Accepted Manuscript Molecular evolution and expression of the CRAL_TRIO protein family in insects Gilbert Smith, Adriana D. Briscoe PII:

S0965-1748(15)00026-0

DOI:

10.1016/j.ibmb.2015.02.003

Reference:

IB 2669

To appear in:

Insect Biochemistry and Molecular Biology

Received Date: 1 December 2014 Revised Date:

29 January 2015

Accepted Date: 3 February 2015

Please cite this article as: Smith, G., Briscoe, A.D., Molecular evolution and expression of the CRAL_TRIO protein family in insects, Insect Biochemistry and Molecular Biology (2015), doi: 10.1016/ j.ibmb.2015.02.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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ACCEPTED MANUSCRIPT Ms Manduca sexta Dp Danaus plexippus Bm Bombyx mori

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Insect CRAL_TRIO domain proteins

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Tc Tribolium castaneum Am Apis mellifera Ag Anopheles gambiae Dm Drosophila melanogaster

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Molecular evolution and expression of the CRAL_TRIO protein family in insects

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Gilbert Smith1* and Adriana D. Briscoe1

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*Corresponding author

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Department of Ecology and Evolutionary Biology, University of California, Irvine, CA 92697

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Keywords: Alpha-tocopherol transfer protein; retinoid-binding protein; CRAL_TRIO; SEC14; gene duplication

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Abstract CRAL_TRIO domain proteins are known to bind small lipophilic molecules such as

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retinal, inositol and Vitamin E and include such gene family members as PINTA, α-tocopherol

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transfer (ATT) proteins, retinoid binding proteins, and clavesins. In insects, very little is known

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about either the molecular evolution of this family of proteins or their ligand specificity. Here we

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characterize insect CRAL_TRIO domain proteins and present the first insect CRAL_TRIO

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protein phylogeny constructed by performing reciprocal BLAST searches of the reference

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genomes of Drosophila melanogaster, Anopheles gambiae, Apis mellifera, Tribolium castaneum,

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Bombyx mori, Manduca sexta and Danaus plexippus. We find several highly conserved amino

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acid residues in the CRAL_TRIO domain-containing genes across insects, and a gene expansion

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resulting in more than twice as many gene family members in lepidopterans than other surveyed

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insect species, but no lepidopteran homolog of the PINTA gene in Drosophila. In addition, we

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examined the expression pattern of CRAL_TRIO domain genes in M. sexta heads using RNA-

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Seq data. Of the 42 gene family members found in the M. sexta reference genome, we found 30

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expressed in the head tissue with similar expression profiles between males and females. Our

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results suggest this gene family underwent a large expansion in Lepidoptera, making the

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leptidopteran CRAL_TRIO domain family distinct from other holometabolous insect lineages.

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1. Introduction The CRAL_TRIO domain is an N-terminal ligand binding region within a larger protein

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domain (SEC14) that takes its name from common sequence elements in cellular retinaldehyde

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binding protein (CRALBP) and the signaling domain of the TRIO guanine exchange factor

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(Panagabko et al., 2003). The domain was first characterized three-dimensionally from structures

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of yeast phosphatidylinositol transfer protein SEC14p (Sha et al., 1998) and human supernatant

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protein factor (SPF; Stocker et al., 2002) but is present in most organisms and is likely to be of

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ancient evolutionary origin (Saito et al., 2007). The domain structure is comprised of alternating

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α-helices and β-strands (Fig. 1) that bind small lipophilic molecules such as retinal, inositol,

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squalene, and Vitamin E, the latter of which is composed of α-, β-, γ-, and δ-tocopherols

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(Panagabko et al., 2003). CRAL_TRIO domains are found alongside many other domains in

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proteins, including the Golgi dynamics (GOLD), protein tyrosine phosphatase (PTP), RasGAP,

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RhoGAP, RhoGEF, pleckstrin homology (PH), macro, spectrin repeats (SPEC), Src homology

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(SH3) and protein kinase domains (Gupta et al.,2012; Saito et al., 2007).

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Relatively few members of the large CRAL_TRIO gene family have been studied for

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ligand specificity. Those that have been studied include human α-tocopherol transfer protein (α-

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TTP), SPF (which binds squalene), cellular retinaldehyde binding protein (CRALBP, which

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binds retinal) and Saccharomyces cerevisiae phosphatidylinositol transfer protein (SEC14p,

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which binds phosphatidylinositol and phosphatidylcholine). Ligand specificity of these related

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proteins has been compared in one study that sought to explain why mammals are able to

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selectively reuptake α-tocopherol regardless of the mixture of dietary tocopherols ingested

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(Panagabko et al., 2003). In humans, mutations in α-TTP can result in a neurodegenerative

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disease called ataxia with isolated vitamin E deficiency (AVED) that causes a variety of

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neurological symptoms (Ouahchi et al., 1995). While, of the four proteins compared, α-TTP

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exhibited the highest binding affinity for α-tocopherol, promiscuity in ligand binding was

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observed in the other three proteins, suggesting that caution should be exercised when attempting

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to infer function of these proteins from single ligand studies. The SEC14 domain contains a large

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flexible lipid binding hydrophobic pocket that is closed off by the N-terminal domain, and

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docking studies suggest different lipids may bind SEC14 in several different ways (Saito et al.,

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2007). Few studies have identified key residues that interact with particular ligands. However,

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structural and mutational analysis of yeast SEC14 suggests that residues E207 and K239 are

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critical for phosphatidylinositol head group binding (Sha et al., 1998), and AVED causing

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mutations in human α-TTP are located in the binding pocket (Bromley et al., 2013; Min et al.,

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2003).

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Besides α-tocopherol binding and transport by α-TTP, and phosphatidylinositol/

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phosphatidylcholine exchange and transfer observed in SEC14p, it has been proposed that other

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gene family members are involved in lipid-mediated regulatory functions in organelles and in

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intracellular traffic (Saito et al., 2007). For example, members of the clavesin (clathrin vesicle-

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associated SEC14 proteins; Katoh et al., 2009) gene family are found abundantly in mammalian

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brain tissue on clathrin-coated vesicles that originate from the trans-Golgi network and form the

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major protein transport pathway from the secretory system to endosomes/lysosomes (Katoh et

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al., 2009). Experimental work by Katoh et al. (2009) indicates that clavesins specifically function

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in neurons in a transport pathway between early endosomes and mature lysosomes and that one

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of the molecules they bind is phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2). Mutations in

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mice that deplete PtdIns(3,5)P2 produce neurodegeneration phenotypes with little effect on other

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tissues (Zhang et al., 2007).

