Accepted Manuscript Insect midgut α-mannosidases from family 38 and 47 with emphasis on those of Tenebrio molitor Nathalia R. Moreira, Christiane Cardoso, Alberto F. Ribeiro, Clelia Ferreira, Walter R. Terra PII:

S0965-1748(15)30027-8

DOI:

10.1016/j.ibmb.2015.07.012

Reference:

IB 2743

To appear in:

Insect Biochemistry and Molecular Biology

Received Date: 2 April 2015 Revised Date:

8 June 2015

Accepted Date: 10 July 2015

Please cite this article as: Moreira, N.R., Cardoso, C., Ribeiro, A.F., Ferreira, C., Terra, W.R., Insect midgut α-mannosidases from family 38 and 47 with emphasis on those of Tenebrio molitor, Insect Biochemistry and Molecular Biology (2015), doi: 10.1016/j.ibmb.2015.07.012. 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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Man1 Man2

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(TmMan2 codes for Man2)

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Man1 and Man2 hydrolase α1,2 α1,3 mannobiose α1,6

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cDNA Library Tm Man2

Peptide synthesis

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Insect midgut α-mannosidases from family 38 and 47 with emphasis on those of

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Tenebrio molitor

Nathalia R. Moreiraa, Christiane Cardosoa, Alberto F. Ribeirob, Clelia Ferreiraa

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a

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and Walter R. Terraa,*

Departamento de Bioquímica, Instituto de Química, Universidade de São

Paulo, C.P. 26077, 05513-970 São Paulo, Brasil b

Departamento de Genética e Biologia Evolutiva, Instituto de Biociências,

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Universidade de São Paulo, C.P. 11461, 05513-970 São Paulo, Brasil

* Corresponding author. Fax.: +55 11 3091 2186.

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E-mail address: [email protected] (W.R. Terra)

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ACCEPTED MANUSCRIPT Abstract

α-Mannosidases are enzymes which remove non-reducing terminal residues from glycoconjugates. Data on both GH47 and GH38 (Golgi and lysosomal) enzymes are available. Data on insect midgut α-mannosidases acting in digestion are preliminary and do not include enzyme sequences. T. molitor

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midgut α-mannosidases were separated by chromatography into two activity peaks: a major (Man1) and a minor (Man2). An antibody generated against a synthetic peptide corresponding to a sequence of α-mannosidase fragment recognizes Man2 but not Man1. That fragment was later found to correspond to

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TmMan2 (GenBank access KP892646), showing that the cDNA coding for Man2 is actually TmMan2. TmMan2 codes for a mature α-mannosidase with 107.5 kDa. Purified Man2 originates after SDS-PAGE one band of about 72 kDa

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and another of 51 kDa, which sums 123 kDa, in agreement with gel filtration (123 kDa) data. These results suggest that Man2 is processed into peptides that remain noncovalently linked within the functional enzyme. The physical and kinectical properties of purified Man1 and Man2 are similar. They have a molecular mass of 123 kDa (gel filtration), pH optimum (5.6) and response to

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inhibitors like swainsonine (Man1 Ki, 68 nM; Man2 Ki, 63 nM) and deoxymannojirimycin (Man1 Ki, 0.12 mM; Man2 Ki, 0.15 mM). Their substrate specificities are a little different as Man2 hydrolyzes α-1,3 and α-1,6 bonds better than α-1,2, whereas the contrary is true for Man1. Thus, they pertain to

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Class II (GH38 α-mannosidases), that are catabolic α-mannosidases similar to lysosomal α-mannosidase. However, Man2, in contrast to true lysosomal α-

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mannosidase, is secreted (immunocytolocalization data) into the midgut contents. There, Man2 may participate in digestion of fungal cell walls, known to have α-mannosides in their outermost layer. The amount of family 38 αmannosidase sequences found in the transcriptome (454 pyrosequencing) of the midgut of 9 insects pertaining to 5 orders is perhaps related to the diet of these organisms, as suggested by a large number of lysosomal α-mannosidase in the T. molitor midgut.

Key words: alpha-mannosidase, substrate specifity, imunocytolocalization, posttranslational processing.

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ACCEPTED MANUSCRIPT 1. Introduction

α-Mannosidases (EC 3.2.1.24) are enzymes which remove non-reducing terminal residues from glycoconjugates. Mammalian α-mannosidases are classified under two classes (I and II) on the base of functional characteristics

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and sequence homology (Gonzalez and Jordan, 2000). Class I α-mannosidases are of family 47 of glycosyl hydrolases (GH47), only hydrolyse α-1,2 mannose bonds and are localized either in endoplasmic reticulum or Golgi complex. Class II α-mannosidases are of family 38 of glycosyl hydrolases (GH38). They

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hydrolyze α-1,2, α-1,3, and α-1,6 mannose bonds from the nonreducing termini of N-glycans during biosynthesis in the endoplasmic reticulum and Golgi

2000).

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complex and catabolism in the lysosomes and cytosol (Gonzales and Jordan, GH38 α-mannosidases are activated by Zn++ and require two carboxylate groups to catalyze hydrolysis: one serving as a nucleophile and the other as a proton donor (Davies and Henrissat, 1995). The Golgi enzyme has a dual specificity

for

α-1,6

and

α-1,3-linked

mannoses,

whereas

lysosomal

mannosidases (LAM) have broad specificity, cleaving α-1,2, α-1,3, and α-1,6

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linked mannoses, or narrow specificity, hydrolyzing only α-1,6 linked mannoses (Rose, 2012). The LAM is post-translationally modified during maturation and cellular transport by proteolytic processing and by N-linked glycosylation

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(Tollersrud et al., 1997).

Insect α-mannosidases have been studied in detail with recombinant enzymes. Data on both GH47 (Ren et al., 1995; Kawar and Jarvis, 2001) and

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GH38 (van den Elsen et al., 2001; Rose, 2012; Nemcovicova et al., 2012) Golgi enzymes are available and lysosomal α-mannosidases (Nemcovicova et al., 2012), as well. There have been also reports on insect midgut α-mannosidases acting in digestion (Chipoulet and Chararas, 1985; Terra et al., 1988; Silva and Terra, 1994; Foster and Roberts, 1997; Bedikou et al., 2009). Those papers, however, are preliminary characterizations of the enzymes and none of them describe the enzyme sequence. In this paper we describe the purification, characterization, sequencing and processing of a T. molitor α-mannosidase and show that it is similar to

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lysosomal α-mannosidase, but it is secreted into the midgut contents, probably acting in fungi digestion.

2. Materials and Methods

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2.1. Animals

Stock cultures of T. molitor were maintained under natural photoregime conditions on wheat bran at 24–26 ºC and 70–75% r.h. Fully grown larvae of both sexes (each weighing about 0.12 g), having midguts full of food, were

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used.

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2.2. Preparation of T. molitor midgut and wheat bran samples

Larvae were immobilized by placing them on ice after which they were rinsed in water and blotted with filter paper. The larvae were dissected in cold iso-osmotic saline (342 mM NaCl solution) and the midguts removed. The isolated midguts were further divided into two sections: one corresponding to the anterior two-thirds and another to the posterior third and the tissue and

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contents corresponding to each section were recovered. The midgut tissue sections were then homogenized in cold double-distilled water using a Potter– Elvehjem homogenizer, filtered through glass wool (to remove fat) and centrifuged at 25,000 xg for 30 min at 4 °C. The supernatant (“soluble fract ion”)

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was recovered, whereas the sediment (“membrane fraction”) was dispersed in double distilled water. Peritrophic membranes and contents were homogenized

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like tissue preparations and centrifuged at 10,000 xg for 10 min at 40C. Centrifugation is necessary to remove undigested food fragments from the homogenates of peritrophic membrane and contents. Wheat bran was treated like the peritrophic membrane and contents. All

samples were stored at -200 C until used. No enzyme inactivation was detected on storage for as long as 6 months.

2.3. Protein determination, enzyme assays, and kinetic parameters

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Protein was determined according to Smith et al. (1985), as modified by Morton and Evans (1992) using ovalbumin as a standard. The activity of αmannosidase was routinely determined with 2.5 mM p-nitrophenyl-α-Dmannopiranoside (NPαMan) as substrate in 20 mM citrate-phosphate buffer pH 6.0 at 30 0C. The activity was determined by measuring the release of p-

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nitrophenolate at 420 nm as described previously (Terra et al., 1979). The activity of α-mannosidase in 2.5 mM 2α-mannobiose, 3α-mannobiose and 6αmannobiose was determined by measuring the increase in reducing groups (Noelting and Bernfeld, 1948). All assays were carried out in triplicate under

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conditions such the activity was proportional to protein concentration and to time. Incubations were carried out for at least four different time periods and the initial rates of hydrolysis were calculated. Controls without enzyme or without hydrolyzes 1 µmol substrate/min.

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substrate were included. One unit of enzyme (U) is defined as the amount that

The effect of substrate concentration on the activity of T. molitor purified digestive α-mannosidase was determined by using 15 different substrate concentrations (range 0.25-23 mM). Km values (mean ± SEM) were determined

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by a weighted linear regression using the software SigmaPlot (Jardel Scientific, USA). For Ki value determinations, purified α-mannosidase was incubated with at least 5 different concentrations of the substrate (0.2-2 times the Km value) at each of 5 different concentrations of the inhibitor (1-5 times the Ki value). Ki

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values were determined from the replots of Lineweaver-Burk plots against inhibitor concentration (Segel, 1975).

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The effect of pH on enzyme activity was measured in the presence of the following buffers (0.2 M): citrate-sodium phosphate (pH 2.6-7.0) and Tris-HCl (pH 7-8). pH stability was evaluated by maintaining enzyme samples at the same buffers as before at 30 ºC for 2 h before assaying in standard conditions.

2.4. Purification of the digestive α-mannosidase from T. molitor larvae

The soluble fraction of T. molitor midgut homogenates (1000 mg protein corresponding to 500 insects) was loaded onto a Hitrap Q XL column (5.0 mL), (FPLC system, Amersham Biosciences) equilibrated with 20 mM Tris-HCl buffer, pH 7.0. Elution was accomplished with a gradient of NaCl from 0 to 1M

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in the same buffer. At all performed purification steps, the flow was set at 0.5 mL/min and fractions of 0.4 mL were collected. Fractions active on NPαMan were pooled, passed through a Superdex 75 HR 10/30 column (Pharmacia Biotech) equilibrated with 20 mM Tris-HCl buffer, pH 7.0, containing 1M NaCl. The active fractions were pooled, the pool buffer was changed with the aid of a

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High Trap desalting column and then it was passed through a Phenyl Superose column HR 5/5 (AKTA System) equilibrated with 50 mM phosphate buffer, pH 7.0, containing 2M (NH4)2 SO4. Elution was accomplished with a (NH4)2SO4 gradient from 2 to 0 M in the same buffer. The fractions corresponding to each

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of the two activity peaks were used as a source of α-mannosidases (peak one named Man1; peak two, Man2).

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2.5. Gel Filtration for molecular weight determination

Gel filtration was performed in a FPLC System (Amersham Biosciences) using a Superdex 200 column equilibrated and eluted with 20 mM Tris-HCl buffer pH 7.0 containing 1 M NaCl. Fractions of 0.3 mL were collected. Molecular weights were calculated by using the following proteins as standards: cytochrome C (12.4 kDa), ovalbumin (43 kDa), bovine serum albumin (67 kDa),

Dextran (2000 kDa).

