Biochemistry and evolution of OBP and CSP proteins

For Insect Pheromone Biochemistry and Molecular Biology Biochemistry and Evolution of OBP and CSP proteins Jean-François Picimbon University of Lund, Department of Ecology, Solvegatan 37, SE 62-223 Lund, Sweden I. Introduction One perceives from the world only what one has been prepared to perceive. In humans and in most mammals, all different senses are used to make sense of life. In contrast, in insects, chemical senses involving odorants and contact chemosensory molecules play the vital role. The olfactory system is the primary sense insects use in analyzing the environment, in crucial tasks such as finding food, nest, mates and conspecifics. Contact chemosensation is specialized to analyze specific substrates to assist in the identification of suitable oviposition sites, the recognition of host-plants, the selection of tastants and the search for further nutrient chemicals. Dedicated to survival, both olfactory and contact chemosensory systems in insects have developed to extremely high levels of sensitivity and selectivity. The sensitivity and selectivity of olfaction and contact chemosensation are due 1) in the brain, to the existence of neuronal network of neurons tuned to a specific chemical stimulus, and 2) in the periphery, to the existence of olfactory/chemosensory receptor neurons housed in sensory micro-organs called sensilla. The sensilla can best be viewed as simple cuticular porous extrusions that increase the surface that captures airborne odorants or chemicals dissolved in water droplets. They contain the receptive olfactory or chemosensory structures (Schneider, 1969). The olfactory sensilla are most numerous on the antennae and mediate the reception of 1 sex pheromones and plant volatiles, as well as other odorants. Low volatility pheromones may also be detected by contact chemoreceptors on the front legs (Xu et al. , 2002; Park et al. , 2002). In contrast, general chemosensory receptor sensilla are distributed over the whole insect body, but mainly occur on the legs, and contain neurons responding to hydrophobic tastants, CO2, temperature, humidity or a combination of different modalities. The antennae, legs and their sensillar complement represent a wide spectrum of structures, shapes and lengths, but the cellular organization of sensilla follows a universal scheme based on conserved morphological features. Most prominent is the presence of a lymphatic fluid, the sensillar lymph, that entirely fills the sensillar lumen into which the dendrites of the sensory neurons extend. Thus, the pores that penetrate the surface of the sensilla are not in direct contact with the receptor proteins which reside on the sensory dendritic membrane, and chemical molecules have to cross the sensillar lymph before to interact with the dendritic receptors. The problem of chemical reception is dual: 1) the chemical molecules (largely hydrophobic in nature) face a hydrophilic environment after penetrating the sensillum, 2) the chemicals in the lymph are exposed to a high concentration of chemical degrading enzymes. Specific binding mechanisms are therefore required, not only to solubilize but also to protect and transport the odorant molecules in the sensillar lymph, upstream to the sensory receptors (Vogt and Riddiford, 1981; 1986; Vogt et al. , 1985; Vogt, 1987). The Odorant Binding Proteins (OBPs) and the ChemoSensory Proteins (CSPs) are proteins from the lymph that are thought to accomplish these tasks, solubilizing and protecting the odorant and contact chemosensory molecules. This chapter describes the biochemical and evolutionary aspects of these two families of peripheral sensory proteins of insects. Particular attention will be paid to the sub-classification of binding proteins, the diversity of gene structures 2 and the phyletic and molecular relatedness between binding proteins from different insect species. II- The family of odorant-binding proteins A- The concept of pheromone-binding protein About twenty years ago, Vogt proposed that small water-soluble proteins called OBPs might aid in odor reception, by keeping the lipophilic odorants soluble and active in the lymph, thus allowing their transport and integrity through the aqueous barrier. The first insect OBP identified as such was the pheromone-binding protein (PBP) found in the male antennae of the large silkmoth Antheraea polyphemus (Vogt and Riddiford, 1981; Table 1). In pioneer binding studies, pheromone alone in a glass vial containing water quickly absorbed to the glass wall (Kaissling et al. , 1985). However, when PBP was added a certain amount of pheromone remained in solution. The degraded pheromone molecule was not held in solution by PBP, indicating a degree of specificity with respect to which odorants are solubilized. The antibody of an OBP-related protein from the blowfly Phormia regina blocks the response of the taste receptor cell to a stimulant containing hydrophobic molecules (Ozaki et al. , 1995). The mutation of one OBP gene from Drosophila, lush, results in abnormal chemoattractive behavior to ethanol (Kim et al. , 1998). These results indicate that reception of tastants and chemical molecules soluble in water requires transport of these molecules by OBPs. It is generally assumed that at, the molecular level, the nature of the diverse chemosensory modalities is similar to that of odorant and tastant reception and that multiple types of binding proteins are involved in the diverse chemosensations (Vogt et al. , 1991a,b; Shanbhag et al. , 2001; Koganezawa and Shimada, 2002). 