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In insects, the function of only one member of the CRAL_TRIO domain protein, PINTA,

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has been determined. PINTA or prolonged depolarization afterpotential (PDA) is not apparent

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was discovered in a screen for Drosophila eye mutants that are deficient in rhodopsin biogenesis

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(Wang and Montell, 2005). Visual pigments or rhodopsins are composed of an opsin protein

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covalently linked to derivatives of Vitamin A (all-trans-retinol), such as 11-cis-retinal. Although

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the cells that compose the vertebrate and invertebrate eye differ significantly, the function of

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PINTA is somewhat reminiscent of human CRALBP, which is involved in the transport of the

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visual pigment chromophore, 11-cis-retinal, in the photoreceptors of the human eye (Crabb et al.,

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1988). Humans that carry mutations in the CRALBP gene may develop retinitis pigmentosa or

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retinitis punctata albescence (Fishman et al., 2004). In Drosophila, PINTA is localized to the

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retinal pigment cells, where all-trans 3-hydroxyretinol is converted to 11-cis 3-hydroxyretinal

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(Pak et al., 2012; Wang and Montell, 2005). Ligand-binding assays indicate that PINTA

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preferentially binds all-trans-retinol (Wang and Montell, 2005). It has been proposed that PINTA

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either sequesters the 3-hydroxy version of all-trans-retinol in retinal pigment cells, generating a

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concentration gradient that promotes uptake of Vitamin A in these cells, and/or that PINTA

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facilitates the presentation of all-trans-retinol to proteins that participate in the oxidation of all-

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trans-retinol to all-trans-retinal (Pak et al., 2012; Wang and Montell, 2005).

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In the course of annotating vision-related proteins of the hawkmoth (Manduca sexta)

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genome, we discovered that lepidopteran genomes appear to contain a large number of

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CRAL_TRIO domain proteins. While many of these gene family members are given names in

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GenBank such as 'α-tocopherol transfer (ATT) protein', 'clavesin' or 'retinaldehyde binding

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protein', in fact nothing is known experimentally about the function of any of these proteins in

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insects, except for PINTA. To help begin to bridge this gap in our knowledge, here we

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characterize for the first time the CRAL_TRIO domain in insects and its molecular phylogeny.

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Specifically, we determine phylogenetic relationships between gene family members from

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insects with reference genomes, including the newly sequenced genome of M. sexta. We find a

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number of conserved sites within the SEC14 domain. Further, we see large variation in the size

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of the gene family, with lepidopterans (butterflies and moths) having the highest number of gene

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family members among the species examined. We also make use of RNA-Seq data to examine

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the expression of gene family members in M. sexta.

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2. Materials and methods

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We searched the predicted proteins of the reference genomes of M. sexta and Danaus

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plexippus for all homologs of the CRAL_TRIO domain family using tblastn and query sequences

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from Drosophila melanogaster, Anopheles gambiae, Apis mellifera, Tribolium castaneum and

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Bombyx mori homologs, as well as reciprocal tblastn searches of the nr/nt or RefSeq RNA

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databases (top hits with e-values of < 1 × 10-10). We then performed tblastn searches of a de novo

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assembly of the M. sexta transcriptome (ArrayExpress accession number E-MTAB-2066; Smith

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et al., 2014) to find mRNA contigs that were identical to the predicted peptides. Comparison of

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predicted peptide sequences from the reference genome against the predicted peptide mRNA

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sequences permitted us to identify and correct errors in the predicted protein sequences. Isoforms

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found in GenBank were excluded from further analysis, with the first annotated transcript

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selected for analysis. The identity of the CRAL_TRIO domain-containing proteins was

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confirmed through a comparison of their SEC14 domains to the Pfam database of HMM-based

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gene family models using HMMER (Finn et al., 2011). Amino acid sequences were aligned

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using MUSCLE with default settings. Neighbor-joining analysis of 455 amino acid sites was

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performed using the Poisson model and pairwise deletion in MEGA (Tamura et al., 2011).

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Robustness of the phylogeny was tested using 500 bootstrap replicates.

3 2.1. 3D modeling and sequence logo of the insect CRAL_TRIO/SEC14 domain

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The three-dimensional protein structure of the SEC14 domain (containing the

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CRAL_TRIO domain) was predicted using I-TASSER (Roy et al., 2010), which uses both

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multiple threading alignments and ab initio modeling with further refinement to obtain the most

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likely 3D structure. Several shorter sequences were omitted from this analysis in order to

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minimize gaps in the alignment. Thus the 3D structure was predicted from the consensus

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sequence of 159 insect SEC14 domains, producing a top model with a confidence score (C-

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score) of 0.87. The three dimensional structure was visualized in Jmol (Jmol Team, 2002) and

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secondary structures were predicted using Jalview (Waterhouse et al., 2009) from the alignment.

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A sequence logo was created in WebLogo (Crooks et al., 2004) to examine conservation of the

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SEC14 domain across insects. Highly conserved amino acid positions were determined as those

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with a bit score of > 2.5 with a frequency of > 70% at a particular position.

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2.2. RNA-Seq Analysis

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Bar plots were made using the R package ggplot2 (Wickham, 2009) to visualize the

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expression levels of members of the CRAL_TRIO domain gene family in M. sexta. This data

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was obtained from Smith et al. (2014) and consisted of mRNA extracted from the heads of four

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male and four female M. sexta adults sequenced on the Illumina platform (50 bp single-end

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reads), followed by de novo transcriptome assembly using Trinity (Grabherr et al., 2011) and

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RSEM (Li and Dewey, 2011) to assemble contigs that represent mRNA transcripts and obtain

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expression levels. Raw count levels were normalized by transcript length using the FPKM

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(Fragments per kilobase of exon per million fragments mapped). FPKM was further normalized

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between libraries in the R package NOIseq (Tarazona et al., 2011), using the trimmed mean of

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M-values (TMM) normalization method. Differences in gene expression between males and

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females were tested using ANOVA, and p-values were corrected for multiple tests using a False

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Discovery Rate (FDR) calculated in the R package qvalue (Dabney and Storey, 2014). Genes

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were defined as significantly differentially expressed when the FDR was < 0.05.

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3. Results and discussion

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Our GenBank searches yielded 43 predicted proteins in the CRAL_TRIO domain family

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from B. mori, 12 from D. melanogaster, 14 from A. gambiae and 18 from T. castaneum. We also

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found 6 from A. mellifera, although this number may have been underestimated due to the poor

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initial annotation of this genome (Elsik et al., 2014). Our searches of whole-genome sequences

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yielded 42 predicted CRAL_TRIO domain proteins in the Manduca sexta genome (Table S1)

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and 38 in the Danaus plexippus genome. A variety of isoforms were also evident in each species.

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A comparison of the blast-identified CRAL_TRIO proteins to existing Pfam HMM protein

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family models showed the top hit for each to be the CRAL_TRIO domain family (Pfam

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accession PF00650). All but four proteins had top hit e-values of < 1 × 10-10, the remaining four

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having e-values of < 1 × 10-5. M. sexta e-values of matches to the CRAL_TRIO domain family

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are shown in Table S1.

