Introduction

The number of sequenced arthropodan lipocalins adds up to over eighty (see Table 1). From our currently fragmented knowledge of arthropodan genomes, the last common ancestor of this phylum is proposed to possess two lipocalins (see Chapter 3). Intra-lineage duplications enlarged the number of lipocalins, with some large family expansions occurring independently in blood-feeding insects and arachnids.

Table 1

Summary of properties of all known arthropodan lipocalins.

Most arthropodan lipocalins share the common signature and structural properties with the rest of the family. They are single modular proteins of around 200 amino acids that fold tightly in a β-barrel with potential for binding small hydrophobic molecules in a central pocket. They usually have two α-helices at the N- and C-terminal sides, packed against the outer surface of the barrel. Nonetheless, we will encounter in this phylum variations around this main theme. While maintaining the lipocalin fold, the arthropodan lipocalins call our attention because of the panoply of interesting structural and functional explorations that have arisen throughout evolution. Because of the recent extensive review on arthropodan lipocalins by Kayser,1 with special emphasis in their structure and function, it is our aim to offer a review focusing on the peculiarities of these lipocalins while framing them in an evolutionary context.

As a first step of analysis, we can simply look at an alignment of amino acid sequences of arthropodan lipocalins. A schematic representation of such an alignment is shown in Figure 1. The location of all important gaps is coincident with the loops (L) of the β-barrel. While L1, L4 and L6 are relatively well conserved, all other loops accommodate significant variations in size. Particularly large extensions in L5 are present in one of the Pallidipin sequences, and in other lipocalins related to Nitrophorins (see below). Other expanded loops (L3 and L7) are also expected in these lipocalins. These three loops (L3, L5 and L7) face the entrance of the binding pocket (‘the open side of lipocalins’, see Chapter 2), and their variations could condition the ligand binding properties of these lipocalins. Other lipocalins such as the Drosophila lipocalins GLaz and Karl show unique extensions in L2. Because this loop faces the closed side of the β-barrel, it is most probably involved in protein-protein interactions. Loop L3 of GLaz is also long, but there is no sequence similarity with the L3 loop of R. prolixus Pallidipin.

Figure 1

Amino acid sequence alignment of arthropodan lipocalins. The alignment was performed with Clustal X (1.8) using a Gonnet series scoring matrix. We used a gap penalty mask based on the resolved secondary structure of some lipocalins, represented schematically (more...)

Variations are also observed in the length of the N- or C- terminal segments. Most lipocalins can be classified roughly in tree groups, which differ in the length of their C-termini after the last cysteine residue. The longest of these C-terminal extensions have a size similar to the hydrophobic GPI anchoring signal unique to the grasshopper Lazarillo (shown as a zigzag line in Fig. 1). However, the sequence signatures for GPI anchorage are absent in all other cases. In addition, Karl shows a uniquely long N-terminal extension, while some of the insect saliva lipocalins are shorter than average (not shown).

Therefore, we find in arthropods a wide range of variation in particular loops, and in the Nand C-terminal regions of the proteins. These changes are not expected to alter significantly the β-barrel core, and thus provide a substrate of variation for potential functional diversification and specialization.

Using the amino acid sequences, we have reconstructed a phylogeny of arthropodan lipocalins (Fig. 2) including the group of chordate lipocalins most related to them (Apolipoprotein D, ApoD), and using lipocalins from other kingdoms (plants, fungi and protoctists lipocalins) as the outgroup. However, the arachnid saliva lipocalins were not included. Because of their highly divergent sequences, the reliability of the alignment and the tree-reconstruction methods decreases due to long-branch attraction artifacts.² Instead, we have superimposed the branch of arachnid saliva lipocalins (dashed lines) as deduced from the analysis of their intron-exon structure³ (see Chapter 3).

Figure 2

Protein sequence based phylogenetic tree of arthropodan lipocalins and their most related vertebrate group (the Apolipoprotein D) resumed to a single node (black dot). The tree was built by a maximum likelihood-based method that has been used for lipocalin (more...)

Several groups of arthropodan lipocalins are supported in the tree, and these relationships will guide us through the following sections of the chapter.

At the most basal position stands the group of Lazarillo (Laz) related lipocalins, which, in turn, closely relates to the chordate ApoDs currently found in many species ranging from tunicates to humans. The Lazarillo clade also shows, so far, the widest species representation within insects, with one lipocalin found in Orthoptera and Lepidoptera, and two in several Diptera species (fruitflies and mosquitoes) and hymenoptera (bees). Some of the new sequences retrieved from mosquitoes and bees can be ascribed as orthologous to one of the four lipocalins found in the fruitfly Drosophila melanogaster. However, divergence rates are either unequal or very high, so that the sequence of an older Drosophila species, D. yakuba, is set apart in the phylogeny.

The group of tick saliva lipocalins might have stemmed out of an ancestor similar to these Lazarillo lipocalins, since their gene structure is most similar to the Drosophila Neural Lazarillo (NLaz).³ This group represents a big expansion of the lipocalin family through gene duplication and divergence, probably associated to the adaptation to blood-feeding habits. They are all expressed in the salivary glands. Whether more “conventional” lipocalins are also present in other tissues of ticks, remains to be investigated.

A clade relates the only fully sequenced lipocalins reported in crustaceans: the Crustacyanins (CRCs). They have been well characterized in the lobster Homarus gammarus, but proteins with very similar biochemical properties are now found in more species of lobsters, crayfish and crabs. Two CRCs per species are found at the most (only the fully sequenced CRCs have been included in the tree).

