Identification and Characterization of C-type Lectins in Ostrinia furnacalis (Lepidoptera: Pyralidae)

Identification and Characterization of C-type Lectins in Ostrinia furnacalis (Lepidoptera:... C-type lectins (CTLs) are a large family of calcium-dependent carbohydrate-binding proteins. They function primarily in cell adhesion and immunity by recognizing various glycoconjugates. We identified 14 transcripts encoding proteins with one or two CTL domains from the transcriptome from Asian corn borer, Ostrinia furnacalis (Guenée; Lepidoptera: Pyralidae). Among them, five (OfCTL-S1 through S5) only contain one CTL domain, the remaining nine (OfIML-1 through 9) have two tandem CTL domains. Five CTL-Ss and six OfIMLs have a signal peptide are likely extracellular while another two OfIMLs might be cytoplasmic. Phylogenetic analysis indicated that OfCTL-Ss had 1:1 orthologs in Lepidoptera, Diptera, Coleoptera and Hymenoptera species, but OfIMLs only clustered with immulectins (IMLs) from Lepidopteran. Structural modeling revealed that the 22 CTL domains adopt a similar double-loop fold consisting of β-sheets and α-helices. The key residues for calcium-dependent or independent binding of specific carbohydrates by CTL domains were predicted with homology modeling. Expression profiles assay showed distinct expression pattern of 14 CTLs: the expression and induction were related to the developmental stages and infected microorganisms. Overall, our work including the gene identification, sequence alignment, phylogenetic analysis, structural modeling, and expression profile assay would provide a valuable basis for the further functional studies of O. furnacalis CTLs. Key words: C-type lectin, Ostrinia furnacalis (Guenée), carbohydrate recognition domain, innate immunity Unlike vertebrates, insects lack adaptive immune system and Kanost 2000, Yu et al. 2006, Zhu et al. 2010). In other arthropods, mainly depend on effective innate immune system to defend against PGRPs, βGRPs, C-type lectins (CTLs), galectins, Leucine-rich repeat the attack from bacteria, fungi and other pathogens or parasites proteins (LRRPs), fibrinogen-related proteins (FREPs), hemocytins, (Kingsolver and Hardy 2012, Kanost and Jiang 2015). Insect innate Nimrods, scavenger receptors (SCRs), thioester proteins (TEPs), immune response is induced by recognition of common components down syndrome cell adhesion molecule (DSCAM), Draper, and bearing on the microbial surfaces but absent from itself, known as Eater were reported to function as PRRs (Christophides et al. 2004, pathogen-associated molecular patterns (PAMPs), by insect pattern Watson et  al. 2005). Among these, CTLs comprise a large super- recognition receptors (PRRs) (Steiner 2004, Takahashi et al. 2015). family of proteins, and exist not only in insects, but in other inver- This type of recognition of non-self leads to the activation of intra- tebrates, vertebrates and plants (Zhang et al. 2013b, Dambuza and cellular signaling pathways to finally produce a battery of effector Brown 2015, Drickamer and Taylor 2015). molecules for sequestering and eliminating the invading microorgan- CTLs, known as calcium-dependent lectins (or calcium-depend- isms (Basbous et al. 2011, Takahashi et al. 2015). ent carbohydrate binding proteins), are defined by the possession PRRs, soluble or membrane-bound, lack the binding specificity of at least one carbohydrate recognition domain (CRD) also called of antibodies and instead perform surveillance function by binding as CTL domain (Cambi et  al. 2005, Geijtenbeek and Gringhuis to polysaccharids, glycoproteins, and glycolipids which are common 2009). Each CTL domain typically consists of 110–130 amino acid to the groups of microorganisms (Steiner 2004, Basbous et al. 2011). residues, and adopts a canonical fold composed of β-sheets, α-heli- PRRs have been experimentally investigated in several insects. In ces, and loops which is stabilized by two or three pairs of disulfide Manduca sexta, hemolin, immulectin 1-4 (IML1-4), peptidoglycan bonds (Weis et al. 1992, Zelensky and Gready 2005). CTL domain recognition protein 1 (PGRP1), β-1,3-glucanase-related protein contains several conserved residues which are coordinated with a 2+ 1-3 (βGRP1-3), microbe binding protein (MBP), and leureptin-1 Ca to form the basis of a primary sugar-binding site and, there- were characterized as PRRs (Ladendorff and Kanost 1991, Ma and fore, determine the sugar-binding specificity of CTL. CTLs with a © The Author(s) 2018. Published by Oxford University Press on behalf of Entomological Society of America. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/2/24/4924850 by Ed 'DeepDyve' Gillespie user on 16 March 2018 2 Journal of Insect Science, 2018, Vol. 18, No. 2 Glu-Pro-Asn (EPN) motif in the CTL domain are characteristic of of these 14 CTLs. Their sequence features and gene phylogeny rela- mannose-binding and thus called mannose-type CTLs. Similarly, tionships were investigated thoroughly, and their developmental and CTLs with a Gln-Pro-Asp (QPD) motif in the CTL domain are char- induced expression profiles were examined. acteristic of galactose-binding and thus called galactose-type CTLs (Drickamer 1992, Zelensky and Gready 2005). In human Mincle (a Materials and Methods 2+ 169 171 172 macrophage inducible Ca -dependent CTL), Glu , Asn , Asn , 193 194 169 171 Biological Materials Asn and Asp are involved in calcium binding. Glu and Asn Asian corn borers (O.  furnacalis) were reared on an artificial diet are also participated in the EPN motif (Furukawa et al. 2013). In rat 185 187 193 205 206 at 28°C under a relative humidity of 70–90% and a photoperiod mannose-binding lectin A, Glu , Asn , Glu , Asn , and Asp 2+ 185 187 of 16:8 (L:D) h (Zhang et  al. 2013a). Beauveria bassiana (strain constitute the Ca -binding site 2, in which Glu and Asn belong 252) was cultured on potato dextrose agar (PDA) plates at 25°C and to the EPN motif (Weis et al. 1992). Most CTLs only contain a sin- a relative humidity of 80%. gle CTL domain. Some CTLs identified from lepidopteran insects, such as M. sexta IML1-4, Bombyx mori lipopolysaccharide-binding protein (LBP), MBP and LEL1-3, and Helicoverpa armigera CTL1-8, Identification and Feature Prediction of have two tandem CTL domains (Koizumi et al. 1999, Yu et al. 2006, O. furnacalis CTLs Takase et al. 2009, Wang et al. 2012). CTLs with dual CTL domains A transcriptome dataset containing 62,382 unigenes were obtained are also reported in coleopteran insect Tribolium castaneum (Zou from O.  furnacalis larvae with ‘next generation’ high-throughput et  al. 2007) and crustacean Fenneropenaeus chinensis (Xu et  al. sequencing (Liu et  al. 2014). All unigene sequences were searched 2010). It is still unknown why dual CRDs arise in these CTLs. against CTLs from M. sexta and other insects, using BLASTX algo- –5 As a category of important PRRs, CTLs play various roles through rithm with an E-value cut-off of 10 . The BLASTX results were recognizing and binding to a diverse range of sugar ligands. Mammalian used to extract coding region sequences from corresponding unigene CTLs have been revealed to function in anti-fungal/bacterial immu- sequences. The predicted coding region sequences were further trans- nity, homeostasis, antoimmunity, allergy, the recognition of dead cells lated into peptide sequences using the Translate tool provided by and tumors, and complement activation (Brown 2015, Drickamer and the Swiss Institute Bioinformatics. Analysis of deduced amino acids Taylor 2015, and references within). Insect CTLs perform relatively less sequences, including prediction of signal peptide, molecular weight, functions and mainly participate in innate immune responses including and isoelectric point, were performed in the Expert Protein Analysis phagocytosis (Jomori and Natori 1992), nodule formation (Koizumi et al. System (EXPASY) (http://www.expasy.org). Conserved domains and 1999), encapsulation and activation of prophenoloxidase (Yu and Kanost transmembrane regions were predicted in SMART (http://smart. 2004). For example, M. sexta IML-1 and IML-4 can induce the agglutin- embl-heidelberg.de/smart/set_mode.cgi). The domain architectures 2+ ation of bacteria and yeast in a Ca -dependent manner (Yu and Kanost were plotted with IBS 1.0 (http://ibs.biocuckoo.org/). 2001, Yu et al. 2006). Recombinant CRD2 of IML-2 directly binds to Caenorhabditis elegans and enhances its encapsulation and melanization Sequence Alignment and Phylogenetic Analysis in vivo (Yu and Kanost 2004). B. mori LBP binds to a variety of Gram- Multiple sequences alignment of CTLs from O.  furnacalis and negative bacteria and MBP principally binds to Gram-positive bacteria other insects (https://www.ncbi.nlm.nih.gov/) were carried out using and yeast. They both trigger hemocyte aggregation and microbe clear- MUSCLE module of MEGA 6.0 (http://www.megasoftware.net/) at the ance (Koizumi et al. 1999, Takase et al. 2009). Recombinant Drosophila following parameters: refining alignment, gap opening penalty = −2.9, melanogaster DL2 and DL3 (Drosophila lectin) can accelerate the agglu- gap extension penalty = 0, hydrophobicity multiplier = 1.2, Maximum tination of Escherichia coli in the presence of calcium and enhance cellu- iterations = 100, clustering method (for iterations 1, 2) = UPGMB, and lar encapsulation and melanization by directly recruiting hemocytes (Ao Minimum diagonal length  =  24. The resulted alignments were used et al. 2007). However, current knowledge about the function and binding to construct the phylogenetic tree using the neighbor-joining method. mechanism of insect CTLs is still limited and incomplete. For neighbor-joining method, gaps were treated as characters, and sta- Recent genome-wide analysis has helped to identify a number of tistics analysis was performed by bootstrap trails with 1,000 replica- genes encoding proteins with one or more CTL or CTL-like domains. tions, Poisson model, uniform rates, and complete deletion of gaps or Aedes agypti, Anopheles gambiae, D.  melanogaster, B.  mori, missing data (Tamura et al. 2013). M.  sexta, and T.  castaneum have 39, 25, 34, 23, 34, and 17 such genes, respectively (Christophides et al. 2002, Waterhouse et al. 2007, Structure Modeling of 22 O. furnacalis CTL Domains Zou et al. 2007, Rao et al. 2015a, Rao et al. 2015b). Development of The deduced amino acid sequences of the 22 O.  furnacalis CTL high through put sequencing and de novo assembly strategies resulted domain were submitted to the Iterative Threading ASSEmbly in the identification of more CTL or CTL-like transcripts from the Refinement (I-TASSER) server (http://zhanglab.ccmb.med.umich. transcriptomes of non-model insects. For example, 8, 9, and 24 tran- edu/I-TASSER/) for