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The insect SEC14 consensus protein structure prediction included four β-strands (Fig. 1A

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and B), compared to six in the yeast phosphatidylinositol transfer protein (Sha et al., 1998) and

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five in the human supernatant protein factor (Stocker et al., 2002). The overall insect SEC14

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structure resembled that of human and yeast SEC14 domain structures, which includes a deep

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hydrophobic pocket with the β-strands making up the floor of the pocket. Sequence logo analysis 8

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of the SEC14 domain, in which the CRAL_TRIO_N domain is embedded (Fig. 1C), indicates

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the presence of 18 highly conserved amino acid residues 12 of which are conserved compared to

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yeast SEC14p and 6 of which appear to be conserved across insects (Fig. 1C). The majority of

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these conserved residues (12 out of 18) were associated with secondary structures (α-helices or

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β-strands). Gupta et al. (2012) surveyed CRAL_TRIO domains across a broad range of species,

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including yeast, plants, fish and humans, yet only one residue (position D178 in yeast SEC14p)

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is conserved in both their study and here, providing further evidence that the six conserved

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residues found here could be insect specific.

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Phylogenetic analysis of the gene family (excluding isoforms) suggests numerous

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lineage-specific gene duplications in insects but especially in lepidopterans (Fig. 2). Many gene

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duplicates are in adjacent positions on the same genomic scaffold suggesting they are tandem

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arrays of gene duplicates (e.g., M. sexta genes Msex005959-Msex005962). In particular, nine

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genes from the lepidopteran expansion are tandemly arrayed on a single scaffold in M. sexta

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(Msex010487-Msex010499), of which four were expressed in the head tissues (Table S1).

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Similar patterns were seen for B. mori and D. plexippus genes within the lepidopteran expansion,

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and duplication patterns suggest independent expansion events in each species (Fig. 2). This

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suggests an important role for gene duplication during the evolution of lepidopteran

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CRAL_TRIO proteins. Orthologs of the PINTA gene in Drosophila were not found in any of the

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three lepidopteran genomes searched. Additionally, we found evidence of mRNA expression in

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M. sexta heads for 30 CRAL_TRIO domain genes (Fig. 3). The majority of these genes had

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appreciable levels of expression and one in particular, comp17579_c0 (Msex0051227; Table

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S1), was expressed at a very high level. Few differences in the expression of CRAL_TRIO

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domain

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genes

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(comp20575_c0/Msex014871) showed significant differential expression after FDR correction,

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having a higher expression level in males (Fig. 3). Clavesins, which are predominantly associated with clathrin-coated vesicles in

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mammalian brain tissue, contain a clathrin box motif, LLALD, which is putatively involved in

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clathrin binding (Katoh et al. 2009). We found this motif, which is the result of a four amino acid

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insertion in the C-terminal domain, to be conserved in three orthologous lepidopteran proteins:

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M. sexta Msex016323, B. mori XP_004922581 and D. plexippus DPOGS213497 (Fig. 2).

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Msex016323 (comp35578_c0) is also one of the 30 CRAL_TRIO domain genes expressed in

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heads, though at relatively low levels compared to other CRAL_TRIO genes (Fig. 3). We did not

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find this insertion and precise motif in any of the lepidopteran proteins found in GenBank named

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clavesin-1-like or clavesin-2-like except for B. mori XP_004929842 and Msex010326

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(comp20338_c0), which contained a related motif, VLALD/N, in a similar position in the

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protein. Unlike comp35578_c0 (Msex016323), comp20338_c0 is expressed at appreciable levels

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in M. sexa heads.

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It is notable that we did not find an ortholog of the PINTA gene in lepidopterans (Fig. 2).

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Currently, the identities of the retinoid binding proteins (RBPs) required for transformation of

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vitamin A to the chromophore remain unclear in most insects. In Drosophila, all-trans 3-

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hydroxyretinal can be converted into 11-cis 3-hydroxyretinal by light but it is also the case that

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11-cis 3-hydroxyretinal can be made from dietary Vitamin A. The fly chromophore does not

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rapidly dissociate from the opsin after light excitation, whereas in butterflies it can (Bernard,

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1983a; 1983b; Stavenga and Hardie, 2011). This suggests that in lepidopteran species,

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production of the chromophore may be even more dependent on enzymatic processes than in

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Drosophila. It is therefore possible that some of the newly-identified CRAL_TRIO domain

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proteins identified here have taken on that role as retinoid-binding proteins involved in the visual

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cycle. We surveyed insect proteins containing the lipid binding CRAL_TRIO domain, including

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the newly sequenced M. sexta genome. We found that the CRAL_TRIO domain gene family has

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undergone a considerable expansion in lepidopteran species and that a number of amino acid

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residues within the wider SEC14 domain appear to demonstrate insect-specific conservation.

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Further, the majority of these genes are expressed in the head tissue in M. sexta. Our results

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suggest that the evolution of CRAL_TRIO domain-containing proteins might play an important

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role in insect evolution and in particular in the evolution of lepidopteran species, which is

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potentially linked to the evolution of visual systems.

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We would like to thank two anonymous reviewers for their constructive comments. This project

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was funded by a National Science Foundation (NSF) grant: IOS-1257627.

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References

2

Bernard, G.D., 1983a. Bleaching of rhabdoms in eyes of intact butterflies. Science. 219, 69-71.

3

Bernard, G.D., 1983b. Dark-processes following photoconversion of butterfly rhodopsins.

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1

Biophys. Struct. and Mech. 9, 277-286.

Bromley, D., Anderson, P.C., Daggett, V., 2013. Structural consequences of mutations to the α-

6

tocopherol transfer protein associated with the neurodegenerative disease ataxia with

7

vitamin E deficiency. Biochemistry 52, 4264-4273.

SC

5

Crabb, J.W., Goldflam, S., Harris, S.E., Saari, J.C., 1988. Cloning of the cDNAs encoding the

9

cellular retinaldehyde-binding protein from bovine and human retina and comparison of

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the protein structures. J. Biol. Chem. 263, 18688-18692.

Crooks, G.E., Hon, G., Chandonia, J.M., Brenner, S.E., 2004. WebLogo: a sequence logo generator. Genome Res. 14, 1188-1190.

Dabney, A., Storey, J.D., 2014. qvalue: Q-value estimation for false discovery rate control. R

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package version 1.36.0.

