Serine protease-related proteins in the malaria mosquito, Anopheles gambiae

Abstract

Insect serine proteases (SPs) and serine protease homologs (SPHs) participate in digestion, defense, development, and other physiological processes. In mosquitoes, some clip-domain SPs and SPHs (i.e. CLIPs) have been investigated for possible roles in antiparasitic responses. In a recent test aimed at improving quality of gene models in the Anopheles gambiae genome using RNA-seq data, we observed various discrepancies between gene models in AgamP4.5 and corresponding sequences selected from those modeled by Cufflinks, Trinity and Bridger. Here we report a comparative analysis of the 337 SP-related proteins in A. gambiae by examining their domain structures, sequence diversity, chromosomal locations, and expression patterns. One hundred and ten CLIPs contain 1 to 5 clip domains in addition to their protease domains (PDs) or non-catalytic, protease-like domains (PLDs). They are divided into five subgroups: CLIPAs (22). are clip1–5-PLD; CLIPBs (29), CLIPCs (12) and CLIPDs (14) are mainly clip-PD; most CLIPEs (33) have a domain structure of PD/PLD-PLD-clip-PLD0–1. While expression of the CLIP genes in group-1 is generally low and detected in various tissue- and stage-specific RNA-seq libraries, some putative GPs/GPHs (i.e. single domain gut SPs/SPHs) in group-2 are highly expressed in midgut, whole larva or whole adult libraries. In comparison, 46 SPs, 26 SPHs, and 37 multi-domain SPs/SPHs (i.e. PD/PLD-PLD≥1) in group-3 do not seem to be specifically expressed in digestive tract. There are 16 SPs and 2 SPH containing other types of putative regulatory domains (e.g. LDLa, CUB, Gd). Of the 337 SP and SPH genes, 159 were sorted into 46 groups (2–8 members/group) based on similar phylogenetic tree position, chromosomal location, and expression profile. This information and analysis, including improved gene models and protein sequences, constitute a solid foundation for functional analysis of the SP-related proteins in A. gambiae.

The CLIP-domain serine protease CLIPC9 regulates melanization downstream of SPCLIP1, CLIPA8, and CLIPA28 in the malaria vector Anopheles gambiae

“…Moreover, the depletion of full-length CLIPC9 240 min post-E. coli challenge was not evident under non-reducing conditions suggesting that the disulfide-linked p30 and p12 fragments comprise the full-length protein. While amino acid sequencing of the p30 and p12 fragments will be necessary to fully characterize CLIPC9 limited proteolysis, our data support a cleavage event at or near the cut site predicted by Cao et al [51]. We note faint cleavage products in naïve and PBS injected samples on extended blot exposure, suggesting low levels of basal and injury-associated CLIPC9 cleavage ( Fig 6B).…”

“…CLIPC9 is required for melanization in Anopheles gambiae p12 fragment was consistently apparent following extended blot exposures and is shown to the right of the compilation. These fragment sizes are comparable to the 27.3 and 8.4 kDa products expected to be generated following CLIPC9 cleavage at its predicted cut site [51]. The p30 and p12 fragments were apparent at both 60 and 240 min post-challenge, with the latter timepoint characterized by the depletion of full-length CLIPC9.…”

“…CLIPC2 was excluded due to poor silencing efficiency, and we were unable to generate dsRNA against CLIPC5. Following the inception of this project, 3 additional CLIPCs were identified (CLIPC12-14) which were not included in this screen and CLIPC7 was reclassified as a CLIPE [51]. Notably, CLIPC9 was the only screened candidate with a significant effect on MelASA, as dsCLIPC9 treatment reduced total spot area 3.7-fold 12 h after E. coli challenge relative to control (Fig 3A).…”

“…TEP1 cut opsonization drives a convertase mechanism that amplifies TEP1 cut accumulation from circulating TEP1-Full and ultimately promotes microbial melanization [4,[45][46][47][48][49][50]. SPCLIP1 (also called CLIPA30) localizes to microbial surfaces in a TEP1-dependent manner and positively regulates melanization by promoting TEP1 convertase activity [46,51]. SPCLIP1 is also required for the cleavage-induced activation of CLIPA8 and CLIPA28, which collectively form the core of a sequentially activated CLIP-SPH cascade essential for melanization [46,52,53].…”

Identification of Immune Response to Sacbrood Virus Infection in Apis cerana Under Natural Condition

“…Serine proteases were significantly inhibited by CSBV infection. SPs with 60-400 members in insects form a large family of enzymes that hydrolyze peptide bonds at different rates (Rawlings and Barrett, 1993;Cao et al, 2017). SPs and SPHs, especially extracellular SPs, have a great influence on insect development and innate immunity (Ahola et al, 2015;Dudzic et al, 2019).…”