The biliproteins (BPs) and their relatives are monophyletically related. All biliproteins so far established as lipocalins are found in Lepidoptera, a relatively young and very diverse group of insects. BPs have also been isolated from other insect orders but their sequences are not yet known and hence their lipocalin nature is still open (reviewed in ref. 1). A few of the lepidopteran lipocalins from this clade are well characterized by having a bilin bound as a specific ligand resulting in a blue protein. The ligands, if any, of the related members of this clade are not known; we therefore refer to them as biliprotein-related proteins (BPRPs). As it is the case for CRCs, a maximum of two lipocalins have been found in each species sampled.

The insect saliva lipocalins relate to the biliproteins clade. This group represents another independent expansion of lipocalins associated with hematophagy adaptation in insects. Interestingly, among them are the Nitrophorins (NPs) that are heme-containing lipocalins. The branches of the insect saliva lipocalins clade are very long, reflecting their high sequence divergence. Caution should thus be taken, since mistaken relationships due to long branch attraction are not ruled out: sequences that are more dissimilar tend to group together. Finding more lipocalins in the same species of blood-sucking insects, as well as the use of other phylogenetically informative characters, should help to establish their relationship to the rest of the family.

In the next sections we will review each of the five groups of lipocalins. We have found big contrasts in the level of knowledge about arthropodan lipocalins. From very fine details on protein structure, ligand-protein interactions and physiological functions, to the vastly unexplored functional and biochemical properties of new sequenced lipocalins. In any case, arthropods are an excellent arena in which to scrutinize and admire the enormous versatility of such an apparently simple protein fold.

The Lazarillo/Apolipoprotein D Related Lipocalins

According to the consensus pattern of gene structure and protein sequence motifs of the family, Lazarillo is a conventional lipocalin with the three conserved protein motifs of the family (see Chapter 2) and its gene ORF intervened by three introns,⁴ two of which are conserved in every metazoan lipocalin found so far (see Chapter 3). As shown in Figure 2, phylogenetic analyses support the orthologous relationship of Laz and the vertebrate ApoD lipocalin.^4-6 In addition, Laz molecular features, expression pattern and function closely resemble those of ApoD.

Crustacyanins: Blue Lipocalins in Crustaceans

As the name suggests, Crustacyanin (CRC) denotes a blue protein from a crustacean. Proteins with biochemical properties of CRC have been isolated from several species of lobsters, crabs and crayfishes, although the CRCs from Homarus gammarus are by far the best known. By contrast to the insect biliproteins discussed below, the blue color of this protein is not due to a bilin, but to a carotenoid. Hence, CRC represents a blue carotenoprotein. Over the past 50 years, many attempts have been undertaken to uncover the building principle and the physical basis of the blue color of CRC, but only recent work succeeded to solve some key features of its structure. The many facets of this long-standing endeavor has been well reviewed recently.¹⁶

CRC represents a protein complex with astaxanthin (3,3'-dihydroxy-β,β-carotene-4,4'-dione), a red carotenoid derived from β-carotene by metabolic oxidation that takes place in many marine arthropods. In the native protein complex, however, the absorption spectrum of the carotenoids is red-shifted by about 150 nm, resulting in an absorption maximum of 632 nm and hence in a blue carotenoprotein. The red color of the free ligand comes back upon protein denaturation, what happens when the lobster is cooked.

The native CRC complex with a molecular mass of ˜320 kDa is called a-Crustacyanin. It is composed of eight β-Crustacyanin subcomplexes that are dimers of 20-kDa monomers, the ultimate protein subunits. As each subunit contains one molecule of astaxanthin, the 16-mer α-Crustacyanin complex contains 16 molecules of astaxanthin. Unusually, the dissociation into the β-Crustacyanin dimers is irreversible. Even more surprising is the shift of the absorption maximum of astaxanthin from 472 nm (in hexane) in the unbound form to about 585 nm in β-Crustacyanin and further to 632 nm in α-Crustacyanin. While the resolved structure of β-Crustacyanin provides a reasonable basis to explain the spectral properties of the carotenoid in the dimer (a 100-nm shift), the further shift of about 50 nm awaits the crystal analysis of the α-complex.^16,17 According to a very recent study, the tight packing of the subunits (yet unknown in detail) allowing exciton coupling between excited states of astaxanthin molecules is the likely cause for this impressive bathochromic effect.¹⁸

When Crustacyanin protein is separated electrophoretically, it appears as five distinct monomers. However, they are encoded by only two genes (CRC1 and CRC2). Recent results suggest that the nonencoded differences result from nonphysiological alterations during purification and storage of α-crustacyanin.¹⁹ The protein sequences of CRC1 and 2 predict a typical lipocalin folding with two conserved cysteine bridges. The structures of four different β-Crustacyanin complexes have been solved,^19-22 with two bound astaxanthin per dimer. In fact, the crystal structures demonstrate a β-barrel made up of eight antiparallel strands and the presence two helices. In the dimers, the monomers are oriented in such a way that their cavity openings are facing each other. Even more surprisingly, each of the two astaxanthin ligands is bound to both cavities by bridging both protein subunits thus sharing each cavity. This unusual mode of ligand binding is a consequence of the large extended conformation of the carotenoid that does not fit into a single lipocalin cavity. The specific arrangement of the two carotenoid chromophores positioned fairly in parallel and close together, explains the high stability of the dimeric pigment complex. The structures further reveal a conformational change of the bound carotenoid that, together with protein contacts, is thought to result in the observed shift of the absorption spectrum of the dimeric β-Crustacyanin versus free astaxanthin.