Elsik, C.G., Worley, K.C., Bennett, A.K., Beye, M., Camara, F., Childers, C.P., de Graaf, D.C.,

16

Debyser, G., Deng, J.X., Devreese, B., Elhaik, E., Evans, J.D., Foster, L.J., Graur, D.,

17

Guigo, R., Hoff, K.J., Holder, M.E., Hudson, M.E., Hunt, G.J., Jiang, H.Y., Joshi, V.,

19 20

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18

EP

15

Khetani, R.S., Kosarev, P., Kovar, C.L., Ma, J., Maleszka, R., Moritz, R.F.A., MunozTorres, M.C., Murphy, T.D., Muzny, D.M., Newsham, I.F., Reese, J.T., Robertson, H.M., Robinson, G.E., Rueppell, O., Solovyev, V., Stanke, M., Stolle, E., Tsuruda, J.M., Van

21

Vaerenbergh, M., Waterhouse, R.M., Weaver, D.B., Whitfield, C.W., Wu, Y.Q.,

22

Zdobnov, E.M., Zhang, L., Zhu, D.H., Gibbs, R.A., HGSC Production Teams, Honey

12

ACCEPTED MANUSCRIPT

1

Bee Genome Sequencing Consortium, 2014. Finding the missing honey bee genes:

2

lessons learned from a genome upgrade. BMC Genomics 15, 86.

4

Finn, R. D., Clements, J., Eddy S. R., 2011. HMMER web server: interactive sequence similarity

RI PT

3

searching. Nucleic Acids Res. 39(Web Server issue): W29-W37.

Fishman, G.A., Roberts, M.F., Derlacki, D.J., Gimsby, J.L., Yamamoto, H., Sharon, D.,

6

Nishiguchi, K.M., Dryja, T.P., 2004. Novel mutations in the cellular retinaldehyde-

7

binding protein gene (RLBP1) associated with retinitis punctata albescens: evidence of

8

interfamilial genetic heterogeneity and fundus changes in heterozygotes. Arch.

9

Ophthalmol. 122, 70-75.

M AN U

SC

5

Grabherr, M.G., Haas, B.J., Yassour, M., Levin, J. Z., Thompson, D. A., Amit, I., Adiconis, X.,

11

Fan, L., Raychowdhury, R., Zeng, Q., Chen, Z., Mauceli, E., Hacohen, N., Gnirke, A.,

12

Rhind, N., di Palma, F., Birren, B. W., Nusbaum, C., Lindblad-Toh, K., Friedman, N.,

13

Regev, A., 2011. Full-length transcriptome assembly from RNA-Seq data without a

14

reference genome. Nat. Biotechnol. 29, 644-652.

17 18 19 20 21

http://www.jmol.org/.

EP

16

Jmol Team, 2002. Jmol: an open-source Java viewer for chemical structures in 3D.

Katoh, Y., Ritter, B., Gaffry, T., Blondeau, F., Hönig, S., McPherson, P.S., 2009. The clavesin

AC C

15

TE D

10

family, neuron-specific lipid- and clathrin-binding Sec14 proteins regulating lysosomal morphology. J. Biol. Chem. 284, 276464-27654.

Li, B., Dewey, C.N., 2011. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics. 12, 323.

13

ACCEPTED MANUSCRIPT

1

Min, K.C., Kovall, R.A., Hendrickson, W.A., 2003. Crystal structure, of human alpha-tocopherol

2

transfer protein bound to its ligand: Implications for ataxia with vitamin E deficiency. P.

3

Natl. Acad. Sci. USA 100, 14713-14718. Ouahchi, K., Arita, M., Kayden, H., Hentati, F., Ben Hamida, M., Sokol, R., Arai, H., Inoue, K.,

5

Mandel, J.L., Koenig, M., 1995. Ataxia with isolated vitamin E deficiency is caused by

6

mutations in the alpha-tocopherol transfer protein. Nat. Genet. 9, 141-145.

RI PT

4

Pak, W.L., Shino, S., Leung, H.T., 2012. PDA (prolonged depolarization afterpotential)-

8

defective mutants: the story of nina's and ina's--pinta and santa maria, too. J.

9

Neurogenet. 26, 216-237.

M AN U

SC

7

10

Panagabko, C., Morley, S., Hernandez, M., Cassolato, P., Gordon, H., Parsons, R., Manor, D.,

11

Atkinson, J., 2003. Ligand specificity in the CRAL-TRIO protein family. Biochemistry

12

42, 6467-6474.

16 17 18 19 20 21

TE D

15

structure and function prediction. Nat. Protoc. 5, 725-738. Saito, K., Tautz, L., Mustelin, T., 2007. The lipid-binding SEC14 domain. Biochim. Biophys. Acta. 1171, 719-726.

EP

14

Roy, A., Kucukural, A., Zhang, Y., 2010. I-TASSER: a unified platform for automated protein

Sha, B., Phillips, S.E., Bankaitis, V.A., Luo, M., 1998. Crystal structure of the Saccharomyces cervisiae phosphatidylinositol-transfer protein. Nature. 391: 506-510.

AC C

13

Smith, G., Chen, Y.R., Blissard, G.W., Briscoe, A.D., 2014. Complete dosage compensation and sex-biased gene expression in the moth Manduca sexta. Genome Biol. Evol. 6, 526-537.

Stavenga, D.G., Hardie, R.C., 2011. Metarhodopsin control by arrestin, light-filtering screening

22

pigments, and visual pigment turnover in invertebrate microvillar photoreceptors. J.

23

Comp. Physiol. A. 197, 227-241.

14

ACCEPTED MANUSCRIPT

1 2

Stocker, A., Tomizaki, T., Schulze-Briese, C., Baumann, U., 2002. Crystal structure of the human supernatant protein factor. Structure 10, 1533-1540. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S., 2011. MEGA5:

4

molecular evolutionary genetics analysis using maximum likelihood, evolutionary

5

distance, and maximum parsimony methods. Mol. Biol. Evol. 28, 2731–2739.

7

Tarazona, S., Garcia-Alcalde, F., Dopazo, J., Ferrer, A., Conesa, A., 2011. Differential expression in RNA-seq: a matter of depth. Genome Res. 21, 2213-2223.

SC

6

RI PT

3

Voolstra, O., Oberhauser, V., Sumser, E., Meyer, N.E., Maguire, M.E., Huber, A., von Lintig, J.,

9

2010. NinaB is essential for Drosophila vision but induces retinal degeneration in opsin-

10 11 12

M AN U

8

deficient photoreceptors. J. Biol. Chem. 285, 2130-2139.

Wang, T., Montell, C., 2005. Rhodopsin formation in Drosophila is dependent on the PINTA retinoid-binding protein. J. Neuroscience. 25, 5187-5194.

Waterhouse, A.M., Procter, J.B., Martin, D.M.A., Clamp, M., Barton, G.J., 2009. Jalview

14

Version 2-a multiple sequence alignment editor and analysis workbench. Bioinformatics

15

25, 1189-1191.

TE D

13

Wickham, H., 2009. ggplot2: elegant graphics for data analysis. New York, Springer.