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β-amylase (200 kDa). The void volume (VO) was calculated with the aid of Blue

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2.6. Sodium dodecyl sulfate (SDS)-Polyacrylamide Gel Electrophoresis (PAGE), Western blotting, and in gel assays Samples containing approx. 2 mg protein were combined with sample

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buffer containing 60 mM Tris–HCl buffer, pH 6.8, 2.5% (w/v) SDS, 0.36mM βmercaptoethanol, 0.5 mM EDTA, 10% (v/v) glycerol and 0.005% (w/v) bromophenol blue. The samples, heated for 5 min at 95 ºC, were loaded onto polyacrylamide gel slabs. The gel slabs and the running buffer contained 0.1% SDS (Laemmli, 1970), and electrophoresis was run at constant voltage of 200 V. Non-denaturating electrophoresis was conducted as described above, but without heating and neither SDS nor β-mercaptoethanol were added in gels. Protein was silver stained (Blum et al., 1987). Molecular-weight markers used: hen egg white ovoalbumin (45 kDa), bovine serum albumin (66.2 kDa),

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rabbit muscle phosphorylase b (97 kDa), Escherichia coli β-galactosidase (113.5 kDa) and rabbit skeletal muscle myosin (200 kDa). Western blotting was performed essentially as previously described (Cristofoletti

et

al.,

2001).

After

SDS-PAGE,

the

proteins

were

electrophoretically transferred onto a nitrocellulose filter, which after a blocking

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step reacted with the anti-α-mannosidase serum and then with anti-rabbit IgG coupled with peroxidase. After washings, the strips were incubated with chloronaphtol as described by Cristofoletti et al. (2001). The transfer efficiency was evaluated using pre-stained molecular weight markers (BioRad or Sigma,

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USA). Pre-immune serum was used in control experiments to show that antiserum was specific.

Prior to in gel detection of enzymatic activity, the gels (prepared without

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SDS and β-mercaptoethanol) were washed three times for 15 min with 20 mM citrate-sodium phosphate buffer pH 6. α-Mannosidase activity was assayed in gel at 30°C as described in Manchenko (1994), using

0.25 mM 4-

methylumbellipheryl α-D-mannopyranoside in the washing buffer.

2.7. Molecular cloning of a fragment (Tm000) of a cDNA coding α-mannosidase

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Tm000 was obtained by polymerase chain reaction (PCR) amplification of a midgut cDNA library (Cristofoletti et al., 2005) as template. For this we used the primers designed basing in a partial sequence obtained in our laboratory

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(Contig 5, Ferreira et al., 2007): Man reverse, 5’ ATTCTTGCCTCCTCGCGAAC 3’; Man forward, 5’ ACCATCAGCTCCACCTCTCC 3’ and of the T3 promoter, ATTAACCCTCACTAAAGGGA;

T7

universal

primer,

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TAATACGACTCACTATAGGG. The PCR was performed using TAQ DNA polymerase (5 units) in 20 mM Tris-HCl buffer, pH 8.4, with 50 mM KCl, 0.2 mM dNTP, and 40 mM MgCl2. The amplification was reached using 30 cycles at the following conditions: 30 s at 94 0C, 45 s at 55 0C and 1.5 min at 72 0C. At the end, the reaction mixture remained a further 10 min at 72 0C. The product of PCR was cloned in pGEM-T Easy Vector (Promega, USA) and sequenced by the Sanger Procedure, resulting in a fragment similar to α-mannosidase sequences deposited in GenBank. The sequence was named Tm000 and is shown in Supplementary Table 1.

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ACCEPTED MANUSCRIPT 2.8 Preparation of midgut α-mannosidase antiserum

One section of the translated sequence of Tm000 was chosen to be synthesized to generate antibodies in a rabbit. The ideal sequences should be hydrophilic, which are expected to be exposed to the medium. With the aid of the Hopp and Woods scale of mean hydrophilicity (www.expasy.org/proscale/)

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the following sequence was selected: TDSNGREVLQRTRNSRPDYDY. The peptide was synthesized (90% pure) and linked to KLH (Keyhole Limpet Hemocyanin) by Invitrogen (Life Technologies, Brazil). The synthesized peptide was used to raise antibodies in a rabbit as detailed elsewhere (Jordão et al.,

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1996). For each injection, one mg of protein was mixed with Freund’s adjuvant and subcutaneously applied into rabbits. Pre-immune serum was collected as

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control.

2.9. Ultrastructural immunocytolocalization

T. molitor larvae were dissected in their own hemolymph and tissue sections were fixed (paraformaldehyde/ glutaraldehyde), embedded in L.R. White acrylic resin, incubated with the primary (α-mannosidase antiserum) and

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secondary (goat anti-rabbit IgG coupled to 15nm gold particles) antibodies and examined in a Zeiss EM 900 electron microscope as detailed elsewhere (Silva et al., 1995). As controls, sections were incubated with pre-immune serum

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under the same conditions.

2.10. Cloning and sequencing the cDNA coding Man 2 and expression analysis

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of selected T. molitor midgut α-mannosidases

Larvae immobilized on ice were dissected in cold 342 mM NaCl with

gloves,

sterile

forceps

and

glassware

previously

treated

with

diethylpyrocarbonate. The dissected tissues were maintained in an ethanol-ice bath and eventually were stored at -80 ºC. RNA were extracted from the tissues with Trizol following the instructions of the manufacturer, Invitrogen, which are based on Chomczynski and Sacchi (1987), and used to synthesize the corresponding cDNA with the aid of a reverse transcriptase present in the kit

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Superscript (Invitrogen). The resulting cDNA was used as a template for amplifying sequences by PCR with specific primers. The following procedure was employed to clone the cDNA coding for Man 2. Tm000 is contained in contig Tm408 (see next item). This contig lacks the 3’-terminal end of the gene. A search among all contigs which are similar to

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α-mannosidase, even if did not meet the criteria to be accepted as a true αmannosidase (see next item), revealed contig TmXXX (see Supplementary Table 1) as a probable 3’-end of the α-mannosidase 2 gene. With the aid of primers designed from contig TmXXX (forward 5’ ATGAGCTGCTGGTGTTT 3’)

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and contig Tm408 ( reverse 5’ GTTATAGCTCACGGTCGC 3’) and with a midgut cDNA library prepared from midgut mRNA by reverse transcription, a cDNA clone coding for the Man 2 full length sequence was obtained (Fig. 5) by

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PCR. This was carried out using TAQ DNA polymerase (Invitrogen) in reaction buffer containing 1.5 mM MgCl2. PCR conditions were: 35 cycles of 30 s at 94 ºC (denaturation), 45 s at 55 ºC (annealing) and 2.5 min at 72 ºC (synthesis). At the end, the reaction mixture remained a further 10 min at 72 0C for completing elongation. The full length cDNA coding for Man2 was named TmMan2 and was deposited in GenBank with the accession number KP892646.

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For tissue expression analysis, cDNA prepared from the different tissues were used with the following primers: Contig Tm000 (forward 5´ GAT GTG GCT GCT TGT TGG TCA 3´; reverse 5´ GAC ATT CTC TTC CAA GAG CAT 3´); 1774

(forward

5´

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Contig

GTTGTAGTCTGGACTGTA

AGATTCGTCCAAGTGGAA3´,

3´),

Contig

602

reverse

(forward

5´ 5´

TGGCAGATAGATCCTTTC 3´, reverse 5´ GCTGACGATTTCTGTTCC 3´).

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PCR reaction was realized using TAQ DNA polymerase (Invitrogen) in

reaction buffer containing 1.5 mM MgCl2. PCR conditions were: 25 cycles of 1 min at 94 ºC (denaturation), 1 min at 52 ºC (annealing) and 1 min at 72 ºC (synthesis). The number of cycles was chosen after several trials so that the amplification was in log phase and resulting in a clearly visible amplified band. The sizes of the products of RT-PCR were those predicted, thus discounting a possible contamination with genomic DNA.

2.11. Annotation of contigs obtained by cDNA pyrosequencing of midgut samples and computational analysis of sequences

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Total messenger RNA for cDNA transcription was extracted from the midgut

of

(Hemiptera:

Periplaneta

americana

Heteroptera)

and

(Dyctioptera),

Bucephalonia

Dysdercus xanthophis

peruvianus (Hemiptera:

Auchenorrhyncha) adults and from the larval midgut of T. molitor (Coleoptera:

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Tenebrionidae), Zabrotes subfasciatus (Coleoptera: Bruchidae), Rhynchosciara americana (Diptera:Nematocera), Musca domestica (Diptera:Cyclorrhapha), Diatraea saccharalis (Lepidoptera:Crambidae) and Spodoptera frugiperda (Lepidoptera:Noctuidae). cDNA pyrosequencing of all samples, except those

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from Z. subfasciatus was performed using a platform 454 Genome Sequencer FLX (454 Life Sciences/Roche), and the contigs were assembled as described in Ferreira et al.(2014). cDNA sequencing of samples from Z. subfasciatus was

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performed in the HiScan sequencer of Illumina, as described in Gomez et al. (2013). The contigs were annotated with the aid of the dCAS software (http://exon.niaid.nih.gov), which performs BLASTx in databanks (nr, pfam, GO, KOG and Swiss-Prot). The hit with higher score was annotated. The sequences were searched in our transcriptomes using the words αmannosidase or α-mannopyranosidase and the annotation of selected

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sequences was manually curated by multiple sequence alignments (Bioedit version 7.1.11, Hall, 1999) with reference sequences. The prediction of sequence features was done as follows: signalP

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(Emanuelsson et al., 2007), big-PI Predictor (Eisenhaber et al., 2000), NetNGlyc (Gupta et al., 2004), NetOGlyc (Steentoft et al., 2013). The presence of transmembrane regions was analysed by TMHMM Server v. 2.0

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(http://www.cbs.dtu.dk/services/TMHMM-2.0); HMMTOP (Tusnády and Simon, 2001). Selected amino acid sequences of α-mannosidases from different families were aligned using the ClustalW Multiple Alignment tool in BioEdit Sequence Aligment Editor (http://www.mbio.ncsu.edu/BioEdit/BioEdit.html). Phylogenetic analysis was performed with the program MEGA 6 (Tamura et al., 2013). The cladograms of chosen sequences were inferred using the maximum likelihood algorithm and confidence estimated with bootstrap (1,000 replicates) (Felsenstein, 1985; Hillis and Bull, 1993). The bootstrap consensus tree inferred from 1,000 replicates is taken to represent the evolutionary history

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of the taxa analyzed (Felsenstein, 1985). Branches corresponding to partitions reproduced in less than 50% bootstrap replicates were collapsed.

3. Results

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3.1. Distribution of α-mannosidase activity along the midgut Most α-mannosidase activity is soluble and is found in luminal contents of the first two-thirds of the midgut, with a specific activity of the same order as that in cells of the same region (Table 1). This means that α-mannosidase

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activity is secreted into the midgut contents; otherwise specific activities in contents would be significantly lower. The small amounts of α-mannosidase activity recovered in pellets of the centrifuged samples of midgut homogenates

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(membrane fraction) may be the soluble enzyme adsorbed onto the cell surface instead of truly membrane proteins, as shown before (see Terra and Ferreira, 2012, for references). This was not further studied. The possibility that part of the luminal α-mannosidase activity originated from the ingested food was discounted. The activity of α-mannosidase recovered from 20 mg wheat bran

mU/animal).

