The fibrinogen domain immunolectin FBN gene family

Members of the FBN gene family, also known as fibrinogen-related proteins (FREPs), share a fibrinogen-like domain (FBG) that is evolutionarily conserved and extends from invertebrates to mammals (Gokudan et al., 1999; Fujita, 2002; Wang et al., 2005). In mammals, fibrinogen participates in both the cellular and fluid phases of coagulation. This soluble plasma protein is composed of six polypep-tide chains, two each of the Aa, Bp, and y chains (Gorkun et al, 1997). The FBG domain consists of approximately 200 amino acid residues and shows high sequence similarity to the C-termini of the fibrinogen p and y chains. FBG domains have been widely identified and well defined in mammals and invertebrates (Gorkun et al, 1997; Kairies et al, 2001; Fujita, 2002; Zhang et al, 2004).

In mammals, three distinct FBN proteins have been identified, Of these, the ficolins are the most important and have been identified in many vertebrate species, including human, rodent, pig, hedgehog, and Xenopus, as well as in the ascidians (Urochordata; reviewed by Fujita, 2002). Ficolins have been seen to participate in both phagocytosis and complement activation and to act as PRRs as part of the innate immune system (Erickson, 1993; Miller et al, 1993; Kobayashi et al, 1994; Lu and Le, 1998; Teh et al, 2000; Fujita, 2002; Lu et al, 2002; Matsushita and Fujita, 2002; Endo et al., 2007). Ficolins are a group of lectins that are each composed of an FBG domain attached to collagenous domain at the N-terminus. All of the mammalian FBG domain proteins contain a common pathogen-binding FBG domain at their C-terminus, while the N-terminal sequences vary from one organism to another; the FBG domain of ficolins is involved in binding N-acetyl-D-glucosamine (GlcNAc) and other sugars; this activity resembles that of the carbohydrate-recognition domain (CRD) of C-type lectins and provides evidence for a PRR role for these molecules (Miller et al, 1993; Lu and Le, 1998; Lu et al, 2002; Endo et al, 2007). L-Ficolin has a globular structure similar to that of a CRD, a bouquet structure that is composed of 12 fibrinogen-like domain subunits that form a tetramer consisting of four triple helices produced through multimeriza-tion of collagen-like domains. Thus, multimeriza-tion of the N-termini of these molecules may help the FREPs to form multimeric protein bundles with potentially increased affinity and specificity for particular pathogens (Fujita, 2002).

Several different FREPs have been described in various species of invertebrates, with the earliest described being two tachylectins (TL5A and -5B) from the horseshoe crab Tachypleus tridentatus (Gokudan et al., 1999). Structural and functional characterization of TL5A has revealed its ability to specifically recognize acetyl-group-containing substances, such as GlcNAc, including non-carbohydrates; it is capable of agglutinating all types of human erythrocytes and Gram-positive and Gram-negative bacteria (Gokudan et al., 1999). Therefore, TL5A probably functions as a host defence protein on the front line. Both TL5A and -5B have similar fibrinogen-like structures, but they lack the typically collagen-like domain of ficolins at their N-terminus. Solution of the structure of TL5A within the GlcNAc-TL5A complex at 2.0 A resolution has yielded insights into the lec-tin activity of TL5A and the evolutionary relationship of TL5A to fibrinogen y chains (Kairies et al, 2001). Four aromatic side chains (Tyr-210, Tyr-236, Tyr-248, and His-220) form a funnel ligand-binding pocket specifically for the acetyl group.

Sequence and structural alignments of the fibrinogen y fragment, ficolins, and TL5A have demonstrated that the overall three-dimensional structure, Ca2+-binding site, and acetyl group ligand-binding pocket are essentially conserved.

Human ficolin is more closely related to TL5A and TL5B than to the fibrinogen y chain, emphasizing the fact that tachylectins have functions that are more closely related to innate immunity than to anti-coagulation; moreover, this relationship indicates the high degree of similarity between mammalian and invertebrate innate immunity. Electron microscopic analysis of negatively stained TL5A and TL5B has provided high-resolution images of their oligomeric structures and has demonstrated that TL5A and TL5B form a three- or four-bladed and a two-bladed propeller structure, respectively. Each blade corresponds to a dimer formed through inter- and intrachain disulphide linkages involving conserved cysteine residues (Gokudan et al, 1999).

