Elsevier

Gene

Volume 251, Issue 1, 13 June 2000, Pages 9-17
Gene

Sp22D: a multidomain serine protease with a putative role in insect immunity

https://doi.org/10.1016/S0378-1119(00)00181-5Get rights and content

Abstract

Serine proteases play critical roles in a variety of insect immune responses; however, few of the genes that code for these enzymes have been cloned. Here, we describe the molecular characterization of a serine protease gene from the mosquito Anopheles gambiae. Sp22D codes for a 1322 amino acid polypeptide with a complex domain organization. In addition to the carboxy terminal serine protease catalytic domain, Sp22D contains two putative chitin binding domains, a mucin-like domain, two low density lipoprotein receptor class A domains, and two scavenger receptor cysteine rich domains. A typical signal peptide sequence and a lack of potential transmembrane helices suggest that Sp22D is secreted. Sp22D is expressed constitutively in three immune-related cell types: adult hemocytes, fat body cells, and midgut epithelial cells. Wounding induces no changes in transcript abundance, but within 1 h after injection of bacteria, Sp22D mRNA increases 1.5-fold. Based on domain organization, tissue distribution, and transcriptional up-regulation in response to immune challenge, we suggest that Sp22D has an immune function. In addition, we predict that Sp22D is secreted into the hemolymph where it may interact with pathogen surfaces and initiate an immune response.

Introduction

Serine proteases play critical roles in a variety of invertebrate immune processes, mainly in the activation of other proteins by site-specific cleavage. Examples of serine protease mediated defense responses include hemolymph (blood) coagulation, activation of antimicrobial peptide synthesis, and melanin synthesis. Unfortunately, despite the importance of these enzymes in invertebrate immunity, few of their genes have been cloned.

Decidedly the best understood invertebrate defense response is hemolymph coagulation in the horseshoe crab (Iwanaga et al., 1998). This immune response is initiated in the presence of two types of pathogen surface molecules, the bacterial lipopolysaccharides (LPS) and the fungal β-1,3-glucans. LPS is bound by Factor C, a protein with both an LPS binding region and a serine protease catalytic domain. LPS binding causes a conformational change in Factor C, which undergoes autoactivation. Activated Factor C cleaves a second serine protease, Factor B, which then cleaves a third serine protease, proclotting enzyme (PCE). PCE cleaves coagulogen to form coagulin, which self-aggregates to create a gel-like clot that can trap pathogens. PCE is also activated by Factor G, whose serine protease subunit becomes activated after another subunit binds to β-1,3-glucan.

An insect defense response that has been under intense study recently is the activation of antimicrobial peptide synthesis in Drosophila. The best characterized of the antimicrobial peptide pathways is one that controls synthesis of drosomycin, an antifungal agent (Imler and Hoffmann, 2000). The presence of certain pathogens somehow activates an unknown, extracellular serine protease. This protease activates Spätzle, a ligand for the transmembrane receptor, Toll. A signal transduction cascade follows Toll activation, and ends with the up-regulation of the drosomycin gene. Surprisingly, this pathway is related to the embryonic dorsal–ventral patterning pathway, in which a protease cascade ends with the activation of Spätzle by Easter. The drosomycin pathway and the horseshoe crab coagulation pathways share some interesting similarities: Spätzle is structurally similar to coagulogen, and Factor B, PCE, and Easter are members of the clip domain family of serine proteases (Jiang and Kanost, 2000).

A third example of an insect immune pathway involving serine proteases is the synthesis of melanin (Ashida and Brey, 1997, Jiang and Kanost, 2000). Melanin formation is typically associated with cellular encapsulation of large pathogens, but melanization is its own form of encapsulation in insects, including mosquitoes, that have small numbers of circulating hemocytes (blood cells). Pathogen recognition molecules that can induce melanization have been discovered in the hemolymph of two large moth larvae, Bombyx mori and Manduca sexta, but unlike the recognition molecules of the horseshoe crab coagulation pathways, they are not serine proteases. Recognition is followed by the activation of a protease cascade, and the last protease in the cascade (the only one to be cloned) is a clip domain serine protease that activates prophenoloxidase, a principal enzyme in the melanin synthesis process.