It is obvious, that the presence of the blue CRC in the carapace of the lobster significantly contributes to camouflage coloration in its marine habitat that is dominated by blue to blue-green light. This is why other marine arthropods (and also other animals) have developed a similar protective coloration in their exoskeleton or underlying tissues. Representative species are the brine shrimp Artemia, some other shrimps and the water flea Daphnia, from which a blue high-molecular weight glycoprotein, called artemocyanin, has been purified.^23,24 In contrast to CRC, however, the blue color of artemocyanin is not due to astaxanthin or any other carotenoid but due to a bilin, reported to be similar to biliverdin IXα. An N-terminal sequence obtained from one of these biliproteins did not reveal any relationship to insect biliproteins that have been identified as lipocalins. So, these crustacean biliproteins seem to have evolved independently as functional equivalent means of protection.

The Bilin-Binding Lipocalins and Their Relatives

The term biliproteins denotes proteins that are specifically associated with a bilin as ligand. They are widespread among insects though mostly known from lepidopteran species. A number of these typically blue proteins have been purified and characterized in more or less detail, but crystal structures have been obtained only for the two proteins discussed below in detail (for an overview, see the review by Kayser in ref. 1). However, other lipocalins, for which the ligand binding properties are not yet known, appear grouped with the biliproteins in a strongly supported clade (Fig. 2). Whether they represent cases of functional divergence without much sequence variations will be discussed below.

Bilin-Binding Protein of Pieris brassicae

The name ‘bilin-binding protein’ (BBP) specifically refers to the biliprotein from Pieris brassicae, the large white cabbage butterfly. BBP is mainly found in the hemolymph of the last larval instar and in the wings of the adult stage, while it is also present in other tissues such as fat body and epidermis. The blue protein in the wings is masked by the large amounts of white pterins in the overlaying scales.

BBP was isolated from whole butterflies by one of us, crystallized and unexpectedly identified as a prototypic lipocalin, the first from an invertebrate source.^25,26 This contrasts to the well known cyanobacterial biliproteins, which are of mainly helical structure and function in photosynthetic light harvesting.^27,28 BBP is found predominantly as a ˜19.8 kDa monomer composed of 174 amino acids,^29,30 is not associated with sugar or lipid, and has one molecule of biliverdin IXγ noncovalently bound.

BBP crystallizes as a tetramer, i.e., as a dimer of dimers, with the monomers showing the typical lipocalin folding of an eight-stranded antiparallel β-barrel with two cysteine bridges at positions known to be conserved in lipocalins.^25,26 Besides of two short helical structures before the first β-strand and close to the C-terminus, respectively, there is one long α-helix attached to a side of the barrel (Fig. 3). The bilin ligand of BBP is bound in the cavity of the barrel and displays the cyclic helical, porphyrin-like structure that is normally adopted also by open-chain tetrapyrroles (for a review, see ref. 31). The orientation of the bilin is such that the two carboxyl groups at the outer pyrrole rings point to the opening of the cavity, i.e., to the solvent. Most remarkably, the bilin, whose two enantiomeric cyclic conformations are rapidly interconverted in solution and therefore optically inactive, is fixed by the protein in one enantiomeric form (Fig. 3) resulting in an optically active bound state.³² This specific mode of binding affects also the visible absorption spectrum of the bilin, which shows a red shift of about 25 nm to a plateau around 670 nm in the bound state; the UV-absorption band of the bilin in BBP is at 383 nm.

Figure 3

Crystal structure of Pieris brassicae bilin-binding protein (BBP), a prototypic lipocalin. Ribbon drawing, in magenta, of the eight antiparallel β-strands forming the core barrel structure. The bound ligand biliverdin IXγ is shown in pale (more...)

According to studies employing radiolabeled precursors for the bilin and the apoprotein, respectively,^33,34 there are two major developmental periods of BBP synthesis, one in the last larval instar during the feeding period, and another one during development of the adult insect starting at mid-pupal stage. These studies further revealed that the bilin and the protein are made in parallel and that opening of the porphyrin ring must immediately follow its synthesis. This developmental pattern of biosynthesis has been confirmed by Northern analysis of BBP messenger RNA, which also identified the fat body as the major site of BBP synthesis in the larva.²⁹ Interestingly, holo-BBP in the developing butterfly is produced in the wings, as revealed in isolated wings in culture (Sehringer and Kayser, unpublished results).

The fact that BBP biosynthesis is under strict developmental and tissue control and, furthermore, that most of the tetrapyrrole precursor 5-aminolevulinate is used for bilin synthesis³⁴ suggest a vital role of this lipocalin in this insect. However, the functional implications of this program remain to be discovered. While the blue biliproteins of insects are generally said to play a role in camouflage coloration, which is certainly true in many species (see below for Insecticyanin), this is not obvious in P. brassicae, for example, where the coloration of larvae and adults is dominated by other pigments (for details, see ref. 1). A hypothetical role of BBP based on light absorption, as in cyanobacterial biliproteins,²⁸ can be excluded as it lacks the required photochemical properties.³⁵ This is likely true for all insect biliproteins. More realistic, though unproved roles of insect biliproteins related to metabolic regulation are discussed in a recent lipocalin review (see ref. 1), and they include prevention of cellular damage by reactive oxygen and nitrogen species, CO signaling cascades, and regulation of soluble guanylyl cyclase by biliverdin.

Expansions of the Lipocalin Family in Blood-Feeding Arthropods

We have described so far many interesting functional explorations that lipocalins have undergone through arthropod evolution. Now, two independent expansions of the family have been found when exploring proteins or mRNA expressed in the salivary gland of two distantly related arthropods, insects and arachnids, that share a common way of life: feeding on vertebrate's blood.

Their assignment to the lipocalin family has been a difficult task, since they share little or almost undetectable protein sequence similarity with other lipocalins. However, evidence of their common ancestry keeps accumulating, and these data are helping to frame the following evolutionary hypothesis: A history of intra-lineage duplications accompanied by high divergence, while both groups of lipocalins (from insects and arachnids) converge to control the hemostatic system of their host to make the most out of a blood meal.