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(which corresponds to the fresh weight of T. molitor midgut) is negligible (0.18

3.2. Purification of midgut soluble α-mannosidase

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The soluble fraction of midgut contents was applied onto a Hitrap Q column and a single major activity peak of α-mannosidase was found (Fig. 1A). The eluted fractions active fractions were pooled and passed through a

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Superdex 75 column and again a single activity peak was recovered (Fig. 1B). Finally, the pooled active fractions were applied onto a Phenyl Superose column and a major (Man 1) and a minor (Man 2) peak of activity were eluted (Fig. 1C).

SDS-PAGE of the fractions corresponding to Man 1 (Fig. 1D, lane 2) and Man 2 (Fig. 1D, lane 4) showed several bands. Man1 originates major bands of about 75, 54, and 43 kDa; whereas Man2 forms bands with 72 and 51 kDa. Nevertheless, Man1 and Man2 result in a single activity peak in gel filtration with 123 kDa (Fig.2) and correspond to a single α-mannosidase activity in gel assays (not shown). The fact that Man1 and Man2 originate single active bands

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assays) and smaller peptides in SDS-

PAGE suggests that the detected fragments remain noncovalently linked in the active form.

3.3. Kinectical characterization of purified α-mannosidases

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Purified Man 1 and Man 2 were separately assayed at different conditions with NPαMan as substrate for determining kinetic parameters. Both enzymes were stable between pH 4 and 7 and have a pH optimum of 5.6 (Table 2). Their Km values towards NPαMan are close (Table 2).

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Man 1 is competitively inhibited by deoxymannojirimycin (Fig.3) and by Swainsonine (Table 2). The same is true for Man 2 (Table 2). Ki values for the two inhibitors do not significantly differ between Man 1 and Man 2. As expected,

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inhibition by Swainsonine occurs in the nanomolar range, whereas for deoxymannojirimycin, in the milimolar range.

Substrate specificity was evaluated for both Man 1 and Man 2 regarding 3 substrates (Table 3). The results showed that Man 2 hydrolyzes α-1,3 and α1,6 bonds better than α-1,2, whereas the contrary is true for Man 1 (Table 3).

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3.4. Immunocytochemical localization of Man 2 in T. molitor midgut cells. Purified Man 1 and Man 2 were not good enough to generate antibodies to be used in immunocytochemical studies. For this, an alternate strategy was

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employed.

A fragment of a cDNA-coding α-mannosidase from T. molitor midgut was cloned by PCR amplification of a midgut cDNA expression library with

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appropriate primers. The sequence obtained (Tm000, Supplementary Table 1) was used in selecting a peptide (TDSNGREVLQRTRNSRPDYDY, see details in Material and Methods) that once synthesized was used to raise antibodies in a rabbit.

The α-mannosidase antiserum recognizes Man 2 (Fig. 1D, lane 5), but not Man 1 (Fig.1D, lane 3). Furthermore, the antiserum recognizes a single protein band both in midgut contents (Fig. 1D, lane 6) and in midgut tissue (Fig. 1D, lane 7) with the same migration as Man2. Thus, the antibody is specific for Man 2 and can be used to study its midgut localization.

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Man 2 labeling in T. molitor midgut is observed in large vesicles and outside the anterior midgut cells (Fig. 4A, arrow), whereas in posterior midgut, labeling is seen outside and rarely inside the cells (Fig.4B, arrow). The data suggest that Man 2 is secreted by an apocrine route in the anterior midgut, as previously described for amylase (Cristofoletti et al., 2005). The posterior

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midgut does not show significant labeling inside the cells, discounting a major Man 2 secretion by these cells. These results agree with the midgut distribution of α-mannosidase activity (Table 1).

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3.5. Cloning and sequencing the cDNA coding Man 2

Tm000 is contained in contig Tm408 (see next item). This contig lacks the 3’-terminal end of the gene. A search among all contigs which are similar to

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α-mannosidase (as compared to XP_968517.2 from Tribolium castaneum), even if did not meet the criteria to be accepted as a true α-mannosidase (see next item), revealed contig TmXXX (see Supplementary Table 1) as a probable 3’-end of the α-mannosidase 2 gene. With the aid of primers designed from contig TmXXX and contig Tm408 and with a midgut cDNA library prepared from midgut mRNA by reverse transcription, a cDNA clone coding for the Man 2 full

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length sequence was obtained (Fig. 5). This sequence (TmMan2) was deposited in GenBank with the accession number KP892646. The putative TmMan2 precursor sequence has a signal peptide

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(corresponding to 18 amino acids) and codes for a mature protein with 954 amino acids, with an estimated molecular mass of 107.511 kDa and an isoelectric point of 4.33. TmMan2 amino acid sequence has the catalytical

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residues (D166 and D288, precursor numbering), the residues responsible for Zn++ coordination (H50, H52, D166, H416) and the Arg residue (R190) that modulates the pKa of D166 (Suits et al., 2010). TmMan2 sequence also has 6 Cys residues homologous to Bos taurus α-mannosidase (AAC48763.1) that form disulphide bonds (C33-C329, C237-C242, C466-C474), but lacks Cys in positions 387 and 442 that form the fourth disulphide bond in the bovine αmannosidase (Heikinheimo et al., 2003). There are three putative Nglycosylation sites (N470, N577 and N895) and 8 O-glycosylation sites (S328, S634, T722, S725, S934, S938, S939, and T940). From the putative N-

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glycosylation sites, B. taurus is supposed to be glycosylated in the residues homologous to TmMan2 N470, N895 (Tollersrud et al., 1997).

3.6 Identification of α-mannosidase sequences in the transcriptome of different

insects

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Transcriptomes annotated as detailed in Material and Methods were searched for α-mannosidases. At first, 54 coding sequences were recovered, translated and the predicted sequences were manually curated.

Forty one sequences identified as pertaining to family GH38 (Henrissat

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and Davies, 1997) were checked for the presence of catalytic and zinc-binding residues by alignment with Streptococcus pyrogens α-mannosidase (GenBank AAK34381.1). All accepted sequences had the catalytic and zinc-binding

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residues, except for few sequences that lacked one of the zinc binding residues because they ended before attaining the site of one of those residues. α-Mannosidases sequences from GH47 family have 3 acid residues important for enzyme catalysis (E330, D463 and E599 in human αmannosidase AAF03215.1) and a conserved Arg residue (R334 in the human enzyme). The 13 GH47 sequences found in our transcriptomes were aligned

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with the human sequence. Seven sequences having at least 2 of the 4 residues mentioned above were accepted for further analysis. The features of the curated sequences of α-mannosidases are listed in

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Tables 4 and 5 and the corresponding FASTA sequences are presented in Supplementary Table 1.

Following the criteria described above, 13 GH38 and 6 GH47 α-

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mannosidase sequences were found in the transcriptome of the 9 insects. In B. xanthophis, D. peruvianus and R. americana only GH47 enzymes were found, whereas in P. americana, only GH38 mannosidases are present. Unexpectedly, no α-mannosidases were found in the intestine of Z. subfasciatus, D. saccharalis and S. frugiperda. On the other hand, T.molitor has a high number of α-mannosidases (Tables 4 and 5). As the enzymes have about 1000 amino acid residues, it is likely that true mannosidase sequences have been discarded because they do not have the catalytical and/or zinc binding residues. The enzymes are not expressed in

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large quantities, especially those belonging to family 47, which can make it difficult to obtain a large extension of their sequences. Family 47 α-mannosidase sequences from our transcriptomes plus sequences from characterized animal α-mannosidases were used to make a cladogram (not shown). The sequences form 2 monophyletic groups. One

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(bootstrap 100) contains all the sequences retrieved from data bases plus the contigs Md1922 and Tm2371. The other contigs from our transcriptomes branched together in the other group. More studies are needed to disclose the reason why the sequences form these 2 branches.

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Among the sequences of family 47, only Bx2085 has a transmembrane helix predicted and no potential GPI-modification site was found in the sequences. The position of the transmembrane helix present in Bx2085 does

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not coincide with that presented by H. sapiens enzyme. The N-terminal portion of all the insect mannosidases from our transcriptomes initiated after the transmembrane helix from the H. sapiens enzyme.

Fig. 6 shows a cladogram of family 38 α-mannosidases found in our transcriptomes plus sequences of α-mannosidases present in lysosomes, Golgi and cytosol from different organisms. The cytosol enzymes (CAM) form one

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monophyletic grouping with bootstrap 100. The AMII α-mannosidases from D. melanogaster, M. musculus and H. sapiens are Golgi enzymes and branch together with the S. frugiperda enzyme, indicating that the later enzyme is

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probably also present in Golgi apparatus. The sequences found in our transcriptomes are all in the same group of the lysosomal enzymes (LAM) (bootstrap 75), indicating that the sequences found by us are similar to

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lysosomal enzymes.

3.7. Expression analysis by RT-PCR of α-mannosidases genes in T. molitor midgut

RT-PCR data (Fig. 7) shows that the mRNA coding for Tm602 is expressed along the whole midgut and Malpighian tubules (here very faintly) whereas Tm000 (which corresponds to TmMan2) and Tm1774 are expressed only along the midgut. The data suggest that TmMan2, Tm602 and Tm1774 have a midgut function.

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4.1. Distribution of α-mannosidase activity in T. molitor midguts Most α-mannosidase activity in larval midguts of T. molitor is found in luminal contents of the anterior two thirds of the midgut, which midgut luminal

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pH is 5.6. The luminal pH of posterior midgut is 7.9 (Terra et al., 1985). The contribution of α-mannosidase activity coming with ingested bran is negligible, meaning that all activity is actually secreted by the midgut cells.

Digestive enzymes are secreted by midgut cells according to 3

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mechanisms. One is apocrine secretion, where the secretory vesicles undergo fusions, leading to larger vesicles (aposomes) that after release, involving the loss of at least 10% of the apical cytoplasm, eventually free their contents by

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solubilization. If the loss of cytoplasm is very small, the secretory mechanism is called microapocrine. The other mechanism is the classical exocytic route, where secretory vesicles fuse with the midgut cell apical membrane, emptying their contents without any loss of cytoplasm (Terra and Ferreira, 2012). Coleopterans usually have apocrine secretion in anterior and exocytic secretion in posterior midgut (Cristofoletti et al., 2001).

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Immunocytochemical data showed that Man2, one of the two αmannosidases chromatographically purified from T. molitor midguts, was found associated with an aposome in anterior midgut cells, as described previously for

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amylase (Cristofoletti et al., 2001), meaning that it is secreted by an apocrine mechanism. α-Mannosidase labeling observed at the microvilli of posterior midgut cells mostly correspond to enzyme molecules synthesized in anterior

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midgut but displaced toward the posterior midgut with the food bolus. Occasionally Man2 labeling is observed in posterior midgut cells. This suggests that Man2 is secreted by a minor exocytic secretory route at this region. It should be noted, however, that the high expression of TmMan2 (RT-PCR data) in posterior midgut is not accompanied by a remarkable Man2 labeling and enzyme activity there. It is not clear the meaning of this.

4.2. Purification, properties and functions of T. molitor α-mannosidases T.

molitor

midgut

α-mannosidase

activity

was

separated

by

chromatography into two activity peaks: a major (Man1) and a minor (Man2). An

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antibody generated against a synthetic peptide corresponding to a sequence of a α-mannosidase fragment (later shown to correspond to TmMan2, GenBank access KP892646) recognizes Man2 but not Man1. Thus, the cDNA coding for Man2 is actually TmMan2. TmMan2 codes for a mature α-mannosidase with 107.5 kDa, whereas

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purified Man2 has 123 kDa, as determined by gel filtration. Bovine lysosomal αmannosidase has 104 kDa, as deduced from the cDNA sequence, whereas the purified enzyme has 119 kDa, which is reduced to 106 kDa on deglycosilation (Tollersrud et al., 1997). Likewise, the difference found between the mass of the

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protein coded by TmMan2 and purified Man2 may be accounted by glycosylation. There are several putative glycosylation sites along the Man2

1997, Heikinheimo et al., 2003).

