Identification and Characterization of a Novel Intelectin in the Digestive Tract

Developmental and Comparative Immunology 59 (2016) 229e239

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Developmental and Comparative Immunology

journal homepage: www.elsevier.com/locate/dci

Identiﬁcation and characterization of a novel intelectin in the digestive tract of Xenopus laevis

Saburo Nagata

Department of Chemical and Biological Sciences, Faculty of Science, Japan Women's University, Mejirodai 2-8-1, Bunkyoku, Tokyo 112-8681, Japan article info abstract

Article history: The intelectin (Intl) family is a group of secretory lectins in chordates that serve multiple functions, þ Received 9 December 2015 including innate immunity, through Ca2 -dependent recognition of carbohydrate chains. Although six Received in revised form Intl family lectins have so far been reported in Xenopus laevis, none have been identified in the intestine. 3 February 2016 Using a monoclonal antibody to the Xenopus embryonic epidermal lectin (XEEL or Intl-1), I identified Accepted 3 February 2016 cross-reactive proteins in the intestines. The proteins were purified by affinity chromatography on a Available online 5 February 2016 galactose-Sepharose column and found to be oligomers consisting of N-glycosylated 39 kDa and 40.5 kDa subunit peptides. N-terminal amino acid sequencing of these peptides, followed by cDNA cloning, Keywords: fi e Lectin identi ed two novel Intls (designated Intl-3 and Intl-4) that showed 59 79% amino acid identities with Omentin known Xenopus Intl family proteins. From the amino acid sequence, immunoreactivity, and properties of Bacterial lipopolysaccharide (LPS) the recombinant protein, Intl-3 was considered the intestinal lectin identified by the anti-XEEL antibody. Goblet cells The purified Intl-3 protein could potentially bind to Escherichia coli and its lipopolysaccharides (LPS), and þ Innate immunity to Staphylococcus aureus and its peptidoglycans, depending on Ca2 . In addition, the Intl-3 protein þ Xenopus agglutinated E. coli cells in the presence of Ca2 . Intraperitoneal injection of LPS increased the intestinal and rectal contents of Intl-3 and XCL-1 (or 35K serum lectin) proteins within three days; however, unlike XCL-1, Intl-3 was detectable in neither the sera nor the other tissues regardless of LPS stimulation. Immunohistochemical analyses revealed accumulation of the Intl-3 protein in mucus secretory granules of intestinal goblet cells. The results of this study suggest that Xenopus Intl-3 is involved in the innate immune protection of the digestive tract against bacterial infections. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction expression is reportedly up-regulated in response to infestation with parasitic worms or allergen-induced inflammation Lectins are structurally and functionally diverse proteins that (Pemberton et al., 2004; Datta et al., 2005; Gu et al., 2010). Human recognize carbohydrates and which have attracted the attention of Intl-1, one of the most intensively characterized Intls, has been immunologists because of their roles as pathogen recognition reported to have remarkable selectivity in the recognition of mi- molecules in the innate immune system (Sharon and Lis, 2004). A crobial over mammalian cell surface glycans (Wesener et al., 2015) þ group of Ca2 -dependent lectins that have a conserved fibrinogen- and thereby enhance phagocytosis of bacteria by macrophages like domain was designated as the intelectin (Intl) family, because (Tsuji et al., 2009). In addition, a mouse strain with a genetic defect the human lectin was found in intestinal epithelia (Lee et al., 2001; in Intl-2 production is unable to eliminate parasitic nematodes Tsuji et al., 2001). This group has also been referred to as the X-type effectively, as compared to those having the wild type gene lectin family, as the first lectin was reported in Xenopus laevis oo- (Pemberton et al., 2004). Accordingly, the Intl family lectins are cytes (Wyrick et al., 1974; Lee et al., 2004). Two or three closely considered to act as microbial recognition molecules in the innate related Intls have been identified in various mammalian tissues, immune system. including intestinal epithelia, vascular tissues, mesothelial mucus, Other studies have identified and characterized Intl-1 as a lac- and blood plasma (Lee et al., 2001; Tsuji et al., 2001; French et al., toferrin receptor, a glycosylphosphatidyl inositol (GPI)-anchored 2009; Tsuji et al., 2009; Washimi et al., 2012) and their iron transporter-binding protein on the membrane of the human intestinal brush border (Suzuki et al., 2001; Chatterton et al., 2013). In addition, human Intl-1 has been alternatively designated as E-mail address: [email protected]. http://dx.doi.org/10.1016/j.dci.2016.02.006 0145-305X/© 2016 Elsevier Ltd. All rights reserved. 230 S. Nagata / Developmental and Comparative Immunology 59 (2016) 229e239 omentin, because it has been characterized as one of the adipocy- amphibian phosphate-buffered saline APBS (70% PBS) at a dose of tokines secreted from visceral adipose tissues (Kralisch et al., 2005). 4 mg/g body weight; on day 3 following injection, they were Omentin has been reported to regulate the differentiation and anesthetized, blood was collected by cardiac puncture, and sera function of vascular endothelial cells (Tan et al., 2010; Maruyama were isolated by centrifugation at 3000g for 10 min. The resected et al., 2012) and modify the action of insulin on target cells (Yang tissues of frogs were either frozen in liquid nitrogen and stored et al., 2006). Moreover, human Intl-1 has been suggested to act as at 80 C for sodium dodecyl sulfate-polyacrylamide gel electro- a tumor suppressor in prostate cells (Mogal et al., 2007) and as a phoresis (SDS-PAGE), immersed in an RNA conservation medium modulator of steroidogenesis in ovarian granulosa cells (Cloix et al., (RNAlater; Qiagen, Tokyo, Japan) and stored at 4 C for RNA isola- 2014). Thus, despite their nomenclature, these studies suggest that tion, or fixed with 4% paraformaldehyde in 0.1 M 3-(N-morpholino) mammalian Intls are involved not only in intestinal innate immu- propanesulfonic acid (MOPS) buffer (pH 7.4) for further preparation nity but also in multiple physiological functions of various tissues. of frozen sections. As previously described, bacterial recombinant Intl family proteins have also been reported in various tissues of proteins were used as immunogens to prepare the monoclonal several chordate species, including ascidian plasma (Abe et al., antibodies (mAbs) 5G7 to XEEL, 4C8 to XCL-1, and 4G12 to all 1999), several tissues of amphioxus (Yan et al., 2013), and the known Xenopus Intl family proteins (Nagata, 2005; Nagata et al., epidermal mucus of catfish (Tsutsui et al., 2011). In the amphibian, 2013). X. laevis, expression of six distinct Intl family genes has so far been reported in different adult or embryonic tissues (Nagata et al., 2.3. Purification of Intls 2003; Lee et al., 2004; Ishino et al., 2007; Nagata et al., 2013). Among them, the oocyte cortical granule lectins (XCGL1 and Intestines of 20e28 LPS-stimulated frogs (total wet weight, XCGL2) are major components of oocyte cortical granules and upon 5.1e6.8 g) were