the prediction of tertiary structure. The repre- scripts for potential CTLs were found in H.  armigera, Nilaparvata sentative structural template was identified from PDB by a locally lugens, and Tenebrio molitor, respectively (Wang et  al. 2012, Bao installed LOMETS meta-threading server. Models were constructed et al. 2013, Zhu et al. 2013). In the previous work, we have obtained by iterative TASSER simulations. The generated PDB files were then a transcriptome dataset from an important insect pest, Asian corn visualized with Pymol Molecular Graphics System. borer, Ostrinia furnacalis (Guenée; Lepidoptera: Pyralidae), and iden- tified 14 possible CTL transcripts (Liu et  al. 2014). After carefully Expression Profile Analysis manual validation, we clarified that 3 of 14 transcripts lacked one or more key amino acid residues critical for CTL structure or binding To investigate the transcriptional changes of CTLs during the various activity. Meanwhile, we identified another three potential CTL tran- stages of O. furnacalis, total RNA samples were individually prepared from scripts. Therefore, we finally obtained 14 O. furnacalis CTLs, desig- three different stages including egg, larva, and pupa using TRNzol Reagent nated as OfCTL-S1 through S5, and OfIML1 through 9. In this study, (TIANGEN, Beijing, China). One μg of RNA equally from five individual we reported the identification, characterization, expression analysis RNA samples in each stage was treated with DNase I (TIANGEN, Beijing, Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/2/24/4924850 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Journal of Insect Science, 2018, Vol. 18, No. 2 3 China) and converted into first-strand cDNA from an oligo (dT) primer Among these, 14 ones were predicted to encode potential CTLs following the instructions for QuantScriptRT Kit (TIANGEN, Beijing, (Liu et al. 2014). After careful validation, we found three of them, China). The cDNA products independently from three biological replicates unigene2023, unigene31526 and CL321.contig3, were possibly not were diluted 10-fold for use as template in RT-PCR experiments. Specific real CTLs. Meanwhile, we identified another three transcripts, uni- primers were designed and listed in Supplementary Table S1. O. furnacalis gene4110, CL4301.contig1 and CL321.contig1, as potential CTLs. ribosomal protein L8 (rpL8) was used as an internal standard to adjust the Thus, we still obtained totally 14 CTL transcripts, which encode template amounts in a preliminary PCR experiments. The thermal cycling proteins with single or dual CTL domains (Table  1). Five of the conditions were 94°C for 3 min, then 28 cycles of 94°C for 30 s, 58°C for protein products (OfCTL-S1 ~ S5) only contain one CTL domain, 30 s, and 72°C for 30 s followed by incubation at 72°C for 10 min. The and the remaining nine (OfIML-1 ~ 9)  belong to the IML family PCR products were separated by electrophoresis on a 1.5% agarose gel. with two tandem CTL domains (Fig. 1). None has other conserved To check the expression profiles of O. furnacalis CTLs under differ - structural units such as complement C1r/C1s (CUB) domain, com- ent inducement conditions, day 0 fifth instar larvae from the same batch plement control protein (CCP/Sushi) domain, epidermal growth were injected into the hemocoel with 2 μl of sterile phosphate-buff- factor (EGF) -like domain, and coagulation factor 5/8 C-terminal ered saline (PBS) containing formaline-killed Escherichia coli DH5α (FA58C) domain, which were reported in B.  mori CTL-X1 ~ X5 (5 × 10 cells/μl), dried Micrococcus luteus (5 μg/μl), B. bassiana sus- and M.  sexta CTL-X1 ~ X6 (Rao et  al. 2015a, Rao et  al. 2015b). pension (3 × 10 conidia/μl), B. bassiana conidia suspension was pre- O.  furnacalis CTL-S1 ~ S5, IML-1 ~ 4, IML7, and IML-8 have a pared as described previously (Zhang et al. 2013a), or sterile PBS as a secretion signal peptide at the amino-terminus and are likely secreted control. After 24 h, each three larvae from challenged or control group into plasma. O.  furnacalis IML-5 and IML-6 lack the N-terminal were collected, and total RNA samples were individually prepared as secretion signals and may be located in the cytoplasm. It is unsure described above. DNase I–treated RNA (1 μg) was converted into first- whether OfIML9 has a signal peptide because the translation start strand cDNA using FastQuant RT Kit (TIANGEN Biotechnology Co., codon preceding the CTL domain is unavailable in its current incom- Ltd, Beijing, China). The cDNA products were diluted 10-fold for use plete transcript (Table 1 and Fig. 1). as template. The quantitative real-time PCR (qRT-PCR) was performed O.  furnacalis CTLs with a simple structure, CTL-S1 ~ S5, con- with SYBR Premix EX Taq (TaKaRa, Japan) on Applied Biosystems sist of 180–220 amino acid residues, and has the calculated molecular 7500 Real-Time PCR System (Life Technologies, Grand Island, NY), mass of around 20 kDa (Table 1). The isoelectric points of the mature according to the manufacturer’s instructions. O. furnacalis ribosomal proteins are around 6.0 with the exception of CTL-S3 and CTL-S5. protein L8 (rpL8) was used as an internal standard to normalize the O. furnacalis CTL-S3 has 21 Arginine residues, 7 Histidine residues, expression level. The thermal cycle conditions for qRT-PCR were 95°C and 5 Lysine residues in the deduced protein sequences, and, therefore, for 2 min followed by 40 cycles of 95°C for 15 s, 55°C for 30 s and results in an isoelectric point of 8.47. It is the only negatively charged 68°C for 30 s ending with a melting curve generation (60 to 95°C in lectin among all 14 identified O. furnacalis CTLs. Mature OfCTL-S5 increment of 0.5°C very 5 s). The relative expression level of genes was contains 10 Aspartic acid residues and 15 Glutamic acid residues, and, −ΔΔCt calculated with the 2 method (Livak and Schmittgen 2001). therefore, has an isoelectric point of low to 4.90 (Table 1). Compared to O. furnacalis CTLs with a single CTL domain, 9 OfIMLs with dual CTL domains consist of more amino acid residues, around 300–330 Results ones with the exception of OfIML-9 which is currently incomplete Overview and General Properties of at the amino-terminus. The calculated molecular mass and isoelec- O. furnacalis CTLs tric point of the mature protein of OfIML-1 ~ 8 are approximately With next generation high-throughput sequencing, we obtained an 33.0 kDa and 5.0, respectively (Table 1). Neither N- nor C-terminal O.  furnacalis transcriptome dataset containing 62,382 unigenes. extension rich in certain amino acid residues, which are existed in Table 1. Structural features of 14 O. furnacalis CTLs a c Designated name Unigene ID Protein length Signal peptide Mr pI Glycosylation CRD numbers Motif (aa) (kDa) sites N-linked O-linked OfCTL-S1 CL4786.Contig1 220 1-19A*Q 23.4 6.33 0 3 1 QPD OfCTL-S2 Unigene6545 223 1-19A*Q 23.7 5.99 0 1 1 QPD OfCTL-S3 CL8286.Contig1 221 1-21A*Q 23.1 8.47 0 6 1 QPD OfCTL-S4 Unigene9847 207 1-21A*Q 21.3 6.20 1 5 1 APQ OfCTL-S5 CL321.Contig1 184 1-21T*V 19.1 4.90 0 5 1 EPN OfIML-1 Unigene22572 301 1-19A*Q 32.6 5.32 1 3 2 APW / EPN OfIML-2 Unigene14484 314 1-22G*R 32.9 5.42 0 2 2 EPD / EPN OfIML-3 CL4301.Contig2 304 1-20S*N 32.5 4.74 1 1 2 VPL / EPN OfIML-4 CL106.Contig3 328 1-21T*D 33.9 5.13 1 2 2 EPD / QPD OfIML-5 Unigene5701 322 35.7 4.89 N/A N/A 2 EPD / QPD OfIML-6 Unigene2411 319 36.5 5.89 N/A N/A 2 DIS / SPD OfIML-7 CL1725.Contig1 307 1-20S*S 32.3 6.20 3 3 2 DVS / VPD OfIML-8 Unigene5307 321 1-21S*Q 34.1 4.98 2 0 2 EPN / QPD OfIML-9 Unigene4110 >205 N/A >23.2 4.44 ≧1 ≧2 ≧1 ? / QPD The molecular weight (Mr) and isoelectric point (pI) are for mature proteins without signal peptides. OfIML-5 and OfIML-6 lack signal peptide, and, therefore, the predication for the numbers of glycosylation sites is not applicable (N/A). The canonical motifs (QPD and EPN) are in bold. Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/2/24/4924850 by Ed 'DeepDyve' Gillespie user on 16 March 2018 4 Journal of Insect Science, 2018, Vol. 18, No. 2 Fig.  1. Domain architectures of 14 O.  furnacalis CTLs. The numbers above the boxes indicates the location of the corresponding amino acid residues in the deduced protein sequences. Signal peptide is in magenta. The C-type lection domains (CTLD) are in green. The ellipsis in OfIML-9 means the amino-terminus is incomplete. B. mori CTL-S4 ~ S6, -S8 ~ S10, and M. sexta CTL-S3 ~ S5 and -S8 be mannose-type lectins. OfIML-8 contains both motifs simultane- (Rao et al. 2015b, Rao, 2015a), is observed in all 14 identified O. fur - ously, with the EPN motif in the first CTL domain and the QPD motif nacalis CTLs. Numbers of putative N-linked and O-linked glycosyla- in the second CTL domain. However, other CTL domains, including tion sites are variant in each O. furnacalis CTL, from 1 in OfCTL-S2 OfCTL-S4, the first CTL domain in OfIML-1 through 7 and the sec- to 6 in OfCTL-S3 and OfCTL-S4 (Table 1). ond CTL domain in OfIML-6 and OfIML-7, contain the non-canon- ical motifs. Their binding specificities need to be further investigated. Conserved Residues and Structural Features of The typical CTL domain is stabilized by three pairs of disulfide O. furnacalis CTLs bonds between Cys-1 and Cys-2, Cys-3 and Cys-6, Cys-4 and Cys- All CTLs contain several conserved amino acid residues which deter- 5, although no all CTL domains contain all six cysteine residues mine the sugar-binding specificity of CTL. Gln-Pro-Asp (QPD) and (Weis et al. 1992, Furukawa et al. 2013). We aligned all 22 O. fur- Glu-Pro-Asn (EPN) motif in the CTL domain suggest potential nacalis CTL domain sequences including 5 ones from OfCTL-S1 galactose and mannose binding specificities of CTLs, respectively through S5, 8 ones from the first CTL domain in OfCTL-1 through (Drickamer 1992, Zelensky and Gready 2005). The QPD motif is 8 (OfCTL-1A ~ -8A, the first CTL domain in OfCTL-9 is currently found in the CTL domain in OfCTL-S1 through S3 and the second unavailable), and 9 ones from the second CTL domain in OfCTL-1 CTL domain in OfIML-4, OfIML-5, and OfIML-9 (Table 1, Fig. 2). through 9 (OfCTL-1B ~ -9B) with well-studied human Mincle (PDB: Therefore, they may be galactose-type lectins. The EPN motif is found 3WHD). These six Cysteine residues mentioned above are absolutely in the CTL domain in OfCTL-S5 and the second CTL domain in conserved in nine CTL domains (OfIML-1B through 9B). Cys-1 OfIML-1 through 3 (Table 1, Fig. 2). These O. furnacalis CTLs may is present but Cys-2 is absent in the CTL domains in OfCTL-S1, Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/2/24/4924850 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Journal of Insect Science, 2018, Vol. 18, No. 2 5 Fig. 2. Sequence alignment of the 22 O. furnacalis