Clip-domain serine proteases as immune factors in insect hemolymph

Abstract

CLIP proteases are non-digestive serine proteases present in hemolymph of insects and other arthropods. They are composed of one or more amino-terminal clip domains followed by a linker sequence and a carboxyl-terminal S1A family serine protease domain. The genes for CLIP proteases have evolved as four clades (CLIPA, CLIPB, CLIPC, CLIPD), each present as multigene families in insect genomes. CLIP proteases in hemolymph function in innate immune responses. These include proteolytic activation of the cytokine Spätzle, to form an active Toll ligand leading to synthesis of antimicrobial peptides, and specific activation of prophenoloxidase, required for the melanization response. CLIP proteases act in cascade pathways. In the immune pathways that have been characterized, microbial surface molecules stimulate activation of an initiating modular serine protease, which then activates a CLIPC, which in turn activates a CLIPB. The active CLIPB then cleaves and activates an effector molecule (proSpätzle or prophenoloxidase). CLIPA proteins are pseudoproteases, lacking proteolytic activity, but some can function as regulators of the activity of other CLIP proteases and form high molecular weight immune complexes. A few three dimensional structures for CLIP proteases are now available for structure-function analysis of these immune factors, revealing structural features that may act in specific activation or in formation of immune complexes. The functions of most CLIP proteases are unknown, even in well studied insect species. It is very likely that additional proteins activated by CLIP proteases and acting in immunity remain to be discovered.

Figure 1. Domain architecture of CLIP serine proteases

CLIP proteases contain one or more amino-terminal clip domains connected by a linker sequence to a carboxyl-terminal serine protease domain. The protease zymogen is activated by specific cleavage at the beginning of the catalytic domain. After this cleavage, the clip domain and protease domain remain connected by an interchain disulfide bond. CLIP proteases that have active sites with an intact catalytic triad (H, D, S) fall into three groups based on sequence alignments, known as clades B, C, and D. CLIPB proteases contain one or two amino-terminal clip domains from sequence type 2. CLIPB proteases include Manduca PAP1, PAP2, PAP3, HP8, Bombyx PPAE and BAEEase, Holotrichia PPAF1 and PPAF3, Tenebrio SPE, Drosophila SPE, MP1, MP2, easter, and Grass, Aedes IMP1, IMP2, TMP, B5, B29, B35, and Anopheles B4, B8, B9, B14. Manduca PAP2, PAP3, and Bombyx PPAE have two clip domains and two extra Cys residues in the linker (shown in light blue). CLIPC proteases, containing a single clip domain from group 1a include Manduca HP6 and Drosophila Persephone and Spirit. CLIPD proteases contain one clip domain from type 1b or 1c. At this time, there are no members of the CLIPD family with a known function. CLIPA pseudoproteases, known as serine protease homologs, have an amino terminal clip domain from type 3 and a protease-like domain in which the active site serine residue is changed to glycine, and therefore these proteins lack protease activity. CLIPA proteins are apparently activated by a specific cleavage in the clip domain. Manduca SPH1a, SPH2, and Anopheles CLIPA8 are examples of CLIPAs. (For simplicity, additional Cys residues in some of the linkers and protease domains are not indicated.)

Figure 2. Protease cascades in insect immune responses

Microbial stimuli lead to activation of CLIP proteases, organized in pathways that result in activation of proPO or proSpätzle. For protease names shown in boxes, genetic evidence indicates participation in an immune pathway, but the activating protease and the protease's substrate are not yet known. Dashed arrows indicate putative steps that have not been verified experimentally. The diagrams summarize data from the following: Aedes aegypti [55,56], Anopheles gambiae [36,45,57-59], Tenebrio molitor [24,29,60,61], Drosophila melanogaster [25-28,62-68], Manduca sexta [19,21-23,30,35,42,43,54,69-75] and unpublished results from the authors' laboratories.

Figure 3. Three dimensional representations of insect CLIP proteases

(A) Drosophila melanogaster Grass zymogen structure solved by x-ray crystallography (2XXL) [38]. The ribbon diagram shows the clip domain (cyan), linker (magenta), and the catalytic domain (yellow). Loops 30, 60, and 140 in the catalytic domain are shown in green; the signature loop 75 of CLIPBs is in gray; a calcium ion is shown as a gray sphere. Side chains of the Cys and catalytic residues (S, H and D) are colored red and blue, respectively. (B) Manduca sexta PAP2 structural model based on the NMR structure of its clip domains [41] and a homology model of the PAP2 catalytic domain. The first clip domain is in gray, the second clip domain is in cyan, and the rest of the molecule is colored using the same scheme described in (A). (C) Holotrichia diomphalis PPAF2 crystal structure (2B9L) [39]. In the PPAF2 structure, regions I, II and III (purple) of the protease-like domain interact with the clip domain (cyan). Region IV (gray) may interact with another protein. The amino-terminal extension (orange) and the linker (magenta), including the unstructured parts (dashed line) and the “interchain” disulfide bond, further stabilize the association of the clip domain with the protease-like domain.

Figure 4. Conservation of a protease cascade module

In pathways in which clip domain substrates have been determined experimentally, a conserved pattern is evident. A non-CLIP modular serine protease activates a penultimate CLIP protease from clade C with a type 1 clip domain, which activates a terminal CLIP protease from clade B with a type 2 clip domain, which then activates an effector protein.

Figure 5. Comparison of insect CLIP proteases known to participate in specific pathways

The phylogenetic tree is based on an alignment of the protease domain sequences, with horseshoe crab proclotting enzyme (Limulus PCE) as an outgroup. Numbers at branches indicate bootstrap value, as a percent of 1000 repetitions. There are two clades that correspond with groups of proteases that are either the terminal protease in a known pathway (CLIPB proteases) or the penultimate proteolytic step of a pathway (CLIPC protease, labeled in red). The tree derived from the protease domain sequences correlates with the type of associated N-terminal clip domain, with terminal proteases having type 2 clip domains and penultimate proteases having type 1a clip domains. The activation site P1 residue is the amino acid residue (determined experimentally or predicted based on sequence alignment) on the amino-terminal side of the peptide bond that is cleaved to activate the protease zymogen.