3 The question thus is to what extent PBP and other binding proteins participate in the recognition of odor messages? The PBPs have been so called by virtue of sex pheromone binding, specific association with sex-pheromonal sensilla (Vogt and Riddiford, 1981; Vogt et al., 1989; Du et al. , 1994; Steinbrecht, 1996; Steinbrecht et al. , 1992, 1995; Laue et al. , 1994). A PBP from Bombyx mori has been crystallized with the pheromone Bombykol packed into a hydrophobic binding pocket (Sandler et al. , 2000). So far, no binding studies have really assessed the degree of specificity of PBPs, i.e. the ability of the carrier-protein to bind to one specific compound. Rather, the binding spectra of PBPs seem broad. The PBPs, like other binding proteins, will bind many chemicals but differently (Vogt et al. , 1989; Du et al. , 1994; Feng and Prestwich, 1997; Wojtaseck et al. , 1999; Campanacci et al. , 2001a; Bette et al. , 2002). Differential binding representing a fine tuning of PBP-pheromone interaction has been documented by different studies of various insect species. A PBP from male moths will bind the chemical component of the female pheromone better than any other components. PBPs may act as selective filters, since PBPs differentially bind specific pheromone components; pheromonePBP interactions may be based on the recognition of the chain hydrocarbons of the pheromone molecules (Du and Prestwich, 1995; Feixas et al. , 1995; Maïbèche-Coisné et al. , 1997; Maida et al. , 2000; Picimbon and Gadenne, 2002). The odor recognition by PBPs might be as sensitive as that exhibited by sensory receptors. In the gypsy moth, Lymantria dispar, two PBPs have been shown to discriminate between two enantiomeric forms of the pheromone (Vogt et al. , 1989; Plettner et al. , 2000). Given such a potential for greater binding and pheromone specificity allowed by a duplication of PBPs, the evolution of PBP genes may have followed the diversification of the pheromone systems. 4 B. The repertoire of PBPs in moths The most prominent examples of pheromone diversification are found in Noctuidae. The sex pheromones of the Noctuidae species are mixtures of at least three compounds that differ mainly in carbon chain length (Lofstedt et al. , 1982; Teal et al. , 1986; Attygale et al. , 1987; Picimbon et al. , 1997). In many noctuid moth species, multiple PBPs has been reported (Vogt et al. , 1989; Merritt et al. , 1998; Maïbèche-Coisné et al. , 1998; Picimbon and Gadenne, 2002; Abraham et al. , 2002). Based on sequence homology and phylogenetic analysis, a subclassification of noctuid PBPs has been proposed (Picimbon and Gadenne, 2002; Abraham et al. , 2002). A neighbor joining tree of selected PBPs from moths shows that noctuid PBPs segregate into two sub-classes (Fig. 1). Sub-class 1 (group 1 or Grp1) corresponds to Grp1-PBPs from Agrotis ipsilon, A. segetum, Heliothis virescens, H. zea and Mamestra brassicae (Aips-1, Aseg1, Hvir-1, Hzea-1 and Mbra-2). The Grp1-PBPs show about 86% identity between each other. The sub-class 2 (group 2 or Grp2) corresponds to Grp2-PBPs from Agrotis, Heliothis and Mamestra species (Aips-2, Aseg-2, Hvir-2, Mbra-1; Fig. 1A) as well as proteins from nonnoctuid species such as Manduca sexta (Msex-2, Msex-3; Robertson et al., 1999; Picimbon and Gadenne, 2002). Sub-class 2 also contains Ycag PBP, a PBP from Yponomeuta cagnagellus (Robertson et al. , 1999; Willett, 2002; Picimbon and Gadenne, 2002). The noctuid Grp2-PBPs show about 72% identity between each other, about 50% to non-noctuid Grp2-PBPs and only 32-47% to other PBPs. The specific grouping of orthologous PBPs strongly suggest that different types of PBP are likely utilized by most species of moth and that within a species multiple subtypes are expressed, perhaps for binding a large repertoire of pheromone molecules. 5 Yponomeutidae and Noctuidae are phylogenetically distant. It is very likely that Grp2PBPs are expressed by other Lepidoptera lineages that share common ancestry with these two Families, and that these PBPs are tuned to a pheromone structures conserved across the different member species. In contrast, the Grp1-PBPs have been reported only in Noctuidae species. However, Antheraea pernyi, Ostrinia nubilalis, Manduca sexta and Lymantria dispar use pheromone components structurally similar to the main pheromone components of Noctuidae (Klun et al. , 1973; Bestmann et al. , 1987; Tumlinson et al. , 1989; Gries et al. , 1996). Therefore, it cannot be excluded that pyralid, sphingid and lymantrid species also express Grp1 types of PBP and that these have simply not yet been identified. The PBPs so far identified in these species correspond to very specific groups of protein (Merritt et al. , 1998; Willett and Harrisson, 1999; Robertson et al. , 1999; Vogt et al. , 1999; Picimbon and Gadenne, 2002). In particular, the two PBPs from L. dispar, Ldis-1 and Ldis-2, are very divergent from other moth PBPs. Interestingly, they both preferentially associate to enantiomers of disparlure, an epoxyde component used as primary pheromone by L. dispar (Plettner et al. , 2000). It could well be that in L. dispar, primary PBPs bind to disparlure and secondary PBPs binds to minor pheromone components. An electrophoretic analysis of antennal proteins has failed to find secondary PBPs and only Ldis-1 and Ldis-2 appear to be detectable by a biochemical approach. Similarly, in the noctuid M. brassicae, only protein bands corresponding to Grp1 and Grp2 PBPs have been identified (Nagnan-Le Meillour, 1996). In the bombycid B. mori, only one PBP protein could be found using either a biochemical approach or homology screening of a cDNA library (Maida et al. , 1993; Krieger et al. , 1996). The failure to find additional PBPs in these species may be due to the fact that the degree of expression is markedly different for each PBP. A primary PBP 6 tuned to high concentrations of a primary pheromone component may be expressed more than secondary binding proteins tuned to lower concentrations of secondary pheromone compounds. Analyzing the presence of Grp-1 and Grp-2 PBPs in non-noctuid insects will be a first step to investigate PBP diversity with respect to recognition of multicomponent pheromone blends. The Grp1 and Grp2 PBPs have been identified in two closely related noctuid species, Agrotis ipsilon and A. segetum, whose females emit pheromone blends that consist of three main components and minor components that vary locally. The major pheromone component of A. ipsilon is (Z)-11-hexadecenyl acetate (Z11-16:Ac), while the major pheromone component in A. segetum is (Z)-5-decenyl acetate (Z5-10:Ac). The antennae from A. ipsilon and A. segetum both have neurons responding to (Z)-7-dodecenyl acetate (Z7-12:Ac) and (Z)-9-tetradecenyl acetate (Z9-14:Ac), two of their major pheromone compounds (Löfstedt et al. , 1982; Toth et al. , 1992; Picimbon, 1995,1998; Picimbon et al. , 1997; Gadenne et al. , 1997; Gemeno and Haynes, 1998; Wu et al. , 1999). The Grp-1 PBPs of these two species, Aips-1 and Aseg-1, are virtually identical (Laforest et al. , 1999; Picimbon and Gadenne, 2002). The Grp-2 PBPs from these species are more divergent: Aips-2 and Aseg-2 show only 76% identity. We could speculate that the conserved Aips-1/Aseg-1 proteins bind either Z7-12:Ac or Z9-14:Ac and that the more variable Aips-2 and Aseg-2 bind to Z11-16:Ac and Z5-10:Ac respectively. C- Gene structures encoding OBPs To explore the functions of Grp-1 and Grp-2 PBPs in depth, cutting edge protein expression, ligand-binding, structural analysis and immunocytochemistry experiments are required. However, determination of the gene structures might also be informative since the 7 diversification of species and of their pheromone systems may have led to specific gene regulations of PBP expression. The genes encoding Aips-1, Aips-2, Aseg-1 and Aseg-2 have been characterized from genomic DNA (Abraham et al. , 2002; Fig. 2). All four share the same two introns - three exons structure but differ in length. The three exons encode similar portions of the protein. The first exon (exon 1) is the shortest and has the same size in all Agrotis PBP genes. This exon pattern may be a general feature across the Grp-PBP genes. The first intron of these genes exhibit little variability. However, intron 2 of both A. ipsilon PBPs are significantly longer than the introns of the corresponding A. segetum PBPs. Phylogenetical analysis of the introns from the four Agrotis genes suggests that the Aips-1/Aseg-1 and Aips-2/Aseg-2 are respectively closely related, and that these Grp1 and Grp2 genes may have evolved from gene duplication that occurred before the divergence of A. ipsilon and A. segetum. This duplication may have occurred even earlier, before the split of Yponomeutoidea, Sphingiodea and Noctuoidea lineages as suggested by the presence of Grp2-PBPs in Y. cagnagellus and M. sexta. In the sphingid M. sexta, Msex-1 is similar to the PBP from B. mori and divergent from the Grp2 proteins (Vogt et al. , 2002). The Msex-1 gene displays the same structure of three exons - two introns as the noctuid PBP genes encoding Aseg-1/Aips-1 and Aseg-2/Aips-2 as well as other non-noctuid PBP genes (Krieger et al. , 1991; Willett and Harrisson, 1999; Willett, 2000; Abraham et al. , 2002). Therefore, the lepidopteran PBP genes have a conserved pattern of two introns and three exons with a variability being observed in the intron length. 8 In contrast, the PBP-related proteins (PBPRPs) from Drosophila melanogaster are highly variable with respect to exon-intron structure. The gene encoding the protein PBPRP5 has a single coding exon, while the genes encoding PBPRP1 and PBPRP2 have four and five coding exons, respectively. The gene encoding the OS-F protein has 4 exons of varying size and a very long second intron (McKenna et al. , 1994; Pikielny et al. , 1994; Hekmat-Scaffe et al. , 1997, 2000). The genes encoding the olfactory proteins OS-E and LUSH exhibit intron-exon patterns similar to those of lepidopteran PBP genes, suggesting that OS-E and the ethanolbinding protein LUSH from D. melanogaster and the moth PBPs may have a common ancestor. D- Relationships of Moth and Drosophila melanogaster OBPs This hypothesis may imply the existence of OS-E and LUSH orthologs in some moth species. A protein similar to LUSH has been identified from the antennae of A. ipsilon (Picimbon, unpublished data). In addition, the PBPRPs from D. melanogaster display significant similarities to a specific subclass of moth OBPs that includes binding proteins whose function is unknown, the so-called Antennal Binding Proteins-X (ABPX). The ABPX proteins have highly conserved amino acid sequences across different moth species and the overall ABPX sequence displays significant similarity with DmelPBPRP1 (Fig. 3). In particular, the proteins AipsABPX-1 and PBPRP1 share common amino acid residues including the six cysteines characteristic of OBPs and the motifs 23-TGA-25, 89-SCGTQ-93 and 99-CDTA-102. The ABPX-1s and PBPRP1 may then represent an OBP-1 type of protein defined by key residues that may underlie specific functions. The ABPX/PBPRP-specific amino acid residues Arginine at position 16, Lysine at position 47, Proline at position 76, Threonine at position 92 are replaced respectively by Leucine, Glycine, Lysine and Lysine residues that are 9 conserved in the different types of binding protein from B. mori (Krieger et al. , 1993; Picimbon, 2001). These replacements may be relevant to support the function of ABPX. Based on the crystal structure of the bombykol-PBP complex, the Tryptophane at position 101 and the Valine 105 have been shown to contact the molecule of Bombykol (Sandler et al. , 2000). These are replaced by two Threonine residues characteristic of OBP-1. Therefore, the Threonine residues characteristic of OBP1s may be of crucial importance for the binding specificities of these proteins. The notion of relatedness between ABPXs of moths and the PBPRPs of D. melanogaster (DmelPBPRPs) is strongly supported by the identification of additional ABPXs in the moth species A. ipsilon: AipsABPX-2 and AipsABPX-3. The protein ABPX2 has significant similarity to DmelPBPRP2 and DmelPBPRP5 on the basis of specific amino acids that may represent an OBP-2 group, while AipsABPX-3 appears more similar to DmelPBPRP4 and may represent an OBP-3 group (Picimbon et al. , unpublished). These relationships suggest the existence of a multiplicity of ABPX in moths similar to that of PBPRP in flies and may indicate a unique importance in insect olfactory behaviors. Alternatively one could speculate that ABPXs may represent "intermediary" molecules between dipteran PBPRPs and lepidopteran PBPs. A phylogenetic analysis of insect OBPs focusing on moth ABPXs and DmelPBPRPs shows the ABPXs falling outside the groups of DmelPBPRPs, reflecting the phylogenetic distance between Lepidoptera and Diptera (Fig. 