The first GNBP was identified in Bombyx mori through a search for insect haemolymph proteins with properties similar to those of the mammalian LPS-binding protein CD14. This GNBP has significant homology to polysaccharide-binding motifs of bacterial P-1,3-1,4-glucanases, has been shown to have strong affinity for the cell wall of Gramnegative bacteria (Lee et al, 1996), and has been found to be upregulated following bacterial challenge. Although GNBP was isolated as a soluble protein from haemolymph, the hydrophobic nature of the C-terminal portion of the molecule, together with the existence of a putative glycosylphosphati-dylinositol anchor site, suggest that a membrane-bound form of this protein may exist.
Other GNBP homologues with a conserved ß-1,3-glucan-binding domain were later identified in D. melanogaster, A. gambiae, and Aedes aegypti (Dimopoulos et al., 1997; Richman et al., 1997; Kim et al., 2000; Christophides et al., 2002; Waterhouse et al, 2007). The A. gambiae GNBPs were shown to be responsive not only to bacterial infection but also to Plasmodium infections (Dimopoulos et al., 1998; Tahar et al, 2002; Rosinski-Chupin et al, 2007; Warr et al., 2008).
Of the three D. melanogaster GNBPs, DGNBP1 has been shown to have a high affinity for micro-bial immune elicitors such as LPS and ß-1,3-glucan, suggesting that it functions as a PRR (Kim et al., 2000). Over-expression of DGNBP1 in Drosophila immunocompetent cells led to an enhanced LPS-and ß-1,3-glucan-induced expression of nuclear factor KB-dependent antimicrobial peptide genes, and this induction could be specifically blocked by an anti-DGNBP1 antibody. These results pointed to a role for DGNBP1 as a PRR for LPS and ß-1,3-glucan that could activate the Toll immune-signalling pathway, leading to the induction of antimicrobial peptide genes (Kim et al., 2000).
The A. gambiae genome harbours six GNBP genes that fall into two distinct sequence groups, together with their known moth and fruit fly homologues (see Figure 5.1 for GNBP domain organization) (Christophides et al., 2002; Waterhouse et al, 2007). Subfamily A includes all known fruit fly and moth GNBPs, as well as two mosquito GNBPs (AgGNBPAl and A2). The GNBPA2 gene of Anopheles and the GNBP3 Drosophila gene are orthologues. The new subfamily B, which is mosquito-specific (AgGNBPB1-B4), has three of its four members tightly clustered in chromosomal subdivision 13E (Christophides et al, 2002). All six members of the A. gambiae GNBP protein family have a signal peptide sequence at the N-terminal end. Three A. gambiae GNBPs (AgGNBPB1, B2, and B4) contain putative glycosylphosphatidylinositol (GPI)-anchor sequences, and three other A. gam-biae GNBPs (AgGNBPA1, B1, and B3) have several potential N-linked glycosylation sites, suggesting that GNBPs are cell-surface molecules or secretory proteins that are involved in cell-cell adhesion or recognition (Warr et al, 2008). However, it is to be noted that AgGNBPA2 has neither the GPI-anchor nor N-linked glycosylation sites. Several studies have provided detailed information on the transcription of mosquito GNBP in various tissues: AgGNBPBl is mainly expressed in the thorax and salivary gland and to a lesser extent in other tissues (Dimopoulos et al, 1997, 1998, 2000). All six AgGNBP transcripts display higher levels of expression in the posterior region of the female midgut than in the cardia itself (Warr et al, 2008). At the protein level, the AgGNBPB4 protein is more abundant in thorax, fat-body tissue, and abdomen than in the head or midgut compartments (Warr et al, 2008).
AgGNBP mRNAs have been shown to be induced in the midgut, carcass, abdomen, and salivary gland in response to infection with various bacteria
ID AgGNBPBl, B2, B4
— Amino acid backbone  GPI-anchor site
■ Signal peptide □ P-1,3-Glucan-binding domain
Figure 5.1 The domain organization of the GNBP protein family of A. gambiae is shown; the thin horizontal black bar indicates the length of each protein while boxes indicate specific domains. Subfamily A includes three GNBPs (AgGNBPAl, A2, and B3) and subfamily B also includes three GNBPs (AgGNBPBl, B2, and B4). GPI, glycosylphosphatidylinositol.
(Gram-positive and Gram-negative) or Plasmodium (Richman et al, 1997; Dimopoulos et al, 1998; Tahar et al, 2002; Dong et al, 2006a; Rosinski-Chupin et al, 2007; Warr et al, 2008). Gene silencing of GNBPA2 had the strongest effect on P. falciparum infection, while GNBPB3 and GNBPB4 silencing had the strongest effect on permissiveness with regard to P. berghei infection (Warr et al, 2008). These results suggest that the GNBP family members show differences in the specificity of their interactions with various microbes and in their defensive activity against those microbes.
As has previously been shown for the D. mela-nogaster DGNBP1, the mosquito GNBPB4 regulates the expression of a number of immune genes (Gambicin, Defensinl, Cecropin 3, LRIM1, CLIPB14, and PGRPLC3) (Warr et al, 2008). The bias in this immune gene regulation toward the Imd pathway has suggested that GNBPs may be playing a dual role in activating both the Toll and Imd pathways in response to different microbial challenges.
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