Identification and expression analysis of a novel R-type lectin from the coleopteran beetle, Tenebrio molitor

Journal of Invertebrate Pathology 114 (2013) 226–229

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Short Communication

Identification and expression analysis of a novel R-type lectin from the coleopteran beetle, Tenebrio molitor Dong Hyun Kim a, Bharat Bhusan Patnaik a, Gi Won Seo a, Seong Min Kang b, Yong Seok Lee c, Bok Luel Lee b, Yeon Soo Han a,⇑ a Division of Plant Biotechnology, Institute of Environmentally-Friendly Agriculture (IEFA), College of Agriculture and Life Sciences, Chonnam National University, Gwangju 500-757, Republic of Korea b National Research Laboratory of Defense Proteins, College of Pharmacy, Pusan National University, Jangjeon Dong, Kumjeong Ku, Busan 609-735, Republic of Korea c Department of Life Science and Biotechnology, College of Natural Sciences, Soonchunhyang University, Asan city 336-745, Republic of Korea

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Article history: Received 14 March 2013 Accepted 6 August 2013 Available online 17 August 2013 Keywords: R-type lectin Tenebrio molitor Immune elicitors Peptidoglycan Listeria monocytogenes

a b s t r a c t We have identified novel ricin-type (R-type) lectin by sequencing of random clones from cDNA library of the coleopteran beetle, Tenebrio molitor. The cDNA sequence is comprised of 495 bp encoding a protein of 164 amino acid residues and shows 49% identity with galectin of Tribolium castaneum. Bioinformatics analysis shows that the amino acid residues from 35 to 162 belong to ricin-type beta-trefoil structure. The transcript was significantly upregulated after early hours of injection with peptidoglycans derived from Gram (+) and Gram () bacteria, beta-1, 3 glucan from fungi and an intracellular pathogen, Listeria monocytogenes suggesting putative function in innate immunity. Ó 2013 Elsevier Inc. All rights reserved.

1. Introduction Lectins are a family of highly homologous sugar-binding proteins that bind to microbial antigens, consisting of repeating saccharide units such as beta-1, 3 glucans which are abundant in yeast cell walls, lipopolysaccharides (LPS) of Gram negative bacteria and peptidoglycan of Gram positive bacteria (Revillard, 2002; Patnaik et al., 2012). Though multidomain proteins, the sugarbinding activity is linked to a single module within the lectin polypeptide, known as the carbohydrate-recognition domain (CRD). Based on the structural attributes of CRD, the lectin family has been grouped into L-type lectins, galectins (both have b-sandwich type structure), C-type lectins (unique mixed a/b structure), P-type lectins (unique b-rich structure), I-type lectins (immunoglobulin superfamily), calnexin and R-type lectins (beta-trefoil structure) (Dodd and Drickamer, 2001). Among these lectins containing CRDs, calnexin and L-type lectins are localized in endoplasmic reticulum, whereas P-type lectins functions in the sorting of proteins in the Golgi complex. Other lectins function largely outside the cell and are either secreted or localized to the plasma membrane (Pace and Baum, 2004).

⇑ Corresponding author. Fax: +82 62 530 2069. E-mail address: [email protected] (Y.S. Han). 0022-2011/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jip.2013.08.002

The R-type lectin family exists ubiquitously in nature and contains short conserved and repeated (QxW)3 motifs (where x is any amino acid), found in animals, plants and bacteria (Rutenber et al., 1987; Hazes, 1996). These proteins possess the common betatrefoil fold structures arranged in a characteristic pseudo-threefold symmetry by the three (a, b and c) subdomains of two homologous domains. The sugar-binding affinity differs according to their ligand specificities. In addition, they are mainly seen to bind to the galactose and/or N-acetylgalactosamine units of the sugar chains (R-type lectins were formerly known as Gal/GalNAc-binding lectins) (Suzuki et al., 2009). Additionally, some of these proteins have binding preferences towards sialic acids (Neu5Ac) (Kaku et al., 2007). The R-type domain has been reported from a galactosebinding lectin of the earthworm, Lumbricus terrestris, where both homologous N- and C-terminal domains (14,500 Da) are R-type (Hemmi et al., 2013). R-type domain is also found in pierisin-1, a cytotoxic protein from Pieris rapae and the homologous protein pierisin-2, from Pieris brassicae (Watanabe et al., 1998). We report for the first time an R-type lectin with a conspicuous beta-trefoil domain and QxW motif by sequencing of random clones from the cDNA library of a coleopteran beetle, Tenebrio molitor. Although R-type lectins have not been seriously implicated in cellular recognition functions, the novel lectin deciphered, showed upregulated transcript expression after challenge with peptidoglycans and glucans from bacteria and fungi, as well as an intracellular pathogen, Listeria monocytogenes.