Both, blood-feeding insects and ticks, produce in their saliva a cocktail of proteins specialized in the control of their host hemostatic machinery (reviewed by refs. 51,52). In Table 2, we relate each antihemostatic activity with the protein that plays that role in the cocktail. In Hemiptera and Acari, most of these functions are carried out by an independently evolved set of lipocalins. In Diptera (mosquitoes) these roles have been taken by completely unrelated proteins. ^53,54 This is certainly a striking case of convergent evolution, in which each parasite organism has adopted a different solution to fulfill the same task, probably evolving in parallel to the evolution of hematophagy (dating around 90 My⁵⁵). It also represents a case of coevolution with the host hemostasis and inflammation systems, which have evolved very complex and redundant reactions.

Table 2

Different strategies for the control of the host's hemostatic machinery by blood feeding arthropods.

More Expansions of the Lipocalin Family? The Case of Cockroach Milk

Recently, Williford et al⁹⁶ have described a new set of lipocalins that are secreted by the brood sack epithelium of the viviparous cockroach Diploptera punctata as part of a nutritive secretion or “milk” for the embryos. In species of ovoviviparous cockroaches, embryos develop within an infolding of the intersegmental membrane at the posterior end of the abdomen and are born as first instar larvae. This poach provides protection against desiccation and parasitism. The appearance of viviparity is linked to the production of “milk”, now known to be full of lipocalins. Although only one of them has been included in our phylogenetic tree (Dpun.Milk in Fig.2), a total of 22 distinct lipocalin protein sequences have been identified and the intron-exon structure of one of them has been described.⁹⁶

The presence of four introns intervening the coding sequence of this cockroach milk lipocalin would relate their gene structure to the tick saliva lipocalins³ and to NLaz⁴ (see Fig.4 in Chapter 3). However, their protein sequence becomes grouped with the insect saliva lipocalins and other highly divergent lipocalin sequences like Drosophila yakuba lipocalin, or the cockroach lipocalins of Blatella germanica (with allergenic activity⁹⁷) and of Leucophaea maderae. Curiously, the later is part of an aphrodisiac secretion from the male tergal gland,⁹⁸ another infolding of the intersegmental membrane that could be considered as a morphological and physiological equivalent to the brood sac of D. punctata, but serving a different function at the organismal level.

Multiple gene copies of the milk lipocalins are detected in the genome of D. punctata, therefore representing one more case of gene duplication associated to a recent cooption event:⁷⁴ the acquisition of the new nutritive function which requires the production of these proteins in large amounts, and that embryos develop the ability to drink. The analysis of this new expansion of the lipocalin family should help to understand the transition of ovoviviparity to viviparity in this group of arthropods, in which morphological, physiological and molecular changes are linked and result in a completely new reproductive strategy.

Concluding Remarks

The main conclusion of this work is that lipocalins show and extraordinary ability to acquire novel functions, even though they are apparently simple, single modular proteins. The pattern of duplication and divergence of this protein family in the phylum Arthropoda is characterized by an initial low number of lipocalins maintained in most species sampled, in contrast with large intra-lineage multiplication of genes clearly linked to the appearance of new functions (e.g., feeding or reproductive strategies). This pattern is the result of an evolutionary pathway independent and quite distinct from the one followed by lipocalins in the other best sampled phylum, the chordates (see Chapter 3). However, the properties of lipocalins that grant evolutionary access to such an amazing panoply of functions are the same: their robust folding combined with a flexibility for change which allows lipocalins to interact with different ligands in their binding pocket and make contact with different molecules through their protein surface.

References

1.

Kayser H. Lipocalins and structurally related ligand-binding proteins. In: Gilbert LI, Iatrou K, Gill S, eds. Comprehensive Molecular Insect Science. Oxford: Elsevier. 2005;4:267–306.

2.

Philippe H, Laurent J. How good are deep phylogenetic trees? Curr Opin Genet Dev. 1998;8:616–623. [PubMed: 9914208]

3.

Mans BJ, Neitz AWH. Exon-intron structure of outlier tick lipocalins indicate a monophyletic origin within the larger lipocalin family. Insect Biochem Mol Biol. 2004;34(6):585–594. [PubMed: 15147759]

4.

Sanchez D, Ganfornina MD, Gutierrez G. et al. Exon-intron structure and evolution of the Lipocalin gene family. Mol Biol Evol. 2003;20(5):775–783. [PubMed: 12679526]

5.

Ganfornina MD, Gutierrez G, Bastiani M. et al. A phylogenetic analysis of the lipocalin protein family. Mol Biol Evol. 2000;17(1):114–126. [PubMed: 10666711]

6.

Gutierrez G, Ganfornina MD, Sanchez D. Evolution of the lipocalin family as inferred from a protein sequence phylogeny. Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology. 2000;1482(1-2):35–45. [PubMed: 11058745]

7.

Ganfornina MD, Sanchez D, Bastiani MJ. Lazarillo, a new GPI-linked surface lipocalin, is restricted to a subset of neurons in the grasshopper embryo. Development. 1995;121:123–134. [PubMed: 7867494]

8.

Sanchez D, Ganfornina MD, Bastiani MJ. Developmental expression of the lipocalin Lazarillo and its role in axonal pathfinding in the grasshopper embryo. Development. 1995;121:135–147. [PubMed: 7867495]

9.

Sanchez D, Ganfornina MD, Bastiani MJ. Lazarillo, a neuronal lipocalin in grasshoppers with a role in axon guidance. Biochimica Et Biophysica Acta. 2000;1482(1-2 SU-):102–109. [PubMed: 11058752]

10.