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sequence and α-mannosidases are known to be glycosylated (Tollersrud et al.,

Man2 originates after SDS-PAGE one band of 72 kDa and another of 51 kDa, which sums 123 kDa, in agreement with gel filtration data. Only the band of 72 kDa is recognized by the α-mannosidase antiserum. These results suggest that Man2 is processed into peptides that remain noncovalently linked within the functional enzyme. Since the α-mannosidase antiserum was prepared

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using a peptide corresponding to the C-terminal end of the molecule (see Fig. 6), the band with 72 kDa should correspond to the C-terminal peptide and that with 51 kDa, to the N-terminal end.

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α-Mannosidase processing into two peptides that remain noncovalently linked in the functional enzyme has been described before for lysosomal αmannosidases (Heikinheimo et al., 2003, Nemcovicova et al., 2012).

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The physical and kinectical properties of purified Man1 and Man2 are

similar. They have a molecular mass of 123 kDa (gel filtration), pH optimum (5.6) and response to inhibitors like swainsonine (Man1 Ki, 68 nM; Man2 Ki, 63 nM) and deoxymannojirimycin (Man1 Ki, 0.12 mM; Man2 Ki, 0.15 mM). Their substrate specificities are a little different as Man2 hydrolyzes α-1,3 and α-1,6 bonds better than α-1,2, whereas the contrary is true for Man1. These characteristics permit to classify Man1 and Man2 as α-mannosidases pertaining to Class II (GH38 α-mannosidases), that are catabolic α-mannosidases similar to the lysosomal α-mannosidases (Rose, 2012).

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The identification of Man2 as a lysosomal enzyme on the basis of its kinectical properties agrees with that resulting from its post-translational processing which is typical from lysosomal enzymes. Furthermore, most residues in the relevant positions of the holding and anchor sites described for D. melanogaster Golgi α-mannosidase (V61, Q64, Y267, H273, P298 and

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W299) (Shah 2008; Zhong et al, 2008) differ in TmMan2. This was based in an alignment of the sequences from lysosomal enzymes from B. taurus (AAC48763), Capra hircus (AEZ02307.1), Cavia porcellus (AAL58982.1), Felis catus (AAB97672.1), H. sapiens (AAC51362.1), Mus musculus (NP_034894.2)

musculus

(CAA43480.1),

H.

sapiens

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and D. melanogaster (AAD38576.1) plus the Golgi enzymes from Mus (AAC50302.1),

D.

melanogaster

(CAA54732.1) and TmMan2. The branching of TmMan2 together with

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lysosomal α-mannosidases in a cladogram of GH38 α-mannosidases also lends support to a lysosomal origin of the protein coded by TmMan2, which is Man2. It is important to stress, however, that Man2, in contrast to true lysosomal αmannosidases, is secreted into the midgut contents. There, Man2 may participate in digestion. Similarly, cathepsin L2 and cathepsin L3 are proteinases that have counterparts in the lysosomes but are also secreted into

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luminal contents (Beton et al., 2012).

It is known that T. molitor food is rich in fungal cells that are absent from the midgut (Genta et al., 2006a). T. molitor midgut has a laminarinase with

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high lytic power against fungal cell walls (Genta et al., 2007) and the specific activity of its chitinase is higher than in other insects (Genta et al., 2006b), indicating that T. molitor is adapted to digest fungal cell walls. α-Mannosides are

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present in the outermost layer of fungi cell walls (Martinez-Esparza et al., 2006). Thus, it is possible that TmMan2 is used to remove mannose units during fungal digestion.

Man1 is a lysosomal α-mannosidase regarding its kinetic properties

and may have the same digestive function as Man2. T. molitor midgut expresses other major GH38 α-mannosidases, in addition to TmMan2, all of them branching together with lysosomal α-mannosidases in a cladogram. One of these is contig Tm602 that is expressed along the whole midgut and Malpighian tubules and may code Man1, thus participating in luminal fungal digestion.

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More work is necessary to propose a function for Tm2371 (GH47 αmannosidase).

4.3. Comparison of insect midgut α-mannosidase sequences The amount of family 38 α-mannosidase sequences found in the

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transcriptome of midgut of insects is perhaps related to the diet of these organisms. This is suggested by the fact that there is a larger number of lysosomal-like α-mannosidases in the T. molitor midgut than in any other. As shown above, T. molitor midgut α-mannosidases are supposed to be involved in

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fungal digestion.

In the active site of family 47 α-mannosidase there are 3 acid residues (E330, D463 and E599, human endoplasmic reticulum mannosidase,

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AAF03215.1), plus an Arg (R334) that could interact with E330 and an His (H524) (Karaveg et al., 2005). Mutagenesis of H334 to an Ala residue reduced the kcat value 4 times and slightly increased Km value, indicating that H524 has no significant role in enzyme catalysis (Karaveg et al., 2005). It is interesting to note that all sequences obtained in our transcriptomes have one Ala residue

Acknowledgments

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instead of His in this position. It is not clear the meaning of this.

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This work was supported by the Brazilian research agencies FAPESP (Tematico) and CNPq. We are indebted to W. Caldeira, and M.V. Cruz for technical assistance and to Dr. Ariel M. Silber for his help in selecting peptides

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to be synthesized to raise antibodies against α-manosidase. Nathalia R. Ramalho was a graduate fellow of FAPESP. C. Ferreira and W.R. Terra are staff members of their respective departments, research fellows of the CNPq, and members of the Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular (INCT-EM).

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Anterior + Middle Posterior Soluble Membrane Soluble Membrane 27 ± 3 29 ± 3 7±1 15 ± 2 10.5 11.7 2.8 6.1 120 ± 20 130 ± 20 57 ± 7 46 ± 3 230 ± 20 240 ± 10 140±20 380 ± 50

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Total activity (mUnits/animal) % of total Protein (µg/animal) Specific activity (mUnits/mg)

Whole midgut Soluble Membrane 220 ± 10 50 ± 3 81.5 18.5 1100 ± 100 160 ± 10 280 ± 30 360 ± 30

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Table 1. Distribution of α-mannosidase activity along the midgut of T. molitor Midgut Tissue

Midgut contents Anterior + Middle Posterior Soluble Soluble 149 ± 9 20 ± 1 60.3 8.2 560 ± 40 145 ± 9 280 ± 10 144 ± 3

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Sum or total activities 270 ±20 250 ± 20 (mU/animal) Values are mean and SEM calculated from determinations carried out in 4 different preparations obtained from 7 animals each. The substrate used was p-nitrophenyl α-D-mannoside. One unit of enzyme is the amount of enzyme that cleaves one µmol of substrate per min.

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Table 2. Kinetic properties of Man1 and Man2 Parameter Man1 Man2 pHo 5.6 5.6 Km (mM) 0.84 ± 0.05 0.62 ± 0.02 Ki deoxymannojirimycin (mM) 0.120 ± 0.005 0.150 ± 0.008 Ki swainsonine (nM) 68 ± 5 63 ± 4 Substrate: NPαMan. The inhibitors are of the linear competitive type for both Man1 and Man2.

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Table 3. Substrate specificity of Man1 and Man2 (relative Vmax/Km) Substrate Man1 Man2 2 α-mannobiose 100 ± 1 50 ± 2 3 α-mannobiose 24.4 ± 0.5 100 ± 3 6 α-mannobiose 34.1 ± 0.5 87 ± 4 Relative Vmax/Km were calculated from Km and Vmax values determined with 15 different concentrations of substrate (range 0.25 - 23 mM).

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Table 4. Features of the manually curated sequences of Family 38 α-mannosidases from the transcriptome of 4 insects from 3 orders. Contig GenBank access Insect Reads/contig Length SP OBS D. maculatus Dm609 75 661 Y N-Fragment D. maculatus Dm1611 97 965 Y N-Fragment P. americana Pa172 75 966 Incomplete P. americana Pa174 7 334 Incomplete M. domestica Md33 XP_005189986.1 1021 1009 Y Complete T. molitor Tm345 67 1030 Y Complete T. molitor Tm404 12 896 Incomplete T. molitor Tm406 17 382 Incomplete T. molitor Tm408 160 963 C-Fragment T. molitor Tm602 653 854 Y N-Fragment T. molitor Tm603 289 590 Incomplete T. molitor Tm1774 433 1001 C-Fragment T. molitor Tm1776 10 343 Y N-Fragment Length is the number of amino acids in the protein putatively coded by the contig. Y indicates a predicted signal peptide in the protein, whereas a dash means that the presence of SP could not be evaluated due to the lack of the N-terminal portion. CFragment or N-Fragment indicates that the sequence misses the part coding for N- or C-terminal portion, respectively. Incomplete sequences have both ends missing. Contig Tm408 corresponds to TmMan2, GenBank access KP892646.

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Table 5. Features of the manually curated sequences of Family 47 α-mannosidases from the transcriptome of 6 insects from 3 orders. Contig Gen Bank Insect Reads/contig Length SP OBS TH access B. xantophis Bx2085 16 432 Incomplete Y D. peruvianus Dp1634 10 476 Incomplete N D. peruvianus Dp3988 6 160 Incomplete N D. maculatus Dm6182 6 207 Incomplete N T. molitor Tm2371 11 326 Y N- Fragment N 7 845 N Complete N Md1922 XP_005188141.1 M. domestica R. americana Ra766 12 365 Incomplete N TH, transmembrane helix. Other details as in Table 4.

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Figure 1. Purification of the digestive α-mannosidases from T. molitor larvae. The soluble fraction of T. molitor midgut homogenates was loaded onto a Hitrap Q XL column (A) equilibrated with 20 mM Tris HCl buffer, pH 7.0. Elution was

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accomplished with a gradient of NaCl from 0 to 1M in the same buffer. At all performed purification steps the flow was set at 0.5 mL/min and fractions of 0.4 ml were collected. Fractions active on the substrate NPαMan were pooled, passed through a Superdex 75 HR 10/30 column (Pharmacia Biotech) (B)

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equilibrated with 20 mM Tris HCl buffer, pH 7.0, containing 1M NaCl. The active fractions were pooled, the pool buffer was changed with the aid of a High Trap

M AN U

desalting column and then it was passed through a Phenyl Superose column HR 5/5 (C) equilibrated with 50 mM phosphate buffer, pH 7.0, containing 2M (NH4)2 SO4. Elution was accomplished with a (NH4)2SO4 gradient from 2 to 0 M in the same buffer. The fractions corresponding to each of the two activity peaks were used as a source of α-mannosidases (Man1 and Man2). Electrophoresis of proteins in SDS-12% polyacrylamide gel slabs (D). Lanes 1,

TE D

2 and 4, SDS-PAGE after silver staining for protein detection. 1, midgut contents; 2, Phenyl Superose column eluate corresponding to Man 1; 4, Phenyl Superose eluate corresponding to Man 2. Lanes 3, 5, 6, and 7, western blots with anti α-mannosidase serum: 3, purified Man 1; 5, purified Man 2; 6, T.

EP

molitor midgut contents; 7, T. molitor midgut tissue homogenate. The lanes were combined from different runs as follows: 1, 2 and 4, 3 and 5, 6 and 7.

AC C

Other details in Material and Methods.