homogenized on ice in 30 mL Tris-buffered saline fertilization, are secreted to the extracellular space to form a (TBS) (100 mM NaCl, 20 mM Tris-HCl, pH 7.4) containing 1% Triton fertilization layer that prevents polyspermic fertilization and X-100, 0.5 mM phenylmethylsulfonyl fluoride, and the ethyl- pathogen invasion (Wyrick et al., 1974; Nishihara et al., 1986). The enediaminetetraacetic acid (EDTA)-free protease inhibitor cocktail Xenopus embryonic epidermal lectin (XEEL) is secreted from the (GE Healthcare Japan, Tokyo, Japan), using a Polytron homogenizer epidermis during a limited period of embryonic stages, while the (Kinematica Japan, Tokyo). The homogenates were centrifuged for adaptive immune system is still immature, suggesting a possible 60 min at 20,600g. The supernatants were supplemented with role in the protection of embryos against pathogenic microbes in 300 mL of 1 M CaCl2 and placed on ice for 15 min. The tubes were the environmental water (Nagata, 2005). Recently, XEEL is shown centrifuged again for 60 min at 20,600g and the supernatants to recognize bacterial surface glycans using its carbohydrate bind- were mixed with the same volume of TBS containing 5 mM CaCl2 ing domain that has architecture highly conserved between XEEL (TBS-Ca) and fractionated on a galactose (Gal)-Sepharose column as and human Intl-1 (Wangkanont et al., 2016). In addition, our recent previously described (Nagata et al., 2013). The fractions eluted with study demonstrated that XCL-1 (35K serum lectin) could bind to TBS containing 5 mM EDTA (TBS-EDTA) were pooled and centri- extracellular carbohydrate components of Gram-negative and fuged on a Centricon YM 100 centrifugal filter device (Merck Mil- Gram-positive bacteria, and that intraperitoneal injection of the lipore, Darmstadt, Germany) to enrich high molecular weight bacteria or their cell wall substances induced an acute increase of (>100K) proteins. Protein concentrations of the samples were the serum XCL-1 content (Nagata et al., 2013). The expression of determined using a Bradford assay reagent (Bio-Rad Laboratories, XCL-2 (Intl-1-like lectin) and XCL3 (Intl-2) genes has also been Hercules, CA). reported in various adult tissues, although their physiological roles remain unclear. Collectively, these findings suggest that amphibian 2.4. SDS-PAGE, western blotting, and peptide sequencing Intl family proteins play even broader physiological functions than mammalian Intls. The present study reports two additional mem- Samples were fractionated by SDS-PAGE and the proteins in the bers of the Xenopus Intl family, Intl-3 and Intl-4, both expressed in gel were visualized using a silver staining kit (Kanto Chemicals, the digestive tract. I also present data suggesting that Intl-3 is Tokyo, Japan) or Coomassie brilliant blue. In western blot analyses, involved in antimicrobial innate immunity of the digestive tract of the proteins in gels were transferred to a polyvinylidene fluoride the host. (PVDF) membrane (Bio-Rad) and the lectins on the blots were visualized with the mAbs, as previously described (Nagata, 2005). 2. Materials and methods Chemiluminescence signals of the western blots were quantified as previously described (Nagata et al., 2013), and the data were pre- 2.1. Bacteria, bacterial components, and sugars sented as mean ± S.D. of each group and values of p < 0.05 in the Student's unpaired t-test were accepted as statistically significant. Formaldehyde-fixed Staphylococcus aureus cells (Cat. No. S2014), Protein bands on the blot were excised from the PVDF mem- their lipoteichoic acids (LTA) (Cat. No. L2515), peptidoglycan (PGN) brane and sent to Nippi Inc. (Tokyo, Japan) for determination of the (Cat. No. 77140), and protein A (PrtA) (Cat. No. P6031), and LPS from N-terminal amino acid sequences. Escherichia coli 0111:B4 (Cat. No. L2630) were obtained from Sigma- Aldrich (St. Louis, MO). Formaldehyde-fixed E. coli cells were pre- 2.5. Reverse transcription-polymerase chain reaction (RT-PCR) and pared as previously described (Nagata, 2005; Nagata et al., 2013). cDNA cloning The monosaccharides, disaccharides, and amino sugars were pur- chased from Sigma-Aldrich and Wako Chemicals (Tokyo, Japan). The total RNA fraction free of contaminant genomic DNA was isolated from the intestines using an RNeasy Plus Total RNA Isola- 2.2. Animals and antibodies tion Kit (Qiagen) and single strand cDNAs were synthesized using a PrimeScript 1st strand cDNA synthesis kit (Takara Biotechnologies, Purchase, care and usage of X. laevis were performed with Tokyo, Japan). Using the cDNAs as templates, cDNA fragments standardized protocols (Sive et al., 2000) under the regulations of encoding novel Intls were amplified with the PrimeSTAR Max DNA the Experimental Animal Committee of Japan Women's University. polymerase (Takara) and degenerate oligonucleotide primers Frogs aged 1.5e2 years were injected intraperitoneally with LPS in (Supplementary Table S1). The sequences of primers were deduced S. Nagata / Developmental and Comparative Immunology 59 (2016) 229e239 231 from the highly conserved amino acid sequences among Intl family sections were treated in methanol containing 3% H2O2 for 5 min to proteins (WTLVASVH and DDYKNPGY). The amplified cDNA frag- inhibit endogenous peroxidase activities, and were incubated with ments were cloned into the plasmid vector, and the sequences were the primary mAbs (1:2000), anti-XEEL 5G7, anti-XCL1 4C8, or anti- determined and analyzed with the GENETYX-MAC software (Gen- Xenopus T cell XT-1 (control). In some experiments, the 5G7 mAb etyx Co., Osaka, Japan). From the sequence data obtained, specific was preabsorbed with rIntl-3 before usage. Gal-Sepharose beads primers were designed (Supplementary Table S2) and applied to were incubated for 30 min with the culture supernatant of 293T the rapid amplification of cDNA ends (RACE) procedure using the cells transfected with the vacant vector or the recombinant vector Smarter RACE 50/30 kit (Clontech Laboratory, Mountain View, CA). inserted with Intl-3 cDNA. After washing in TBS-Ca, the beads were The amplified DNA fragments were cloned and ligated to obtain the reincubated with the 5G7 mAb solution (1:1000), centrifuged for cDNA clones containing complete open reading frames. 1 min at 500g, and the supernatants were used as the pre- For RT-PCR assays, total RNA fractions were isolated from absorbed or control mAbs. After incubation with the primary mAbs, various tissues or whole embryos and the single strand cDNAs were the sections were washed in APBS, incubated with goat anti-mouse synthesized as described above. The following transcripts were IgG-horseradish peroxidase (HRPO) conjugate (Santa Cruz Bio- amplified using a HotStarTaq DNA polymerase (Qiagen) and specific technologies, Dallas, TX) as a secondary antibody, and color primers (Supplementary Table S3): XCL-1, XCL-2, XCL3a/b, XEEL, development was conducted in diaminobenzidine (DAB)/H2O2 so- and ornithine decarboxylase (ODC). Amplification was performed lution. Some sections were incubated with fluorescein isothiocya- by 25e30 cycle reactions, the products were electrophoresed on nate (FITC) -labeled anti-mouse IgG antibody as a secondary agarose gels and visualized with ethidium bromide stain. antibody.