D.H. Kim et al. / Journal of Invertebrate Pathology 114 (2013) 226–229

2. Materials and methods A cDNA library was constructed with whole larvae of the coleopteran beetle, T. molitor, using the SMART cDNA Library Construction Kit (Clontech laboratories, Inc, Mountain view, CA). Based on the ESTs obtained, gene specific primer pairs were designed as 50 -GGGGCAGTACTTTCTTTG-30 (forward primer) and 50 CTTCTCCACCTGTTCTAG-30 (reverse primer) for Tm-R-type lectin. PCR was performed, and a cDNA fragment was generated, including the 30 -untranslated (UTR) region with poly-A tail. The fragment was sequenced using Model 3730 XL sequence analyzer (Applied Biosystems, Foster City, CA, USA). In order to obtain the full-length cDNA sequence of the gene, 50 -RACE was performed using 50 -gene specific (50 -GTAGAGTCACAAGAGTCGATCCGCCATT-30 ) and nested primer (50 -GATACTGGCTCGCGTCAAGCACCATAC-30 ). The PCR procedure was: 94 °C for 3 min; 25 cycles at 94 °C for 40 s, 55 °C for 40 s, and 72 °C for 90 s; and finally 72 °C for 10 min. The cDNA and deduced amino acid sequence of Tm-R-type lectin was analyzed using UltraEdit-32 Professional Text/HEX editor (version 12.00) software package and deduced amino acid sequence was predicted by ORF finder online in NCBI (http:// www.ncbi.nih.gov). The protein domains were predicted with the simple modular architecture research tool (SMART) version 4.0 (http://www.smart.embl-heidelberg.de/). Multiple sequence alignment of the deduced amino acid sequence was performed using the ClustalX (version 1.83) program. Expression of R-type lectin mRNA was examined after T. molitor larvae were challenged by immune elicitors. Third instar larvae were injected with 1 ll (2 lg/ll) of Listeria, peptidoglycans (PGN)

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and beta-1, 3 glucan, while the control group was only injected with buffer (0.14 M sodium chloride, 3 mM potassium chloride, 8 mM disodium hydrogen phosphate dodecahydrate, 1.5 mM potassium phosphate monobasic, pH 7.4). Quantitative real-time PCR (qRT-PCR) was performed to determine the time-course expression profiles after immune elicitors challenge. Two genespecific primers (50 -TCCAGACTTACACAGGATGC-30 and 50 GCAACTCTTTGCACCACCCT-30 , forward and reverse primers respectively) were used to amplify a partial fragment of the gene. TmL27a was used as internal control, and amplified with specific primers (50 -TCATCCTGAAGGCAAAGCTCCAGT-30 and 50 -AGGTTGGTTAGGCAGGCACCTTTA-30 forward and reverse primers respectively). qRT-PCR was performed using Exicycler™ 96 Real-Time Quantitative Thermal Block (Bioneer, Daejeon, Korea), in a final volume of 50 ll reaction mixture including 1 ll of cDNA, 10 pmol of each primer and 25 ll of 1X LightCycler 480 SYBR Green (Takara Corp., Japan). The amplification condition was as follows: initial denaturation at 95 °C for 20 s, followed by 40 cycles at 95 °C for 15 s, 60 °C for 1 min and 72 °C for 10 s. The comparative cycle threshold (Ct) values were used to analyze the relative expression level. Statistical differences between non-infected and infected samples were determined with multiple comparison tests. Values were considered to be significant at P < 0.05. 3. Results and discussion Sequencing of random clones from T. molitor cDNA library identified a single expressed sequence tag (EST), homologous to the already characterized lectin genes. The clones corresponding to the