CTL domains. Based on the domain predications with SMART, the amino acid sequences for 22 O. furnacalis CTL domains are aligned with that of human Mincle (PDB: 3WHD). The conserved Cys residues are numbered and shown in red. They are predicted to form three disulfide bonds (1–2, 3–6, 4–5) based on the determined structure in human Mincle. The regions C(D/N)F(K/A)GC in OfCTL-S1 through S3 may contain a unique disulfide linkage and are shaded in grey. The canonical QPD and EPN motifs are shaded in green and cyan, respectively. Residues involved in calcium binding 2+ and carbohydrate binding are in blue and purple, respectively. Residues involved in both Ca and sugar binding are in bold. The secondary structure elements (α, α-helix; β, β-strand; T, turn) of human Mincle are shown above or below the sequences. The asterisks above the sequences indicate the residues involved in calcium binding in human Mincle. -S2, -S5 and OfIML-2A. Both Cys-1 and Cys-2 are missing in the of 22 O.  furnacalis CTL domain with human Mincle indicated remaining CTL domains including OfCTL-S3, -S4, OfIML-1A, -3A that CTL-Ss with the exception of CTL-S4 and most of the IML through 8A (Fig.  2). Additionally, the cysteine residues in C(D/N) A  domains lack these residues corresponding to those in Mincle F(K/A)GC (shaded in grey in Fig. 2) of OfCTL-S1 through S3 may whereas IML B domains other than OfIML-7B possess them (Fig. 2). form a unique disulfide linkage. We further performed structural modeling of 22 CTL domains 2+ CTLs usually recognize carbohydrates via a Ca -mediated to explore the putative calcium and sugar binding sites. The over- binding network (Zelensky and Gready 2005). In a canonical CTL, all structures of all 22 O. furnacalis CTL domains are predicted to the EPN or QPD motifs on the loops of CTL cooperate with the be closely similar (Fig. 3). They all may bind to carbohydrates in a Asn-Asp (ND) residues on the adjacent β-sheet to bind the calcium calcium-dependent manner since five of the models (-S5, -2B, -3B, 2+ (Furukawa et  al. 2013). For example, E169, N171, N172, N193, -4A, and -8A) contain two Ca and the remaining 17 CTL domains 2+ 2+ and D194 in human Mincle constitute the Ca -bound sites (marked contain one Ca (Table  2, Figs. 2 and 3). Further experiments are 2+ by red asterisks in Fig.  2) (Furukawa et  al. 2013). The alignment required to determine whether Ca indeed enhances the binding Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/2/24/4924850 by Ed 'DeepDyve' Gillespie user on 16 March 2018 6 Journal of Insect Science, 2018, Vol. 18, No. 2 Fig. 3. Structural models of O. furnacalis CTL-S1 (A), IML-8A (B), and IML-8B (C). The tertiary structures predicted from I-TASSER server are exhibited as cartoons. The calcium ions are shown as red spheres and indicated by red arrows. The mannose-type EPN motif and galactose-type QPD motif are shown as blue sticks and indicted by blue arrows. The Cys residues for the formation of disulfide bonds are represented as orange sticks. Table 2. Structural features of the 22 O. furnacalis CTL domain models 2+ 2+ a b Domain Ca Putative Ca coordinators Motif Sugar Putative sugar binding residues Template C-score 2+ 2+ [Ca #1] [Ca #2] OfCTL-S1 1 [48,50,54,168] QPD GQ2 69,125,127,128,142,155,156,157,163 4C9F 0.52 -S2 1 [47,49,53,172] QPD MMA 129,131,132,142,159,160,161 1KWU 0.53 -S3 1 [40,42,46,159] QPD GQ2 61,117,119,120,133,146,147,148,154 4C9F 0.58 -S4 1 [43,45,49,167] APQ PI 100,101,111,112,113,122,123,124,125,126 2ORK 0.25 -S5 2 [49,51,55,145] [79,83,86,107,116,117] EPN TRE 72,104,106,116,122,132,133,138 4ZRV 0.65 OfIML-1A 1 [40,42,46,121] APW GAL 88,90,92,96,108,109,110 1JZN 0.37 -1B 1 [43,45,49,142] EPN TRE 71,108,110,112,116,122,129,130,135 4KZV 0.72 OfIML-2A 1 [38,40,44,126] EPD MAN 93,95,101,113,114,116 2VUZ 0.72 -2B 2 [43,45,49,140] [80,84,87,115,128] EPN TRE 73,107,109,111,115,121,127,128,133 4KZV 0.64 OfIML-3A 1 [40,42,46,122] VPL BM3 89,91,93,97,109,110,111 2ORJ 0.51 -3B 2 [43,45,49,141] [79,84,87,115,129] EPN TRE 72,107,109,115,121,128,129,134 4ZRV 0.69 OfIML-4A 2 [43,45,49,131] [72,76,79,101,106,107] EPD MAN 64,98,100,106,118,119,121 2VUZ 0.66 -4B 1 [43,45,49,143] QPD GQ2 72,106,108,110,121,129,130,131,138 4C9F 0.59 OfIML-5A 2 [45,47,51,131] [72,76,79,101,106,107] EPD NGA 65,98,100,102,106,110,112,118,119 1WMZ 0.66 -5B 1 [43,45,49,145] QPD GQ2 74,108,110,112,123,131,132,133,140 4C9F 0.61 OfIML-6A 1 [40,42,46,122] DIS MAN 94,96,98,109,110,111 1KWZ 0.39 -6B 1 [42,44,48,137] SPD TRE 71,105,107,111,117,123,124,129 4ZRV 0.59 OfIML-7A 1 [45,47,51,126] DVS NGA 96,98,101,105,113,114,115 1BCH 0.34 GQ4 26,62,64,65,96,98,101,105,115,119,121 4C9F 0.25 -7B 1 [42,44,48,139] VPD MBG 105,107,109,113,125,126 1AFA 0.34 OfIML-8A 2 [40,42,46,128] [69,73,76,98,103,104] EPN MAN 95,97,103,115,116,118 2VUZ 0.74 -8B 1 [43,45,49,137] QPD TRE 71,105,107,112,118,124,125,130 4ZRV 0.66 OfIML-9B 1 [43,45,49,136] QPD TRE 72,104,106,111,117,123,124,129 4ZRV 0.61 EPN and QPD motifs are in bold. The residues involved in both calcium and sugar binding are in bold. GQ2, a viral ligand to human herpesvirus 6 (HHV-6); MMA, methyl α-D-mannopyranoside; PI, phosphatidylinositol; TRE, trehalose dimycolate; GAL, β-D-galactose; MAN, α-D-mannose; BM3, N-acetyl-α-D-mannosamine; NGA, N-acetyl-D-galactosamine; MBG, methyl-β-galactose. C-score is the confidence score representing the quality of the generated models. It is calculated based on the Z-score of LOMETS threading alignments and 2+ the convergence of I-TASSER simulations. C-score ranges 0–1, where a higher score indicates a more reliable prediction. Ca sites with C-score > 0 and sugar sites with C-score > 0.2 are shown in the table. C-scores shown here are for putative sugar binding sites. strength. On the other hand, three-dimensional structure modeling ranging from 0.64 to 0.74 (Table  2). The exceptions are CTL-S2, also allows us to predict the sugar binding specificities of 22 O. furn- IML-8B and -9B contain the QPD motif, but are predicted to bind acalis CTL domains. The results from the above sequence alignment galactose (Table  2 and Fig.  3). OfCTL-S4, IML-1A through -7A, and molecular modeling are mostly consistent (Table 2, Figs. 2 and -6B, and -7B have the non-canonical motifs, but also show the sugar 3). The high C-scores of the models suggest that OfCTL-S1, -S3, binding potentials in structural modeling. More experiments will be IML-4B, and -5B may form stable complexes with galactose. They required to validate the binding predictions. Beside, homology mod- just contain the QPD motif. OfCTL-S5, IML-1B, -2B, -3B, and -8A eling also indicated that two amino acid residues (Glu in CTL-S5, 115 128 115 129 106 has the EPN motif, and may bind to mannose tightly with C-scores Glu and Asp in IML-2B, Glu and Asp in IML-3B, Glu Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/2/24/4924850 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Journal of Insect Science, 2018, Vol. 18, No. 2 7 106 103 in IML-4A, Glu in IML-5A, and Glu in IML-8A) are involved led to the significant up-regulation for both CTL-S1 and IML-2; in both calcium and carbohydrate binding (Table 2 and Fig. 3). B. bassiana-injection resulted in the significant increase for all four transcripts other than CTL-S1 (Fig. 5B). Phylogenetic Analysis of O. furnacalis CTLs To investigate the evolutionary relationships between CTLs of Discussion O.  furnacalis and other insects, we retrieved 77 CTL protein CTLs acts as a category of important PRRs, and play various physi- sequences from 19 insect species by BLASTP searches in GenBank, ological roles in animals including human and insects (Brown 2015, VectorBase, and Insect Innate Immunity Database. Since no CTLs Dambuza and Brown 2015). In this study, we have identified 14 with other complicated structural unites than CTL domains are CTLs (OfCTL-S1 ~ S5, and OfIML-1 ~ 9) from O. furnacalis tran- currently identified in O.  furnacalis, we did not included M.sexta scriptome. We performed comprehensive sequence analysis for these CTL-Xs and their orthologs, which contain CTL domain(s) together 14 CTLs and investigated their expression profiles in different devel- with other conserved domains in the alignment. The phylogenetic opmental stages and upon different infections. tree (Fig. 4) shows that all CTLs are generally divided into two cate- CTLs are characterized by processing one or more carbohy- gories based on the numbers of CTL domains. OfCTL-S1 through S5 drate-recognition domains (also known as CTL domains) (Cambi formed tight, monophyletic groups with their respective orthologs et al. 2005, Geijtenbeek and Gringhuis 2009). Among 14 identified from the other species in Lepidoptera and Diptera. Moreover, the O.  furnacalis CTLs, 5 ones only has a single such domain, and 9 orthologs of OfCTL-S1 and OfCTL-S4 were also found in the red contain two tandem CTL domains (Table 1 and Fig.1). So far, CTLs flour beetle T. castaneum (Coleoptera) (Fig. 4). with one CTL domain have been identified from almost all animals Nine O.  furnacalis IMLs are grouped with the CTLs with dual including human (Furukawa et  al. 2013), rats (Weis et  al. 1992), CTL domains, with a bootstrap value of high to 99 (Fig.  4). All crustaceans (Zhang et al. 2013b) and insects (Rao et al. 2015a, Rao the orthologs in this group are from lepidopteran insects including et al. 2015b). For example, D.  melanogaster, A.  gambiae, B.  mori, B.  mori, M.  sexta, H. armigera, Hyphantria cunea, Danaus plexip- M. sexta, and T. castaneum have 34, 25, 12, 9, and 10 genes encod- pus, Danaus plexippus, and Pieris rapae. Unlike MsIML-1, 18, 19 ing proteins with one CTL domain, respectively (Christophides et al. and BmIML-1, 4~6 which had 1:1 orthologs, no O.  furnacalis IML 2002, Waterhouse et  al. 2007, Zou et  al. 2007, Rao et  al. 2015a, formed 1:1 orthologous group. OfIML-4 and OfIML-5, as well as Rao et  al. 2015b). We only identified five transcripts for encoding OfIML-6 and OfIML-7, showed lineage-specific expansions (Fig. 4). proteins with one CTL domain. Therefore, it is highly possible there They may evolve by multiple gene duplication. Additionally, we con- are other unidentified transcripts to encode O.  furnacalis CTL-Ss. structed the phylogenetic tree based on the alignment of O.  furna- At least, there should be one CTL-S for ortholog in CTL-S5 group calis CTL domains alone, instead of the alignment of entire protein since other insects than O.  furnacalis have clearly 1:1 orthologous sequences. Twenty-two CTL domains formed three separate branches: relationship in this group (Fig.  4). Compared to the wide distribu- CTL-S, CTL domain A, and CTL domain B (Supplementary Fig. S1). tion of short CTL-Ss, IML family with two tandem CTL domains was found mainly in lepidopteran