2.6. Recombinant Intls 3. Results

The cDNA fragments encoding XEEL, XCL-1, XCL-2, XCL-3a, Intl- 3.1. Isolation and characterization of Intl protein 3, and Intl-4 proteins were amplified using cDNA clones as tem- fi plates and primer pairs speci c to each lectin (Supplementary Preliminary western blot analyses of intestinal extracts of frogs Table S4), and inserted into the pCEP4 plasmid vector (Clontech). showed that the anti-XEEL mAb 5G7 identified protein bands at The recombinant vectors were transfected to the human epithelial about 40 kDa, which are typical sizes of the Intl family lectins. kidney cell line 293T and the culture supernatants or cell lysates Hence, an attempt to purify the proteins from intestinal extracts by were obtained as previously described (Nagata et al., 2013). Re- affinity chromatography on a Gal-Sepharose column was per- fi fi combinant lectins were puri ed by af nity chromatography on a formed, according to the methods previously outlined for the Gal-Sepharose column as described above. isolation of the XEEL and XCL-1 proteins (Nagata, 2005; Nagata et al., 2013). Extracts from 20 to 28 LPS-stimulated frogs were 2.7. Intl-3 binding assay applied on the column and yielded 20.2e32.9 mg of the protein sample. In an SDS-PAGE analysis under reducing conditions, the Lectin binding assays were performed as previously described purified lectin protein exhibited two major bands at 39 and fi (Nagata et al., 2013). The puri ed Intl-3 protein was mixed with a 40.5 kDa, both of which were identified by western blotting using suspension of formaldehyde-fixed E. coli or S. aureus cells, LPS- Sepharose, PGN-Sepharose, or Gal-Sepharose in TBS-Ca and incubated for 30 min at 24 C with continuous rotation. In the competitive binding assays, various saccharides, amino sugars, or bacterial cell surface substances were added to the Intl-3 binding reaction. Following incubation, the bound Intl-3 was examined by western blotting. In the competitive elution assays, Gal-Sepharose beads with bound Intl-3 were incubated with various concentrations of competitors in TBS-Ca and the eluted lectin was examined by western blotting. Lectin-mediated bacterial agglutination assays were performed using recombinant Intl-3 (rIntl-3) and the Intl-3 protein fraction purified from frog intestines. GFP-fluorescent E. coli (GFP-E. coli) was generated by transformation of XL1 MRF' strain E. coli with the plasmid pAcGFP (Clontech) and fixed in paraformaldehyde in TBS. The same volume of GFP-E. coli suspension (OD600 ¼ 0.5) in TBS-Ca and lectin solutions (20 mg/ml) were mixed in wells of a microplate, vibrated for 30 min on a microplate mixer (Sanko Junyaku, Tokyo, Japan) and then incubated for 3 h at room temperature. After incubation, the suspensions were observed under the fluorescent microscope.