17

Zhang, Y., Zolov, S.N., Chow, C.Y., Slutsky, S.G., Richardson, S.C., Piper, R.C., Yang, B., Nau,

18

J.J., Westrick, R.J., Morrison, S.J., Meisler, M.H., Weisman, L.S., 2007. Loss of Vac14, a

20 21

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regulator of the signaling lipid phosphatidylinositol 3,5-bisphosphate, results in neurodegeneration in mice. Proc. Natl. Acad. Sci. USA 104, 17518-17523.

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Fig. 1. Protein structure and conservation of the SEC14 domain in insects. The three-dimensional protein structure was predicted using I-TASSER and visualized in Jmol from two different viewpoints (a & b) using the consensus sequence of 159 insect sequences (α-helices shown in pink, β-strands in yellow and 310 helices purple). The sequence logo (c) was created in WebLogo from 159 insect sequences and includes the approximate positions of α-helices (blue bars) and βstrands (red arrows) numbered from the N- to C-terminus, the majority of which are labeled in a & b. The height of each stack is proportional to the sequence conservation, measured in bits, and the height of each letter is proportional to the frequency of residues at that position. The position of the CRAL_TRIO_N domain is shown (dashed red line). Black arrows denote highly conserved amino acid positions with a bit score of > 2.5 and frequency of > 70% at that site. Numbers above arrows show the position of that amino acid within the yeast SEC14p protein (NCBI accession: NP_013796). Red stars indicate conserved sites between the insect consensus and yeast SEC14p sequences.

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Figure and table legends

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Fig. 2. Phylogeny of the insect CRAL_TRIO domain proteins. The neighbor-joining tree was constructed in MEGA with 500 bootstrap replicates. Bootstrap support values of > 80% are shown as dots on branches.

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Fig. 3. Expression levels of members of the CRAL_TRIO domain gene family in the head tissue of Manduca sexta. Expression level is the mean normalized FPKM for female (red) and male (blue) RNA sequencing libraries. Error bars are the standard errors of mean female and male expression across four biological replicates of each sex. Contig IDs are from the Trinity assembly. Significant sex differences are denoted by asterisks, with significance defined as a FDR of < 0.05.

Table S1. Top BLAST hits of M. sexta genome annotations, and corresponding de novo assembled mRNA transcripts, to B. mori and D. plexippus. Table includes the BLAST hit description, e-value, percent identity, accession and the top-hit e-value from the HMMER search of the Pfam database.

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co

0

_c

44 2

23

mp

co

0

_c

57 5

20

mp

co

0

_c

38 7

20

mp

co

0

_c

33 8

20

mp

0

_c

89 6

_c

0

300

co

mp

co

87 5

18

mp

co

17

0

_c

81 3

17

mp

0

_c

AC C

200

co

mp

0

_c

57 9

17

mp

co

51 2

17

100

co

0

0

_c

42 8

17

mp

co

9_ c

23

73

0

_c

11 6

17

mp

co

mp 1

co

0

_c

49 6

16

mp

co

0

_c

14 7

16

mp

co

_c

0

0

5_ c

49 0

13

mp

co

mp

co

19

6_ c

99

24

mp 1

03

mp 1

co

co

Expression level (FPKM)

ACCEPTED MANUSCRIPT

500 Female Male

*

0

ACCEPTED MANUSCRIPT

Highlights

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The insect CRAL_TRIO domain containing proteins contain 18 highly conserved amino acid residues. We present the predicted 3D structure of the consensus insect CRAL_TRIO containing SEC14 domain. The CRAL_TRIO domain containing gene family shows a large expansion in lepidopterans with lineage specific duplication events. Thirty of the CRAL_TRIO domain containing proteins are expressed in the head tissue of Manduca sexta.

RI PT

•

1.00E-134 2.00E-133 1.00E-179 0.00E+00 4.00E-161 5.00E-158 2.00E-156 4.00E-117 5.00E-106 2.00E-165 5.00E-131 2.00E-92 0.00E+00 0.00E+00 0.00E+00 0.00E+00 0.00E+00 0.00E+00 0.00E+00 6.00E-173 2.00E-83 1.00E-65 1.00E-97 5.00E-72 3.00E-100 2.00E-141 2.00E-86 1.00E-121 1.00E-83 4.00E-129 0.00E+00 0.00E+00 6.00E-132 3.00E-66 0.00E+00 7.00E-174 0.00E+00 5.00E-32 2.00E-76 8.00E-101

Identity Accession 85% XP_004929634 61% XM_004929578.1 65% XP_004929635.1 65% XP_004929639.1 78% XM_004928914.1 88% EHJ76881.1 71% XP_004929378 69% XP_004929248 75% AK382842.1 54% XP_004931422.1 54% XP_004922757.1 71% XP_004922758.1 60% XP_004922800.1 53% XM_004922706.1 93% XP_004928836 85% NP_001040355 78% XP_004928931 92% XP_004928929 81% XM_004928871.1 90% XP_004930908 91% XP_004930906.1 71% XM_004929785.1 42% XP_004928863.1 38% XM_004928806.1 46% XM_004928806.1 37% XM_004928806.1 45% XP_004928863.1 58% XP_004928863.1 43% XM_004928806.1 53% XP_004929052.1 44% XM_004928806.1 56% EHJ79293.1 80% XM_004925582.1 84% XP_004925625.1 61% XP_004927864.1 43% XP_004928863.1 82% XP_004925626.1 75% XM_004930854.1 92% XM_004922524.1 88% XP_004933182.1 52% XM_004929189.1 45% EHJ78511