Ganfornina MD, Sanchez D, Bastiani MJ. Embryonic development of the enteric nervous system of the grasshopper Schistocerca americana. J Comp Neurol. 1996;372:581–596. [PubMed: 8876455]

11.

Graf S, Ludwig P, Boyan G. Lazarillo expression reveals a subset of neurons contributing to the primary axon scaffold of the embryonic brain of the grasshopper Schistocerca gregaria. J Comp Neurol. 2000;419(3):394–405. [PubMed: 10723013]

12.

Boyan G, Reichert H, Hirth F. Commissure formation in the embryonic insect brain. Arthropod Structure & Development. 2003;32(1):61–77. [PubMed: 18088996]

13.

Boyan GS, Braunig P, Posser S. et al. Embryonic development of the sensory innervation of the clypeo-labral complex: Further support for serially homologous appendages in the locust. Arthropod Structure & Development. 2003;32(4 SU-):289–302. [PubMed: 18089013]

14.

Sanchez D, Ganfornina MD, Torres-Schumann S. et al. Characterization of two novel lipocalins expressed in the Drosophila embryonic nervous system. Int J Dev Biol. 2000;44(4):349–359. [PubMed: 10949044]

15.

Bailey UM. The drosophila lipocalin, Karl, is specifically expressed in the blood cell compartment. Paper presented at: Benzon symposium No. 50: The lipocalin protein superfamily. Copenhagen, Denmark. 2003

16.

Chayen NE, Cianci M, Grossmann JG. et al. Unravelling the structural chemistry of the colouration mechanism in lobster shell. Acta Crystallogr D Biol Crystallogr. 2003;59(Pt 12):2072–2082 (Epub 2003 Nov 2027). [PubMed: 14646064]

17.

Dellisanti CD, Spinelli S, Cambillau C. et al. Quaternary structure of alpha-crustacyanin from lobster as seen by small-angle X-ray scattering. FEBS Letters. 2003;544(1-3):189–193. [PubMed: 12782314]

18.

van WijkAA, Spaans A, Uzunbajakava N. et al. Spectroscopy and quantum chemical modeling reveal a predominant contribution of excitonic interactions to the bathochromic shift in alpha-crustacyanin, the blue carotenoprotein in the carapace of the lobster homarus gammarus. J Am Chem Soc. 2005;127(5):1438–1445. [PubMed: 15686376]

19.

Habash J, Helliwell JR, Raftery J. et al. The structure and refinement of apocrustacyanin C2 to 1.3 Å resolution and the search for differences between this protein and the homologous apoproteins A1 and C1. Acta Crystallogr D Biol Crystallogr. 2004;60(Pt 3):493–498 (Epub 2004 Feb 2025). [PubMed: 14993674]

20.

Gordon EJ, Leonard GA, McSweeney S. et al. The C1 subunit of alpha-crustacyanin: The de novo phasing of the crystal structure of a 40 kDa homodimeric protein using the anomalous scattering from S atoms combined with direct methods. Acta Crystallogr D Biol Crystallogr. 2001;57(Part 9):1230–1237. [PubMed: 11526314]

21.

Cianci M, Rizkallah PJ, Olczak A. et al. The molecular basis of the coloration mechanism in lobster shell: Beta-crustacyanin at 3.2-Å resolution. Proc Natl Acad Sci USA. 2002;99(15):9795–9800. [PMC free article: PMC125020] [PubMed: 12119396]

22.

Cianci M, Rizkallah PJ, Olczak A. et al. Structure of lobster apocrustacyanin A1 using softer X-rays. Acta Crystallogr D Biol Crystallogr. 2001;57(Pt 9):1219–1229. [PubMed: 11526313]

23.

Krissansen GW, Trotman CNA, Tate WP. Identification of the blue-green chromophore of an abundant biliprotein from the haemolymph of Artemia. Comp Biochem Physiol. 1984;77B:249–252.

24.

Peeters K, Brendonck L, Moens L. The occurrence of artemocyanin in Branchiopoda (Crustacea). Comp Biochem Physiol A Physiol. 1994;109(3):773–779. [PubMed: 8529016]

25.

Huber R, Schneider M, Epp O. et al. Crystallization, crystal structure analysis and preliminary molecular model of the bilin binding protein from the insect Pieris brassicae. J Mol Biol. 1987;195(2):423–434. [PubMed: 3656419]

26.

Huber R, Schneider M, Mayr I. et al. Molecular structure of the bilin binding protein (BBP) from Pieris brassicae after refinement at 2.0 Å resolution. J Mol Biol. 1987;198(3):499–513. [PubMed: 3430616]

27.

Schirmer T, Bode W, Huber R. Refined three-dimensional structures of two cyanobacterial C-phycocyanins at 2.1 and 2.5 Å resolution. A common principle of phycobilin-protein interaction. J Mol Biol. 1987;196(3):677–695. [PubMed: 3119857]

28.

MacColl R. Cyanobacterial phycobilisomes. J Struct Biol. 1998;124(2-3):311–334. [PubMed: 10049814]

29.

Schmidt FS, Skerra A. The bilin-binding protein of Pieris brassicae. cDNA sequence and regulation of expression reveal distinct features of this insect pigment protein. Eur J Biochem. 1994;219(3):855–863. [PubMed: 8112337]

30.

Suter F, Kayser H, Zuber H. The complete amino-acid sequence of the bilin-binding protein from Pieris brassicae and its similarity to a family of serum transport proteins like the retinol-binding proteins. Biol Chem Hoppe Seyler. 1988;369(6):497–505. [PubMed: 3202956]

31.

Kayser H. Pigmens. In: Kerkut GA, Gilbert LI, eds. Comprehensive insect biochemistry, physiology and pharmacology. Pergamon Press. 1985;10:367–415.

32.