2.8. Immunohistochemistry

Digestive tracts were removed from anesthetized frogs, Fig. 1. Purification of Intl proteins. Intl proteins (Intls) were purified from the intestinal dissected into five parts, i.e., stomach; proximal, intermediate, and extracts by affinity chromatography and fractionated along with the extract (Ext) by distal small intestines; and rectum, and fixed overnight in 0.1 M SDS-PAGE under reducing conditions (R) or fractionated alone under non-reducing conditions (NR). Proteins in the gels were visualized by silver staining (Prt), and the MOPS buffer containing 4% paraformaldehyde. The specimens were Intls, by western blotting using the 5G7 mAb (W). The numbers on the left of gels embedded in a resin (OCT compound, Sakura Finetek, Tokyo, Japan) represent protein sizes in kDa. The triangles on the right panel indicate major bands at and the frozen sections were made using a cryomicrotome. The about 420 kDa, 210 kDa, 70 kDa, 35.5 kDa, and 34 kDa. 232 S. Nagata / Developmental and Comparative Immunology 59 (2016) 229e239

Fig. 2. Analyses of Intl-3 and Intl-4 tranlates. (A) Alignment of Intl-3 and Intl-4 with human Intl-1. The entire amino acid sequences are aligned with the human Intl-1 (HIntl-1). Identical amino acids, gaps, and potential N-glycosylation residues are shown as dots, bars, and asterisk, respectively. The underlines and shaded boxes show signal peptides and fibrinogen-like domains, respectively. The amino acids important to carbohydrate recognition are boxed. Inverted color highlights the sequence that corresponds to N-terminal amino acid sequencing of the intestinal lectin. (B) Phylogenetic relationship of Intl family proteins. The tree is constructed by the neighbor-joining method using whole amino acid sequences of Xenopus, amphioxus, lamprey zebrafish, mouse, and human Intls. The numbers at nodes indicate bootstrap values and the scale bar indicates the number of substitutions per residue. GenBank accession numbers for the amino acid sequences are as follows: human Intl-1 (HIntl-1, AB036706); human Intl-2 (HIntl-2, NM_080878); X. laevis XEEL (AB105372); XCL-1 (AB061238); XCL-2 (AB061239); XCGL-1 (X82626); XCGL-2 (AAI70085); zebrafish Intl-1 (ZIntl-1, ACC62155.1); ZIntl-2 (ACC62157.1); and ZIntl-3 (ACC62156.1). the 5G7 mAb (Fig. 1). Analysis of the protein under non-reducing performed with the Basic Local Alignment Search Tool (BLAST) on conditions showed a major protein band at about 420 kDa and the X. laevis protein database of the National Center for Biotech- minor bands at 210, 70, 34, and 35.5 kDa that reacted with the 5G7 nology Information (NCBI) webpage (http://blast.ncbi.nlm.nih.gov/ mAb. This finding suggests that the major form of the lectin protein Blast.cgi?PROGRAM¼blastp&PAGE_TYPE¼BlastSearch&LINK_ is a dodecamer (12-mer) of subunit peptides that exhibit apparent LOC¼blasthome). The search returned several similar sequences, sizes of 34 and 35.5 kDa under non-reducing conditions. The dif- including XEEL and other Intl family proteins but no identical ferences in apparent sizes of the subunit peptides under reducing matches, suggesting novelty of the purified lectin protein. and non-reducing conditions were probably due to the protein folding involving intra-polypeptide disulfide bridges. 3.2. Cloning and analysis of Intl cDNA Strips of the 39 and 40.5 kDa peptide bands were cut out of the PVDF membrane blot and the N-terminal amino acid sequences To clone the cDNA encoding the novel lectin protein in frog in- were determined. The resulting sequences of two peptides were testines, RT-PCR/RACE procedures were applied using degenerate identical, REXDQASVSE, where X was an unidentified residue. Us- oligonucleotide primers, the sequences of which were determined ing this amino acid sequence as a query, a protein search was based on the highly conserved amino acid sequences among known S. Nagata / Developmental and Comparative Immunology 59 (2016) 229e239 233