ACCEPTED MANUSCRIPT

Top hit E-value of hit to Pfam CRAL_TRIO domain Amino acid sequence PREDICTED: alpha-tocopherol transfer protein-like [Bombyx mori] 4.40E-23 MPDQEIMQCVDLVTIKDWLSKQPHLPHDVDDILLLRFLTSCKFSLERTKRTIDFFFTIRHNAPELFCKRDPWAPEIRRVFEITDMLPLPNKTKENYKVFIYRLKSPELD PREDICTED: Bombyx mori uncharacterized LOC101746774 (LOC101746774), mRNA 2.00E-19 MSNITSQDIQYIRDWMAKEDYLPKDFSDMMIMKFLHSCYGSLEQTKQCIEKFCVGRLNMPEIFTNRDPLSPKLQTAFSITNMASYNAGECEILLYQIDDPTLENFDFY PREDICTED: uncharacterized protein LOC101746774 [Bombyx mori] 6.30E-20 MAVEMTNISNNDIQFIRDWMAKEDHLPKDFTDMMIKKFLHSCYGSLEQTKQCIEKFCAGRQNMPELYTNRDPLSPRVLTANSITTVATYNIEGDEILIHQLYDPNLE PREDICTED: alpha-tocopherol transfer protein-like [Bombyx mori] 4.80E-26 MDDKISDEVRKQLEDMKQWASTQPLIPKDIEDRILLRFIHSCYYDLEKAKVAADLFFSIRSASSELFTNRDPLAPAMQKTLQIVNLAQYVTSKNRNIWIWQLNDPGL PREDICTED: Bombyx mori alpha-tocopherol transfer protein-like (LOC101736577), mRNA 2.80E-24 MPVELEFDYDEATASMDKFSQDDIDELRAWTEKLDKSKCVPKDLTDKQLLLFYNACYGDMDKTKTCMEKYYACRKNAPEFFDNRVFNTQEMKSAQDVLEFSFL cellular retinaldehyde-binding protein [Danaus plexippus] 1.30E-34 MTEYAEYDWKLHQMQNERLCEAARKRLTDGDRQRALVALKDTVDSSLNLALRDKSRCQDDNFLRRFLYARKHDVQQSFELLVRYHQHRREHPELWATADGGV PREDICTED: alpha-tocopherol transfer protein-like [Bombyx mori] 1.40E-28 MSIRPLNPQLAEKAKKELFEDPSRIAADVQHLKEWIAKQPHLRARTDDQWLVTFLRGCKYSLERTKEKFDLYYSVRSTAPELFRIHINHAMFDEILSLGSMLILPKLA PREDICTED: clavesin-1-like isoform X1 [Bombyx mori] >ref|XP_004929249.1| PREDICTED: clavesin-1-like isoform X2 [Bombyx mori] 4.30E-28 MIRELPSDLAKIAKEELNEKPKRTKDDVKHLKDWIAKQPHLNARTDDQWLVAMLRGCKFSLERVKQKLDLYYTLRTTAPEVTLRILPTEDKFVEFFKLGSCLVLPK Bombyx mori mRNA, clone: fepM03J23 3.90E-20 MAFLQGPSLKQAEVIKQELGEGPGDLERGVRDLRSMLAASPHLPQPEAFDRHLLELFVRGCRMDLDRARSKLEAFCLARARHSDLYEHRSFTEPPLNDICKFMDIA PREDICTED: uncharacterized protein LOC101744959 [Bombyx mori] 1.40E-24 MPQDTQDLVQLNPDSVKYIRKLYGFEDEKKLEEAIDILDAWIKKQNHFTKKDFPRKYLESTILANKGSIERSKSKLDKLCTLRAILGFFFKFSNIRTDFSNFFKATTLIP PREDICTED: clavesin-2-like [Bombyx mori] 5.60E-13 MALKQISIQREYEKNPEISPEDIAQLREWLVTQPHLPGELLTDLDLLLVYHCCERSMQVSKQVLDLHYTLRTMFTQFFHNRSVDKKIEFTLDTVLICPLPTRSTDGNK PREDICTED: alpha-tocopherol transfer protein-like [Bombyx mori] 1.60E-21 MTADVVQPLPLEEEYKKKTRITPEDIKKFRQWLETQPHLPSDYITDMDLILTYHCCNRSSEVSKQVLDLNFTLRTLFTNLFKDRHPDDKMMKAFSYYLLLPLETRAH PREDICTED: clavesin-1-like [Bombyx mori] 1.30E-19 MTIINSYQQKINMSRLESFPLEEEYKKPTGITPQDIARLRQWLATQPHLPHEHITDLDLILLYHRYGCSIEVSKQELDNQYTLRTLFASFFKDRVLDEKIETVLNTVLIT PREDICTED: Bombyx mori alpha-tocopherol transfer protein-like (LOC101746971), mRNA 2.10E-19 MNSYVARSFPLEEEYKKPTGITPQDIARLRKWLATQPHLPQHITDLDLILSFHSCKRCMETTKKLLDTHYTMKTNFDAIFKNRIVDDKIELVLKRVLLNPLPTRTKDG PREDICTED: uncharacterized protein LOC101741307 [Bombyx mori] 3.50E-22 MGCWMKSQPHLPHIPDEYIFLFLHSNYYKLKETKDTIECYFTLRANTPDLFTNRDPLTPKNKTIMDITHMVALPNKTTEGYHVLLYRLADFDYSKLNFADAVRVFC cellular retinaldehyde-binding protein [Bombyx mori] >gb|ABF51246.1| cellular retinaldehyde-binding protein [Bombyx mori] 1.20E-28 MCNMKIKQELLVQPTGELWKAIRVELNEDVANNDRDLAAIREWLKKQPHLPDDWETGPLMTFLRGSSFSLEKCKRKLDMHFTMRAACPEFFTKRDASSPELSEIL PREDICTED: alpha-tocopherol transfer protein-like [Bombyx mori] 3.20E-29 MGINETSKFCGRQARKTNEPKMAVSLVQPSGEMWKKIRVELNEDINTRERDLATIKEWLRKQPHLPNEWDDNCLMTFLRGSSFSLEKCKRKLDMYFTMRAACPE PREDICTED: alpha-tocopherol transfer protein-like [Bombyx mori] 4.10E-28 MTLEQPAGEMWQKIREELNENVDTRDADLAHIKEWLKKEPHLPDEFDDQRIMTFLRGCKFSLEKTKRKLDMYFTMRTAVPEFFTNRDVARPELQEILNIVQMPPLP PREDICTED: Bombyx mori uncharacterized LOC101742743 (LOC101742743), mRNA 3.10E-09 MFGIVFNKQHNITHLSPKSFRMSPAASFAERWARDNLTRDPTPDELEVLGLFPITCDGDEIEASKRARRYYKARGPGGLTELWHKRHPDDEDILSSCQDVYIVPLRA PREDICTED: alpha-tocopherol transfer protein-like isoform X1 [Bombyx mori] 7.00E-44 MPAEAETRIVLRDVTPEISQHMLTDLEKLPGVQLGDFLLQFELDEPRESVREIARTELRETPEVVKPAIEELRRLLQEDKDLRVPLDSEAWMIRFLRPCKFYPESSYDL PREDICTED: clavesin-1-like [Bombyx mori] 5.40E-41 