As a result of the availability of the full gen-omic sequences of D. melanogaster, A. gambiae, and A. aegypti, and more recently of 12 distinct species of Drosophila sequences, genome-wide comparisons of FBG domains from various species have identified FREP gene families in all these organisms (Waterhouse et al, 2007; Middha and Wang, 2008). There is a significant expansion in the FREP gene family in A. gambiae, which has 61 members; in contrast, the corresponding gene family in A. aegypti has 37 members, and that in D. melanogaster has only 14 members. The FREP gene family exhibits species-specific expansion in A. gambiae, with only three orthologous pairs having been identified in this species. This finding is consistent with a previous prediction that FBG domains may function by binding to pathogens as part of the host's immune response (Dimopoulos et al, 2000, 2001; Christophides et al, 2002; Wang et al, 2005; Waterhouse et al, 2007; Y. Dong and G. Dimopoulos, unpublished results).

Of the putative immune gene families that have been identified in A. gambiae, the FBN gene family is one of the largest, and phylogenetic analysis indicates that the gene expansion of the FBN gene family occurred after divergence of this species from others, in agreement with the hypothesis that recent gene duplications have occurred more often in Anopheles than in D. melanogaster (see Figure 5.5 for FBN domain organization) (Christophides et al, 2002). The predicted structures of the FBNs are closely related to that of TL5A, and the acetyl-group-binding sites, Ca2+-binding sites, and cyst-eine residues involved in disulphide linkages are all conserved (Wang et al., 2005; Y. Dong and G. Dimopoulos, unpublished results). Correlation of sequence data with the chromosomal location of the FBG domains within the FBN gene family proteins has suggested that the expansion of the FBN gene family in A. gambiae is mainly accounted for by a major expansion of the FBG domains, and both tandem duplication and shuffling have been involved in this expansion. There is a strong correlation between phylogeny, chromosomal location, and the expression pattern of FBN genes in A. gambiae, pointing to conserved functions among the duplicated family members (Y. Dong and G. Dimopoulos, unpublished results).

Microarray-hybridization-based transcriptomic analysis has showed that FBN gene family members in A. gambiae are involved in the mosquito's

100 amino acids FBN9

■■■■■■■■■■■■■■ ^^H h Other FBNs

Amino acid backbone I I Fibrinogen domain I"! Disulphide bridge

Figure 5.5 The domain organization of the fibrinogen domain immunolectin (FBN) gene family in A. gambiae. All the members of the FBN family contain a fibrinogen domain and the disulphide bridge is conserved in the majority of members. FBN9 represents the member without a signal peptide, and majority of the proteins, like FBN25, contain a signal peptide. The dotted line indicates the variation of the lengths of different FBN proteins.

immune response to both bacteria and malaria parasites. RNAi-mediated gene-silencing assays have indicated that FBN8, FBN9, and FBN39 are involved in the anti-Plasmodium defence, with FBN39 showing specificity in regulating the mosquito's resistance only to the human malaria parasite, P. falciparum (Dimopoulos et al, 2000, 2001; Christophides et al, 2002; Dong et al, 2006a). In a more recent study, the functions of 38 members of the FBN gene family in A. gambiae have been characterized, and their involvement in antimicrobial and anti-Plasmodium activity has been corroborated by RNAi-mediated gene silencing, which has revealed that FBN has complementary and syner-gistic activities mediated by the association of different members of the gene family. FBN9 interacts with both Gram-negative and Gram-positive bacteria and is strongly colocalized with both rodent and human malaria parasites in the mosquitoes' midgut epithelium. Interestingly, in vitro bacterial binding assays with FBN9 antibody shows that FBN9 appears to form dimers and specifically bind to the bacterial surfaces with different affinity, suggesting that FBN might use a multimerization mechanism to form homo- or hetero-multimers as a means of increasing the mosquito's PRR repertoire, but the molecular basis for this mechanism still remains unknown (Y. Dong and G. Dimopoulos, unpublished results). Besides, the ability to FBN members to form homo- or hetero-multimers has not been investigated, and further detailed studies with more antibodies against different members of the FBN family will help to elucidate this mechanism.

FREPs have also been characterized in other invertebrates. FREPs from the snail Biomphalaria glabrata are composed of two functional domains, an N-terminal IgSF domain that may be repeated in tandem, and a C-terminal FBG domain (Adema et al, 1997; Zhang and Loker, 2003; Zhang et al, 2004). This gene family has at least 13 members; FREP2 is involved in immune responsiveness and plays a role in host-parasite interactions (Jiang et al, 2006). FREPs have also been identified in the solitary ascidian Halocynthia roretzi (Kenjo et al, 2001), a tachylectin-related protein in the sponge Suerites domuncula (Schroder et al, 2003), and aslectin in the mosquito Armigeres subalbatus (Wang et al., 2004).

All of these FREPs contain a common C-terminal FBG domain, but their N-termini have no typical conserved IgSF structure like that seen in snails. These FREPs probably play an important role in the innate immune response against bacteria and parasites (Gokudan et al, 1999; Schroder et al, 2003; Wang et al, 2004; Jiang et al, 2006).

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