To estimate the evolutionary relationships among Intl-3, Intl-4, and the Intl family proteins in Xenopus, amphioxus, lamprey, zebrafish, mice, and humans, a neighbor-joining tree was constructed (Fig. 2B). Xenopus, mouse, and human Intls were split into two main branches: one branch contained Xenopus Intl-3, XEEL, Intl-4, XCL-2, and mouse and human Intls; the other branch contained the remaining four Xenopus Intls. This suggests that the expansion of the Intl family occurred before divergence of these species and mammalian Intls evolved from one of these branches. Search for the Intl-3 and Intl-4 genes in the Xenbase J-strain X. laevis genome database 9.1, identified single genome sequences at chr7S:24354410-24366743 and chr8L:99454479-99444250, respectively. To characterize the protein encoded by the isolated Intl-3 cDNA, the expression vector containing the entire coding region was transfected to human epithelial kidney cell line, 293T cells and the rIntl-3 protein was affinity-purified from the culture supernatants Fig. 3. Characterization of Intl-3 protein. (A) SDS-PAGE and Western blot analyses of using a Gal-Sepharose column. When the purified rIntl-3 protein rIntl-3. The purified rIntl-3 fraction was separated by SDS-PGE under reducing (5% 2- ME), partially reducing (0.1% 2-ME), or non-reducing (0% 2-ME) conditions and the was analyzed by SDS-PAGE under non-reducing conditions, the proteins were visualized by silver staining (Prt), and the rIntl-3, by western blotting rIntl-3 protein was found as a major band at about 420 kDa and a (W). The triangles indicate the rIntl-3 protein and the asterisks, the non-relevant minor band at 70 kDa that were both reactive to the 5G7 mAb proteins. (B) Intl-3 is the predominant Intl in Xenopus intestines. Intestine extracts (Fig. 3A, left lane). In contrast, under reducing conditions (right and rXEEL, rXCL-1, rXCL-2, and rXCL-3a proteins were examined by western blotting fi using the 5G7 or 4G12 mAbs. (C) N-glycosylation of Intl-3. The purified Intls, rIntl-3, lane), it was identi ed as a predominant 39 kDa protein. Under and rXCL-2 were incubated with (þ) or without () protein N-glycosidase (PNGase) partially reducing conditions (middle lane), two major rIntl-3 and changes in the size were examined by western blotting. bands appeared at 35e39 kDa and 78 kDa. These results indicate that the rIntl-3 protein consisted of a major 12-mer and a minor dimer of subunit peptides encoded by the cloned cDNA, suggesting Intl family proteins. Sequence analyses of the isolated cDNA clones that the Xenopus Intl-3 protein could be produced from a single revealed those encoding the known Intl family proteins, XCL-1, gene. XCL-2, and XCL-3; a novel splicing isoform of XCL-3, designated Similarly, the recombinant Intl family proteins were produced XCL-3a (Supplementary Fig. S1); and two novel Intls, now desig- by 293T cells following transfection of the recombinant expression nated Intl-3 and Intl-4 (Supplementary Figs. S2 and S3). Fig. 2A il- vectors and compared with Intls purified from the frog intestinal lustrates a comparison of the amino acid sequences of deduced Intl- extracts by western blotting. The results showed that among the 3 and Intl-4 translates with that of the human Intl-1, the molecular recombinant Intl family proteins recognized by the 4G12 mAb, the structure of which has been best correlated with its carbohydrate rXEEL, rXCL-2, and rIntl-3 proteins were recognized by the 5G7 recognition properties (Wesener et al., 2015). The Intl-3 and Intl-4 mAb (Fig. 3B) and that Intl-3 is predominant among the intestinal translates consisted of 340 and 335 amino acids that showed 67% Intls. When the purified intestinal Intl-3 was treated with N- and 63% sequence identity with that of human Intl-1, respectively. glycosidase, the sizes were reduced from 39 kDa and 40.5 kDa to Both had a possible signal peptide, N-glycosylation residue, and 36 kDa and 37.5 kDa, respectively (Fig. 3C), indicating that the fi brinogen-like domain. In the amino acid residues forming the subunit peptides were N-glycosylated. The same treatment reduced carbohydrate recognition structure in human Intl-1 (Asn-243, Glu- the size of rIntl-3 from 39 kDa to 36 kDa, whereas it did not affect 244, Asn-260, Glu-262, His-263, Glu-274, Trp-288, and Tyr-297), the size of the rXCL-2 protein at 33/34 kDa as estimated from its both Intl-3 and Intl-4 had minor amino acid substitutions that amino acid sequence, which was deduced from cDNA possibly did not alter the basic carbohydrate recognition structure (Supplementary Fig. S4). The 40.5 kDa subunit that was absent in (Wesener et al., 2015). The Intl-3 and Intl-4 translates showed the rXIntl-3 protein might be a splicing isoform or a product of e e 61 79% and 59 68% amino acid sequence identities to known alternative post-translational modification of the 39 kDa peptide. Xenopus Intl family proteins, respectively (Supplementary Fig. S4). Notably, the Intl-3 translate contained an amino acid sequence, 3.3. Expression of Intl-3 and Intl-4 in adult tissues and embryos RECDQASVSE, following the signal peptide that showed a perfect match with the N-terminal amino acid sequences of the 39 and To examine Intl-3 and Intl-4 expression in adult tissues and 40.5 kDa peptides identified in intestines with 5G7 mAb. This fact developing embryos, RT-PCR assays were performed using specific indicates that Intl-3 was the major Intl in the intestines of X. laevis. primers. Intl-3 expression was strong in the intestine; at

Fig. 4. Expression of Intl-3 and Intl-4 in adult tissues and embryos. RT-PCR assays were performed using total RNA fractions isolated from the tissues indicated and embryos (stage 35 embryos on the left panel). The primers used and the expected sizes of the cDNA fragments ampliﬁed are shown in Supplementary Table S1. The assays without RT reaction resulted in no DNA ampliﬁcation. ODC was a loading control. 234 S. Nagata / Developmental and Comparative Immunology 59 (2016) 229e239

binding of Intl-3 to bacterial cells, LPS, and PGN is dependent on the þ presence of Ca2 . When Intl-3 was incubated with fixed E. coli cells in TBS-Ca containing various competitors, the binding was blocked most effectively with xylose (Xyl), and partially with LPS and PGN (Fig. 5B). However, the binding was not affected by glucose (Glc), LTA, PrtA, N-acetyl-N-galactosamine (GalNAc), and N-acetyl-N- glucosamine (GlcNAc). Intl-3 was incubated with Gal-Sepharose in TBS-Ca, and after washing, the beads were incubated in TBS-EDTA or TBS-Ca containing 100 mM of various saccharides or amino sugars. The competitively eluted Intl-3 was examined by western blotting and the intensities of the 39e40.5 KDa protein bands on the blot were quantified by densitometry. The bound Intl-3 was most efficiently released with EDTA and the pentoses Xyl and ribose (Rib), whereas arabinose (Ara) and the hexose Gal were less effective (Fig. 5C). The other hexoses such as fructose (Frc), Glc, and mannose (Man), disaccharides such as lactose (Lac), maltose (Mal), and melibiose (Mel), and amino sugars (GalNAc and GlcNAc) were even less competitive for elution. To examine whether the Intl-3 binding could cause bacterial agglutination, fixed GFP-E. coli cells were incubated with the purified Intl-3 protein or rIntl-3 (both 10 mg/ml) and observed under a fluorescence microscope. The results showed that as compared with the control (Fig. 6A), both the purified Intl-3 and rIntl-3 preparations agglutinated the bacteria in TBS-Ca (Fig. 6B and E). This agglutination was abolished by addition of EDTA (Fig. 6C and F) or Xyl (Fig. 6D and G).