insects (Yu and Kanost 2001). Six, Expression Profiles of O. furnacalis CTL Genes 19, and eight such CTLs have been reported in B.  mori, M.  sexta, We analyzed the mRNA levels of O.  furnacalis CTLs in various and H.  armigera, respectively (Wang et al. 2012, Rao et al. 2015a, development stages using semi-quantitative RT-PCR methods. As Rao et  al. 2015b). Unlike CTL-Ss which normally have clear ort- shown in Fig.  5A, the 14 O.  furnacalis CTLs exhibited distinct hologs in different insects, some immulection genes show extensive expression patterns. The transcripts of OfCTL-S5, IML-4, IML-5, lineage-specific expansions. For instance, M.  sexta IML-3, 4, 6–8, and IML-8 were detected in all examined developmental stages, IML-9~12, and IML-13~17 and H.  armigera CTL-1, 2, and 6 are including egg, larval and pupal stages. OfCTL-S1 was also detected four groups of related IMLs (Fig.  4) (Wang et  al. 2012, Rao et  al. in all stages, but with obviously higher level in the stage of egg and 2015a). As to the formation of IMLs with dual CTL domains, it may young larva and low level in the stage of the fifth instar larva and be a result of the duplication of CTL-S genes because the first CTL pupa. OfCTL-S2 was clearly expressed in all tested developmental domains in B. mori and M. sexta IMLs seem to evolve from common stages other than egg. IML-6, -7 and -9 were expressed in the second ancestors of BmCTL-S11 and MsCTL-S7, respectively (Rao et  al. to fifth instar larval stage and pupal stage, but not in eggs and the 2015a, Rao et  al. 2015b). We speculated that the event of entire first instar larvae. CTL-S3 mRNA was detectable in the old larvae gene duplications and sequence divergence resulting in the appear- and pupae, and the mRNA level was lower in the pupal stage. The ance of tandem CTL domains in moths and butterflies took place remaining three OfCTLs exhibited irregular expression patterns: before the radiation of Lepidoptera. In addition, there exists another OfCTL-S4 was detected in egg and pupa; IML-2 in egg and the fifth type of IMLs containing one or two CTL domains along with other instar larva; and IML-3 in egg and the fourth instar larva (Fig. 5A). structural units, such as D.  melanogaster furrowed, uninflatable, To check the O. furnacalis CTL expression profiles after exposure and contactin (Stork et al. 2008, Xie et al. 2012, Chin and Mlodzik to microbial elicitors, we randomly selected eight CTLs and analyzed 2013), B. mori CTL-X1~5 (Rao et al. 2015b), M. sexta CTL-X1~6 their transcript level after O.  furnacalis larvae were injected with (Rao et al. 2015a), and T. castaneum CTL-X1~5 (Zou et al. 2007). E. coli, M. luteus, B. bassiana, or PBS as a control. The result from However, we did not identify any similar IMLs in O. furnacalis tran- quantitative RT (qRT) -PCR assay indicated that the expression pat- scriptome. The possible reason is that the assembly quality of cur- terns of these eight CTLs could be divided into three groups: the tran- rent transcriptome is not high enough to find the long extensions for script levels of IML-8, CTL-S2, and CTL-S5 remained unchanged; encoding the other structural modules. We believed that there also CTL-S4 decreased significantly upon challenge; and CTL-S1, IML- existed similar complex CTL-Xs in O. furnacalis, but the identifica- 2, IML-3, and IML-9 increased after infection (Fig. 5B). There still tion requires more sequence resources. existed some differences although CTL-S1, IML-2, IML-3, and The CTL domain adopts a characteristic double-loop fold IML-9 mRNA levels all increased upon challenge: E.  coli-injection consisting of five β -sheets and two α-helices: the closely located only caused the significant increase for IML-9; M.  luteus-injection N- and C-termini (β1, β5) make the overall domain a loop, and Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/2/24/4924850 by Ed 'DeepDyve' Gillespie user on 16 March 2018 8 Journal of Insect Science, 2018, Vol. 18, No. 2 Fig.  4. Phylogenetic analysis of insect CTLs. The amino acid sequences of 91 CTLs from 20 insect species were examined. Lepidopteran-specific CTLs were indicated with bracket. The branches specific for CTL-S1 through -S6 are shaded. Numbers at the nodes were bootstrap values as percentage. Only bootstrap values greater than 70 were shown. Of, O.  furnacalis; Aa, A.  aegypti; Ag, A.  gambiae; Ap, Antheraea pernyi; Bm, B.  mori; Bt, Bombus terrestris; Cq, Culex quinquefasciat; Dm, D.  melanogaster; Dp, D.  plexippus; Ha, H.  armigera; Hc, H.  cunea; Hv, Heliothis virescens; Mr, Megachile rotundata; Ms, M.  sexta; Nv, Nasonia vitripennis; Pr, P. rapae; Ppo, Papilio polytes; Pxu, Papilio xuthus; Pxy, Plutella xylostella; and Tc, T. castaneum. 2+ the region between β2 and β3 strands forms the second loop also were predicted to have Ca -binding site 2 (Ca-2), and no O. fur- 2+ called the long loop region (Zelensky and Gready 2005, Furukawa nacalis CTL domain has other potential Ca -binding sites (Ca-3 et al. 2013, Feinberg et al. 2016). Canonical CTL domain has the or Ca-4) observed in human macrophage C-type lectin (MCL) long loop region and the compact one lacks it. All identified 22 and Mincle (Furukawa et al. 2013). The region surrounding Ca-2 O.  furnacalis CTL domains possess the long loop region. It sug- in O.  furnacalis CTL domains is more similar to that in human gests that they all belong to canonical CTLs. On the other side, DC_SIGNR than to that in MCL and Mincle. The roles of calci- 2+ the long loop region is reported to be involved in Ca -dependent um-binding sites are various in individual CTL, either involved in carbohydrate binding and in domain-swapping dimerization of carbohydrate binding or just playing structural roles. For example, some CTLs (Liu and Eisenberg 2002, Zelensky and Gready 2005). the calcium-binding site in human tetranectin is known not to Structural modeling also indicated that 22 CTL domains with long bind carbohydrates, but interact with kringle domain-containing loop region had potential to bind one or two calcium ions, sug- proteins leading to changes in the long loop region conformation 2+ gesting they might function in a calcium-mediated manner. Ca - (Nielbo et al. 2004). The functional impact of calcium-binding sites binding site 1 (Ca-1) close to the N- and C-terminus is present in all in O. furnacalis CTL domains is still unclear. 22 CTL domains (Fig. 3). It is consistent with the previous report Relative to calcium-binding sites, the characteristic motif, such as that Ca-1 is a highly conserved structural feature in virtually all mannose-type EPN motif or galactose-type QPD motif, is possibly CTL domains (Zelensky and Gready 2003, 2005). However, only 6 more important for carbohydrate binding of CTLs. For example, out of 22 CTL domains (CTL-S5, IML-2B, -3B, -4A, -5A and -8A) the EPN motif in rat MBP-A (E185, P186, N187) provides carbonyl Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/2/24/4924850 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Journal of Insect Science, 2018, Vol. 18, No. 2 9 Fig. 5. Expression profile analysis of O. furnacalis CTLs. (A) Expression profiles of O. furnacalis CTLs at different stages of development. RNA was extracted from the whole bodies collected from eggs, first-instar (L1), second-instar (L2), third-instar (L3), fourth-instar (L4), fifth-instar (L5) larvae, and pupae. The rpL8 was used as an internal control. (B) Expression profiles of O. furnacalis CTLs upon microbial challenge. Day 0, fifth instar larvae were infected with PBS, E. coli, M. luteus, or B. bassiana. RNA was prepared from the whole bodies 24 h after injection. qRT-PCR was used to assess the transcript change of OfCTLs with rpL8 as an internal standard to indicate a consistent total mRNA amount. The bars represent mean ± SD (n = 3). Bars labeled with different letters are significantly different (one-way ANOVA, followed by the Newman-Keus test, P < 0.05). sidechains to form hydrogen bonds with the monosaccharide and CTLs. Further experiments are undergoing to determine the binding determine its binding specificity (Zelensky and Gready 2005). specificities of O. furnacalis CTLs. Replacing the EPN sequence in MBP-A with a QPD sequence was The developmental and induction profiles of the CTL genes enough to switch the specificity to galactose (Drickamer 1992). reflect their functional importance to some extent (Fig.  5). For Twelve out of 22 O.  furnacalis CTL domains contain canonical instance, CTL-S4 transcripts were detected in eggs and pupae, but EPN or QPD motif (Tables 1 and 2), suggesting these O.  furnaca- not in the larvae, suggesting that it may be involved in the devel- lis CTLs have the potential to binding carbohydrates. Additionally, opment. Its mRNA levels surprisingly decreased after immune it is worthy of note that the remaining 10 CTL domains contain challenge (Fig.  5). The orthologous B.  mori CTL-S5 also showed unusual motifs in the long loop region such as EPD, APQ motif down-regulation in the E. coli or S. aureus injected fat bodies (Rao (Tables 1 and 2). Human MCL also has an EPD motif, and it still et al. 2015b). However, the function of B. mori CTL-S5 is currently shows the binding affinity to trehalose dimycolate (Furukawa et al. unknown. On the other side, immulections have been reported to 2013). Sarcophaga lectin from the flesh fly Sarcophaga peregrina participate in the immune responses including agglutination and/or contains an NPD motif, and exhibits the galactose-binding property encapsulation of microorganisms (Zelensky and Gready 2005). The (Kawaguchi et al. 1991). It is more surprising that blood dendritic expression of most IMLs is stimulated after microbial challenge (Yu cell antigen 2 (BDCA-2) contains an EPN motif, but bind galac- and Kanost 2004, Yu et al. 2006). O. furnacalis IML-2, IML-3, and tose-terminated glycans instead of predicted mannose (Jegouzo et al. IML-9 were consistently up-regulated after infection from microor- 2015). Langerin contains an EPN motif, but bind both mannose and ganism, but IML-8 remained unchanged upon infection (Fig. 5). So galactose (Feinberg et al. 2011). Therefore, we infer that there exist far, we only investigated the preliminary function of O.  furnacalis non-canonical sugar-binding sites in CTLs. Another possibility is IML-2. It exhibited the promising potential to bind and agglutinate that contacts outside of the primary sugar-binding site in extended E. coli (data not shown). The functional studies for other O. furna- or secondary binding sites also contribute to the sugar binding of calis CTLs are undergoing. 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Genome Biol. 8: R177. Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/2/24/4924850 by Ed 'DeepDyve' Gillespie user on 16 March 2018 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Insect Science Oxford University Press