MASSEFRLERSVELLPETKEIAEKELRETPERVREALERLRELLKENKDLYFADDDELLTIFLRPCKWYPESALALMRRVAEFKRENASLLDGLLPEHEKDAFLEHK PREDICTED: Bombyx mori clavesin-1-like (LOC101736368), mRNA 4.30E-27 MTTASILMTVTTTWNVTNEKHEKDGGRLGHTLRIVVADAVKELRETSTIKDQTLHIMRDWIQQNNDIVNVRQDEAFLLRFLRHKKYSVPMAQQTLLKYLSLRKYY PREDICTED: alpha-tocopherol transfer protein-like [Bombyx mori] 2.80E-19 MDFFDDVFLKLHPNTVTTVRKLYNLEKREDMDRAIDALEDWLKKQYHFTKTDYTRDFLERSIIMTKGSVERAKTQISNLCSMRAVYPKFFEKCDARNDFNNLDSIA PREDICTED: Bombyx mori alpha-tocopherol transfer protein-like (LOC101745141), mRNA 3.30E-21 MDSINDILLKLHPDTVTTVRKLYNLEKREDLDRAIDALEDWLKMQHHFTKRDFPRDFLERSIIMMKGSVERAKYQISNLCSMRAMYPKFFDKYDAKNDFNKLDYF PREDICTED: Bombyx mori alpha-tocopherol transfer protein-like (LOC101745141), mRNA 1.10E-17 MESLPKDPILEFNDNTFKYMRKEYKFEKNEDMNAAIDAFEDWIAKQNHFTKKKYPRDYLERLIILQKGSVERAKTHMEKLCTLRTLLPQFFEHYDVKKDCIPFINGA PREDICTED: Bombyx mori alpha-tocopherol transfer protein-like (LOC101745141), mRNA 6.00E-19 MDAVPEDKLLRYTPDTMQALRRLYNLDKPGDMDFAITALQDWLKKQHHLTKKDYSRSYLERLIILQKGSLERSKAQIENICAMRTLLPEYFEINGDEEIVKLDGLS PREDICTED: alpha-tocopherol transfer protein-like [Bombyx mori] 9.30E-17 MESLPSDPILEYNDDTFESLRKQYNFEKKEDMDAAIDVFEEWLKKQNHFTRKEYPREHLERLIILHKGSVERAKTHMDKLCTLKTLLPHFFEHYDVRNDCASYIND PREDICTED: alpha-tocopherol transfer protein-like [Bombyx mori] 2.40E-23 MEELPRDKILTYNPDTLKAVRYEFNLENPGRLEEAINILEDWIKKQDHFVKKDFDREYLERTIVTTKGLVERAKARMDKLCTFKTLLPNFFQILEEKRHQSIAEKLSH PREDICTED: Bombyx mori alpha-tocopherol transfer protein-like (LOC101745141), mRNA 5.00E-18 MQAIPKLPILEFTDDALESIRKCHNLHDPGRMEEAVDILCEWIQKQEHFLRKDFPRDYLERIIISTKGSLEKAKVRLDKICTMKTLAPKYFQDQDLRDVKHVLDGFKF PREDICTED: alpha-tocopherol transfer protein-like [Bombyx mori] 7.60E-19 MESLPCNPILKFKPDTLDVIRKSFNLDQRELNEAIDRFEEWLNKQEHFVKKDFSRDYIERTIIQGKGSVEKAKSQMDKLCTLKTLLPKFFKNCDIKTEFKDISQIAWLI PREDICTED: Bombyx mori alpha-tocopherol transfer protein-like (LOC101745141), mRNA 9.20E-20 MEELKPTNILKFNPNQMADVRKVFGYGDIGILKQDIKILEDWINKQNHFIVRDFGRDYLERFLIYNKGSVERAKKHFDRLCTFRNLMPEFLRDFDVRNEFVDLFKCS putative CRAL/TRIO domain-containing protein [Danaus plexippus] 5.60E-18 MERPPHHPLVIVTEDDIKAIREIYNIGDENKINESLNAIQEWIQKEEHLVEASKYLSRRNLERVLLIAKGSVEETKRRIERLLTARGMIPELCLNKTLEEFEDIWDAVNY PREDICTED: Bombyx mori clavesin-1-like (LOC101744403), mRNA 6.30E-27 MKTDIKMWSFEEIAFQAEMNRHEDPDSEEDAFVLCGEDPTTRNKKIVEFRNLIKERNECHPHRTDDAYLLRFLRARQSIPARAHRLLVRYCNFREANPHLWRDVDW PREDICTED: clavesin-1-like [Bombyx mori] 6.70E-31 MPFIDIAFQAEISRYEDPEFEEFAKKNCNENFETRGKAIDELRRMIREKGECCPRRLDDAYCLRFLRCRRFIPALAHKLMVRYEEFRHKNAYLYNCDAFGLQKVKDV PREDICTED: alpha-tocopherol transfer protein-like [Bombyx mori] 1.70E-17 MTVSQIAMEIEYNKETNIKPEVILRLREWLQKQAHMPHDHITELDIILAYHCCDCDAEITKRVIDLNFTARTLFSFYQNREINYSLETALHTWLVTPLDAATNKGYRP PREDICTED: alpha-tocopherol transfer protein-like [Bombyx mori] 2.00E-15 MGSCDDELLKLHSDTVSTLRKLYKLDKREDMDRAIDALEDWLKKQYHFTKREYPREYLERSIIFMKGSVERVKLQISNLCSMRTIFPNFFEKVDAINDFVHYEDPV PREDICTED: clavesin-2-like [Bombyx mori] 2.50E-26 MYECFLEIAYEAELNTEENEEMLELAKELCNENPATRATAITDLRQMIYERGECRPWRTDDAFLLRYLRARNFVVPRAHKLLVRNCVFREQHNQLYEGVDLWGLV PREDICTED: Bombyx mori alpha-tocopherol transfer protein-like (LOC101742992), mRNA 9.70E-36 MPTPDYSVDIDLGEPPPELEDYARQHCGEDPSTKLQAIDELRDMIYERGECTPHRMDDEFLVKFLRARNFIPQRAHRLLVNYYQFKEENPELFDNVYPLDLQGIGDA PREDICTED: Bombyx mori retinaldehyde-binding protein 1-like (LOC101739669), transcript variant X1, mRNA 3.30E-30 MATTVLQPEECRREHEPKPNIIDSPRLTDDERVQILEAKERETGDLPPELREKARMELREEPALREQALAQMRHFIDKHPAIKKCRTDAPFLLRFLRTKKYSIPQACSM PREDICTED: alpha-tocopherol transfer protein-like [Bombyx mori] 7.00E-11 MRHKEIYGVNVHPTIKFALDFGMALISEKIRKRVKLYTSIEDVEIDKSVLPKEYGGVMPMRQMI PREDICTED: alpha-tocopherol transfer protein-like [Bombyx mori] 2.10E-12 DQWFLAFLRGCKHDVEKSKRKLEKYYTLKKSAPDFFANRNPTDEKIQALLKLGLPLRQCVKEDSPRTCLVRVSQLCNSQYSLVDLTKIGFMTAEILLLEDDNFTVV hypothetical protein KGM_17942 [Danaus plexippus] 3.10E-25 MTVRALSSVLRKKAEEEVNETPNVADAAIAQLRDWLHQKPHLRAAKIPDQWLIAFLRGSKFNINKAQEKLDTYYTLKTIAPEIAANRHPFDASIQEILKIGFFLPLRK