3.5. Injection of LPS increases intestinal Intl-3 content

As intraperitoneal injection of LPS has shown to increase XCL-1 contents in the serum and spleen (Nagata et al., 2013), whether or not it would also change Intl-3 content in the digestive tract was investigated. Three days after the LPS injection, digestive tracts were isolated, divided into 5 sections (Fig. 7A), and the extract of þ Fig. 5. Binding of Intl-3 to bacteria and saccharides. (A) Ca2 -dependent binding of each section was examined by western blotting. In control APBS- Intl-3 to bacteria. Purified Intl-3 was incubated with either E. coli or S. aureus (S. aur) injected frogs, Intl-3 content was highest in the intestinal section cells, LPS-Sepharose or PGN-Sepharose in TBS, or TBS containing either 5 mM MgCl2, closest to the stomach and lowest near the distal intestine, whereas CaCl , or 5 mM CaCl and 10 mM EDTA (CaCl þ EDTA). After washing, the bound Intl-3 2 2 2 it was undetectable in the stomach and rectum (Fig. 7B, upper was detected by western blotting. (B) Competitive binding assay of Intl-3. Purified Intl- 3 was incubated with fixed E. coli cells in TBS-Ca containing 2 mg/mL, 0.4 mg/mL, or panels). The injection of LPS increased Intl-3 contents throughout 0.08 mg/mL of competitors, and the bound Intl-3 was examined by western blotting. the intestines and rectum. Quantitative analyses of the blots pre- (C) Competitive elution assay of Intl-3. Purified Intl-3 was adsorbed to Gal-Sepharose, pared from four APBS-injected and four LPS-injected frogs revealed and incubated in TBS containing 10 mM EDTA (EDTA) or 10 mM CaCl2 plus 100 mM of 2.5e4 fold increases of Intl-3 contents in the intestinal and rectal the indicated saccharides. Intl-3 in the eluates was detected by western blotting (inset) and quantified by densitometry (histogram). Each column represents the Intl-3 sections (Fig. 7B, histogram). Although XCL-1 was barely detectable quantity relative to that eluted with EDTA. The figure is representative of three inde- in control frogs, following LPS stimulation, it was identified in all pendent experiments. the sections of the digestive tract. To examine the expression levels of Intl transcripts, total RNA fractions were isolated from the intestines of frogs and RT-PCR assays were performed. The expression intermediate levels in the brain, testis, and stomach; and weak in of XCL-1, XCL-3, and Intl-3 exhibited definite increases following the liver, lung, and kidney (Fig. 4). In contrast, Intl-4 expression was LPS stimulation, whereas the expression of XEEL, XCL-2, and Intl-4 strong in the liver, lung, and kidney, and observed at relatively appeared to remain unchanged (Fig. 7C). lower levels in the ovary, testis, stomach, and intestine. Both Intl-3 Intl-3 contents in the sera and spleens of five APBS-injected and and Intl-4 expression were barely detectable in other tissues and five LPS-injected frogs were compared. The results showed that embryos up to late tailbud stage 35, when XEEL expression reached LPS-stimulation increased XCL-1 contents in the serum and spleen high levels. (Fig. 8A), confirming a previous report (Nagata et al., 2013). How- ever, Intl-3 was undetectable in both serum and spleen, regardless 3.4. Binding of Intl-3 to bacteria and saccharides of LPS injection. When similar analyses were expanded to include other tissues, LPS stimulation resulted in increases in XCL-1 con- The purified Intl-3 protein was incubated with either fixed E. coli tents of the liver, lung, heart, muscle, in addition to the spleen and or S. aureus cells, LPS-Sepharose or PGN-Sepharose in TBS, TBS-Ca, serum (Fig. 8B). In contrast, the Intl-3 protein was detected only in TBS containing 5 mM MgCl2, or TBS-Ca containing 10 mM EDTA. the intestine regardless of LPS stimulation, although mRNA was After washing the bacteria and Sepharose beads, the bound Intl-3 found in the brain, testis, stomach, and liver, as well as the intestine was detected by western blotting (Fig. 5A). The results showed (Fig. 4), suggesting that the tissue Intl-3 contents might be regu- that strong Intl-3 protein bands were seen only when incubation lated by not only transcription, but also by translation or post- was performed in TBS containing CaCl2, suggesting that efficient translational modification steps. S. Nagata / Developmental and Comparative Immunology 59 (2016) 229e239 235

Fig. 6. Intl-3-mediated agglutination of E. coli. Suspensions of fixed GFP-E. coli cells were incubated in TBS-Ca (A), or mixed and incubated with the purified Intl-3 (BeD) or rIntl-3 (EeG) in TBS-Ca (B, E), TBS-Ca containing 10 mM EDTA (C, F), or TBS-Ca containing 100 mM Xyl (D, G). After incubation, GFP-E. coli cells were observed under a fluorescence microscope. The pictures show representatives of those obtained in duplicate experiments using different protein and GFP-E. coli preparations. Scale bars, 20 mm.