4). The ABPXs from Agrotis ipsilon cluster with the ABPX proteins from other moth species. In particular, ABPX-1s from A. ipsilon and H. virescens group with ABPXs of B. mori, A. pernyi and M. sexta and not with other identified noctuid PBPs. There are well supported separations between the branches containing A. ipsilon 10 proteins. One could speculate that these A. ipsilon genes represent multiple rearrangements or duplications of the ABPX ancestral gene and that the events which produced the ABPX diversity are common in noctuid species. Similar duplications may have occurred to produce diverse PBPRPs in D. melanogaster but it seems that ABPX and PBPRP genes have evolved independently, despite established similarities in there sequences. As the PBPRP genes are quite different in sequence from the moth PBP genes, and as the Drosophila and moth olfactory genes diverged very early in the evolutionary course of these insects, moth PBPs in moths may have evolved for binding pheromones, while ABPX may have persisted and developed for binding more generalist odorants. Identification of the genes encoding AipsABPX and studies of the structure/activity relationships of ABPX/PBPRP need to expand clarify the evolutionary and functional relationships between PBPRP, ABPX and PBP proteins. III- The family of chemosensory proteins A- The concept of Chemosensory Protein In the context of evolution of pheromone olfaction and general chemosensation, we can speculate that general chemosensory proteins may be more highly conserved than olfactory PBPs when compared across species. Indeed, little sequence diversification would be expected to occur among genes encoding chemosensory proteins tuned to bind chemicals of common importance to all species. In insects, a class of putative general chemosensory proteins has been described and has gained increasing interest over the last few years, expanding our understanding of the complexity 11 of the repertoire of sensory binding proteins. The first member of this novel class of proteins was found in Drosophila melanogaster and called OS-D (Olfactory Specific-protein type D) or A10 (McKenna et al. , 1994; Pikielny et al. , 1994); OS-D is abundant in sensory appendages and contains 4 cysteines. Similar proteins (Table 2) have been since identified in several species and variously referred to as OS-Ds, SAPs (Sensory Appendage Proteins) or CSPs (Chemosensory Proteins) (Danty et al. , 1998; Angeli et al. , 1999; Picimbon and Leal, 1999; Robertson et al. , 1999; Picimbon et al. , 2000a). The first strong evidence that these proteins have a role in chemosensation came from immunocytochemistry experiments in the grasshopper Schistocerca gregaria that demonstrated OS-D protein in the lymph of the contact chemosensory sensilla (Angeli et al., 1999). B- Comparison of CSPs to OBPs On the basis of the immunocytochemical localization of CSP, Angeli et al. (1999) have suggested that CSPs have an OBP-like function. However, the CSPs cannot be regarded as OBPs considering the basic definition of an OBP: a lymphatic, acidic α-helical, mainly hydrophobic carrier protein of 14-16 Kds characterized by six cysteines linked by three disulfide bridges, a flexible structure and an expression pattern restricted to the olfactory sensilla of the antennae (Vogt and Riddiford, 1981; Breer et al. , 1992; Prestwich et al. , 1995; Steinbrecht, 1996; Wojtasek and Leal, 1999; Scaloni et al. , 1999; Leal et al. , 1999; Campanacci et al. , 1999; Sandler et al. , 2000; Kowcun et al. , 2001; Horst et al. , 2001). The CSPs are lymphatic acidic α-helical proteins, but 1) they have a molecular weight in the range of 12-13 Kds, 2) they show a set of only four conserved cysteines and two disulfide bridges, 3) they are highly hydrophilic, 4) they show high structural stability, and 5) they are not specific to the antennae but also found in 12 other parts of the insect body, more particularly in the legs (Angeli et al. , 1999; Picimbon et al. , 2000a,b, 2001; Picone et al. , 2001; Campanacci et al. , 2001b). The developmental patterns and structures of CSPs and OBPs are also different. The CSPs are produced synchronously to the shedding of the cuticle, very early during adult development, in contrast to OBPs which are produced late during adult development. This demonstrates that the chemosensory CSPs and olfactory OBPs are controlled by independent mechanisms (Vogt et al., 1993; Picimbon et al., 2001; Gavillet and Picimbon, 2002). Moreover, X-Ray structure analysis of CSPs has revealed a novel type of α-helical fold with six helices connected by α-α-loops and a narrow channel expanding over the protein hydrophobic core. This structural feature may confer specific binding properties to CSPs, in particular the ability to interact with long linear acyl chains of hydrophobic components (Lartigue et al. , 2002). C- The repertoire of CSPs A phylogenetic analysis of currently known insect CSPs, based on amino acid sequence comparisons, is shown in Fig. 4B. With few exceptions, proteins from different insect Orders segregate to different branches, consistent with the phylogenetic distance between these Orders. The CSPs from moths segregate into 3 groups, noted as CSP1, CSP2 and CSP3; each group includes taxa from multiple lepidopteran Families. Three CSPs from H. virescens, which segregate to these 3 groups, share about 50% amino acid identity between each other; similar between-group identities are seen for proteins of M. sexta and M. brassicae (Robertson et al. , 1999; Nagnan-Le Meillour et al. , 2000; Picimbon et al. , 2001; Jacquin-Joly et al. , 2001). Within-group identities for the M. brassicae are unusually high. The CSP1 proteins CSPMbraA4 and CSPMbraA1 differ by only two residues, and are virtually identical to 13 CSPMbraA3/A6, CSPMbraA2 and CSPMbraA5. Similarly, the CSP2 M. brassicae proteins MbraCSPB1 and MbraCSPB3 differ by only one amino acid. These proteins may actually be alleles representing the same locus (Picimbon et al., 2000a; Nagnan- Le Meillour et al. , 2000; Jacquin-Joly et al. , 2001). The CSP1 group, and especially BmorCSP1, attracted a protein from the phasmid E. calcarata ( 