Fig. 1. Sequence features of the novel R-type lectin from T. molitor. (A) Nucleotide and deduced amino acid sequence of cDNA encoding putative R-type lectin from Tenebrio molitor. Deduced amino acids are denoted as a one-letter code, initiation codon (ATG) is represented by arrow, ⁄ Translation stop codon. Polyadenylation signal (AATAA) is represented in bold and polyadenylation site is underlined. The CRD formed from presumed gene triplication (Ricin-type beta-trefoil) analyzed by SMART program is boxed (amino acid residue position 35 to 162). Putative signal peptide region is not detected. (B) Amino acid sequence comparison of putative R-type lectin from Tenebrio molitor with 29-kDa galectin from Tribolium castaneum (NCBI Ref-XP_001816090). The alignment was generated using ClustalX (version 1.83). The CRD formed from presumed gene triplication (Ricin-type beta-trefoil) is boxed (amino acid residue position 35 to 162). The (QxW)3 domains are boxed in red. The putative sugar-binding sites have been represented with green arrow heads. Gaps are shown as ‘_’, ‘⁄’ identical, ‘:’ highly conserved residues, ‘.’ conserved residues.

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D.H. Kim et al. / Journal of Invertebrate Pathology 114 (2013) 226–229

EST were re-sequenced and yielded cDNA sequence of 495 bp, encoding a polypeptide of 164 amino acid residues and a predicted molecular weight of 18.5 kDa (Fig. 1A). Prediction of conserved domains showed that the amino acids from 35 (R) to 162 (T) belonged to a CRD formed from presumed gene triplication (ricin-type beta tre-foil). The most characteristic, though not completely conserved, sequence feature was the presence of (QxW)3 pattern. Consequently, the ricin B lectin domain has also been referred as the (QxW)3 domain and three homologous regions as (QxW)3 repeats (Barondes et al., 1994). The lectin family member from T. molitor also showed the typical pattern at amino acid residue Nos. 67–69 (–QLW–) and 113–115 (–QQW–) within its CRD. Interestingly, the third (QxW)3 domain at amino acid residue number 157–159 (–QKF–) lack the true character. Similar domains are a characteristic feature in many carbohydrate-recognition proteins like plant and bacterial AB-toxins, glycosidases or proteases (Hazes, 1996; Hirabayashi et al., 1998). The domain analysis and characterization of the novel lectinlike protein from T. molitor led us to report it as an R-type lectin. This contribution is the first report of an R-type lectin from a coleopteran beetle. The sequence of Tm-R-type lectin has been deposited in European Nucleotide Archive-European Molecular Biology Laboratory (ENA-EMBL) under accession No. HF935082. Multiple alignment of the putative R-type lectin from T. molitor showed that the deduced amino acid sequence had an identity of 49% with galectin sequence from another model coleopteran beetle Tribolium castaneum (Fig. 1B), and lower identity with other vertebrate galectins. It therefore is reasonable to speculate the galactose binding affinity of the novel R-type lectin.

Expression analysis of Tm-R-type lectin in immune elicitor challenged T. molitor larvae were investigated with qRT-PCR. In larvae challenged with bacterial cell-wall components namely Lys- and DAP-type peptidoglycan, the transcript was maximally expressed after 12 h of challenge (about 6–8-fold) compared with uninjected control, followed by a sharp decline (Fig. 2A and B). It was also noticed that the lysine-type PGN showed a marginally stronger elicitor activity compared to DAP-type PGN. It is categorical, that the invertebrate galectins exhibit wide spectrum activity after stimulation with microbes and pathogen associated molecular patterns (PAMPs). Similar induction patterns were reported in Solen grandis galectin-1 (SgGal-1) after stimulation with PAMPs from Gram-negative bacteria, Gram-positive bacteria and fungi (Wei et al., 2012). Pattern Recognition Receptors (PRRs) such as hemolin, C-type lectin, beta-1, 3 glucan recognition protein and PGN-recognition protein (PGRP) have also been identified in lepidopteran Manduca sexta (Kanost et al., 2004). The Tm-R-type lectin transcript showed a 6-fold increase at 3 h post-challenge with L. Monocytogenes in comparison to other immune elicitors used in the present study (Fig. 2C). The sharp rise in the transcript was followed by a gradual depletion in the transcript level with increasing time from the point of injection. In larvae challenged with fungal cell wall component, beta-1, 3 glucan, the R-type lectin transcript showed a stable increase after an initial upregulation at 6 h post-injection (Fig. 2D). However, there was no appreciable decline in the transcript level with steady levels maintained till about 24 h post-immune challenges. Though the response time and the pattern of recognition were varied to the immune elicitors, it is justified that the novel ricin-

Fig. 2. Expression analysis of Tm-R-type lectin by qPCR. Tenebrio larvae were injected with immune elicitors such as (A) Lysine-type peptidoglycan, (B) DAP-type peptidoglycan, (C) Listeria cells and (D) beta-1, 3 glucan. Total RNA was extracted from whole larvae after 3, 6, 12, 18 and 24 h post-injection, and profiled by qPCR. Ribosomal protein L27a (Tenebrio molitor) was used as internal control. Results of triplicate experiments have been presented with standard errors. ⁄ P < 0.05, ⁄⁄ P < 0.01.