3.6. Immunohistochemical localization of Intl-3 members to the list of X. laevis Intl family lectins and present previously unidentified counterparts of Intls expressed in the in- Immunohistochemical and immunofluorescent examination testines of mammals (Komiya et al., 1998; Tsuji et al., 2001; French using the 5G7 mAb were performed to localize the Intl-3 protein in et al., 2009), fish (Russell et al., 2008), and amphioxus (Yan et al., frozen transverse sections of the digestive tract. The Intl-3 protein 2012). Intl-3 was shown to be the major Intl in Xenopus digestive was not observed in the stomach (Fig. 9A); however, in comparison tracts, by virtue of its cross-reactivity with the anti-XEEL mAb 5G7. to the control section (Fig. 9I and J), it was clearly visible in the Although Intl-4 transcripts were distributed among various tissues, epithelium of intestinal and rectal villi (Fig. 9BeF). Immunoreac- analyses of the Intl-4 protein were not performed in the present tivity was stronger in the anterior intestine (Fig. 9B) and weaker study because the specific probe was unavailable. The Intl-3 protein toward the distal portion of the rectum (Fig. 9C), which appeared to is highly homologous to other chordate Intl family proteins in terms be consistent with the results of western blot analyses (Fig. 7B). of overall amino acid sequences and basic structural characteristics, Detailed observation showed more intense Intl-3 staining at the i.e., an oligomeric (probably 12-meric) structure consisting of N- base of villi (Fig. 9D) in comparison to the apical portions (Fig. 9E). glycosylated subunit peptides containing a conserved fibrinogen- Higher magnification of the labeled cells revealed an accumulation like domain (Lee et al., 2004). Human Intl-1 is a trimer of subunit of immunoreactivity in apical granules of the goblet cells and weak peptides that are linked by disulfide bonds with Cys-31 and Cys-46 labeling in the cytoplasm (Fig. 9D). In the intestines of LPS- in the N-terminal region (Tsuji et al., 2009; Wesener et al., 2015). stimulated frogs, stronger Intl-3 immunoreactivity was observed Xenopus Intl-3 probably uses Cys-21, Cys-39, or Cys-68 for oligo- in granules of the goblet cells (Fig. 9F). Preabsorption of the 5G7 merization within a similar region. mAb with rIntl-3 abolished the staining of granules and cytoplasm Consistent with a previous report on XCL-1 (Nagata et al., 2013), of the goblet cells (Fig. 9G), whereas the preabsorption control gave Intl-3 binds to the surface of bacterial cells depending on the þ the staining similar to the non-preabsorbed mAb (Fig. 9H), sup- presence of Ca2 and this binding is blocked by saccharides such as porting that the 5G7 mAb visualized Intl-3 on the intestinal sec- Xyl. In addition, similar to XEEL (Wangkanont et al., 2016), the Intl- tions. Staining with the anti-XCL-1 mAb 4C8 or the control mAb XT- 3 binding caused agglutination of the bacterial cells. These results 1 yielded no noticeable labeling in the intestine of a LPS non- suggest that Intl-3 crosslinks bacterial surface glycans through its stimulated frog (Fig. 9I and J). Similar to the immunohistochem- multivalent carbohydrate binding. Immunohistochemical analysis ical assay, the 5G7 mAb specifically visualized the Intl-3 immuno- demonstrated an accumulation of Intl-3 protein in secretory mu- reactivity in immunofluorescent staining (Fig. 9K and L). In this cous granules of intestinal and rectal goblet cells and its levels staining, multiple fluorescent granules were clearly visible in apical increased shortly after intraperitoneal injection of LPS. Thus, as has cytoplasm of the goblet cells (Fig. 9K). been suggested with mammalian Intls, Intl-3 is most likely involved in the innate immune function of mucus secretions that protect the 4. Discussion walls of the digestive tract against pathogenic bacteria and para- sites (Komiya et al., 1998; Pemberton et al., 2004). The present findings of Intl-3 and Intl-4 transcripts add novel Mammalian Intl expression has been shown to increase in 236 S. Nagata / Developmental and Comparative Immunology 59 (2016) 229e239

Fig. 8. Changes in tissue XCL-1 and XIntl-3 contents following LPS stimulation. Frogs were intraperitoneally injected with APBS or LPS, and the sera and tissues indicated were harvested three days later. XCL-1 and Intl-3 proteins were examined by western blotting. (A) LPS injection caused increases of Intl-3 contents in the intestine, but not in the serum and spleen. Intl-3 and XCL-1 contents of the sera (Ser), spleens (Spl), and intestines (division 2) were examined in five APBS-injected and five LPS-injected frogs. Positive controls for the respective mAbs were rXCL-1 and purified Intl-3. The IgY heavy chain and actin were the loading controls. (B) Effects of LPS injection on Intl-3 and XCL-1 contents of various tissues. The figure shows representative blots of trip- licate analyses showing similar results.

from local inflammation sites. Increasing knowledge has been accumulated on detailed signaling mechanisms in intestinal Fig. 7. LPS stimulates Intl-3 production in intestines. Frogs were injected intraperito- epithelia that are triggered by the recognition of pathogenic mi- neally with APBS or LPS, and their digestive tracts were dissected three days later and crobes and lead to the induction of inflammatory and innate im- subjected to western blotting or RT-PCR analyses. (A) A picture of the digestive tract showing divisions 1e5 for preparation of samples for SDS-PAGE and RT-PCR: 1, mune responses (Wells et al., 2011). In particular, a recent study stomach; 2e4, small intestine; 5, rectum. (B) LPS stimulation increases intestinal XIntl- (Courth et al., 2015) reported that monocyte-derived cytokines are 3 contents. Western blot analysis of XCL-1 and Intl-3 contents in digestive tract di- potential stimulators of defensin gene expression in intestinal e þ e visions 1 5 of LPS-injected (LPS ) or APBS injected (LPS ) frogs. Actin was a loading Paneth cells that are known to produce Intls in mice and humans control. Intensities of Intl-3 bands on western blots were quantified by densitometry and the relative values were presented as a histogram. Each column shows the mean (Komiya et al., 1998; Wrackmeyer et al., 2006). Since the present value of four frogs with the standard deviation (bar). *, P < 0.05 for LPS-injected versus data show that variation in the levels of Intl family gene transcripts APBS-injected frogs. (C) LPS stimulation increases intestinal Intl-3 mRNA contents. does not necessarily parallel with the levels of corresponding Total RNA fractions were isolated from small intestine division 2, of 4 APBS-injected proteins, tissue or cellular Intl contents are probably regulated at and 4 LPS-injected frogs, and the mRNAs of XEEL, XCL-1, XCL-2, XCL-3a/b Intl-3, and various steps, including transcription, translation, and post- Intl-4 were examined by RT-PCR. Ornithine decarboxylase (ODC) was a loading control. translational modification. A recent study employing X-ray crystallography established the intestinal and airway epithelia following parasitic infection fine structural basis of carbohydrate recognition by the human Intl- (Pemberton et al., 2004; Datta et al., 2005) and allergen-induced 1(Wesener et al., 2015). For carbohydrate binding, human Intl-1 inflammation (Gu et al., 2010; Kerr et al., 2014), respectively. In uses the C-terminal region of each subunit peptide, but does not the present study, intraperitoneal injection of LPS, a prominent use the fibrinogen-like domain as had been previously assumed inflammogenic substance from the outer membrane of Gram- (Lee et al., 2004). In the C-terminal region, several critical amino 2þ negative bacteria, caused increased production of Xenopus Intl-3 acids and a Ca coordinate to form a flexible target recognition protein in the intestinal goblet cells and XCL-1 protein in the structure that interacts to a terminal acyclic 1,2-diol, which is a spleen, serum, and several other tissues (Nagata et al., 2013). common feature unique to bacterial glycans. This provides Although little is known about the signals regulating tissue Intl remarkable selectivity of the human Intl-1 in the recognition of levels, these findings suggest that Intl production is stimulated by microbial over mammalian cell surface glycans. The C-terminal cytokines or chemokines secreted by inflammatory cells migrating region of Xenopus Intl-3, as well as those of XEEL and XCL-1, are S. Nagata / Developmental and Comparative Immunology 59 (2016) 229e239 237

