Immune novelties revealed through comparative genomics the LRIM and Nimrod examples

Leucine-rich repeat (LRR) sequence motifs are present in many proteins implicated in immune responses, and have emerged as powerful immune-recognition domains in multicellular organisms. The discovery that the Toll receptor is important in Drosophila innate immunity was the catalyst for elucidating the roles of Toll-like receptors (TLRs) in mammalian immunity. The LRR ectodomain of Drosophila Toll recognizes and binds to a pro-teolytically activated cytokine, Spätzle, resulting in intracellular NF-kB signalling and immune-effector production. Activation of the homologous mammalian TLRs also leads to immune transcrip-tional responses through NF-kBs. However, TLR ectodomains bind directly to components of infectious agents. Similarly, LRRs encoded by plant R genes can confer resistance either by interacting directly with pathogen virulence factors, or indirectly by binding to perturbed host proteins (Bent and Mackey, 2007). Many R proteins combine a variable number of LRRs with a nucleotide-binding domain (NBD) and an N-terminal Toll-interleukin-1 receptor (TIR) homology region or a coiled-coil domain. These R proteins show striking structural and functional similarities to animal nucleotide-binding leucine-rich repeat (NLR) proteins, which combine LRRs and NBDs with a variable N-terminal domain associated with apoptosis and/or signalling: they sense PAMPs such as peptidoglycan or flagellin, initiating immune responses through activation of NF-kBs and mitogen-activated protein kinases (Shaw et al, 2008). The power of forming recognition domains from a variably repeating, structure such as the LRR, is also the basis of the adaptive immune system of the primitive jawless vertebrates, the lamprey and hagfish. Instead of using immuno-globulin gene segments, these organisms create a repertoire of variable lymphocyte receptors built from highly diverse combinatorial assemblies of gene segments from a library of LRR cassettes (Pancer et al, 2004).

Novel discoveries and insights from extensive functional analyses continue to reveal genes and families that make up the insect immune repertoire. Microarray studies in A. gambiae identified numerous novel genes with putative roles in defence and immunity (Dimopoulos et al, 2002). Two LRR-containing genes were differentially activated in cell cultures by septic, but not sterile injury and massively upregulated by heat-killed bacteria and microbial components. One of these genes, later named LRIM1, was also highly upregulated during mosquito infection by P. berghei (Dimopoulos et al., 2002; Vlachou and Kafatos, 2005). RNAi-mediated silencing of LRIM1 revealed a striking increase in P. berghei oocyst numbers, identifying LRIM1 as the prototype antagonist of the development of this rodent malaria parasite (Osta et al., 2004). The same study identified two CTLs that act as inhibitors of parasite melanization, and revealed that LRIM1 acts upstream of the CTLs in initiating the melanization reaction. However, the LRIM1/CTL4 genetic module appeared to not have an effect against the human parasite Plasmodium falciparum (Cohuet et al, 2006). The second LRR gene was later found in a population survey of West African A. gambiae mosquitoes and mapped to the APL1 genetic locus, with major effects on P. falciparum development and melanization (Riehle et al, 2006). RNAi silencing of APL1 in laboratory mosquitoes produced a similar phenotype to that observed for LRIM1, with dramatically increased numbers of P. berghei oocysts. Furthermore, a recent study showed that orthologues of LRIM1 and APL1 in the malaria non-vector mosquito, Anopheles quadriannulatus species A, are involved in the melanization response that these mosquitoes mount naturally against P. berghei (Habtewold et al, 2008).

The important functions of LRIM1 and APL1 in Anopheles innate immunity, and specifically the wide-ranging recognition roles of LRR domains, provide a basis for bioinformatic characterization of a novel immune-related gene family (Povelones et al, 2009). LRIM1 and APL1 both have signal pep-tide sequences followed by a stretch of LRRs that create an alternating a-helix/P-strand pattern, with some irregularities that likely translate into subtle structural variations of their characteristic horseshoe fold. The C-terminal sequences of both these genes exhibit characteristic seven-residue (heptad) repeats that define the primary structure of coiled-coil domains; a distinctive cysteine-rich pattern can be identified in the hinge region between the LRR and the coiled-coils. Comprehensive scans employing these LRIM features identified over 20 regions encoding potential LRIM-like genes in each of the three available mosquito genomes. They encompass short LRIMs with six to seven LRRs and long LRIMs with 10-13 LRRs, such as LRIM1 and APL1 (Povelones et al., 2009).

The structural integrity of the LRR domain rests with a conserved pattern of leucine residues that tolerates only limited substitutions with similar amino acids: the intervening positions are far less constrained. The coiled-coil heptads must maintain a pattern of hydrophobic and polar residues within the repeat, but the identity of individual amino acids is not critical. Thus, sequence conservation within the LRIM family varies considerably, hindering robust phylogenetic analysis for resolving orthologous relationships. Nevertheless, a comparative approach employing synteny among the three mosquitoes does resolve and confirm relationships suggested by initial protein sequence analyses. Indeed, orthologous genomic clusters of both short and long LRIMs can be identified in all three mosquito species. APL1 is found within a cluster of mostly long LRIMs, located between conserved BRACA2-like and zinc-finger genes that delineate the synteny (Figure 6.4a). In A. gambiae, three of these genes, APL1A, APL1B, and APL1C, are very similar in sequence and likely originated from recent gene duplications; only APL1C has an effect against P. berghei (Riehle et al, 2008). A second cluster contains only short LRIMs and the synteny is supported by a gene encoding a guan-ine nucleotide exchange factor in all three species. Both clusters exhibit striking examples of local gene shuffling, duplication, and even a clear case of pseudogenesis after duplication. The identity of LRIM1 orthologues in the culicine mosquitoes is somewhat obscured by high sequence divergence. However, detailed inspection reveals convincing evidence of orthology: the same number of LRRs, matching profiles of coiled-coil heptad repeats, and preservation of synteny (Figure 6.4b).