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type lectin can act as a putative recognition protein and may have implications in insect innate immunity. A detailed analysis of the function of such lectins in host development and immunoregulation is important and are currently been explored using molecular genetics and functional genomics approaches. Additionally, it will be important to note the remarkable evolutionary plasticity shown by the pathogens and parasites in evading such lectins by mimicking their hosts to gain entry into the hosts. Acknowledgment This work was supported by the Next-Generation Bio Green 21 Program (No. PJ008158) of the Rural Development Administration, Republic of Korea. References Barondes, S.H., Castronovo, V., Cooper, D.N.W., Cummings, R.D., Drickamer, K., Felzi, T., Gitt, M.A., Hirabayashi, J., Hughes, C., Kasai, K., Leffler, H., Liu, F.-T., Lotan, R., Mercurio, A.M., Monsigny, M., Pillai, S., Poirer, F., Raz, A., Rigby, P.W., Rini, M., Wang, J., 1994. Galectns: A family of animal b-galactoside binding lectins. Cell. 76, 597–598. Dodd, R.B., Drickamer, K., 2001. Lectin-like proteins in model organisms: implications for evolution of carbohydrate-binding activity. Glycobiology 11, 71–79. Hazes, B., 1996. The (QxW)3 domain: a flexible lectin scaffold. Prot. Sci. 5, 1490– 1501.

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Hemmi, H., Kuno, A., Hirabayashi, J., 2013. NMR structure and dynamics of the Cterminal domain of R-type lectin from the earthworm Lumbricus terrestris. FEBS J. 280, 70–82. Hirabayashi, J., Dutta, S.K., Kasai, K., 1998. Novel galactose-binding proteins in annelida. Characterization of 29-kDa tandem repeat-type lectins from the earthworm Lumbricus terrestris. J. Biol. Chem. 273, 14450–14460. Kaku, H., Kaneko, H., Minamihara, N., Iwata, K., Jordan, E.T., Rojo, M.A., Minami-Ishii, N., Minami, E., Hisajima, S., Shibuya, N., 2007. Elderberry bark lectins evolved to recognize Neu5Aca2, 6Gal/GalNAc sequence from a Gal/GalNAc binding lectin through the substitution of amino acid residues critical for the binding of sialic acid. J. Biochem. 142, 393–401. Kanost, M., Jiang, H., Yu, X., 2004. Innate immune responses of a lepidopteran insect, Manduca sexta. Immunol. Rev. 198, 97–105. Pace, K.E., Baum, L.G., 2004. Insect galectins: roles in immunity and development. Glycoconj. J. 19, 607–614. Patnaik, B.B., Saha, A.K., Bindroo, B.B., Han, Y.S., 2012. Lectins as possible candidates towards anti-microbial defense in silkworm, Bombyx mori L. Afr. J. Biotechnol. 11, 11045–11052. Revillard, J.P., 2002. Innate immunity. Eur. J. Dermatol. 12, 224–227. Rutenber, E., Ready, M., Robertus, J.D., 1987. Structure and evolution of ricin B chain. Nature 326, 624–626. Suzuki, R., Kuno, A., Hasegawa, T., Hirabayashi, J., Kasai, K., Momma, M., Fujimoto, Z., 2009. Sugar-complex structures of the C-half domain of the galactose-binding lectin EW29 from the earthworm Lumbricus terrestris. Acta Cryst. D65, 49–57. Watanabe, M., Kono, T., Koyama, K., Sugimura, T., Wakabayashi, K., 1998. Purification of pierisin, an inducer of apoptosis in human gastric carcinoma cells, from cabbage butterfly, Pieris rapae. Jpn. J. Cancer Res. 89, 556–561. Wei, X., Yang, J., Liu, X., Yang, D., Xu, J., Fang, J., Wang, W., Yang, J., 2012. Identification and transcriptional analysis of two types of lectins (SgCTL-1 and SgGal-1) from mollusk Solen grandis. Fish Shellfish Immunol., 1–9.