39% identity) even though moths and phasmids are quite distant phylogenetically (Picimbon et al. , 2000b; Marchese et al. , 2000). CSPs of the orthopteroids (C. cactorum, Locusta migratoria, S. gregaria) are highly conserved within species and with few exceptions (e.g. CLP1) separate into species specific groups. One CSP group per orthopteroid species is in sharp contrast with moths where CSPs from a given species fall into three groups. However, the S. gregaria protein SgreOS-D1 is attracted to the CSPs of L. migratoria, sharing 79% identity with the L. migratoria CSPs but only 55% identity to the other CSPs from S. gregaria (SgreCSPs), so perhaps there are multiple orthopteroid CSP classes that have simply not been much identified. Analysis of the D. melanogaster and A. gambiae genomes reveals the full repertoire of CSPs within single species. D. melanogaster has 6 highly divergent CSPs; amino acid sequence identities of PEBmeIII, DmelOS-D and RH74005 range from 16-23% and from 14-23% when compared with the moth proteins. DmelOS-D is somewhat more similar, sharing about 45% sequence identity with the moth proteins. Four of the 7 A. gambiae CSPs cluster in a single group and share about 72% identity, but the others segregate by themselves. Although A. gambiae and D. melanogaster are both Diptera, only a single pair of CSPs share significant similarity to be attracted together (agCG5020865 and CG30172, 65% identity); these may be 14 orthologs. Overall, the divergence seen in CSP sequences is intriguing; divergent CSPs may have different and specific binding properties for distinct chemical ligands. D- Structural and binding properties of CSPs Differences in the amino acid sequences of CSPs presumably relate to differences in the ligand binding properties of each protein (Fig. 5). The type-1 and 2 CSPs of moths (CSP1 and CSP2) are proteins with about 107-112 amino acids and all exhibit the diagnostic elements of CSP: Aspartatic acid 6 and 88, Lysine 43, Glutamine 61, Proline 89 and four Cysteines at positions 29, 36, 54 and 57. Overall, the CSP1 proteins are highly conserved, and most have the sequence (1) -D-YTDKYD-----EIL-N-RLL--Y--CV---GKC--EGKELK--L--A---GC-KC---QEG----I--LIKN----W--L----DP---WR-KYEDRA-A-GI-IP-- (110). Six isoforms (alleles?) of CSPMbraA proteins, all CSP1s, differ at only three sites, 59, 69 and 92 (Ala/Thr, Ala/Val and Val/Gly). The CSPMbraA proteins differ from other CSP1 proteins in being one amino acid longer (Glu ) and have the C-terminal sequence DRAKAAGIVIPEE (Nagnan- Le Meillour et al., 2000; Jacquin-Joly et al., 2001). The three dimensional structure has been determined for CSPMbraA6. This CSP protein binds aliphatic molecules with 12-18 carbons, suggesting that CSPs generally bind hydrophobic compounds (Lartigue et al. , 2002). Thus, multiple subtypes of CSP may mediate transport of carbon chains of different lengths. If CSP1s bind specific hydrophobic chains, the large repertoire of proteins may bind the large number of diverse alkyl chain components. We could hypothesize that a small CSP1a protein would bind to a C12 molecule, while the longer CSP1b protein would bind to molecules longer than C12. Other types of CSP may have totally different binding properties. The CSP2s are characterized by diagnostic CSP residues and by other conserved amino acids that may underlie 15 specific functions. Overall, the CSP2 proteins are highly conserved, and most have the sequence (1) ----YTD-YD-V-LDEIL-N-R--VPY-KCILD-GKCAPD-KELKEHI-EALE-ECGKCT--QKGGTRRVI-HLINHE---W-EL--K-DP--K---KYEKEL---K- (108). The amino acid pattern (1) ---YTD-D-----EIL-N-R----Y--C----GKC----KELK----------C-KC---Q--G----------N-----W--L----DP-----K------- (106) is conserved in CSP1 and CSP2. CSP3s are so far only represented by the proteins HvirCSP2 and SAP4 which share 84% sequence identity but are only about 50% identical to other CSP1 or CSP2. These CSP3s have the sequence (1) ASTYTDKWDNINVDEILESXRLXKXYVDCLLDXGRCTPDGKALKETLPDALEXXCS KCTEKQKAGS-KVIR-LVNKR--LWKELSAKYDPNN-YQ--YKDKIXXXKQG- (106/107). The α-helical regions of the moth CSP1, CSP2 and CSP3 proteins differ in amino acid sequence. The α-helix (α1) in the N-terminal region anchors a narrow hydrophobic channel and includes residues 5 (or 12)-18; α1 is conserved in all three CSP groups with the sequence YTD-D-----EIL (Fig. 5). In CSP1s, α1 interacts with residues Tyr 8 and Glu 39 to stabilize the hydrophilic channel; however, CSP2s lack Glu 39. And in CSP3s, a non-polar residue Trp replaces the polar, uncharged Tyr 8. Residue Glu 39 of CSP1s is replaced by Asp in CSP2s and CSP3s. In contrast, the α-helix (α6) in the C-terminal region is highly variable and may support ligand specificity. Furthermore, of thirteen residues of CSPMbraA6 that have been shown to interact with water molecules, only six are conserved in CSP1s (Asp 9, Tyr 26, Leu 43, Leu 47, Glu 63 and Gly 65) and only the residues Asp 9, Tyr 26 and Leu 43 are found in all three types of moth CSP, while the other residues are highly variable (Fig. 5). Lartigue et al. (2002) suggested that Tyr 26 may rotate towards the protein surface upon ligand binding, and that the previous position of its hydroxyl group may then be occupied by the side chain of Leu 43. Since 16 both residues Tyr 26 and Leu 43 are highly conserved in all insect CSPs, this mechanism may be common for all CSPs. Two tryptophan residues from MbraA6 were proposed to interact with ligands; these are conserved in all CSP1s, but only one (Trp 82) is conserved throughout the CSPs, suggesting that different CSPs may bind different ligands. The overall sequence similarities of CSP1s, CSP2s and CSP3s suggest that they all bind to lipid compounds. However, the diversity of CSPs not only in moths but also in other insect species, suggests that CSPs transport diverse types of chemicals. The CSPs of D. melanogaster, A. gambiae, B. mori and E. calcarata are highly variable, sharing only 40% sequence identity (Picimbon et al. , 2000b; Marchese et al. , 2000; this chapter). The CSPs from M. sexta (SAP1 and SAP5) are also very divergent (Robertson et al. , 1999; this chapter). Different types of CSP with multiple isoforms are also found in the acridid species L. migratoria and S. gregaria (Angeli et al. , 1999; Picimbon et al. , 2000a; Picimbon, 2001; see Fig. 5). Analyzing the cysteine arrangements in a CSP from S. gregaria, Angeli et al. have shown that the two adjacent cysteines form two small loops along the main protein chain in a fashion similar to thioredoxins. Thus, CSP may well play a role not only in binding lipid molecules but also in CO2 sensing (Bogner et al. , 1986; Stange, 1992, 1996; Maleszka and Stange, 1997; Angeli et al. , 1999). 