpins. (A) Structure of M. sexta serpin1K(pdb code: 1SEK) [122], showing the conserved serpin fold. The serpin contains three β-sheets (βA, red; βB, blue; βC, yellow) surrounded by nine conserved helices (A-I, green). The Reactive Center Loop (RCL, teal) is located above βC. (B) The structure of M. sexta serpin1B A353K co-crystallized in a Michaelis-Menten complex with rat trypsin (pdb code: 1I99) [123]. The serpin1B RCL (orange) interacts with trypsin (pink) at the active site. (C) Close-up the structural alignment of M. sexta serpin1K (teal) and serpin 1B A353K (orange) RCLs interacting with trypsin (pink) indicating the movement of the serpin in response to interaction with trypsin. (D) Alignment of the hinge region of antithrombin (pink, pdb code: 1ATH) [138], T. molitor SPN48 (red, pdb code: 3OZQ) [35], An. gambiae SRPN2 (blue, pdb code: 3PZF) [125], and M. sexta serpin 1K (teal) demonstrating the partial hinge insertion found in antithrombin, SPN48 and SRPN2. (E) Alignment of the cleaved D. melanogaster Serpin 42 Da (pdb codes: 4P0O) [124] and I. ricinus IRS-2 (pdb code 3NDA) [108] structures indicating complete insertion of the RCL (magenta and purple, respectively), indicated by an arrow. Although neither protein was crystallized in an inhibitory complex with a proteinase, the presumed position of the inactivated proteinase in such a complex is marked with an asterisk. (F) Close-up of the RCL of B. moriserpin18(pdbcode: 4R9I) [126] and M. sexta serpin1K indicating the close proximity of the Serpin18 RCL to the serpin body compared to normal serine proteinase inhibitory serpins.