Identification and Characterization of C-type Lectins in Ostrinia furnacalis (Lepidoptera: Pyralidae)

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Abstract

C-type lectins (CTLs) are a large family of calcium-dependent carbohydrate-binding proteins. They function primarily in cell adhesion and immunity by recognizing various glycoconjugates. We identified 14 transcripts encoding proteins with one or two CTL domains from the transcriptome from Asian corn borer, Ostrinia furnacalis (Guenée; Lepidoptera: Pyralidae). Among them, five (OfCTL-S1 through S5) only contain one CTL domain, the remaining nine (OfIML-1 through 9) have two tandem CTL domains. Five CTL-Ss and six OfIMLs have a signal peptide are likely extracellular while another two OfIMLs might be cytoplasmic. Phylogenetic analysis indicated that OfCTL-Ss had 1:1 orthologs in Lepidoptera, Diptera, Coleoptera and Hymenoptera species, but OfIMLs only clustered with immulectins (IMLs) from Lepidopteran. Structural modeling revealed that the 22 CTL domains adopt a similar double-loop fold consisting of β-sheets and α-helices. The key residues for calcium-dependent or independent binding of specific carbohydrates by CTL domains were predicted with homology modeling. Expression profiles assay showed distinct expression pattern of 14 CTLs: the expression and induction were related to the developmental stages and infected microorganisms. Overall, our work including the gene identification, sequence alignment, phylogenetic analysis, structural modeling, and expression profile assay would provide a valuable basis for the further functional studies of O. furnacalis CTLs. Key words: C-type lectin, Ostrinia furnacalis (Guenée), carbohydrate recognition domain, innate immunity Unlike vertebrates, insects lack adaptive immune system and Kanost 2000, Yu et al. 2006, Zhu et al. 2010). In other arthropods, mainly depend on effective innate immune system to defend against PGRPs, βGRPs, C-type lectins (CTLs), galectins, Leucine-rich repeat the attack from bacteria, fungi and other pathogens or parasites proteins (LRRPs), fibrinogen-related proteins (FREPs), hemocytins, (Kingsolver and Hardy 2012, Kanost and Jiang 2015). Insect innate Nimrods, scavenger receptors (SCRs), thioester proteins (TEPs), immune response is induced by recognition of common components down syndrome cell adhesion molecule (DSCAM), Draper, and bearing on the microbial surfaces but absent from itself, known as Eater were reported to function as PRRs (Christophides et al. 2004, pathogen-associated molecular patterns (PAMPs), by insect pattern Watson et  al. 2005). Among these, CTLs comprise a large super- recognition receptors (PRRs) (Steiner 2004, Takahashi et al. 2015). family of proteins, and exist not only in insects, but in other inver- This type of recognition of non-self leads to the activation of intra- tebrates, vertebrates and plants (Zhang et al. 2013b, Dambuza and cellular signaling pathways to finally produce a battery of effector Brown 2015, Drickamer and Taylor 2015). molecules for sequestering and eliminating the invading microorgan- CTLs, known as calcium-dependent lectins (or calcium-depend- isms (Basbous et al. 2011, Takahashi et al. 2015). ent carbohydrate binding proteins), are defined by the possession PRRs, soluble or membrane-bound, lack the binding specificity of at least one carbohydrate recognition domain (CRD) also called of antibodies and instead perform surveillance function by binding as CTL domain (Cambi et  al. 2005, Geijtenbeek and Gringhuis to polysaccharids, glycoproteins, and glycolipids which are common 2009). Each CTL domain typically consists of 110–130 amino acid to the groups of microorganisms (Steiner 2004, Basbous et al. 2011). residues, and adopts a canonical fold composed of β-sheets, α-heli- PRRs have been experimentally investigated in several insects. In ces, and loops which is stabilized by two or three pairs of disulfide Manduca sexta, hemolin, immulectin 1-4 (IML1-4), peptidoglycan bonds (Weis et al. 1992, Zelensky and Gready 2005). CTL domain recognition protein 1 (PGRP1), β-1,3-glucanase-related protein contains several conserved residues which are coordinated with a 2+ 1-3 (βGRP1-3), microbe binding protein (MBP), and leureptin-1 Ca to form the basis of a primary sugar-binding site and, there- were characterized as PRRs (Ladendorff and Kanost 1991, Ma and fore, determine the sugar-binding specificity of CTL. CTLs with a © The Author(s) 2018. Published by Oxford University Press on behalf of Entomological Society of America. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/2/24/4924850 by Ed 'DeepDyve' Gillespie user on 16 March 2018 2 Journal of Insect Science, 2018, Vol. 18, No. 2 Glu-Pro-Asn (EPN) motif in the CTL domain are characteristic of of these 14 CTLs. Their sequence features and gene phylogeny rela- mannose-binding and thus called mannose-type CTLs. Similarly, tionships were investigated thoroughly, and their developmental and CTLs with a Gln-Pro-Asp (QPD) motif in the CTL domain are char- induced expression profiles were examined. acteristic of galactose-binding and thus called galactose-type CTLs (Drickamer 1992, Zelensky and Gready 2005). In human Mincle (a Materials and Methods 2+ 169 171 172 macrophage inducible Ca -dependent CTL), Glu , Asn , Asn , 193 194 169 171 Biological Materials Asn and Asp are involved in calcium binding. Glu and Asn Asian corn borers (O.  furnacalis) were reared on an artificial diet are also participated in the EPN motif (Furukawa et al. 2013). In rat 185 187 193 205 206 at 28°C under a relative humidity of 70–90% and a photoperiod mannose-binding lectin A, Glu , Asn , Glu , Asn , and Asp 2+ 185 187 of 16:8 (L:D) h (Zhang et  al. 2013a). Beauveria bassiana (strain constitute the Ca -binding site 2, in which Glu and Asn belong 252) was cultured on potato dextrose agar (PDA) plates at 25°C and to the EPN motif (Weis et al. 1992). Most CTLs only contain a sin- a relative humidity of 80%. gle CTL domain. Some CTLs identified from lepidopteran insects, such as M. sexta IML1-4, Bombyx mori lipopolysaccharide-binding protein (LBP), MBP and LEL1-3, and Helicoverpa armigera CTL1-8, Identification and Feature Prediction of have two tandem CTL domains (Koizumi et al. 1999, Yu et al. 2006, O. furnacalis CTLs Takase et al. 2009, Wang et al. 2012). CTLs with dual CTL domains A transcriptome dataset containing 62,382 unigenes were obtained are also reported in coleopteran insect Tribolium castaneum (Zou from O.  furnacalis larvae with ‘next generation’ high-throughput et  al. 2007) and crustacean Fenneropenaeus chinensis (Xu et  al. sequencing (Liu et  al. 2014). All unigene sequences were searched 2010). It is still unknown why dual CRDs arise in these CTLs. against CTLs from M. sexta and other insects, using BLASTX algo- –5 As a category of important PRRs, CTLs play various roles through rithm with an E-value cut-off of 10 . The BLASTX results were recognizing and binding to a diverse range of sugar ligands. Mammalian used to extract coding region sequences from corresponding unigene CTLs have been revealed to function in anti-fungal/bacterial immu- sequences. The predicted coding region sequences were further trans- nity, homeostasis, antoimmunity, allergy, the recognition of dead cells lated into peptide sequences using the Translate tool provided by and tumors, and complement activation (Brown 2015, Drickamer and the Swiss Institute Bioinformatics. Analysis of deduced amino acids Taylor 2015, and references within). Insect CTLs perform relatively less sequences, including prediction of signal peptide, molecular weight, functions and mainly participate in innate immune responses including and isoelectric point, were performed in the Expert Protein Analysis phagocytosis (Jomori and Natori 1992), nodule formation (Koizumi et al. System (EXPASY) (http://www.expasy.org). Conserved domains and 1999), encapsulation and activation of prophenoloxidase (Yu and Kanost transmembrane regions were predicted in SMART (http://smart. 2004). For example, M. sexta IML-1 and IML-4 can induce the agglutin- embl-heidelberg.de/smart/set_mode.cgi). The domain architectures 2+ ation of bacteria and yeast in a Ca -dependent manner (Yu and Kanost were plotted with IBS 1.0 (http://ibs.biocuckoo.org/). 2001, Yu et al. 2006). Recombinant CRD2 of IML-2 directly binds to Caenorhabditis elegans and enhances its encapsulation and melanization Sequence Alignment and Phylogenetic Analysis in vivo (Yu and Kanost 2004). B. mori LBP binds to a variety of Gram- Multiple sequences alignment of CTLs from O.  furnacalis and negative bacteria and MBP principally binds to Gram-positive bacteria other insects (https://www.ncbi.nlm.nih.gov/) were carried out using and yeast. They both trigger hemocyte aggregation and microbe clear- MUSCLE module of MEGA 6.0 (http://www.megasoftware.net/) at the ance (Koizumi et al. 1999, Takase et al. 2009). Recombinant Drosophila following parameters: refining alignment, gap opening penalty = −2.9, melanogaster DL2 and DL3 (Drosophila lectin) can accelerate the agglu- gap extension penalty = 0, hydrophobicity multiplier = 1.2, Maximum tination of Escherichia coli in the presence of calcium and enhance cellu- iterations = 100, clustering method (for iterations 1, 2) = UPGMB, and lar encapsulation and melanization by directly recruiting hemocytes (Ao Minimum diagonal length  =  24. The resulted alignments were used et al. 2007). However, current knowledge about the function and binding to construct the phylogenetic tree using the neighbor-joining method. mechanism of insect CTLs is still limited and incomplete. For neighbor-joining method, gaps were treated as characters, and sta- Recent genome-wide analysis has helped to identify a number of tistics analysis was performed by bootstrap trails with 1,000 replica- genes encoding proteins with one or more CTL or CTL-like domains. tions, Poisson model, uniform rates, and complete deletion of gaps or Aedes agypti, Anopheles gambiae, D.  melanogaster, B.  mori, missing data (Tamura et al. 2013). M.  sexta, and T.  castaneum have 39, 25, 34, 23, 34, and 17 such genes, respectively (Christophides et al. 2002, Waterhouse et al. 2007, Structure Modeling of 22 O. furnacalis CTL Domains Zou et al. 2007, Rao et al. 2015a, Rao et al. 2015b). Development of The deduced amino acid sequences of the 22 O.  furnacalis CTL high through put sequencing and de novo assembly strategies resulted domain were submitted to the Iterative Threading ASSEmbly in the identification of more CTL or CTL-like transcripts from the Refinement (I-TASSER) server (http://zhanglab.ccmb.med.umich. transcriptomes of non-model insects. For example, 8, 9, and 24 tran- edu/I-TASSER/) for the prediction of tertiary structure. The repre- scripts for potential CTLs were found in H.  armigera, Nilaparvata sentative structural template was identified from PDB by a locally lugens, and Tenebrio molitor, respectively (Wang et  al. 2012, Bao installed LOMETS meta-threading server. Models were constructed et al. 2013, Zhu et al. 2013). In the previous work, we have obtained by iterative TASSER simulations. The generated PDB files were then a transcriptome dataset from an important insect pest, Asian corn visualized with Pymol Molecular Graphics System. borer, Ostrinia furnacalis (Guenée; Lepidoptera: Pyralidae), and iden- tified 14 possible CTL transcripts (Liu et  al. 2014). After carefully Expression Profile Analysis manual validation, we clarified that 3 of 14 transcripts lacked one or more key amino acid residues critical for CTL structure or binding To investigate the transcriptional changes of CTLs during the various activity. Meanwhile, we identified another three potential CTL tran- stages of O. furnacalis, total RNA samples were individually prepared from scripts. Therefore, we finally obtained 14 O. furnacalis CTLs, desig- three different stages including egg, larva, and pupa using TRNzol Reagent nated as OfCTL-S1 through S5, and OfIML1 through 9. In this study, (TIANGEN, Beijing, China). One μg of RNA equally from five individual we reported the identification, characterization, expression analysis RNA samples in each stage was treated with DNase I (TIANGEN, Beijing, Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/2/24/4924850 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Journal of Insect Science, 2018, Vol. 18, No. 2 3 China) and converted into first-strand cDNA from an oligo (dT) primer Among these, 14 ones were predicted to encode potential CTLs following the instructions for QuantScriptRT Kit (TIANGEN, Beijing, (Liu et al. 2014). After careful validation, we found three of them, China). The cDNA products independently from three biological replicates unigene2023, unigene31526 and CL321.contig3, were possibly not were diluted 10-fold for use as template in RT-PCR experiments. Specific real CTLs. Meanwhile, we identified another three transcripts, uni- primers were designed and listed in Supplementary Table S1. O. furnacalis gene4110, CL4301.contig1 and CL321.contig1, as potential CTLs. ribosomal protein L8 (rpL8) was used as an internal standard to adjust the Thus, we still obtained totally 14 CTL transcripts, which encode template amounts in a preliminary PCR experiments. The thermal cycling proteins with single or dual CTL domains (Table  1). Five of the conditions were 94°C for 3 min, then 28 cycles of 94°C for 30 s, 58°C for protein products (OfCTL-S1 ~ S5) only contain one CTL domain, 30 s, and 72°C for 30 s followed by incubation at 72°C for 10 min. The and the remaining nine (OfIML-1 ~ 9)  belong to the IML family PCR products were separated by electrophoresis on a 1.5% agarose gel. with two tandem CTL domains (Fig. 1). None has other conserved To check the expression profiles of O. furnacalis CTLs under differ - structural units such as complement C1r/C1s (CUB) domain, com- ent inducement conditions, day 0 fifth instar larvae from the same batch plement control protein (CCP/Sushi) domain, epidermal growth were injected into the hemocoel with 2 μl of sterile phosphate-buff- factor (EGF) -like domain, and coagulation factor 5/8 C-terminal ered saline (PBS) containing formaline-killed Escherichia coli DH5α (FA58C) domain, which were reported in B.  mori CTL-X1 ~ X5 (5 × 10 cells/μl), dried Micrococcus luteus (5 μg/μl), B. bassiana sus- and M.  sexta CTL-X1 ~ X6 (Rao et  al. 2015a, Rao et  al. 2015b). pension (3 × 10 conidia/μl), B. bassiana conidia suspension was pre- O.  furnacalis CTL-S1 ~ S5, IML-1 ~ 4, IML7, and IML-8 have a pared as described previously (Zhang et al. 2013a), or sterile PBS as a secretion signal peptide at the amino-terminus and are likely secreted control. After 24 h, each three larvae from challenged or control group into plasma. O.  furnacalis IML-5 and IML-6 lack the N-terminal were collected, and total RNA samples were individually prepared as secretion signals and may be located in the cytoplasm. It is unsure described above. DNase I–treated RNA (1 μg) was converted into first- whether OfIML9 has a signal peptide because the translation start strand cDNA using FastQuant RT Kit (TIANGEN Biotechnology Co., codon preceding the CTL domain is unavailable in its current incom- Ltd, Beijing, China). The cDNA products were diluted 10-fold for use plete transcript (Table 1 and Fig. 1). as template. The quantitative real-time PCR (qRT-PCR) was performed O.  furnacalis CTLs with a simple structure, CTL-S1 ~ S5, con- with SYBR Premix EX Taq (TaKaRa, Japan) on Applied Biosystems sist of 180–220 amino acid residues, and has the calculated molecular 7500 Real-Time PCR System (Life Technologies, Grand Island, NY), mass of around 20 kDa (Table 1). The isoelectric points of the mature according to the manufacturer’s instructions. O. furnacalis ribosomal proteins are around 6.0 with the exception of CTL-S3 and CTL-S5. protein L8 (rpL8) was used as an internal standard to normalize the O. furnacalis CTL-S3 has 21 Arginine residues, 7 Histidine residues, expression level. The thermal cycle conditions for qRT-PCR were 95°C and 5 Lysine residues in the deduced protein sequences, and, therefore, for 2 min followed by 40 cycles of 95°C for 15 s, 55°C for 30 s and results in an isoelectric point of 8.47. It is the only negatively charged 68°C for 30 s ending with a melting curve generation (60 to 95°C in lectin among all 14 identified O. furnacalis CTLs. Mature OfCTL-S5 increment of 0.5°C very 5 s). The relative expression level of genes was contains 10 Aspartic acid residues and 15 Glutamic acid residues, and, −ΔΔCt calculated with the 2 method (Livak and Schmittgen 2001). therefore, has an isoelectric point of low to 4.90 (Table 1). Compared to O. furnacalis CTLs with a single CTL domain, 9 OfIMLs with dual CTL domains consist of more amino acid residues, around 300–330 Results ones with the exception of OfIML-9 which is currently incomplete Overview and General Properties of at the amino-terminus. The calculated molecular mass and isoelec- O. furnacalis CTLs tric point of the mature protein of OfIML-1 ~ 8 are approximately With next generation high-throughput sequencing, we obtained an 33.0 kDa and 5.0, respectively (Table 1). Neither N- nor C-terminal O.  furnacalis transcriptome dataset containing 62,382 unigenes. extension rich in certain amino acid residues, which are existed in Table 1. Structural features of 14 O. furnacalis CTLs a c Designated name Unigene ID Protein length Signal peptide Mr pI Glycosylation CRD numbers Motif (aa) (kDa) sites N-linked O-linked OfCTL-S1 CL4786.Contig1 220 1-19A*Q 23.4 6.33 0 3 1 QPD OfCTL-S2 Unigene6545 223 1-19A*Q 23.7 5.99 0 1 1 QPD OfCTL-S3 CL8286.Contig1 221 1-21A*Q 23.1 8.47 0 6 1 QPD OfCTL-S4 Unigene9847 207 1-21A*Q 21.3 6.20 1 5 1 APQ OfCTL-S5 CL321.Contig1 184 1-21T*V 19.1 4.90 0 5 1 EPN OfIML-1 Unigene22572 301 1-19A*Q 32.6 5.32 1 3 2 APW / EPN OfIML-2 Unigene14484 314 1-22G*R 32.9 5.42 0 2 2 EPD / EPN OfIML-3 CL4301.Contig2 304 1-20S*N 32.5 4.74 1 1 2 VPL / EPN OfIML-4 CL106.Contig3 328 1-21T*D 33.9 5.13 1 2 2 EPD / QPD OfIML-5 Unigene5701 322 35.7 4.89 N/A N/A 2 EPD / QPD OfIML-6 Unigene2411 319 36.5 5.89 N/A N/A 2 DIS / SPD OfIML-7 CL1725.Contig1 307 1-20S*S 32.3 6.20 3 3 2 DVS / VPD OfIML-8 Unigene5307 321 1-21S*Q 34.1 4.98 2 0 2 EPN / QPD OfIML-9 Unigene4110 >205 N/A >23.2 4.44 ≧1 ≧2 ≧1 ? / QPD The molecular weight (Mr) and isoelectric point (pI) are for mature proteins without signal peptides. OfIML-5 and OfIML-6 lack signal peptide, and, therefore, the predication for the numbers of glycosylation sites is not applicable (N/A). The canonical motifs (QPD and EPN) are in bold. Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/2/24/4924850 by Ed 'DeepDyve' Gillespie user on 16 March 2018 4 Journal of Insect Science, 2018, Vol. 18, No. 2 Fig.  1. Domain architectures of 14 O.  furnacalis CTLs. The numbers above the boxes indicates the location of the corresponding amino acid residues in the deduced protein sequences. Signal peptide is in magenta. The C-type lection domains (CTLD) are in green. The ellipsis in OfIML-9 means the amino-terminus is incomplete. B. mori CTL-S4 ~ S6, -S8 ~ S10, and M. sexta CTL-S3 ~ S5 and -S8 be mannose-type lectins. OfIML-8 contains both motifs simultane- (Rao et al. 2015b, Rao, 2015a), is observed in all 14 identified O. fur - ously, with the EPN motif in the first CTL domain and the QPD motif nacalis CTLs. Numbers of putative N-linked and O-linked glycosyla- in the second CTL domain. However, other CTL domains, including tion sites are variant in each O. furnacalis CTL, from 1 in OfCTL-S2 OfCTL-S4, the first CTL domain in OfIML-1 through 7 and the sec- to 6 in OfCTL-S3 and OfCTL-S4 (Table 1). ond CTL domain in OfIML-6 and OfIML-7, contain the non-canon- ical motifs. Their binding specificities need to be further investigated. Conserved Residues and Structural Features of The typical CTL domain is stabilized by three pairs of disulfide O. furnacalis CTLs bonds between Cys-1 and Cys-2, Cys-3 and Cys-6, Cys-4 and Cys- All CTLs contain several conserved amino acid residues which deter- 5, although no all CTL domains contain all six cysteine residues mine the sugar-binding specificity of CTL. Gln-Pro-Asp (QPD) and (Weis et al. 1992, Furukawa et al. 2013). We aligned all 22 O. fur- Glu-Pro-Asn (EPN) motif in the CTL domain suggest potential nacalis CTL domain sequences including 5 ones from OfCTL-S1 galactose and mannose binding specificities of CTLs, respectively through S5, 8 ones from the first CTL domain in OfCTL-1 through (Drickamer 1992, Zelensky and Gready 2005). The QPD motif is 8 (OfCTL-1A ~ -8A, the first CTL domain in OfCTL-9 is currently found in the CTL domain in OfCTL-S1 through S3 and the second unavailable), and 9 ones from the second CTL domain in OfCTL-1 CTL domain in OfIML-4, OfIML-5, and OfIML-9 (Table 1, Fig. 2). through 9 (OfCTL-1B ~ -9B) with well-studied human Mincle (PDB: Therefore, they may be galactose-type lectins. The EPN motif is found 3WHD). These six Cysteine residues mentioned above are absolutely in the CTL domain in OfCTL-S5 and the second CTL domain in conserved in nine CTL domains (OfIML-1B through 9B). Cys-1 OfIML-1 through 3 (Table 1, Fig. 2). These O. furnacalis CTLs may is present but Cys-2 is absent in the CTL domains in OfCTL-S1, Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/2/24/4924850 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Journal