17 E- Multiple functions of CSPs 1. Tissue-distribution CSPs are found not only in external sensory organs, but also in external and internal nonsensory tissues, further suggesting the proteins may have diverse functions. In moths the distributions of CSPs have been characterized by Northern blot analysis. A DIG-RNA probe encoding the BmorCSP1 hybridized not only with mRNAs from male and female antennae but also with mRNAs from legs and other parts of the insect body. Northern blot analysis using BmorCSP2 showed a very similar distribution pattern with no tissue specificity, as did the three CSPs of H. virescens which showed high levels of expression in legs (Picimbon et al. , 2000a, 2001). These results are consistent with the isolation of CSP cDNAs and proteins. The CSP of C. cactorum was as a cDNA from labial palps. The CSPs of M. Brassicae were isolated as cDNAs from antennae and pheromone glands, and as proteins ( N-terminal sequences) from proteins antennae, proboscis and leg (Malezska and Stange, 1997; Bobhot et al. , 1998; NagnanLe Meillour et al. , 2000; Jacquin-Joly et al. , 2001). CSPs are also broadly distributed in tissues of insects other than moths. In Drosophila melanogaster, OS-D, also called A10, is extremely abundant in the antennal appendages, and the CSP EBSP-III, is expressed in the ejaculatory bulb (McKenna et al. , 1994; Pickielny et al. , 1995; Dyanov and Dzitoeva, 1995). In locusts, CSPs are expressed in male and female from adults as well as 5th instar larvae, and are found in many different tissues including antennae, legs, mouth organs, thorax, abdomen, head and wings (Picimbon et al. , 2000b; in preparation). In the phasmid Eurycantha calcarata, a CSP has been isolated from the cellular layer underlying the cuticle (Marchese et al. , 2000). In the cockroach Periplaneta americana, the CSP p10 is expressed in legs and antennae (Nomura et al. , 1992; Kitabayashi et al. , 1998; Picimbon 18 et al. , 2001), and other CSPs have been detected in tissues including legs, brain and cerci (Picimbon and Leal, 1999; unpublished). Thus, in various insect species, the expression of CSP occurs in many different tissues, especially in the legs and contact sensory organs. The differences between CSPs and OBPs in tissue distribution and developmental expression suggest that the roles of these proteins in chemoreception may also be different. CSPs may have a broader function than OBPs, functioning in more systems than olfaction and taste, perhaps as general molecule carriers but especially involved in the transport of contact sensory molecules. 2. Role in contact chemosensation Immunocytochemistry experiments performed with rabbit antiserum generated against a CSP isoform from Schistocerca gregaria (sgCSP-5) showed selective labeling of the outer lymph of the peg lumen and in the cavity below the peg base of contact sensilla from the antennae and mouth organs, such as maxillary palps and tarsi. Olfactory sensilla were not labeled (Angeli et al. , 1999). This pioneer work showed the localization of CSPs in the lymphatic space surrounding the contact chemosensory receptor neurons and suggested an OBP-like function for CSPs in contact chemosensation. In the context of OBP-like function, the CSPs could well mediate the clearance of hydrophobic odorant molecules absorbed haphazardly by the cuticle as well as the delivery of these odorant molecules to degradative enzymes (Ferkovitch et al. , 1982; Lonergan, 1986; Prestwich et al. , 1989). Clearance and degradation of odorant molecules has to occur, e.g. while the insect flies in a pheromone plume or feeds on nectar. More data regarding 19 the specific localization of CSPs in chemosensory and non-chemosensory organs would provide a better understanding of the OBP-like function of these proteins. 3. Function and evolutionary history In the cockroach Periplaneta americana, CSP have been found that differentially express between the sexes, suggesting a role in delivering conspecific pheromones to olfactory neurons (Picimbon and Leal, 1999). In the phasmid Carausius morosius, a CSP is differentially expressed in only a subset of olfactory sensilla (Monteforti et al. , 2002). Multiple CSPs have been detected in phasmids, locusts and cockroaches, but no proteins related to known PBPs have been found (Tuccini et al. , 1996; Mameli et al. , 1996; Picimbon and Leal, 1999; Picimbon et al. , unpublished). These observations do not exclude the presence of RNA encoding PBPs in these insects, but RNA may not be necessarily translated into proteins (Segal et al. , 2001). Since Phasmatodea, Acridoidea and Blattoidea are among the most primitive insect Orders, one could hypothesize that CSPs perform PBP functions in ancient insects. Later in evolution, CSPs may have developed into general chemosensory proteins in flies and moths, where their original function was replaced by more efficient pheromone-binding proteins. This hypothesis could be tested by analyzing the diversity of CSP genes across a variety of insect species and by comparing the CSP and OBP genes. The genes underlying olfaction and general chemosensation are, indeed, most likely under different evolutionary pressures that may act selectively through defined regulatory elements. Specific intron structures, like the intron 2 of PBPs, may be key targets for regulatory mechanisms that control the differential expression or loss of specific OBPs and CSPs. What 20 gene structures will be found in the various insect species that use different pheromone and chemosensory components? IV- Concluding remarks and perspectives Binding of hydrophobic molecules by specific