Figure 2. Calibration curve for Superdex 200 column. It shows that Man 1 and Man 2 have similar molecular weight (∆) of 123 kDa. The following proteins were used as standards: cytochrome C (12.4 kDa), ovalbumin (43 kDa), bovine serum albumin (67 kDa), β-amylase (200 kDa). Other details in Material and Methods.

31

ACCEPTED MANUSCRIPT

Figure 3. Inhibition of purified Man 1. Lineweaver-Burk plots for different concentrations of deoximannojirimycin. Inset: replots of slopes of LineweaverBurk plots against the concentration of deoximannojirimycin.

Figure 4. Electron transmission immunocytochemical localization of Man 2 in

RI PT

anterior midgut (A) and posterior midgut (B). (A) Note labeling (arrow) associated with a large vesicle, which is actually an aposome and also outside the cell. (B) Note that labeling (arrow) is only seen outside the cell. Mv,

SC

microvilli; SV, secretory vesicle. Bars: 1 µm.

Figure 5. Nucleotide and deduced amino acid sequence of the cDNA (TmMan2) coding for Man2. The signal peptide is in italics. Catalytic residues are in bold.

antibodies in a rabbit are boxed.

M AN U

The amino acid residues of the peptide synthesized and used for raising

Figure 6. Cladogram (maximum likelyhood) of insect G38 family of αmannosidases. Branching corresponding to partitions in under 50% of replicates (1000) were collapsed. Abbreviations followed by numbers correspond to

domestica;

Pa,

TE D

contigs found in our transcriptomes: Dm, Dermestes maculatus; Md, Musca Periplaneta

americana;

Tm,

Tenebrio

molitor.

Other

abbreviations: AMII, α-mannosidase II (Golgi); CAM, cytosol α-mannosidase;

EP

LAM, lysosomal α-mannosidase.

Figure 7. Expression pattern of mRNAs for α-mannosidases in different tissues

AC C

and in different portions of the midgut tissue of T. molitor. A, M, P are anterior, middle, and posterior midgut; C, carcass; FB, fat body; MT, Malpighian tubules. The contig Tm000 is part of TmMan2.

ACCEPTED MANUSCRIPT

A

B A280

0.6

40

0.4

20

0.2

10

20

C

30

40

Fractions

50

60

Phenyl Man 1

60

40

20

0

0

10

20

30

40

50

Man 2

40

1.2 1.0 0.8 0.6 0.4

20

0.2 0

10

20

30

40

50

60

Fractions

70

80

90

0.0

[(NH4)2SO4] (M)

AC C

60

70

80

90

SDS-PAGE western

D 1

2

3

4

1.8

1.4

60

Fractions

1.6

80

0

0.0

A280 2.0

100

Relative activity (%)

70

TE D

0

RI PT

60

80

SC

0.8

Relative activity (%)

80

M AN U

1.0

A280

100

[NaCl] (M)

100

0

Superdex

EP

Relative activity (%)

HTQ

. . .

. .

5

6

7

RI PT

ACCEPTED MANUSCRIPT

2.5

SC

2.5

M AN U

Ve/Vo

2.0 2.0

1.5 1.5

0.5 0.5 0.0

0.0

1.2 1.2

AC C

EP

1.0 1.0

TE D

1.0 1.0

1.4 1.4

1.6 1.6

1.8 1.8

Log (MW, kDa)

2.0 2.0

2.2 2.2

2.4 2.4

RI PT

ACCEPTED MANUSCRIPT

9

SC

8

M AN U

7

1/v

6 5

TE D EP

3

1

-1

0

AC C

2

1

-0.2

Slope

3

5 4

2 1

0

0.2

0.4 0.4

0.6

[I], mM

2 3 -1 1/[S], mM

4

5

6

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT atgagcctgctggtgtttctgttttgctttagcctgtttctgggcgtgaacaccaaaccgtatgaagcggatgcgccgagctgcggctat 30 M S L L V F L F C F S L F L G V N T K P Y E A D A P S C G Y

31

gatgcgtgcccggaagcggatccgagcgcgctgaacgtgcatattgtgccgcatagccatgatgatgtgggctggctgaaaaccctggat 60 D A C P E A D P S A L N V H I V P H S H D D V G W L K T L D

61

cagtattattttcaggatgtgcagaacgtgattagcagcgtgattgtggcgctgaaactgaacccggaacgccgctttgtgcaggtggaa 90 Q Y Y F Q D V Q N V I S S V I V A L K L N P E R R F V Q V E

91

accgcgttttttaaactgtggtggagccgccagaacgatattattaaagaagcggtgcagaacctggtgaacaacggccagctggaattt 120 T A F F K L W W S R Q N D I I K E A V Q N L V N N G Q L E F

121

attaacggcgcgtggagcatgaacgatgaagcggcggtgcattatcagagcaccattgatcagtttaccctgggcctgcgctatattgaa 150 I N G A W S M N D E A A V H Y Q S T I D Q F T L G L R Y I E

151

gataacctgggccgctgcgcgcgcccgaaagtgggctggcagattgatccgtttggccatagccgcgaacaggcgagcattagcgcgcag 180 D

N

L

G

R

C

A

R

P

K

V

G

W

Q

I

D

P

F

G

H

RI PT

1

S

R

E

Q

A

S

I

S

A

Q

ctgggctttgatagcatgttttttgcgcgcctggattatcgcgataaaaaccgccgcatggatgataaaaccatggatctgctgtggcgc 210 L G F D S M F F A R L D Y R D K N R R M D D K T M D L L W R

211

ggcagcgcgaacctgggcaacaacgcggatatttttaccagcgtgctgtatcagcattatagcgcgccgggcggcttttgctttgatatt 240 G S A N L G N N A D I F T S V L Y Q H Y S A P G G F C F D I

241

gtgtgcaacgatgaagtgattattgatgatgaagaagatccggattataacctggaaaaacgcgtgggcgaatttgcggatcagatgcgc 270 V C N D E V I I D D E E D P D Y N L E K R V G E F A D Q M R

271

gatcgcgcggaacattatccgaccaacaacattctggtgaccatgggcgatgattttcgctatgaagcggcgatgaccacctatatgaac 300 D

R

A

E

H

Y

P

T

N

N

I

L

M AN U

SC

181

V

T

M

G

D

D

F

R

Y

E

A

A

M

T

T

Y

M

N

ctggatctgctgattaaaggctttgatctgtttgaacagacctataacgataaacgcattaaagtgttttatagcaccccgagctgctat 330 L D L L I K G F D L F E Q T Y N D K R I K V F Y S T P S C Y

331

accaaagcggtgaacgattatgtgaacagcaacaactataacctggaactgaaaaccgatgatttttttccgtatgcggatggcaccaac 360 T K A V N D Y V N S N N Y N L E L K T D D F F P Y A D G T N

211

ggcagcgcgaacctgggcaacaacgcggatatttttaccagcgtgctgtatcagcattatagcgcgccgggcggcttttgctttgatatt 240 G S A N L G N N A D I F T S V L Y Q H Y S A P G G F C F D I

241

gtgtgcaacgatgaagtgattattgatgatgaagaagatccggattataacctggaaaaacgcgtgggcgaatttgcggatcagatgcgc 270 V C N D E V I I D D E E D P D Y N L E K R V G E F A D Q M R

571

ctgctgaaaaaaaccccgaacctgagcgtgggctatgaagaaaccagctttgaaattagcgaacagaccggcctgctggaaagcattacc 600 L L K K T P N L S V G Y E E T S F E I S E Q T G L L E S I T

601

atgaacggcgtgaccctgcaggtgacccaggattttcagtattataccagccagaacagcagcggcgcgtatatttttgtgccggtggaa 630 M N G V T L Q V T Q D F Q Y Y T S Q N S S G A Y I F V P V E

631

accgatccgagccgcgtggcgggcggcccgattaccaccaccctggtgagcggcgatgtgagccagggcgtgctgcaggaatttggcagc 660 T D P S R V A G G P I T T T L V S G D V S Q G V L Q E F G S

661

tgggcgcgccagtttattaaagtgtataacgatgataaaagctatattgaatttgattggattgtgggcccgctggatattagcgatggc 690 W A R Q F I K V Y N D D K S Y I E F D W I V G P L D I S D G

691

gtgggcaaagaagtggtgagcaaatttagcaccccgctggaaaccaacggccagttttataccgatagcaacggccgcgaagtgctgcag 720 V G K E V V S K F S T P L E T N G Q F Y T D S N G R E V L Q

721

cgcacccgcaacagccgcccggattatgattataccgatgaacagccggtggcgggcaactattatccggtgaccagcaaaattgtgatt 750 R T R N S R P D Y D Y T D E Q P V A G N Y Y P V T S K I V I

751

gaagatgatgatgtggaatttgcggtgctgaccgatcgcagccagggcggcagcagcattaacgatggcgaagtggaactgatggtgcat 780 E D D D V E F A V L T D R S Q G G S S I N D G E V E L M V H

781

cgcgcgtgccagcatgatgatggccgcggcgtgggcgaaaacctgaacgaacaggaatttggcgatggcattcgcgtgcgcggcaaacat 810 R A C Q H D D G R G V G E N L N E Q E F G D G I R V R G K H

811

tttctggtgctgggcccgaaaggcggcaacggcgataaaagcattgcggcggtggaacgcgatgtggcgcagcgcaaactgctgagcccg 840 F L V L G P K G G N G D K S I A A V E R D V A Q R K L L S P

AC C

EP

TE D

301

ACCEPTED MANUSCRIPT 841

tggacctttgtgaccaaacaggtggataacctgaacaacctgcagtttagcggcctgaaaaacagcctgccggataacgtgcagattctg 870 W T F V T K Q V D N L N N L Q F S G L K N S L P D N V Q I L

871

accctggaaccgtggagcgaaaacaccctgctgtttcgcctggaacatgtgctggaaaacggcgaagatgataacctgagccaggaagtg 900 T L E P W S E N T L L F R L E H V L E N G E D D N L S Q E V

901 accgtggatgtgagcgatctgtttaccctgtttagcattaccgaactgaaagaaaccaccctgggcgcgaacatgctgctggaagaaaac 930 T V D V S D L F T L F S I T E L K E T T L G A N M L L E E N gtgcgcctgagctggccgggcagcagcaccaccgatgatcaggtggaaaaacgcgatgtggatgatctgaccgtgaccctgcagccgatg 960 V R L S W P G S S T T D D Q V E K R D V D D L T V T L Q P M

961

cagattcgcacctttctggcgaccgtgagctataac 972 Q I R T F L A T V S Y N

AC C

EP

TE D

M AN U

SC

RI PT

931

ACCEPTED MANUSCRIPT

99 93

Tm602

Tm345

50

Tm1774 98

Tm1776

SC

Dm609

RI PT

Tm603

Tm408 Tm404

M AN U 99

Tm406

75

88

Pa172 Pa174

LAM D.melanogaster AAD38576.1

TE D

91 64

AC C

EP

100

69

Md33 LAM M.musculus NP 034894.2 LAM B taurus AAC48763.1 LAM H.sapiens AAC51362.1 Dm1611 S.frugiperda AAB62719.1 AMII D.melanogaster CAA54732.1

65

AMII M.musculus CAA43480.1

91 99

AMII H.sapiens AAC50302.1 CAM H.sapiens AAC00190.2

100

CAM M.musculus AAH16253.1

Tm 602

EP

AC C

Control

TE D

Tm 1774 Tm 000

M

P

M AN U

A

SC

RI PT

ACCEPTED MANUSCRIPT

C

FB

MT

ACCEPTED MANUSCRIPT Highlights

- Two soluble α-mannosidases (Man1 and Man2) were purified by chromatography from the midgut of Tenebrio molitor

RI PT

- The α-mannosidases have MW 123 kDa (gel filtration) and Man 2 form two bands (72 and 51 kDa) in SDS-PAGE.