Scheer H, Kayser H. Conformational studies of biliproteins from the insects Pieris brassicae and Cerura vinula. Z Naturforsch. 1988;43c:84–90.

33.

Kayser H. De novo synthesis and levels of cytochrome c and a biliprotein during pupal-adult development in a butterfly, Pieris brassicae. Z Naturforsch. 1984;39c:938–947.

34.

Kayser H, Krull-Savage U. Development-specific incorporation of [14C]5-aminolevulinate and [3H]leucine into cytochrome c and biliprotein in the butterfly, Pieris brassicae. Correlation with the ecdysteroid titer in the pupa. Z Naturf. 1984;39c(948-957)

35.

Schneider S, Baumann F, Geiselhart P. et al. Biliproteins from the butterfly Pieris brassicae studied by time-resolved fluorescence and coherent anti-stokes Raman spectroscopy. Photochem Photobiol. 1988;48:239–242.

36.

Cherbas P. Biochemical studies of insecticyanin. Ph.D. thesis, Harvard University, Cambridge, Massachusets. 1973

37.

Riley CT, Barbeau BK, Keim PS. et al. The covalent protein structure of insecticyanin, a blue biliprotein from the hemolymph of the tobacco hornworm, Manduca sexta L. J Biol Chem. 1984;259(21):13159–13165. [PubMed: 6386809]

38.

Holden HM, Rypniewski WR, Law JH. et al. The molecular structure of insecticyanin from the tobacco hornworm Manduca sexta L. at 2.6 Å resolution. EMBO J 1987. 6(6):1565–1570. [PMC free article: PMC553525] [PubMed: 3608987]

39.

Riddiford LM, Palli SR, Hiruma K. et al. Developmental expression, synthesis, and secretion of insecticyanin by the epidermis of the tobacco hornworm, Manduca sexta. Arch Insect Biochem Physiol. 1990;14(3):171–190. [PubMed: 2134175]

40.

Li W, Riddiford LM. Two distinct genes encode two major isoelectric forms of insecticyanin in the tobacco hornworm, Manduca sexta. Eur J Biochem. 1992;205(2):491–499. [PubMed: 1572353]

41.

Li WC, Riddiford LM. The two duplicated insecticyanin genes, ins-a and ins-b are differentially expressed in the tobacco hornworm, Manduca sexta. Nucleic Acids Res. 1994;22(15):2945–2950. [PMC free article: PMC310259] [PubMed: 8065906]

42.

Kiely ML, Riddiford LM. Temporal programming of epidermal cell protein synthesis during the larval-pupal transformation of Manduca sexta. Roux's Arch Devel Biol. 1985;194:325–335.

43.

Li W, Riddiford LM. Differential expression of the two duplicated insecticyanin genes, ins-a and ins-b, in the black mutant of Manduca sexta. Arch Biochem Biophys. 1996;330:65–70. [PubMed: 8651705]

44.

Kang Y, Kulakosky PC, Van Antwerpen R. et al. Sequestration of insecticyanin, a blue hemolymph protein, into the egg of the hawkmoth Manduca sexta. Evidence for receptor-mediated endocytosis. Insect Biochem Mol Biol. 1995;25(4):503–510. [PubMed: 7742835]

45.

Kang Y, Ziegler R, van Antwerpen R. et al. Characterization of the solubilized oocyte membrane receptor for insecticyanin, a biliprotein of the hawkmoth, Manduca sexta. Biochim Biophys Acta. 1997;1324(2):285–295. [PubMed: 9092715]

46.

Saito H. Purification and characterization of two insecticyanin-type proteins from the larval hemolymph of the Eri-silkworm, Samia cynthia ricini. Biochim Biophys Acta. 1998;1380:141–150. [PubMed: 9545563]

47.

Filippov VA, Filippova MA, Kodrík D. et al. Two lipocalin-like peptides of insect brain. In: Suzuki A, Kataoka H, Matsumoto S, eds. Molecular mechanisms of insect metamorphosis and diapause. Tokyo: Industrial Publishing and Consulting, Inc. 1995:35–43.

48.

Sakai M, Wu C, Suzuki K. Nucleotide and deduced amino acid sequences of a cDNA encoding a lipocalin protein in the central nervous system of Bombyx mori. Nippon Sanshi-gaku Zasshi. 2001;70:105–111.

49.

Veiga ABG, Francischetti IMB, Guimaraes JA. A catalog of Lonomia obliqua transcripts and proteins probably involved in the hemorrhagic syndrome [GenBank database]

50.

Reis CV, Ho PL, Ramos CR. r-LOPAP: A member of lipocalin family which shows serine-protease activity [GenBank database]

51.

Montfort WR, Weichsel A, Andersen JF. Nitrophorins and related antihemostatic lipocalins from Rhodnius prolixus and other blood-sucking arthropods. Biochimica Et Biophysica Acta. 2000;1482(1-2 SU-):110–118. [PubMed: 11058753]

52.

Urata J, Shojo H, Kaneko Y. Inhibition mechanisms of hematophagous invertebrate compounds acting on the host blood coagulation and platelet aggregation pathways. Biochimie. 2003;85(5):493–500. [PubMed: 12763308]

53.

Ribeiro JMC, Charlab R, Pham VM. et al. An insight into the salivary transcriptome and proteome of the adult female mosquito Culex pipiens quinquefasciatus. Insect Biochem Mol Biol. 2004;34(6):543–563. [PubMed: 15147756]

54.

Valenzuela JG, Pham VM, Garfield MK. et al. Toward a description of the sialome of the adult female mosquito Aedes aegypti. Insect Biochem Mol Biol. 2002;32(9):1101–1122. [PubMed: 12213246]

55.