protein carriers appears to be a very efficient mechanism to increase both solubility and transport these molecular messengers in a hydrophilic medium. OBPs and CSPs may represent a successful application of this principle. In particular, the molecular mechanisms of transport of hydrophobic molecules may be more ancient than that most ancient of senses, olfaction. The olfactory system may have developed to extract the hydrophobic odorants from the air environment and optimize their transport and delivery to sensory cells. In recent years, studies of diverse insect species have revealed the heterogeneity of the family of binding proteins, and thus challenged the dogmatic concept that odorant-binding proteins are expressed only in olfactory structures. In particular, an increasing number of binding proteins related to OBPs have been identified in various non-sensory organs, such as the hemolymph, brain, accessory and salivary glands (TH12 proteins, sericotropin, B proteins and D7 gene products) (Paesen and Happ, 1995; Kõdrick et al. , 1995; Thymianou et al. , 1998; Rothemund et al. , 1999; Graham et al. , 2001). Such distributions suggest that this large family of carrier proteins may perform diverse functions throughout the insect body, paralleling the distribution and functional breadth of lipocalins from vertebrates (Flower, 2000; Ganfornica et al., 2000). OBPs may have arisen as general transporters of hydrophobic molecules, and later developed to bind and solubilize odorant molecules. The CSPs, which are expressed all over the body, have certainly conserved a functional polyvalence. In contrast, the specific expression of 21 many OBPs in the antennae strongly support an adaptation of these proteins to the reception of hydrophobic odorants. Efforts should be made to utilize the most modern techniques to analyze the binding properties and tissue specificity of all identified proteins, and eventually to rename the proteins on the basis of specific groupings and conserved motifs of amino acid. This suggestion is intended to be neither provocative nor inflammatory, but certainly the most reasonable way to define function of diverse binding proteins with respect to the pheromone systems and life history of the different insect species. Genetic studies will undoubtedly lead to the elucidation of how specific transporter molecules have developed to a fine-tuned function in olfaction. Specific and non-specific transporter molecules might have evolved differently over the course of evolutionary history and the diversification of species. If there is one thing future research should accomplish, it would be the unveiling of the evolution of the families of CSPs and OBPs. In addition, it must be considered that other families of binding proteins may well exist and participate to the reception of the extremely large repertoire of odor and chemosensory molecules. Investigations into these matters would have a strong impact not only in fundamental genetics underlying chemosensation and olfaction but also in applied industry, assuming that gene manipulation is accessible and permits control of the expression of OBP and CSP, and thereby control of the sensory abilities of insect pests (Picimbon, 2001, 2002). Acknowledgements: My heartfelt thanks to Profs. R.G. Vogt and P. 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Res. in press. 42 Table 1: The PBP related family of Odorant Binding Proteins. Protein Insect PBP Aips-1 Aips-2 Aseg-2 Aseg-1 Aper-1 Aper-2 Apol-1 Bmor-1 Hvir-1 Hvir-2 Hzea-1 Ldis-2 Ldis-1 Mbra-2 Mbra-1 Msex-1 ABPX AipsABPX-1 AperABPX BmorABPX HvirABPX-1 MsexABPX DmelPBPRP DmelPBPRP-1 Lepidoptera Agrotis ipsilon A. ipsilon Agrotis segetum A. segetum Antheraea pernyi A. pernyi Antheraea polyphemus Bombyx mori Heliothis virescens H. virescens Heliothis zea Lymantria dispar L. dispar Mamestra brassicae M. brassicae Manduca sexta A. ipsilon A. pernyi B. mori H. virescens M. sexta Diptera Drosophila melanogaster GenBANK Access Number References AF090191 AF007868 AF007867 AF051143 AF05051142 AF323972 Picimbon and Gadenne, 2002 Picimbon and Gadenne, 2002 Abraham et al., 2002 Prestwich et al., 1995; Laforest et al., 1999 Raming et al., 1990 Krieger et al., 1991 Raming et al., 1989 Krieger et al., 1996 Krieger et al., 1993 Abraham et al., 2002 Callahan et al., 2000 Prestwich et al., 1995 Merritt et al., 1998 Maïbèche-Coisne et al., 1998 Maïbèche-Coisne et al., 1998 Gyorgyi et al., 1988; Robertson et al., 1999 CAA05509 CAA64446 CAA05508 AF117577_1/AF117575_1 Picimbon et al., unpublished Krieger et al., 1997 Krieger et al., 1996 Krieger et al., 1997 Robertson et al., 1999 NP_524039/P54191/AAC46474 Pikielny et al., 1994 AF134253-AF134294 X96773 X96860 X17559 X94987 X96861 Table 2: The OS-D-related family of ChemoSensory Proteins. Protein Insect GenBANK Access Number References agCG50175 agCG50200 agCG50208 agCG50210 agCG50220 agCP11484 AgSAP1 AipsCSP BmorCSP1 BmorCSP2 CLP1 DmelOS-D/A10 CG30172 CG9358 PEBmeIII RH74005/CG11390 CSPec1 CSPec2 CSPec3 CSPHarm HvirCSP1 HvirCSP2 HvirCSP3 LmigOS-D1 LmigOS-D2 LmigOS-D3 Anopheles gambiae A. gambiae A. gambiae A. gambiae A. gambiae A. gambiae A. gambiae Agrotis ipsilon Bombyx mori B. mori Cactoblastis cactorum Drosophila melanogaster D. melanogaster D. melanogaster D. melanogaster D. melanogaster Eurycantha calcarata E. calcarata E. calcarata Heliothis armigera Heliothis virescens H. virescens H. virescens Locusta migratoria L. migratoria L. migratoria EAA12703 EAA12591 EAA12601 EAA12338 EAA12353 EAA12322 AAL84186 The Anopheles Genome Sequencing Consortium TAGSC TAGSC TAGSC TAGSC TAGSC Biessmann et al. , 2002 Picimbon, unpublished Picimbon et al. , 2000b Picimbon et al. , 2000b Maleszka and Stange, 1997 McKenna et al. , 1994; Pikielny et al. , 1994 Adams et al. , 2000 Adams et al. , 2000 Dyanov and Dzitoeva, 1995 Stapelton et al. , unpublished; Adams et al. , 2000 Marchese et al. , 2000 Marchese et al. , 2000 Marchese et al. , 2000 Deyts et al. , unpublished Picimbon et al. , 2001 Picimbon et al. , 2001 Picimbon et al. , 2001 Picimbon et al. , 2000a Picimbon et al. , 2000a Picimbon et al. , 2000a AAM34276 AAM34275 AAC47827 AAA21358/NP524121 