-The sequence TmMan2 (KP892646) codes for Man2 that is processed into two peptides that remain linked in the functional enzyme.

SC

- Man2 hydrolyzes α-1,2, α-1,3, and α-1,6-mannobiose and once secreted by an apocrine mechanism (immunocytolocalization) may digest fungal cells.

AC C

EP

TE D

M AN U

- The number of family 38 of α-mannosidases in the midgut of 9 different insects may be related to their diets.

ACCEPTED MANUSCRIPT Supplementary Table 1- FASTA sequences corresponding to the manuallycurated sequences coding for α-mannosidases from 6 insects pertaining to 5 orders plus TmXXX and Tm000. See text for details.

>TmXXX MSLLVFLFCFSLFLGVNTKPYEADAPSCGYDACPEADPSALNVHIVPH

SC

SHDDVGWLKTLDQYYFQDVQNVISSVIVALKLNPERRFVQVETAFFKKWW

RI PT

Contigs Family 38

EQQKDSIKQDVIDLVNNGQFEIINGAWSMNDEAAVLYQCTIDQFTLGLRY

M AN U

LEDRLGACSRPRVGWQIDPFGHSREQASISAQLGFDSIFFARLDYRDKIN

RMGKKTGDLIWRGSSNLGNSSDIFTSVLYNHYSAPPGFCFDIVCDDDPSS TTKKVRITTTKAESKILQTL

TE D

>Tm000

RLTIGRLGPTSHAPGRHGGRGNSIWTGYFTSRATSKHFERQGNNLLQVSKQLAANAQGSY DNEQINTLKEAVGVMQHHDAITGTEKQHVADNYYMRLSRGMQSAGDAAGQVLSNLITGDD

EP

TNLEFDSCLLANVSACTQTESDTFTVAVYNPLSRTQTAIVTLPVFDQQNYQIRDPDDNDV PYQLDASLTDFSYVENTRTSQTTLQFAAKDLPPLGFKVYRFSAADKQPKSNPLLKKTPNL

AC C

SVGYEETSFEISEQTGLLESITMNGVTLQVTQDFQYYTSQNSSGAYIFVPVETDPSRVAG GPITTTLVSGDVSQGVLQEFGSWARQFIKVYNDDKSYIEFGWIVGPLDISDGVGKEVVSK FSTSLETNGQFYTDSNGREVLQRTRNSRPDYDYTDEQPVAGNYYPVTSKIVIEDDDVEFA VLTDRSQGGSSINDGEVELMVHRACQHDDGRGVGENLNEQEFGDGIRVRGKHFLVLGPKG GNGDKSIAAVERDVAQRKLSSPWTFVTKQVDNLNNLQFSGLKNSLPDNVQILTLESWSEN TLLFRLEHVLENGEDDNLSQEVTVDVSDLFTLFSITELKETTLGANMLLEENVRLSWPGS STTDDLVEKRDVDDLTVTLQPMQIRTFLATVSYN

ACCEPTED MANUSCRIPT >Dm1611 NAMKLFAFILFLSFLGLISAKPIEESSKCVSCRRKADPKKLSIHLVPHSHDDVGWLKTVD QYYYGTNNSYQRASVELIIDSVYRALLENKNRTFIQVETYFLHKWWQRQNEYTRKKYREL VNNGQIEIVGGGWSMNDEAVTNYLSLIEQFEFGFRKLDEMLGECGHPKVAWQIDPFGHSK

RI PT

TMAQLFSEIGFDGLFFARLDWRDRMKRRNEAQMEMIWEGFGGNLFTSILYEHYGAPDGFC FDITCNENNPIVDDEDSPLFNQESAVKQFTSIVEKRSKSYRTNNILIPMGDDFNYMHASS YYLNMDMLIEGFNKYPQKTDDGREIEIFYSTPWCYIKAVNEEIGKNSSIQLQKKTDDFLP

LGNDPHTYWSGYYTSRPTSKRLEREGINLMQVFKQLNSFATKPYSTVPIESAMGIMQHHD

SC

AITGTEKQHVANDYHRILTSIIHETTFAVDSIISELLSVNKEKPVVLASSSCPLSNISYC

PITESDEFVVVIYNPFSESVTHHIRLPVNNTNYQVIGPTDEIIKSQIVPAISDFKVLNGT

M AN U

FEHSKYDLVFNADIYPHTITTMKVKKVEENSNTGTIKVNPETNCDEIGDIFKAYFEKDGR LKSVKYKDIDLDLKQELLHYMSAHGNNRNFSQRASGAYIFRPDPEQPDAIPLPNLNFSSK CYKGEVVDEVHQTYNEWVKQIIRVYKNEDHMEFDWLVGPINISDDRGKEIISRFYTTKIN SSDTFYTDSNARQLIKRVRDHRPTFNYTAEEKVAGNYYPVTSRIVLKDDNLEMAVLNDRP

TE D

QGGTSLKSGEVELMVHRRCLNDDAFGVGEALNETEYNVGLIARGRHYLTVNNPDDSHSEH LSQKKLFSPMVFISLIKLDEKLIKRSFSAIDPTILPDGAQILTLEPLSKNEILLRLENIY

EYKLA

AC C

>Dm609

EP

ENGEEKTVKLNKLFKNFNVIKVSELDLGGVRPKGLSKRLQWGEDKGETEKLNSVANEGAF

MLLKIALLIFTFTYCAICVPIAEEIKCGYQSCHPIEDNKLNIHLVPHSHDDVGWLKTVDE YYYGVNNKVQPVGIQYIISSVVQSLQSNPDRRFIQVEGAYFWQWWKRQTEETKSIVKKLV NSGQLEIINGAWSMNDEAATNYQSIIDQFTLGIKMYNDTLGSCSYPKIGWQIDPFGHSKE MANIFSQLGYEAIFFARIDHKDKTKRSQQKALELIWQGSQTRGKASDIFAVIFSDFYIQP LHFCWDVLCFDEPIVDDINSPEYNADKKVKLFMDYVNKHAEHYQTNNILIPMGGDFTYQA AEMYYSNIDKLIQALRKFDKNVNPIYSTPSCYVKAVNDVSRKKKISFPVLTTDFFPYSSD DVSFWSGYYTSRPNSKRLERMGNNILQVTKQLISMHRVENKDFNDTLTLLKESMGIMQHH

ACCEPTED MANUSCRIPT DAITGTEKQAVAQDYARLMKKSIDKVEQNITNILADLSKKEKSKDVSEDKIQFSSCLLAN VSYCEASKKKSFLVAVYNPLSRVVSDYIRLPVQANFSFEITGPNGIVQYDVMKTISPFNY INNVSQTKYELVFHAQNIPPLGLQYYYVKSLNLTQQKNEQIPFAEETIFGNNKTGFAINE RGLLKEITVNGETLEIEQQFYKYMAFEGNNYGQNRSSGAYIFRPSKSNGGIAEPLIPGDK

RI PT

K

>Md33

MKFGVWALSSALLVLSWSYHAEAACGYKACPASKANMINVHLVPHSHDDTGWLKTVDQYY

SC

YGSRNGIQHAGVQYILDSVITELLKDKSRRFIQVESAFFFKWYNEQTAAVKKNVKTLVEE GRLEFTGGAWSMNDEAAVHYQSVIDQFTLGLKSLADTFGSCARPRIGWQIDPFGHSREMA

M AN U

SLFAQMGYDGNFFARMDYMEKNARLNNMTGEMIWKASENLDERSEIFTGVLYNHYSAPPG FCFDNLCSDDPIIDGDSYENNVKAKVDDFLTYINKMGTYYRATDLLVPMGDDFNYENAYV NYKNMDKLIKYVNQRQLQGGKVNVFYSTPACYLNALHNDNKTWPTKTQDFFPYSTDWHTY WTGYYSSRPTQKRFERDGNHFLQVAKQLTTMANLTTTAAFANLDTLRQAMGIMQHHDAVT

TE D

GTEKQHVAQDYDRMLTKAINVAETNTRDALRKLTNLTNGEFVSCLQLNLSICEVSQWSPN NLVVTVVNPLAHPSTQYVRIPVKNGTYIVTDATGRQVKSDVVPVPKELIDLTHLRPNASQ HELVFSAYADKIANYYIKVQPQPREFDEEIQRNEIKLPKRFLRQHLTKLEVAPASETIEP

EP

MAVGDVVVQNSQIKLTFNSNGILYNVQMNGISEDITQQFFYYKGAYGNNAEFKNRSSGAY IFRPNGTEILIGTKANLTIVNGSLVKEVHQRFTDWVSQVIRIYEGINRVEFEWLVGPIPI

AC C

QDNIGKEVITRFTSNLKNSGVFYTDSNGREMLRRVRNQREYFKPNMTESVSGNYYPITSR IALESSTRRMALLNDRAQGGSSLKDGALELMLHRRLLRDDAFGVGEALNETVNGKGLVAR GKVYLMLSTVSKISTTNERLAEREIHLPFWKFFSNATKATNKVLPRIPTFTSLPIGIDVL TLEPYSTNERLLRLEYFFNKNETSSLTFNIRSIFDALGGTEIRETTLDGNLALGSMNRFK FHPDGTVPKKVEYYKSKHTPLAAVQKDAVSKFQITMDPMQIRTFIVKWK

>Pa172 AARAVCGYESCHNVDPTKLNVHLVAHTHDDVGWLKTLDQYYYGSRNDIQNAGVQYIIDSV

ACCEPTED MANUSCRIPT VDSLVANPDRRFIYVETAFFWKWWVEQDEDMQQTVKDLVNEGRLEFIGGAWSMNDEAASH YYSTIDQFTWGLRKLNDTFGACGRPHIGWQIDPFGHSREMASLFAQMGYDGVLFARLDYQ DKDNRLKTKTPEMIWEGSPNLGESADLFTSVLYNFYNAPGGFCFDILCNDQPFIDNVESP DYNVDQKVQDFVNFVKTQGSYYTSSNIILTMGGDFNYQDANTWFKNLDKLIKYVNAADST

RI PT

LNVIYSTPSCYLKAVNDAGLTYTTKQDDFFPYASDPHSYWTGYFTSRPASKYFERLGNNF LQVAKQLEAMTLLGQVGDSSIDDLREAMGVMQHHDAITGTEKQHVAGDYARLQYRAMDEA SVSMETALNQLMENPTTTPIEIHSCLLFNISECHVTERGGSQFLVTVYNPLSHSLDYSVR FPVPSGTYTVQDPTGAELPFQIIPLPDEVLALPERDSSIATHELAFRALSLPPLGFRSYY

SC

VVRVSDDFVEVEPSADTFIGDSLLQVTIDNVTGLVDSLTINGEEIALSQNFFYYDGYIGE

NDNADHRSSGAYIFRPISSVPQTIATSATWKIYKGHIVDEIHQTFSPWVSQVIRIYKQEN

M AN U

HVEFSWLVGPIPIEDGVGKEVVNKFSTDLASDGLFYTDSNGREMLERKRDFRPTWTVDIA EPVAGNYYPVTSKILIRDTTKGQEFAVLNDRAQGGSSLNDGEVELMVHRRLLHDDAFGVG EALNEVAYDQGLVVRGRHYVIAGPTSGLSPSLAAQERELEQKKMLSPWMFFAVAGPSFEY WQSTFKMEFSGLTESLPQNVKIHTLEPWRDQNLLLRLEHILEQDDDPELSQPATVVYEDI