Gaunt MW, Miles MA. An insect molecular clock dates the origin of the insects and accords with palaeontological and biogeographic landmarks. Mol Biol Evol. 2002;19(5):748–761. [PubMed: 11961108]

56.

Andersen JF, Champagne DE, Weichsel A. et al. Nitric oxide binding and crystallization of recombinant nitrophorin I, a nitric oxide transport protein from the blood-sucking bug Rhodnius prolixus. Biochemistry. 1997;36(15):4423–4428. [PubMed: 9109649]

57.

Andersen JF, Weichsel A, Balfour CA. et al. The crystal structure of nitrophorin 4 at 1.5 Å resolution: Transport of nitric oxide by a lipocalin-based heme protein. Structure. 1998;6(10):1315–1327. [PubMed: 9782054]

58.

Andersen JF, Montfort WR. The crystal structure of nitrophorin 2. A trifunctional antihemostatic protein from the saliva of Rhodnius prolixus. J Biol Chem. 2000;275(39):30496–30503. [PubMed: 10884386]

59.

Weichsel A, Andersen JF, Champagne DE. et al. Crystal structures of a nitric oxide transport protein from a blood-sucking insect. Nat Struct Biol. 1998;5(4):304–309. [PubMed: 9546222]

60.

Ribeiro JM, Hazzard JM, Nussenzveig RH. et al. Reversible binding of nitric oxide by a salivary heme protein from a bloodsucking insect. Science. 1993;260(5107):539–541. [PubMed: 8386393]

61.

Ribeiro JM, Schneider M, Guimaraes JA. Purification and characterization of prolixin S (nitrophorin 2), the salivary anticoagulant of the blood-sucking bug Rhodnius prolixus. Biochem J. 1995;308(Pt 1):243–249. [PMC free article: PMC1136869] [PubMed: 7755571]

62.

Ribeiro JM, Walker FA. High affinity histamine-binding and antihistaminic activity of the salivary nitric oxide-carrying heme protein (nitrophorin) of Rhodnius prolixus. J Exp Med. 1994;180(6):2251–2257. [PMC free article: PMC2191789] [PubMed: 7964498]

63.

Champagne DE, Nussenzveig RH, Ribeiro JM. Purification, partial characterization, and cloning of nitric oxide-carrying heme proteins (nitrophorins) from salivary glands of the blood-sucking insect Rhodnius prolixus. J Biol Chem. 1995;270(15):8691–8695. [PubMed: 7721773]

64.

Sun J, Yamaguchi M, Yuda M. et al. Purification, characterization and cDNA cloning of a novel anticoagulant of the intrinsic pathway, (prolixin-S) from salivary glands of the blood sucking bug, Rhodnius prolixus. Thromb Haemost. 1996;75(4):573–577. [PubMed: 8743181]

65.

Ribeiro JMC, Andersen J, Silva-Neto MAC. et al. Exploring the sialome of the blood-sucking bug Rhodnius prolixus. Insect Biochem Mol Biol. 2004;34(1):61–79. [PubMed: 14976983]

66.

Moreira MF, Coelho HSL, Zingali RB. et al. Changes in salivary nitrophorin profile during the life cycle of the blood-sucking bug Rhodnius prolixus. Insect Biochem Mol Biol. 2003;33(1):23–28. [PubMed: 12459197]

67.

Weichsel A, Andersen JF, Roberts SA. et al. Nitric oxide binding to nitrophorin 4 induces complete distal pocket burial. Nat Struct Biol. 2000;7(7):551–554. [PubMed: 10876239]

68.

Gerczei T, Fazekas A, Keseru GM. Theoretical study on the nitric oxide binding to nitrophorin 1, an NO-carrier protein from a blood-sucking insect. J Mol Struct: THEOCHEM. 2000;503(1-2):51–58.

69.

Andersen JF, Ding XD, Balfour C. et al. Kinetics and equilibria in ligand binding by nitrophorins 1-4: Evidence for stabilization of a nitric oxide-ferriheme complex through a ligand-induced conformational trap. Biochemistry. 2000;39(33):10118–10131. [PubMed: 10956000]

70.

Kaneko Y, Yuda M, Iio T. et al. Kinetic analysis on nitric oxide binding of recombinant Prolixin-S, a nitric oxide transport protein from the bloodsucking bug, Rhodnius prolixus. Biochim Biophys Acta. 1999;1431(2):492–499. [PubMed: 10350624]

71.

Ribeiro JC. Salivary thiol oxidase activity of Rhodnius prolixus. Insect Biochem Mol Biol. 1996;26(8-9):899–905. [PubMed: 9014335]

72.

Ribeiro JMC. Rhodnius prolixus salivary nitrophorins display heme-peroxidase activity. Insect Biochem Mol Biol. 1998;28(12):1051–1057.

73.

Andersen JF, Gudderra NP, Francischetti IMB. et al. Recognition of anionic phospholipid membranes by an antihemostatic protein from a blood-feeding insect. Biochemistry. 2004;43(22):6987–6994. [PMC free article: PMC2915585] [PubMed: 15170336]

74.

Ganfornina MD, Sánchez D. Generation of evolutionary novelties by functional shift. Bioessays. 1999;21:432–439. [PubMed: 10376014]

75.

Andersen JF, Francischetti IMB, Valenzuela JG. et al. Inhibition of hemostasis by a high affinity biogenic amine-binding protein from the saliva of a blood-feeding insect. J Biol Chem. 2003;278(7):4611–4617. [PubMed: 12464610]

76.

Francischetti IM, Ribeiro JM, Champagne D. et al. Purification, cloning, expression, and mechanism of action of a novel platelet aggregation inhibitor from the salivary gland of the blood-sucking bug, Rhodnius prolixus. J Biol Chem. 2000;275(17):12639–12650. [PubMed: 10777556]

77.