RI PT

E-value 0.00E+00 2.00E-125

EP

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Manduca sexta Trinity mRNA contig Expressed in head? yes no yes yes yes yes no no yes yes yes yes no no yes yes yes yes yes yes yes yes no no no no no yes yes yes yes yes yes no yes yes yes yes yes no no yes

AC C

Manduca sexta gene

Msex000860 comp16496_c0 Msex000861 N/A Msex000862 comp34451_c0 Msex000863 comp17428_c0 Msex001952 comp17116_c0 Msex002032 comp23442_c0 Msex003804 N/A Msex003805 N/A Msex004308 comp18896_c0 Msex004519 comp8581_c0 Msex005124 comp17875_c0 Msex005127 comp17579_c0 Msex005131a N/A Msex005131b N/A Msex005929 comp8592_c0 Msex005959 comp6560_c0 Msex005960 comp50693_c0 Msex005961 comp45729_c0 Msex005962 comp54527_c0 Msex006294 comp24283_c0 Msex006295 comp6717_c0 Msex010326 comp20338_c0 Msex010487 N/A Msex010488 N/A Msex010489 N/A Msex010492 N/A Msex010495 N/A Msex010496 comp17512_c0 Msex010497 comp17813_c0 Msex010498 comp13490_c0 Msex010499 comp16147_c0 Msex010502 comp20387_c0 Msex010701 comp173239_c0 Msex010702 N/A Msex011959 comp7598_c0 Msex012101 comp124195_c0 Msex014334 comp27439_c0 Msex014871 comp20575_c0 Msex016323 comp35578_c0 Msex018304 N/A MsexScaffold00053 (961518-965039) N/A N/A comp56142_c0

ACCEPTED MANUSCRIPT

RI PT

NDKRWAVDDSLRPLNSKDDRVDKVKELPGSFRSLALD NSVEESREFYLDDNLWKAIPTKKSKAMENAMNGSFRTLSID WKGLAAKKSKSADNAMNGSFRTLSID VFMKKSLDLFSKNREHFLNDNLWRVDKKSKNKDVSTEIGSFRSLAID KFYETVIPRSSLPPDHGGTLPDTQTLHKKCMQQLQLLEPYFKAEEEQRINALPDKKREKAMEKAFKNLDID ERIVLHGNNLSTLHDALPLDILPAELGGEGPSYNSERWLQEFCRSENINPAPVTPVTTAASDDNDLPSAKLDKAYKQKDNANFSFNGSEKSAKSELLRDKD IIDYWKKKVQEYRVFLEEDQTFGTDESKRPGKPKTAEDMFGVEGSFRQLQFD HIPKEIVPTEYGGDGGPLVDIIEYWVSKIKEYQDWMRQEVKFGTDESKRAGKPMVKEMAGLDGSFRKLEID LLEHISASVLPQEWGGEEEPIDVLVRKWRQRIDESREFLRDLNELCCAVPAQIADAEVYGTMGSFRKLDID SYKEIKDSWIELLSSKESMEYLKASREATVNEAYRLKSDYNEHLGLAGTFRTLSID ARPNNVDNIFGIQGSFKKLDID MEALPKEAGGQYKDFHTAQADILAKIKGSPAFFAEENKKRVKEAVRPGKPKTITDIFGGVEGSFKKLEID YKTLEELREETQAMMRANKDFFVEENKKRVKESLRPGKPKTITDIFGSVEGTIFLKQG SFHSCKRCMESTKKLLDTHYT LIETEMFKADEKKRIKKPSKGMFASFTSSFKSLDID DCSTMEALHEQWTMKLQEYRDWFKRQESIKANEELRPGKPTNYDELFGIDGSFRQLTID PFLKEKMKNRIFFHTDITELQKYIPKEIIPIEYGGNGCSMEEVNIAWMKKLEEYIPWFKEQENIKADEALRPGKPTNYDELFGIDGSFRQLSID YVPKDMLPAEYGGNAGPMNDLHNAWVKKLGEYTDWFTENENIKANEALRPGKPTNYDELFGIDGSFRQLVID GAMKDLNDEWTKKLLSNAKWLQTEERKFYDTDLRPEVRTRKNKNAMRTLRSNIGSYDMITRAHSCRSLHTRDDLDEGLYGAYRTLKVKPDKFYV EKLRSRIIFMGNDRDLLHKHISPKCLPESYGGTLTIPSVTGAQWLELLLMCDREFMAINSYGYKKK HGSKMSSLHKHMSPSHLPADYGGELPAIDYSGAEWYPVINDILPHFDKMELQLNEDGKPYVMW FAISKVTPKMAERLHIYTNLKQLHKQTDAECLPTIYGGPIELQNMIVYTKQLLGEQRKKVLALDEMLITNTRGIISSRAKNVKSDTTSVEGSFRKLEID IAVLKEQWKDLLGTKENVNYMQEMSNAVSNEAYKIKDKYNLDGAG MAVLQGMNNGKMFLVRKKTLITCKR ESLQVTHDKWIDVLSSKEFVEFIQVMNQGRTNESLRLNDTVKEQYMGIAGTFRTLSVD ADRHMKWVQALTSKKMTEHLKEMSEAKSTQGYKMKDTISDPDLGISGSFRSLNVD QESLESIYEKWLDVLTTKEFMEYLQVMNQARSREDLRLNDTFKEEYMSIAGTFRTLSVD TLYDYVPKSMLPSEFDGDGKSLRELHQQWIEVITSEEFTQYLKEMNTATTNEAYRQTDKFNEQYLGMPGSFRTLSVD KSIAELHDKWFEVLNSKEHREYIKETVKAGTNESRRLINKLNDEYLGMPGTFRVLKID VEELNKKWMEIISTEEHTAYMKEMNGACTDESRRSKLDFNNEYIGMPGTFRTLTVD KELSEANFKELSTDEHIARLKFLEKAATNEDLRLSCKFNEEYSGLPGAFKTLCVD MSCDKLNELWKETLKSEESQKIIRDTEKLVSDETKRHESKFNEEYMGMPGSFRRLTVD KVYMHGYDITSLHRYIDPECLPKRYGGYRDPVSLERWLTKIKEYKNKSFDNDMRNLGYGVD RHVVTTEEWLAKVYKYRDDFLVQELRDLGFYVK KANKDFFEMESKKRVDETKRTERPKTISSFFPNMEEAFKKLEID

HGSDLKSLQQHISLDCLPPRYGGTCRSHVSFAHWLKKVKKYRDEKFDREMKDLGYVIKE VWPDYPYTIWFESLRKNYHVAKEMVACGYKFREEEISADVVRKLKEDGIQLS CTHFVNIPQYGVRFFEFAVSLLSDKLKDRVMFHRACDDLTKHVDPAILPKEYGGTVPLKDMIEELKRNLLKHREELLALDDLSIDLYALEKNDLTQDIHSTAGSFRKLELD

AC C

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QDLIDHWKMKVETYSDWYAQHDSIRPDESKSSDAKNSANVLGIVNCFKKLDID