of Insect Science, 2018, Vol. 18, No. 2 5 Fig. 2. Sequence alignment of the 22 O. furnacalis CTL domains. Based on the domain predications with SMART, the amino acid sequences for 22 O. furnacalis CTL domains are aligned with that of human Mincle (PDB: 3WHD). The conserved Cys residues are numbered and shown in red. They are predicted to form three disulfide bonds (1–2, 3–6, 4–5) based on the determined structure in human Mincle. The regions C(D/N)F(K/A)GC in OfCTL-S1 through S3 may contain a unique disulfide linkage and are shaded in grey. The canonical QPD and EPN motifs are shaded in green and cyan, respectively. Residues involved in calcium binding 2+ and carbohydrate binding are in blue and purple, respectively. Residues involved in both Ca and sugar binding are in bold. The secondary structure elements (α, α-helix; β, β-strand; T, turn) of human Mincle are shown above or below the sequences. The asterisks above the sequences indicate the residues involved in calcium binding in human Mincle. -S2, -S5 and OfIML-2A. Both Cys-1 and Cys-2 are missing in the of 22 O.  furnacalis CTL domain with human Mincle indicated remaining CTL domains including OfCTL-S3, -S4, OfIML-1A, -3A that CTL-Ss with the exception of CTL-S4 and most of the IML through 8A (Fig.  2). Additionally, the cysteine residues in C(D/N) A  domains lack these residues corresponding to those in Mincle F(K/A)GC (shaded in grey in Fig. 2) of OfCTL-S1 through S3 may whereas IML B domains other than OfIML-7B possess them (Fig. 2). form a unique disulfide linkage. We further performed structural modeling of 22 CTL domains 2+ CTLs usually recognize carbohydrates via a Ca -mediated to explore the putative calcium and sugar binding sites. The over- binding network (Zelensky and Gready 2005). In a canonical CTL, all structures of all 22 O. furnacalis CTL domains are predicted to the EPN or QPD motifs on the loops of CTL cooperate with the be closely similar (Fig. 3). They all may bind to carbohydrates in a Asn-Asp (ND) residues on the adjacent β-sheet to bind the calcium calcium-dependent manner since five of the models (-S5, -2B, -3B, 2+ (Furukawa et  al. 2013). For example, E169, N171, N172, N193, -4A, and -8A) contain two Ca and the remaining 17 CTL domains 2+ 2+ and D194 in human Mincle constitute the Ca -bound sites (marked contain one Ca (Table  2, Figs. 2 and 3). Further experiments are 2+ by red asterisks in Fig.  2) (Furukawa et  al. 2013). The alignment required to determine whether Ca indeed enhances the binding Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/2/24/4924850 by Ed 'DeepDyve' Gillespie user on 16 March 2018 6 Journal of Insect Science, 2018, Vol. 18, No. 2 Fig. 3. Structural models of O. furnacalis CTL-S1 (A), IML-8A (B), and IML-8B (C). The tertiary structures predicted from I-TASSER server are exhibited as cartoons. The calcium ions are shown as red spheres and indicated by red arrows. The mannose-type EPN motif and galactose-type QPD motif are shown as blue sticks and indicted by blue arrows. The Cys residues for the formation of disulfide bonds are represented as orange sticks. Table 2. Structural features of the 22 O. furnacalis CTL domain models 2+ 2+ a b Domain Ca Putative Ca coordinators Motif Sugar Putative sugar binding residues Template C-score 2+ 2+ [Ca #1] [Ca #2] OfCTL-S1 1 [48,50,54,168] QPD GQ2 69,125,127,128,142,155,156,157,163 4C9F 0.52 -S2 1 [47,49,53,172] QPD MMA 129,131,132,142,159,160,161 1KWU 0.53 -S3 1 [40,42,46,159] QPD GQ2 61,117,119,120,133,146,147,148,154 4C9F 0.58 -S4 1 [43,45,49,167] APQ PI 100,101,111,112,113,122,123,124,125,126 2ORK 0.25 -S5 2 [49,51,55,145] [79,83,86,107,116,117] EPN TRE 72,104,106,116,122,132,133,138 4ZRV 0.65 OfIML-1A 1 [40,42,46,121] APW GAL 88,90,92,96,108,109,110 1JZN 0.37 -1B 1 [43,45,49,142] EPN TRE 71,108,110,112,116,122,129,130,135 4KZV 0.72 OfIML-2A 1 [38,40,44,126] EPD MAN 93,95,101,113,114,116 2VUZ 0.72 -2B 2 [43,45,49,140] [80,84,87,115,128] EPN TRE 73,107,109,111,115,121,127,128,133 4KZV 0.64 OfIML-3A 1 [40,42,46,122] VPL BM3 89,91,93,97,109,110,111 2ORJ 0.51 -3B 2 [43,45,49,141] [79,84,87,115,129] EPN TRE 72,107,109,115,121,128,129,134 4ZRV 0.69 OfIML-4A 2 [43,45,49,131] [72,76,79,101,106,107] EPD MAN 64,98,100,106,118,119,121 2VUZ 0.66 -4B 1 [43,45,49,143] QPD GQ2 72,106,108,110,121,129,130,131,138 4C9F 0.59 OfIML-5A 2 [45,47,51,131] [72,76,79,101,106,107] EPD NGA 65,98,100,102,106,110,112,118,119 1WMZ 0.66 -5B 1 [43,45,49,145] QPD GQ2 74,108,110,112,123,131,132,133,140 4C9F 0.61 OfIML-6A 1 [40,42,46,122] DIS MAN 94,96,98,109,110,111 1KWZ 0.39 -6B 1 [42,44,48,137] SPD TRE 71,105,107,111,117,123,124,129 4ZRV 0.59 OfIML-7A 1 [45,47,51,126] DVS NGA 96,98,101,105,113,114,115 1BCH 0.34 GQ4 26,62,64,65,96,98,101,105,115,119,121 4C9F 0.25 -7B 1 [42,44,48,139] VPD MBG 105,107,109,113,125,126 1AFA 0.34 OfIML-8A 2 [40,42,46,128] [69,73,76,98,103,104] EPN MAN 95,97,103,115,116,118 2VUZ 0.74 -8B 1 [43,45,49,137] QPD TRE 71,105,107,112,118,124,125,130 4ZRV 0.66 OfIML-9B 1 [43,45,49,136] QPD TRE 72,104,106,111,117,123,124,129 4ZRV 0.61 EPN and QPD motifs are in bold. The residues involved in both calcium and sugar binding are in bold. GQ2, a viral ligand to human herpesvirus 6 (HHV-6); MMA, methyl α-D-mannopyranoside; PI, phosphatidylinositol; TRE, trehalose dimycolate; GAL, β-D-galactose; MAN, α-D-mannose; BM3, N-acetyl-α-D-mannosamine; NGA, N-acetyl-D-galactosamine; MBG, methyl-β-galactose. C-score is the confidence score representing the quality of the generated models. It is calculated based on the Z-score of LOMETS threading alignments and 2+ the convergence of I-TASSER simulations. C-score ranges 0–1, where a higher score indicates a more reliable prediction. Ca sites with C-score > 0 and sugar sites with C-score > 0.2 are shown in the table. C-scores shown here are for putative sugar binding sites. strength. On the other hand, three-dimensional structure modeling ranging from 0.64 to 0.74 (Table  2). The exceptions are CTL-S2, also allows us to predict the sugar binding specificities of 22 O. furn- IML-8B and -9B contain the QPD motif, but are predicted to bind acalis CTL domains. The results from the above sequence alignment galactose (Table  2 and Fig.  3). OfCTL-S4, IML-1A through -7A, and molecular modeling are mostly consistent (Table 2, Figs. 2 and -6B, and -7B have the non-canonical motifs, but also show the sugar 3). The high C-scores of the models suggest that OfCTL-S1, -S3, binding potentials in structural modeling. More experiments will be IML-4B, and -5B may form stable complexes with galactose. They required to validate the binding predictions. Beside, homology mod- just contain the QPD motif. OfCTL-S5, IML-1B, -2B, -3B, and -8A eling also indicated that two amino acid residues (Glu in CTL-S5, 115 128 115 129 106 has the EPN motif, and may bind to mannose tightly with C-scores Glu and Asp in IML-2B, Glu and Asp in IML-3B, Glu Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/2/24/4924850 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Journal of Insect Science, 2018, Vol. 18, No. 2 7 106 103 in IML-4A, Glu in IML-5A, and Glu in IML-8A) are involved led to the significant up-regulation for both CTL-S1 and IML-2; in both calcium and carbohydrate binding (Table 2 and Fig. 3). B. bassiana-injection resulted in the significant increase for all four transcripts other than CTL-S1 (Fig. 5B). Phylogenetic Analysis of O. furnacalis CTLs To investigate the evolutionary relationships between CTLs of Discussion O.  furnacalis and other insects, we retrieved 77 CTL protein CTLs acts as a category of important PRRs, and play various physi- sequences from 19 insect species by BLASTP searches in GenBank, ological roles in animals including human and insects (Brown 2015, VectorBase, and Insect Innate Immunity Database. Since no CTLs Dambuza and Brown 2015). In this study, we have identified 14 with other complicated structural unites than CTL domains are CTLs (OfCTL-S1 ~ S5, and OfIML-1 ~ 9) from O. furnacalis tran- currently identified in O.  furnacalis, we did not included M.sexta scriptome. We performed comprehensive sequence analysis for these CTL-Xs and their orthologs, which contain CTL domain(s) together 14 CTLs and investigated their expression profiles in different devel- with other conserved domains in the alignment. The phylogenetic opmental stages and upon different infections. tree (Fig. 4) shows that all CTLs are generally divided into two cate- CTLs are characterized by processing one or more carbohy- gories based on the numbers of CTL domains. OfCTL-S1 through S5 drate-recognition domains (also known as CTL domains) (Cambi formed tight, monophyletic groups with their respective orthologs et al. 2005, Geijtenbeek and Gringhuis 2009). Among 14 identified from the other species in Lepidoptera and Diptera. Moreover, the O.  furnacalis CTLs, 5 ones only has a single such domain, and 9 orthologs of OfCTL-S1 and OfCTL-S4 were also found in the red contain two tandem CTL domains (Table 1 and Fig.1). So far, CTLs flour beetle T. castaneum (Coleoptera) (Fig. 4). with one CTL domain have been identified from almost all animals Nine O.  furnacalis IMLs are grouped with the CTLs with dual including human (Furukawa et  al. 2013), rats (Weis et  al. 1992), CTL domains, with a bootstrap value of high to 99 (Fig.  4). All crustaceans (Zhang et al. 2013b) and insects (Rao et al. 2015a, Rao the orthologs in this group are from lepidopteran insects including et al. 2015b). For example, D.  melanogaster, A.  gambiae, B.  mori, B.  mori, M.  sexta, H. armigera, Hyphantria cunea, Danaus plexip- M. sexta, and T. castaneum have 34, 25, 12, 9, and 10 genes encod- pus, Danaus plexippus, and Pieris rapae. Unlike MsIML-1, 18, 19 ing proteins with one CTL domain, respectively (Christophides et al. and BmIML-1, 4~6 which had 1:1 orthologs, no O.  furnacalis IML 2002, Waterhouse et  al. 2007, Zou et  al. 2007, Rao et  al. 2015a, formed 1:1 orthologous group. OfIML-4 and OfIML-5, as well as Rao et  al. 2015b). We only identified five transcripts for encoding OfIML-6 and OfIML-7, showed lineage-specific expansions (Fig. 4). proteins with one CTL domain. Therefore, it is highly possible there They may evolve by multiple gene duplication. Additionally, we con- are other unidentified transcripts to encode O.  furnacalis CTL-Ss. structed the phylogenetic tree based on the alignment of O.  furna- At least, there should be one CTL-S for ortholog in CTL-S5 group calis CTL domains alone, instead of the alignment of entire protein since other insects than O.  furnacalis have clearly 1:1 orthologous sequences. Twenty-two CTL domains formed three separate branches: relationship in this group (Fig.  4). Compared to the wide distribu- CTL-S, CTL domain A, and CTL domain B (Supplementary