IDVAKR

EP

>Pa174

TE D

FSTFTVTSSHETTLAANQWIEDLDRLVWKSDSAREPKEKQKMEKDPKYVTLTPMQIKTYV

VTCFAHPRRSLYRDDRADVTCGYQSCHQVDPTKLNVHLVPHTHDDVGWLKTVDQYYYGAN

AC C

TNYQRAGVQYILDSVIEALLDNPDRRFIYVETAFFWKWWVEQTPERQQEVKDLVNEGRLE FIGGAWSMNDEAASHYQSTVDQFTWGLRKLNDTFGSCGRPHIGWQIDPFGHSREMASIIX ARMGYDGLFFARLDYQDKNTRLAGKTAEMIWEGSPNLGASADMFTSALYNHYSAPSGFCF DILCNTEPFIDNQKSPDYNVDAKVQNFVNFVNGQAEHYTSNNILVTMGDDFNYQDALMYF KNMDKLVKYVNAADKGVNVIYSTPSCYLKAVNDE

>Tm404 HDDLGWLKTMDKYYFQDVQNVIGSVVGALKQNPDRRFVQVETAYFKEWWDRQNDIVRQDV

ACCEPTED MANUSCRIPT IDLVNNGQLEIINGGWCMNDEANTNYQSTIDQYTLGLRFLEDTLGPCGRPRVGWQIDTFG HSREQASISAKLGFDSLFFMRMDYRDKNKRLADKTGDLLWKGSQNLNDSYIFTSIFYGAY SFPEGFCFDIVCQDEPIIDDEESPDYNYDRRVDEFAEFVRGQASQYPTNNILVVMGDDVR YQASLTNFLNIDRLIKGFELFPKTFDDKPIKLLYSTPSCYAKAVNEFVTANDYNLDIKTD

RI PT

DFLPYATDGYGYWSGFYTSRPSKKRFERQGNNFLQIAKQLSAITNQPYESRITRLKEAVA VIQHHDAITGTEKEDVMKDYVRMLDTALEEANQAVDPILSTLIGSTSDDSFKFNTCLLAN ISSCGPSKSDKFTVTVYNPLSRPVSSPIELPVDSQTWMIEDPQGNEVIYQTDPPSXDFSY AEDVPISPYTLLFTAEDLPPLGFKVYTFTKAETVPEEQPHLTVGNDNTSFEIDDDTGLLK

SC

SITMNGVTMDVSQDLAYYRSGSYSGAYIFVPLENEKHRVTEDKVKTTPISGDIYQGVLQE FNSWAKQIIKVYNDDSNYIEIDWIIGPVDVSDGIGREVVSVFTTPLQTEGNFYTDSNGRE

M AN U

MLKRTRNYRPTFDYTNEEPIAGNYHPITSRIVLKDEEQGLELAILNDRAQGGASLEDGQV EIMIQRACTHNDRSQGNVRDSINDQEYGQGVIIRGKHYLILGPSSGNGEKSLAAIQRDVA QKKLLAPWAFVTDQDITQQXXDTREFSSLKTPLSDNVHILTLEPWNENTLLLRLEHIMEK

>Tm406

TE D

DEDENLSQETTVDLSDLFATFTITELEETTLGANIPLEDSVRLSWPGTGDTEDGKN

EADPSALNVHIVPHSHDDVGWLKTLDQYYFQDVQNVISSVIVALKLNPERRFVQVETAFF

EP

KKWWEQQKDSIKQDVIDLVNNGQFEIINGAWSMNDEAAVLYQCTIDQFTLGLRYLEDRLG ACSRPRVGWQIDPFGHSREQASISAQLGFDSIFFARLDYRDKINRMGKKTXDLIWRGSSN

AC C

LGNSSDIFTSVLYNHYSAPPGFCFDIVCDDDPXIDDEESPDYNYQSRVENFANFVKEQAS KFPTNNIIVVMGDDFRYQAALNSYINTDRLIKGFDLFPQTFQGKPSKSSTRLPRVTPKLS TITSQPTITSLKLKPTTFFLTPTELEALXXWDTFTSRPASKRFVYEGNNLLQVLPTYLXP NQSVNLFQVAKQLAVVGQESYD

>Tm408 VFIALSVDGKPVVEDDPICGYEACPEASPDTLNIHIIPHSHDDVGWLKTVDQYFFQDVQNVISSVVDALKQN PERRFVQVETAFFKLWWSRQNDIIKEAVQNLVNNGQLEFINGAWSMNDEAAVHYQSTIDQFTLGLRYIED NLGRCARPKVGWQIDPFGHSREQASISAQLGFDSMFFARLDYRDKNRRMDDKTXGPFVERKRQLGNNAD IFTSVLYQHYSAPGGFCFDIVCNDEVIIDDEEDPDYNLEKRVGEFADQMRDRAEHYPTNNILVTMGDDFRYE

ACCEPTED MANUSCRIPT

RI PT

AAMTTYMNLDLLIKGFDLFEQTYNDKRIKVFYSTPSCYTKAVNDYVNSNNYNLELKTDDFFPYADGTNTYW TGYFTSRATSKHFERQGNNLLQVSKQLAANAQGSYDNEQINTLKEAVGVMQHHDAITGTEKQHVANNYYL RLSRGMQSAADAAGQVLSNLITGDDTNLEFDSCLLANVSACTQTESDTFTVAVYNPLSRTQTAIVTLPVFDQ QNYQIRDPDDNDVPYQLDASLTDFSYVENARTSQTTLQFAAKDLPPLGFKVYRFSATDKQPKSNPLLKKTPN LSVGYEETSFEISEQTGLLESITMNGVTLQVTQDFQYYTSQNSSGAYIFVPVETDPSRVAGGPITTTLVSGDVS QGVLQEFGSWARQFIKVYNDDKSYIEFDWIVGPLDISDGVGKEVVSKFSTSLETNGQFYTDSNGREVLQRTR NSRPDYDYTDEQPVAGNYYPVTSKIVIEDDDVEFAVLTDRSQGGSSINDGEVELMVHRACQHDDGRGVGE NLNEQEFGDGIRVRGKHFLVLGPKGGNGDKSIAAVERDVAQRKLLSPWTFVTKQVDNLNNLQFSALKNSLP DNVQILTLEPWSENTLLFRLEHVLENGEDDNLSQEVTVDVSDLFTLFSITELKETTLGANMLLEENVRLSWPG SSTTDDQAEKRDVDDLTVTLQPMQIRTFLATVSYN

SC

>Tm345

MHFWIVPSVLLLNFHGIISNPIKGDIQAKDSVVCQKACHPLVDGKINVHLVPHSHDDLGW

M AN U

LKTFDQYYEGTGQFNAVENAGVRFILSSTIPALKADPNRRYIQVETGFFWKWWQEQSNET RQDVVDLVNSGQLEMINGGWSMNDEAASHYHSIIDQFTWGFRILEDTVGKCGRPKIGWQI DPFGHSKEHASILKQLGFEGLVVVRIDYRDKNKRRAEKNLDFIWKSNDNLENSEIFTTMF PDFYFEESGYCFDVMCSWDTINEGNLDSKVKGFAKILDGYKEYYKTNNIMMPMGRDFTYQ KAEQNFASMDLFLKGFKDHDKYNVIYSTPSCYIQAVQDEIQKNSIKLEEKTDDFFPYASE

TE D

AHSFWTGYFTSRPTSKRFERTGNNILQSVKQLTAFAKMKSKDYGESIADLRGAMGVMQHH DGITGTEKQAVSNDYAQMLHQAIKEAEEPVGTIIGELLRKNDEDEIDLQLSSCLLANVSI CDTTGKDRFLVVVQNPLSRVVTHYVHLPVEGDNYKVTGPDGEEVYDVFDTLHSFDYINEP

EP

TKPSSKDLVFAARNLPALGINLYYVEKMSESSNLYKPSQPLEAAEDGAFGTETNGFKIDT STGKLASVTINGATQEISQEFLYYPGYTDSTTTDDHRSSGAYIFRPAENEAKPMTTESSI

AC C

DTNCAKGNLVDQCIQIINDEVRQIIKVYKDADDAFVEFDWLVGDLQFVDNKGKEVITRFT VKDFSNSGTFYTDANGRQQVRRELNKRGDFEYDPEEEPVSSNYYPVTSKIVIKDETKKLE VAVLNDRAQGGTSLKEGTVELMIHRRLLGDDDKGVKEALNEIQYDKGLYVRGQHYLTFGS TESKVANGKSTAAFERDLAHRKLLAPWILLSEATDTLATLEKTQQILEFKFEALKKSLPD NVHILTLEPWKNSYLLRLEHALEKNEDDALSTEVTVDLEELFTLFNITEIWETTLGANQV LDESPNLKITLKPMEIRTFIIKTDQNNDDIDNDNDNSAGSHTARVKMGFKVKGIYVINNP ISFLKHVYQL

ACCEPTED MANUSCRIPT >Tm602 MECGRGLIAVFVLFNVWWVNSTPLTKDDATECVSACQGLDEKKVNVHLVPHSHDDVGW LQTFEGYYKGTGDFKEIKNAGVQYILDSTIQALQKNKNRRYIQVETAFFWKWWNNQGRRT PAAPDLVNSGQLEMVGGGWSMNDEAAAHYQSIIDQFTWGFRILDDTVGECGRPKVGWQ

RI PT

IDPFGHSREHASISKQLGFEGLVLGRIDYRDKSQRIEDKNLDFNWVTNPNFEDSTILTTM FPDFYTWPEGLCLDSTCASSQKLINDDNVEAVVANFTAILQEWIGYYKTKNILIPMGHDF TYQKAEDNFNSMDKLIEGFKDSDYNVIYSTPSCYIEAVTAAKPTLTKKEDDFFPYASNAN

EFWTGYFTSRTDSKTIRKPTAKRFERVANNILQSVKQLTTFSRIQGGDYDKNIVDLRMAMGVMQHHDA

SC

ITGTEKQDVANDYTSMLYKGIAKTQDSISKIISDLLKKNDSAVDLALSTCLLANVTICDA

SNKDKFLVAVQNPLSKTVSHHVRLPVNGTNFKITDADGEVAHDVLDAMHTFDFETKFETP

M AN U

SKEIVFLAKDLPPLGVKLYYVETVTDDTKKYKPQDDVVTFGDAKTTEFTIDATTGK

LASVTIKGVEIKVQQDFYYYHGRVDGAYVFEPEEGTSEDATLLGGDFKEKKYVKGDLVEE VHQVISDEATQVIRVYKTEDDAYVEFDWLIGNLQFDKNQSKEVITRFTIDGIANKDIFYT DANGRQQVQRTLNKRSDYEYDATEEPISSNYYPVTSKIVVKDETAKIEVAVLNDRAQGGS