Francischetti IM, Valenzuela JG, Pham VM. et al. Toward a catalog for the transcripts and proteins (sialome) from the salivary gland of the malaria vector Anopheles gambiae. J Exp Biol. 2002;205(Pt 16):2429–2451. [PubMed: 12124367]

78.

Noeske-Jungblut C, Haendler B, Donner P. et al. Triabin, a highly potent exosite inhibitor of thrombin. J Biol Chem. 1995;270(48):28629–28634. [PubMed: 7499380]

79.

Sant AnnaMR, Araujo JG, Pereira MH. et al. Molecular cloning and sequencing of salivary gland-specific cDNAs of the blood-sucking bug Triatoma brasiliensis (Hemiptera: Reduviidae). Insect Mol Biol. 2002;11(6):585–593. [PubMed: 12421416]

80.

Fuentes-Prior P, Noeske-Jungblut C, Donner P. et al. Structure of the thrombin complex with triabin, a lipocalin-like exosite-binding inhibitor derived from a triatomine bug. Proc Natl Acad Sci USA. 1997;94(22):11845–11850. [PMC free article: PMC23629] [PubMed: 9342325]

81.

Noeske-Jungblut C, Kratzschmar J, Haendler B. et al. An inhibitor of collagen-induced platelet aggregation from the saliva of Triatoma pallidipennis. J Biol Chem. 1994;269(7):5050–5053. [PubMed: 8106481]

82.

Haendler B, Becker A, Noeske-Jungblut C. et al. Expression, purification and characterisation of recombinant pallidipin, a novel platelet aggregation inhibitor from the haematophageous triatomine bug Triatoma pallidipennis. Blood Coagul Fibrinolysis. 1996;7(2):183–186. [PubMed: 8735814]

83.

Paddock CD, McKerrow JH, Hansell E. et al. Identification, cloning, and recombinant expression of procalin, a major triatomine allergen. Journal Of Immunology (Baltimore, MD: 1950). 2001;167(5):2694–2699. [PubMed: 11509613]

84.

Mans BJ, Neitz AWH. Adaptation of ticks to a blood-feeding environment: Evolution from a functional perspective. Insect Biochem Mol Biol. 2004;34(1):1–17. [PubMed: 14723893]

85.

Mans BJ, Louw AI, Neitz AWH. The major tick salivary gland proteins and toxins from the soft tick, Ornithodoros savignyi, are part of the tick Lipocalin family: Implications for the origins of tick toxicoses. Mol Biol Evol. 2003;20(7):1158–1167. [PubMed: 12777525]

86.

Paesen GC, Adams PL, Harlos K. et al. Tick histamine-binding proteins: Isolation, cloning, and three-dimensional structure. Mol Cell. 1999;3(5 SU-):661–671. [PubMed: 10360182]

87.

Paesen GC, Adams PL, Nuttall PA. et al. Tick histamine-binding proteins: Lipocalins with a second binding cavity. Biochimica Et Biophysica Acta. 2000;1482(1-2 SU-):92–101. [PubMed: 11058751]

88.

Sangamnatdej S, Paesen GC, Slovak M. et al. A high affinity serotonin- and histamine-binding lipocalin from tick saliva. Insect Mol Biol. 2002;11(1):79–86. [PubMed: 11841505]

89.

Keller PM, Waxman L, Arnold BA. et al. Cloning of the cDNA and expression of moubatin, an inhibitor of platelet aggregation. J Biol Chem. 1993;268(8):5450–5456. [PubMed: 8449907]

90.

Waxman L, Connolly TM. Isolation of an inhibitor selective for collagen-stimulated platelet aggregation from the soft tick Ornithodoros moubata. J Biol Chem. 1993;268(8):5445–5449. [PubMed: 8449906]

91.

Mans BJ, Venter JD, Vrey PJ. et al. Identification of putative proteins involved in granule biogenesis of tick salivary glands. Electrophoresis. 2001;22(9):1739–1746. [PubMed: 11425229]

92.

Mans BJ, Neitz AW. Molecular crowding as a mechanism for tick secretory granule biogenesis. Insect Biochem Mol Biol. 2004;34(11):1187–1193. [PubMed: 15522614]

93.

Mans BJ, Steinmann CM, Venter JD. et al. Pathogenic mechanisms of sand tampan toxicoses induced by the tick, Ornithodoros savignyi. Toxicon. 2002;40(7):1007–1016. [PubMed: 12076655]

94.

Jeffery CJ. Moonlighting proteins. Trends Biochem Sci. 1999;24:8–11. [PubMed: 10087914]

95.

Jeffery CJ. Moonlighting proteins: Old proteins learning new tricks. Trends Genet. 2003;19(8):415–417. [PubMed: 12902157]

96.

Williford A, Stay B, Bhattacharya D. Evolution of a novel function: Nutritive milk in the viviparous cockroach, Diploptera punctata. Evol Dev. 2004;6(2):67–77. [PubMed: 15009119]

97.

Arruda LK, Vailes LD, Hayden ML. et al. Cloning of cockroach allergen, Bla g 4, identifies ligand binding proteins (or calycins) as a cause of IgE antibody responses. J Biol Chem. 1995;270(52):31196–31201. [PubMed: 8537384]

98.

Korchi A, Brossut R, Bouhin H. et al. cDNA cloning of an adult male putative lipocalin specific to tergal gland aphrodisiac secretion in an insect (Leucophaea maderae). FEBS Letters. 1999;449(2-3 SU-):125–128. [PubMed: 10338117]

99.

Thompson JD, Gibson TJ, Plewniak F. et al. The ClustalX windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tool. Nucleic Acids Res. 1997;24:4876–4882. [PMC free article: PMC147148] [PubMed: 9396791]