Immune processes in Anopheles gambiae are of great interest because this mosquito is one of the major vectors of human malaria. The protozoan parasites that cause malaria (Plasmodium species) must survive for longer than a week in the body of the mosquito as they go through three developmental stages, and during this time, they must endure the various immune responses that the mosquito mounts against foreign invaders (Paskewitz and Gorman, 1999). Whether the presence of malaria parasites induces hemolymph coagulation is unknown, but antimicrobial peptide synthesis and melanotic encapsulation have been observed. Also of interest is how the mosquito responds to natural bacterial and fungal pathogens as well as those that may be used in biocontrol strategies to reduce mosquito-borne transmission of disease.

To identify immune-related serine proteases in Anopheles gambiae, we cloned serine protease cDNAs from adult hemolymph and further characterized the genes that appeared most interesting. Three are candidate activators of prophenoloxidase or Spätzle-like ligands, and a fourth is similar to an immune-related protease from M. sexta hemocytes (Gorman et al., 2000, Paskewitz et al., 1999). A much larger serine protease designated Sp22D was not fully characterized (Gorman et al., 2000). The Sp22D transcript is about 4.6 kb. It is expressed at all developmental stages but is much more abundant in adults. Based on semi-quantitative RT–PCR, Sp22D mRNA appears to be slightly up-regulated at about 1 h after immune challenge. The partial translated sequence of Sp22D (158 amino acids (aa)) was most similar to the sequence of Tequila, a gene with unknown function in Drosophila. This was an interesting finding, however, because the Tequila sequence contains a protein domain found in a vertebrate pathogen recognition molecule, the scavenger receptor cysteine rich (SRCR) domain. Since Sp22D is expressed constitutively in hemolymph, is up-regulated after immune challenge, and resembles Tequila, we considered it an interesting candidate immune gene meriting further study. In this report, we describe the complex domain organization of the complete Sp22D protein sequence, quantitative northern blot analysis of the effect of immune challenge, and the tissue specificity of Sp22D mRNA as determined by in situ hybridization.

Section snippets

Mosquito and bacteria stocks and immune challenge

A Plasmodium-susceptible strain (4arr) of A. gambiae was used for all experiments, and mosquitoes were reared as described previously (Paskewitz et al., 1999). Bacteria stocks used for immune challenge were the XL1-Blue strain of Escherichia coli (Stratagene) and the 2001 strain of Micrococcus luteus (gift from John Lindquist, University of Wisconsin). Liquid cultures of the bacteria were grown overnight, combined and centrifuged. Adult females (4 days post-eclosion) were immobilized by chilling

Sp22D coding sequence analysis

A partial Sp22D cDNA was used to screen a cDNA library, and the largest hybridizing clone was chosen for DNA sequencing (accession no. AF117751). An open reading frame coding for a 1322 aa polypeptide was identified (data not shown). The Sp22D protein has a complex domain organization (Fig. 1). A putative signal sequence at the N-terminus suggests that Sp22D is secreted, and a lack of transmembrane helices suggests that it is not an integral membrane protein. The signal sequence is followed by

Conclusions

Sp22D codes for a putative trypsin-like serine protease with a complex domain organization. Three characteristics suggest that it may play a role in immunity. First, most of its domains are similar to regions of immune-related proteins. Second, Sp22D transcript abundance increases after bacterial infection but not wounding. Third, Sp22D mRNA is present in hemocytes, fat body, and midgut epithelium, which are all tissues with important immune functions.

The domain organization of Sp22D is unlike

Acknowledgements

We are grateful to Peter Bryant, Tatsuhiro Shibata, Jules Hoffmann and Fotis Kafatos for sharing unpublished results regarding Tequila, GRAAL, and Sp22D. We thank John Lindquist for the stock of M. luteus. We regret that we were unable to cite all of the relevant primary literature due to a strict citation limitation. This work was supported by NIH Tropical Disease Research Unit grant AI28781.

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