AFERDLAHKKLLAP

EP

>Tm603

TE D

VLEPGVIELMVHRKEVADDHKGVDEVLNEQQFGKGLYARGQHYLTFGSSESKPESGVSTA

DHGVRQRFDSGVCAFQFWWVNSTPLTKDDATECVSACQGLDEKKVNVHLVPHSHDDVGWL

AC C

QTFEGYYKGTGDFKEIKNAGVQYILDSTIQALQKNKNRRYIQVETAFFWKWWNNQGXKDT RSAVRDLVNSGQLEMVGGGWSMNDEAAAHYQSIIDQFTWGFRILDDTVGECGRPKVGWQI DPFGHSREHASISKQLGFEGLVLGRIDYRDKSQRIEDKNLDFNWVTNPNFEDSTILTTMF PDFYTWPEGLCLDSTCASSQKLINDDNVEAVVANFTAILQEWIGYYXNKNILIPMGHDFT YQKAEDNFNSMDKLIEGFKDSDYNVIYSTPSCYIEAVTAAKPTLTKKEDDFFPYASNANE FWTGYFTSRTDSKTXFERVANNILQSVKQLTTFSRIQGGDDDKNIVDLRMAMGVMQHHDA ITGTEKQDVANDYTSMLYKGIAKTQDSISKIISDLLKKNDSAVDLALSTCLLANVTICDA SNKDKFLVAVQNPLSKTVSHHVRLPVNGTNFKITDADGEVAHDVLDAMHTFDFETKFETP

ACCEPTED MANUSCRIPT SKEIVFLAKDLPPLGVKLYYVETVTDDTKKYKPLQDITGRRGYLWRCEDN

>Tm1774 IFTRARSAFAGGFASPVKRPRAQSCQACHPVDPDKINVHLIPHSHDDVGWLKTVDQYYYG

RI PT

SHSDIQRAGVQYIISSTVEALKADPDRRFVQVETAFFWKWWQHQPDNLKQDFIDLVNNGQ LEIINAAWSMNDEAATNYQSTIDQFTYGLRTINDTVGTCGTPRIGWQIDPFGHSREQASI

FAQLGYDGVFFARIDHDDRDQRIADKTMEVIWQGSANLDNANIFTNVFPEFYYPPSGYCF DIECGDEVLNDDEASPDYNIPRKVDDFLSKVQELASYYQTNNLLIPMGGDFQYQSAEKNF

SC

VNMDKLISGFQGNDQINLIYSTPSCYIQAVNDEAASKNIEFTVKTDDFFPYASESHCYWT GYFTSRPNAKRFERTTNNILQAGKQLAAFSKVKGNDQEDSLTLLKQSVGILQHHDAITGT

M AN U

AKAAVANDYARLLAKSIKRAEPSLGNIVTDLLKKDSSSDINLNLQTCLLANVSICEPAAS

DRFVVTVYNPLERPVTDYVRIPVPDGGYTITGPDGEVESDLLDSISSFDYVDDDTGSPNP KELVFAAADLPGFGLRLYYVEKTSSKSKPIKSTPKLKFGTDEIGFEIDDGTGLLKSVTMN GQTVDITQQFFSYNGYNGENDGADNQASGAYIFRPLENSSNAVGDTVSVTSTSGNLVDEV

TE D

RQQVNDWITQIIRVYKGGNNNYIEFDWLVGPIPVDTDNGIGTEIVSRFTVGDFDNGESFY TDSNGRELIKRQLNKRYDYEYDSSLEPIASNYYPVTSKIVIKDETKNLEVALLNDRAQGG ASLQNGQVELMVHRRLLKDDAKGVGEALDDQEFGQGVVARGQIYLVVGSTDSDGGDGKST

EP

AAQERELALKKLLTPLVLVGDASSDDLSLDNIQSTLNLNFDGLAKTLPDNVHILTLEPWQ EDSYILRLEHILENNEDDSLSQAVSVDLSGLFSLFEVTEIRETTLGANQWLDEFEAKEKY

AC C

VWNVKGGGSVVNNKASAAPRADEISLNPMQIRTFVVKVAAN

>Tm1776

MIKYLLAVTVLFVGVYSTPIKLKEAPSCQACHPVDPDKINVHLIAHSHDDVGWLKTVDQY FYGSHPEIQRAGVQYTISSTIEALKGDPDRRYIQVETAFFWKWWQRQPDRIKQDVIDLTN SGQLEFTNAAWTQNDEAGTNYQSIIDQFTDGLRFINETIGQCGAPTIGWQVDPFGHSREQ ASLFAQFGFDGDFFGRIDYSDLKTKRDNKDMEVVWQGSANLDNTNIFTVVFPDGYGPLGG YCYDIQCGDTVFNDDESSPDYNVPQIVGAFQERLDQLITYYRTNNIIIPMGTDFQFQSAE

ACCEPTED MANUSCRIPT KNFINMDRMIAGFKDNDRYNVLYSTPSCYIKAVNEAAAAQDVE

RI PT

Contigs Family 47

>Bx2085

GVLRWYRGQLLHLAKDIGYRLLPAFNTTTGIPHARVNLRYGLKSQKLEWSQETCTACAGT

MVLEMAALSRLTGEPVFEEKAHRAMDELWKLRHRSSDLMGTVLNVQSGTWVRRDSGVGAG

SC

IDSYYEYCLKAYILLGEDRYLSRFNRHYSAVMKYISQGPMLLDVHMHRPHTNSRNFMDAL LAFWPGLQVLKGDLKPAVETHEMLYQVMQRHNFIPEAFTTDFQVHWGQHPLRPEFLESTY

M AN U

FLYRATKDPHYLHVGRDILRALQTFARVPCGYAAVKDVRTNHHEDRMDSFVLAETLKYLY LLFAEDTDLLLNLDEFLFTTEAHLLPLALARSPGNLSFFSLGDELSMDDSEWADQCPSHI RLFPASVRHPLRNMVKGSCPRSVDAKPRKLTASQFQTVEGPKQIHLLTIYLTFFISVALY VKIYIYCTLYRG

TE D

>Dp1634

DMFYHAYRAYMDNAYPADELMPLSCEGRYRGSHPSRGDLDDTLGNFSLSLIDSLDTLVVL GDLAEFEYAVKLVIDNVKFDSDVTVSVFETNIRVLGGLLSAHILADYVKENIGIMHWYRG

EP

ELLEMAKDIGYRLLPAFNTSTGIPQARVNLKQGTKLTKIWNTRETCTACAGTMLLEMAAL SRLTGDPIFEEKADKGMDSLWGLRHRGSGLMGSVLDVESGRWVRRESGVGAGIDSYYEYC

AC C

FKAYVLLGNPKYLSRFNKHYSAVMKYISQGPMLLDVHMHRPHTNSRNFMDALLAFWPGLQ VLSGDLKPAVETHEMLYQVMQRHNFIPEAFTTDFQVHWGEYPLRPEFLESTYFLYRATGD HHYLRVGEKILRGLQTHTKVPCGYAALSDVRSGRHDDRMDSFVLAETFKYLYLLFTDPND LIIDLDDFLFTTEAHLLPLSLSRISVNANTSSYTEGLEDEDGPQQCPSTLKLFLKL

>Dp3988 AAGPLKIMRNSTDPPDSLWAERRRHVVQMTKFAWDNYVRYAWGKNELRPLTKKAHNTGIF GSAALGATIVDGLDTLYIMGLHEEFRQGRAWIQENLTMEDLNGDFSVFETTIRYIGGLLT

ACCEPTED MANUSCRIPT CYSFTGDVMFRDKAENIAKKLLPAFQTITGIPHSLVNFKN

>Dm6182 VFEEKAHRAMDELWKMRHRSSDLMGTVLNVHSGDWVRRDSGVGAGIDSYYEYCLKAYILL

RI PT

GDDRYLNRFNRHYNAVMKYISQGPMLLDVHMHRPHTNSRNYMDALLAFWPGLQVLKGDIK PAVETHEMLYQVMQRHKFIPEAFTTDFQVHWGNHPLRPEFLESTYFLYSATNDPYYLEVG KNVLRSLQKYARVSCGYAAVNDVRTGK >Tm2371

SC

MLSVSEVVVRLLGVCVFIVFCTQCYSEESKAMTREERIRLREETRDMFYHAYRAYMENAY PADELMPLSCKGRYRGLTPNRGDIDDCLGNFSLTLIDTLDSLVVLGDLEEFEHAVKLVIK

M AN U

DVSFDNDVVVSVFETNIRVLGGLLSAHILADYLQQRDGIMTWYKGELLNMAKDVGYRLLP AFNTTTGIPHSRVNMKYGMKSDRLESARETCTACAGSMILEMAALSRLTGEPIFEEKAHK AMDELWKMRHRSSDLMGTVLNVHSGDWVRRDSGVGAGIDSYYEYCLKAYILLGDNKYLHR FNRHYNAVMKYISQGPMLLDVHMHRP

TE D

>Md1922

MPNFCLPRTSHNCRPLSSGPMTGTAEFQRNNISTINSSSYTSFNKLENMAKIVICTLLAL GLFVTLVSSDTIPTQNMSHKEREALREEARDMFYHAYHAYMDNAYPADELMPLSCKGRYR

EP

GVTPSRGDMDDILGNFSMTLVDTLDTLVILGDFAEFEHAVKLVIRDVQFDNDIIVSVFET NIRMMGGLLSAHILAEYIQKNAQVMHWYKGELLEMARDLGYRLLPAFNTSTGIPHARVNL

AC C

RYGMKDELLKKSRETCTACAGTILLEFAALSRLTGDPIFEARAHAAMDALWKLRHRGSDL MGTVLNVHSGDWVRRDSGVGAGIDSYYEYLFKSYVLLGDDKYLARFNRHYNAVMKYVSEG PMLLDVLMHRPHAKSRNFMDALLAFWPGLQVLSGDLKPAVQTHEMLYQVMQMHTFIPEAF TIDFQIHWGQHHLRPEFIESTYFLYRATGDHHYLQVGKKALKALQQHAKVPCGYAAVNDV RTGKHEDRMDSFVLSETFKYLYLLFTEPQDLILNIDEFVFTTEAHLLPLSLAQLGNSTYS FRQSEEHNVLDFMRTCPSSNKLFPEKVRKPLRNFITGSCPRTPATKRLRALDFQASNADH LRAVYDMGITMVSLGDGKVRLFHSFYNAKSPEEGEMGLQFMQEMLELTKLQSLNQLAQLQ AVAYPLDDKSNDWTALMAGPSHFSPELTEDDFVESEVILAEPLRACDERLANAEKAKGKI

ACCEPTED MANUSCRIPT LIAERGDCTFVSKARLAQKSGALALIVCDNVPGSSGETQPMFAMSGDGINDVKIPVVFMY SQEFAKLSKVMQRRPHMKIRIMQMIEFKRWQMAKESKANATETTPSASASTTTTTTKKSK PEKEL >Ra766

RI PT

FAALSRLSGEPIFERKAHASMDALWKLRNRGSDLMGTVLNVHNGDWVRRDSGVGAGIDSY YEYCLKSYVLXGHGKYLARFNRHYSAVMKYISQGPMLLDVHMHRPHAKSRNFMDALLAFW PGLQVLSGDLKPAVQTHEMLYQVMQMHTFIPEAFTFDFSDSLGTASTAXXEFIESTYFLY

KATGDHYYLQVAKKALQTLQKHARVDCGFAAVNDVRTGKHEDRMDSFVLSETIKYLFLIF

SC

ADPSELILDLDSFIFTTEAHLLPLSLGQLTNVTTESIDQDDHVAMDYMRSCPSPNKLFPE

TVRRPLRDLVTGVCPRIPNSKRLRALDFQSSNADHLRTVYDMGISMVSLGEGKVQLMHSF

AC C

EP

TE D

M AN U

YNAKS