The variable repeating units that characterize the LRIM family of putative recognition receptors are thought to provide both the structural and evolutionary flexibility required to facilitate recognition of diverse immune-stimulating pathogen structures. Such repetitive features are also found in a recently identified family of another class of recognition receptors in Drosophila, characterized by additional complex protein-domain architectures. The distinct epidermal growth factor (EGF)-like repeats of the Drosophila phagocytosis receptors Eater (Kocks et al, 2005) and Nimrod C1 (NimC1) allowed the characterization of a novel family of putative receptors (Kurucz et al., 2007). The EGF-like repeats of Eater and NimC1 exhibit a conserved six-cysteine consensus (NIM repeat) separated by loops of variable length. NimC1 neighbours the haemocyte-specific Hemese gene on chromosome 2, where a cluster of genes containing NIM repeats is found. These Nimrod-like genes make up three distinct classes based on their additional domain features. NimA exhibits domain organization similar to Draper, another Drosophila phagocytosis receptor (Manaka et al, 2004). It consists of a large intracel-lular domain, an extracellular EMI domain (with possible protein-protein interaction properties), and eight-cysteine EGF-like repeats. NimBs are probably secreted proteins, but apart from a weakly conserved N-terminal region, they do not have additional identifiable features. Finally, the NimC class encodes transmembrane proteins with a partially conserved N-terminal region upstream of the NIM repeats.

Evolutionary analysis of these NIM-repeat-containing proteins suggests how the Nimrod superfamily may have evolved from an EGF-repeat-containing ancestor via a Draper-like gene, containing both EGF repeats and a single NIM repeat (Somogyi et al., 2008). The genomic cluster of Nimrod genes remains broadly conserved across the 12 sequenced Drosophila genomes, but

BRACA2-like

AGAP007039

Chr.2L

Anopheles 70Kb

7033 7034 7035 7036 7037 APL1C

Zn finger

Scont1.445

Aedes 280Kb

BRACA2-like

AAEL010128 yAAEL010128 MII

If t

10125 10132 10129

Zn finger

Scont3.421 Culex 160Kb

BRACA2-like u/tr^w

12894

Coiled coil a

CPIJ012900

CPIJ012895 CPIJ012896

Zn finger

41 240K

41 310K

620K

340K

160K

320K

Anopheles 200Kb

KCNQ-like potassium channel

Scont 1.674 Aedes [400K 740Kb

1.1022 1.653

640K 300K 200K 1

Folate transporter

LRIM1 AGAP006348

Folate transporter

400K

KCNQ-like potassium channel a o

LRIM1 Protease AAEL012086

Scont3.77

Culex 350K 350Kb

Ribosomal protein o

Folate transporter

KCNQ-like potassium channel a

LRIM1 CPIJ004924

30 170K

30 370K

Figure 6.4 Two genomic loci encoding multiple leucine-rich repeat immune protein (LRIM) genes in three mosquito species. (a) Anopheles gambiae APL1C (AGAP007033) is found in a genomic cluster of LRIM genes where the orthologous genomic regions in Aedes aegypti and Culex pipiens, delineated by conserved neighbouring BRACA2-like and zinc-finger genes, also encode LRIMs. Two Anopheles LRIMs in this cluster (AGAP007035 and AGAP007036) are closely related to APL1C; however, putative orthologues are not found within these synteny regions in Aedes and Culex. The syntenic cluster nevertheless exhibits three LRIM orthologous trios, whose relative orientations indicate the occurrence of genomic shuffling events since the anopheline/culicine divergence. One of the Aedes LRIM genes appears to have undergone a duplication event followed by pseudogenesis leaving a dysfunctional copy, ¥AAEL010128. (b) The preservation of synteny at the LRIM1 gene locus delineated by the neighbouring KCQN-like potassium channel and folate transporter genes helps to confirm the putative LRIM1 orthologues in Aedes and Culex which, at the protein-sequence level, are somewhat obscured by high levels of divergence. Identification of the orthologous genomic region in Aedes was hampered by incomplete assembly and sequence gaps as indicated on the figure, but a better assembly in Culex serves to confirm the synteny among these regions. Chromosomes (Chr) and supercontigs (Scont) are labelled in Anopheles gambiae (white), Aedes aegypti (light grey), and Culex pipiens (dark grey) with start and end positions of the displayed genomic regions. LRIM genes are indicated in bold typeface and neighbouring genes are in grey typeface. For clarity, the species code and leading zeros have been removed from some gene identifiers.

expansions and lineage novelties are also identified (Sackton et al., 2007). Hemese is only found in the melanogaster group, prompting the hypothesis that this gene has originated from a truncated duplication of an ancestral NimCl. Novel NimD genes are found in the Sophophora subgenus and NimE in the Drosophila virilis/Drosophila mojavensis clade. Nimrod-like genes exhibiting most of the family-defining features are identifiable in other insect genomes, including A. gambiae (which also has an Eater homologue) and A. mellifera. NimB-like genes have been described in B. mori and Holotricia diom-phalia. Proteins exhibiting Nimrod-like features have also been identified beyond insects; several of them have been implicated in phagocytosis and/or binding to microbes. The NimA-like Caenorhabditis elegans CED-1 protein is a receptor for phagocytosis of apoptotic cells, and mammalian Ced-1-like genes may perform similar roles (Mangahas and Zhou, 2005).

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