Fig. S1). tion of short CTL-Ss, IML family with two tandem CTL domains was found mainly in lepidopteran insects (Yu and Kanost 2001). Six, Expression Profiles of O. furnacalis CTL Genes 19, and eight such CTLs have been reported in B.  mori, M.  sexta, We analyzed the mRNA levels of O.  furnacalis CTLs in various and H.  armigera, respectively (Wang et al. 2012, Rao et al. 2015a, development stages using semi-quantitative RT-PCR methods. As Rao et  al. 2015b). Unlike CTL-Ss which normally have clear ort- shown in Fig.  5A, the 14 O.  furnacalis CTLs exhibited distinct hologs in different insects, some immulection genes show extensive expression patterns. The transcripts of OfCTL-S5, IML-4, IML-5, lineage-specific expansions. For instance, M.  sexta IML-3, 4, 6–8, and IML-8 were detected in all examined developmental stages, IML-9~12, and IML-13~17 and H.  armigera CTL-1, 2, and 6 are including egg, larval and pupal stages. OfCTL-S1 was also detected four groups of related IMLs (Fig.  4) (Wang et  al. 2012, Rao et  al. in all stages, but with obviously higher level in the stage of egg and 2015a). As to the formation of IMLs with dual CTL domains, it may young larva and low level in the stage of the fifth instar larva and be a result of the duplication of CTL-S genes because the first CTL pupa. OfCTL-S2 was clearly expressed in all tested developmental domains in B. mori and M. sexta IMLs seem to evolve from common stages other than egg. IML-6, -7 and -9 were expressed in the second ancestors of BmCTL-S11 and MsCTL-S7, respectively (Rao et  al. to fifth instar larval stage and pupal stage, but not in eggs and the 2015a, Rao et  al. 2015b). We speculated that the event of entire first instar larvae. CTL-S3 mRNA was detectable in the old larvae gene duplications and sequence divergence resulting in the appear- and pupae, and the mRNA level was lower in the pupal stage. The ance of tandem CTL domains in moths and butterflies took place remaining three OfCTLs exhibited irregular expression patterns: before the radiation of Lepidoptera. In addition, there exists another OfCTL-S4 was detected in egg and pupa; IML-2 in egg and the fifth type of IMLs containing one or two CTL domains along with other instar larva; and IML-3 in egg and the fourth instar larva (Fig. 5A). structural units, such as D.  melanogaster furrowed, uninflatable, To check the O. furnacalis CTL expression profiles after exposure and contactin (Stork et al. 2008, Xie et al. 2012, Chin and Mlodzik to microbial elicitors, we randomly selected eight CTLs and analyzed 2013), B. mori CTL-X1~5 (Rao et al. 2015b), M. sexta CTL-X1~6 their transcript level after O.  furnacalis larvae were injected with (Rao et al. 2015a), and T. castaneum CTL-X1~5 (Zou et al. 2007). E. coli, M. luteus, B. bassiana, or PBS as a control. The result from However, we did not identify any similar IMLs in O. furnacalis tran- quantitative RT (qRT) -PCR assay indicated that the expression pat- scriptome. The possible reason is that the assembly quality of cur- terns of these eight CTLs could be divided into three groups: the tran- rent transcriptome is not high enough to find the long extensions for script levels of IML-8, CTL-S2, and CTL-S5 remained unchanged; encoding the other structural modules. We believed that there also CTL-S4 decreased significantly upon challenge; and CTL-S1, IML- existed similar complex CTL-Xs in O. furnacalis, but the identifica- 2, IML-3, and IML-9 increased after infection (Fig. 5B). There still tion requires more sequence resources. existed some differences although CTL-S1, IML-2, IML-3, and The CTL domain adopts a characteristic double-loop fold IML-9 mRNA levels all increased upon challenge: E.  coli-injection consisting of five β -sheets and two α-helices: the closely located only caused the significant increase for IML-9; M.  luteus-injection N- and C-termini (β1, β5) make the overall domain a loop, and Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/2/24/4924850 by Ed 'DeepDyve' Gillespie user on 16 March 2018 8 Journal of Insect Science, 2018, Vol. 18, No. 2 Fig.  4. Phylogenetic analysis of insect CTLs. The amino acid sequences of 91 CTLs from 20 insect species were examined. Lepidopteran-specific CTLs were indicated with bracket. The branches specific for CTL-S1 through -S6 are shaded. Numbers at the nodes were bootstrap values as percentage. Only bootstrap values greater than 70 were shown. Of, O.  furnacalis; Aa, A.  aegypti; Ag, A.  gambiae; Ap, Antheraea pernyi; Bm, B.  mori; Bt, Bombus terrestris; Cq, Culex quinquefasciat; Dm, D.  melanogaster; Dp, D.  plexippus; Ha, H.  armigera; Hc, H.  cunea; Hv, Heliothis virescens; Mr, Megachile rotundata; Ms, M.  sexta; Nv, Nasonia vitripennis; Pr, P. rapae; Ppo, Papilio polytes; Pxu, Papilio xuthus; Pxy, Plutella xylostella; and Tc, T. castaneum. 2+ the region between β2 and β3 strands forms the second loop also were predicted to have Ca -binding site 2 (Ca-2), and no O. fur- 2+ called the long loop region (Zelensky and Gready 2005, Furukawa nacalis CTL domain has other potential Ca -binding sites (Ca-3 et al. 2013, Feinberg et al. 2016). Canonical CTL domain has the or Ca-4) observed in human macrophage C-type lectin (MCL) long loop region and the compact one lacks it. All identified 22 and Mincle (Furukawa et al. 2013). The region surrounding Ca-2 O.  furnacalis CTL domains possess the long loop region. It sug- in O.  furnacalis CTL domains is more similar to that in human gests that they all belong to canonical CTLs. On the other side, DC_SIGNR than to that in MCL and Mincle. The roles of calci- 2+ the long loop region is reported to be involved in Ca -dependent um-binding sites are various in individual CTL, either involved in carbohydrate binding and in domain-swapping dimerization of carbohydrate binding or just playing structural roles. For example, some CTLs (Liu and Eisenberg 2002, Zelensky and Gready 2005). the calcium-binding site in human tetranectin is known not to Structural modeling also indicated that 22 CTL domains with long bind carbohydrates, but interact with kringle domain-containing loop region had potential to bind one or two calcium ions, sug- proteins leading to changes in the long loop region conformation 2+ gesting they might function in a calcium-mediated manner. Ca - (Nielbo et al. 2004). The functional impact of calcium-binding sites binding site 1 (Ca-1) close to the N- and C-terminus is present in all in O. furnacalis CTL domains is still unclear. 22 CTL domains (Fig. 3). It is consistent with the previous report Relative to calcium-binding sites, the characteristic motif, such as that Ca-1 is a highly conserved structural feature in virtually all mannose-type EPN motif or galactose-type QPD motif, is possibly CTL domains (Zelensky and Gready 2003, 2005). However, only 6 more important for carbohydrate binding of CTLs. For example, out of 22 CTL domains (CTL-S5, IML-2B, -3B, -4A, -5A and -8A) the EPN motif in rat MBP-A (E185, P186, N187) provides carbonyl Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/2/24/4924850 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Journal of Insect Science, 2018, Vol. 18, No. 2 9 Fig. 5. Expression profile analysis of O. furnacalis CTLs. (A) Expression profiles of O. furnacalis CTLs at different stages of development. RNA was extracted from the whole bodies collected from eggs, first-instar (L1), second-instar (L2), third-instar (L3), fourth-instar (L4), fifth-instar (L5) larvae, and pupae. The rpL8 was used as an internal control. (B) Expression profiles of O. furnacalis CTLs upon microbial challenge. Day 0, fifth instar larvae were infected with PBS, E. coli, M. luteus, or B. bassiana. RNA was prepared from the whole bodies 24 h after injection. qRT-PCR was used to assess the transcript change of OfCTLs with rpL8 as an internal standard to indicate a consistent total mRNA amount. The bars represent mean ± SD (n = 3). Bars labeled with different letters are significantly different (one-way ANOVA, followed by the Newman-Keus test, P < 0.05). sidechains to form hydrogen bonds with the monosaccharide and CTLs. Further experiments are undergoing to determine the binding determine its binding specificity (Zelensky and Gready 2005). specificities of O. furnacalis CTLs. Replacing the EPN sequence in MBP-A with a QPD sequence was The developmental and induction profiles of the CTL genes enough to switch the specificity to galactose (Drickamer 1992). reflect their functional importance to some extent (Fig.  5). For Twelve out of 22 O.  furnacalis CTL domains contain canonical instance, CTL-S4 transcripts were detected in eggs and pupae, but EPN or QPD motif (Tables 1 and 2), suggesting these O.  furnaca- not in the larvae, suggesting that it may be involved in the devel- lis CTLs have the potential to binding carbohydrates. Additionally, opment. Its mRNA levels surprisingly decreased after immune it is worthy of note that the remaining 10 CTL domains contain challenge (Fig.  5). The orthologous B.  mori CTL-S5 also showed unusual motifs in the long loop region such as EPD, APQ motif down-regulation in the E. coli or S. aureus injected fat bodies (Rao (Tables 1 and 2). Human MCL also has an EPD motif, and it still et al. 2015b). However, the function of B. mori CTL-S5 is currently shows the binding affinity to trehalose dimycolate (Furukawa et al. unknown. On the other side, immulections have been reported to 2013). Sarcophaga lectin from the flesh fly Sarcophaga peregrina participate in the immune responses including agglutination and/or contains an NPD motif, and exhibits the galactose-binding property encapsulation of microorganisms (Zelensky and Gready 2005). The (Kawaguchi et al. 1991). It is more surprising that blood dendritic expression of most IMLs is stimulated after microbial challenge (Yu cell antigen 2 (BDCA-2) contains an EPN motif, but bind galac- and Kanost 2004, Yu et al. 2006). O. furnacalis IML-2, IML-3, and tose-terminated glycans instead of predicted mannose (Jegouzo et al. IML-9 were consistently up-regulated after infection from microor- 2015). Langerin contains an EPN motif, but bind both mannose and ganism, but IML-8 remained unchanged upon infection (Fig. 5). So galactose (Feinberg et al. 2011). Therefore, we infer that there exist far, we only investigated the preliminary function of O.  furnacalis non-canonical sugar-binding sites in CTLs. Another possibility is IML-2. It exhibited the promising potential to bind and agglutinate that contacts outside of the primary sugar-binding site in extended E. coli (data not shown). The functional studies for other O. furna- or secondary binding sites also contribute to the sugar binding of calis CTLs are undergoing. 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