AAM68292 AAF47307 AAA87058 AAM29645/AAF47140 AAD30550 AAD30551 AAD30552 AAK53762 AAM77041 AAM77040 AAM77042 CAB65177 CAB65178 CAB65179 LmigOS-D4 LmigOS-D5 SAP1 SAP2 SAP3 SAP4 SAP5 CSPMbraA1 CSPMbraA2 CSPMbraA4 CSPMbraA5 CSPMbraA6/A3 CSPMbraB1 CSPMbraB2 CSPMbraB3 CSPMbraB4 p10 CSPsg1 CSPsg2 CSPsg3 CSPsg4 CSPsg5 SgreOS-D1 L.migratoria L.migratoria Manduca sexta M. sexta M. sexta M. sexta M. sexta Mamestra brassicae M. brassicae M. brassicae M. brassicae M. brassicae M. brassicae M. brassicae M. brassicae M. brassicae Periplaneta americana Schistocerca gregaria S. gregaria S. gregaria S. gregaria S. gregaria S. gregaria CAB65180 CAB65181 AAF16696 AAF16714 AAF16707 AAF16721 AAF16716 AAF19647 AAF19648 AAF19650 AAF19651 AAF71289, AAF19649 AAF19652 AAF19653 AAF71290 AAF71291 AAB84283/AAB24286 AAC25399 AAC25400 AAC25401 AAC25402 AAC25403 Picimbon et al. , 2000a Picimbon et al. , 2000a Robertson et al. , 1999 Robertson et al. , 1999 Robertson et al. , 1999 Robertson et al. , 1999 Robertson et al. , 1999 Nagnan-Le Meillour et al. , 2000 Nagnan-Le Meillour et al. , 2000 Nagnan-Le Meillour et al. , 2000 Nagnan-Le Meillour et al. , 2000 Nagnan-Le Meillour et al. , 2000; Jacquin-Joly et al. , 2001 Jacquin-Joly et al. , 2001 Jacquin-Joly et al. , 2001 Jacquin-Joly et al. , 2001 Jacquin-Joly et al. , 2001 Nomura et al. , 1992; Kitabayashi et al. , 1998 Angeli et al. , 1999 Angeli et al. , 1999 Angeli et al. , 1999 Angeli et al. , 1999 Angeli et al. , 1999 Picimbon, unpublished FIGURE CAPTIONS Figure 1: Neighbor joining tree selected members of the PBP protein class, based on 1000 bootstrap replicates (Clustal X 1.8; Saitou and Nei, 1987; Thompson et al. , 1997). Relative branch lengths are indicated by the scale bar. Two groups of PBPs within Noctuidae are clearly revealed. Group 1 (Grp1): Aips-1/Aseg-1/Mbra-2/Hvir-1/Hzea-1; Group 2 (Grp2): Aips-2/Aseg2/Mbra-1/Hvir-2. Figure 2: Exon and intron boundaries of the genes encoding the PBPs from A. ipsilon and A. segetum. The numbers indicate the length of exons and introns (base pairs). The two groups of PBPs (Grp1 and Grp2) correspond to different gene structures. Aseg-1/Aseg-2/Aips-1/Aips-2 (Laforest et al. , 1999; Abraham et al. , 2002; Picimbon and Gadenne, 2002). Figure 3: Alignment of moth ABPX and DmelPBPRPs proteins. Sources of the sequences and accession numbers are reported in table 1. The amino acids conserved in ABPXs and DmelPBPRP-1 are represented in bold. Those amino acids found in DmelPBPRP-1 and ABPX or AipsABPX and DmelPBPRP-1, are represented in italics. Conserved cysteines are underlined. These specific amino acids support the classification of these sequences as OBP1 type of proteins. 43 Figure 4: A. Phylogenetic relationships of moth ABPXs and DmelPBPRPs. The primary sequences of the proteins were aligned in Clustal X 1.8 and processed using PAUP 4.0d65. The tree represents equally most parsimonious trees of 909 steps and consistency index 0.52. The numbers above each branch indicates the percent bootstrap support above 50% for the supported node using maximum parsimony (Felsenstein, 1985). The ABPX related protein PdivOBP1 from the scarab beetle Phylloperta diversa (Acc. Num. BAA88061; Wojtasek et al. , 1999) was used as outgroup to root the tree. Using the well-defined PBP/GOBP clade as outgroup generated the same tree topology. B. Phylogenetic analysis of the CSP protein family. An alignment of the sequences of different CSPs reported in table 2 was used to determine a distance matrix and generate an unrooted tree (Clustal X 1.8; PAUP 4.0d65). This strict consensus tree is the result of trees derived using maximum parsimony of 10,000 steps and consistency index 0.52. The numbers above each branch indicates the percentage of bootstrap above 50% that support the branching pattern. The proteins agCG50208 and CG30172 were declared as the outgroup to build the phylogenetic tree of CSPs. Specific grouping of CSPs is observed, in particular in the CSPs from moths. Group 1 (CSP1):AipsCSP/SAP2/BmorCSP1/CLP1/HvirCSP3/CSPHarm/CSPMbraA; Group 2 (CSP2): HvirCSP1/SAP3/CSPMbraB; (CSP3): HvirCSP2/SAP5; SAP1; BmorCSP2; SAP5. Figure 5: Structural properties of moth CSPs. The amino acid residues conserved in these three types of CSP are shown in bold. Black triangles indicate residues in contact with water molecules in the channel structure of CSP. The position of the six α-helices characteristic of CSP are indicated (α1 to α6). 44 45 Picimbon Figure 1 Figure 2, Picimbon Aseg-1 Aips-1 Aseg-2 Aips-2 Gene: Exon1 - Intron1 - Exon2 - Intron2 - Exon3 Aseg-1: 66 318 - 180 993 - 183 Aips-1: 66 312 - 180 - 1056 - 183 Aseg-2: 66 362 - 180 513 - 180 458 - 180 564 - 180 Aips-2: 66 Picimbon Figure 3 AipsABPX-1 HvirABPX-1 MsexABPX AperABPX BmorABPX DmelPBPRP-1 OBP-1 1 10 20 30 40 50 60 GVVMDEDMAELARMVRESCVDETGADVKLVEAANGGADLME--DDKLKCYIKCTMETAGMAVAMDEDMAELARMVRENCAAETGADVALVERVNAGADLMP--DDKLKCYIKCTMETAGMLALEDEEQAELARMVRENCVHEIGVDEGLLAKVDDGADLMP--DPKLKCYLKCTMEMAGMVASLDGEMAELAKMIRDNCADEIGVDVTLLEQVDAGANLMP--DEKFKCYLKCTLETAGMHGQLDDEIAELAAMVRENCADESSVDLNLVEKVNAGTDLATITDGKLKCYIKCTMETAGMVEINPTIIKQV-RKLRMRCLNQTGASVDVIDKSVKNRILPT--DPEIKCFLYCMFDMFGLI V-----------R--R--C---TGA-V-----------L-T--D---KC-L-C-----G-- 70 80 90 100 110 MSDGEVDIEAVMALLPPEMAEHNGPALKSCGTQRGADDCDTAWKTQVCWQNANKAEYFLI MADGEVDIEAVLALLPPELAEHNAPSLRACGTVRGADHCDTAFRTQQCWQNANKADYFLI ISDGVVDVEAVLGLLPDDVKLRTTDIVRACDTQKGADDCDTAFLTQTCWQQANRADYIFI MSDGVVDIEIVLELLPEDLKTKNENLLRKCDTQKGSDDCDTAFLTQVCWQNGNKADYFLI MSDGVVDVEAVLSLLPDSLKTKNEASLKKCDTQKGSDDCDTAYLTQICWQAANKADYFLI DSQNIMHLEALLEVLPEEIYKTINGLVSSCGTQKGKDGCDTAYETVKCYIAVNGKFFIWEEIIVLLG -S------EA-LE-LPEE-------LV-SCGTQ-G-D-CDTA--T--C--A-N------- -118 -118 -118 -118 -120 -123 Picimbon Figure 4 Picimbon Figure 5 1 CSP1 CSP2 CSP3 10 20 40 50 -D-YTDKYD-----EIL-N-RLL--Y--CV---GKC--EGKELK--L--A----YTD-YD-V-LDEIL-N-R--VPY-KCILD-GKCAPD-KELKEHI-EALE ASTYTDKWDNINVDEILES-RL-K-YVDCLLD-GRCTPDGKALKETLPDALE YTD D EIL R Y C GKC K LK A α2 α1 60 CSP1 CSP2 CSP3 30 70 α3 80 90 100 110 -GC-KC---QE-G-----I--LIKN----W--L----DP---WR-KYEDRA-A-GI-IP--ECGKCT--QK-GGTRRVI-HLINHE---W-EL--K-DP---KYEKEL---K--CSKCTEKQKAG~S-KVIR-LVNKR--LWKELSAKYDPNN-YQ--YKDKI---KGQ C KC Q G I L W L DP α4 α5 α6