Fig. 9. Immunohistochemical and immunofluorescent localization of Intl-3. Frozen sections of stomach (A), small intestine (B, DeL; division 2 in Fig. 2A) and rectum (C) of a normal (AeE, GeJ) or a LPS-stimulated frog (F, I, K, L) were processed for immunohistochemical (AeJ) or immunofluorescent staining (K, L). First antibodies used were as follows: 5G7 mAb without preabsorption (AeF, K), 5G7 mA preabsorbed with rIntl-3 (G), 5G7 mAb preabsorption control (H), anti-XCL1 4C8 (I), and unrelated mouse IgG (J, L). The second antibodies were HRPO-anti-mouse IgG (AeJ) or FITC-anti-mouse IgG (K, L). The sections stained by immunofluorescence were treated with the 40,6-diamidino-2-phenylindole (DAPI) stain for DNA (K, L). highly homologous to that of human Intl-1 and functionally capacity for multivalent binding, detailed functions of Intl-3 and conserve critical amino acids. These facts are consistent with the other intestinal Intls are yet to be determined. Following secretion findings that the profiles of carbohydrate recognition specificity of from the epithelia, Intl-3 may entrap and immobilize microbes on these Xenopus lectins are similar to those of the human Intl-1, i.e., the intestinal mucosa to prevent them from making contact with they bind to pentoses such as Xyl, Rib, and Ara with a higher affinity the surface of epithelial cells. On the other hand, if microbial in- than they do to the hexoses Frc, Gal, and Glc, or to the amino sugars vasion occurs beneath the epithelia, Intl-3 may act as a tag that GalNAc and GlcNAc (Tsuji et al., 2001; Nagata, 2005; Nagata et al., would facilitate the clearance of tagged microbes by the activation 2013). Thus, Xenopus Intl-3, as well as XCL-1, XEEL, and human of phagocytic macrophages (Tsuji et al., 2009). Alternatively, by Intl-1, may function as microbial recognition molecules in the analogy with XCGL, which aggregates carbohydrate chains in the innate immune system. egg jelly coat and forms a layer that is impenetrable to sperm Despite its putative selectivity in microbial recognition and (Wyrick et al., 1974), Intl-3 may crosslink carbohydrate chains of 238 S. Nagata / Developmental and Comparative Immunology 59 (2016) 229e239 mucus to form a dense barrier that prevents microbes from pene- induced steroidogenesis through NAMPT. Biol. Reprod. 91 (50), 51. € trating the mucosa. Courth, L.F., Ostaff, M.J., Mailander-Sanchez, D., Malek, N.P., Stange, E.F., Wehkamp, J., 2015. Crohn's disease-derived monocytes fail to induce Paneth The human Intl-1 (omentin) reportedly regulates vascular cell defensins. Proc. Natl. Acad. Sci. U. S. A 112, 14000e14005. endothelial cell function (Maruyama et al., 2012) and steroido- Datta, R., deSchoolmeester, M.L., Hedeler, C., Paton, N.W., Brass, A.M., Else, K.J., 2005. fi genesis by ovarian granulosa cells (Cloix et al., 2014). It has also Identi cation of novel genes in intestinal tissue that are regulated after infection with an intestinal nematode parasite. Infect. Immun. 73, 4025e4033. been suggested that human Intl-1 modulates the action of insulin French, A.T., Knight, P.A., Smith, W.D., Pate, J.A., Miller, H.R., Pemberton, A.D., 2009. on target cells (Yang et al., 2006), and has tumor suppressor activity Expression of three intelectins in sheep and response to a Th2 environment. (Mogal et al., 2007). In the aforementioned studies, recombinant Vet. Res. 40, 53. Gu, N., Kang, G., Jin, C., Xu, Y., Zhang, Z., Erle, D.J., Zhen, G., 2010. Intelectin is Intl-1 was expressed in vivo, or added to the cell culture in vitro, required for IL-13-induced monocyte chemotactic protein-1 and -3 expression and the biochemical or morphological changes were examined in in lung epithelial cells and promotes allergic airway inflammation. Am. J. the putative target tissues or cells. The receptors for Intl-1, however, Physiol. Lung Cell. Mol. Physiol. 298, L290eL296. fi Ishino, T., Kunieda, T., Natori, S., Sekimizu, K., Kubo, T., 2007. Identification of novel have not been identi ed on its target cells, and it is unknown members of the Xenopus Ca2þ -dependent lectin family and analysis of their whether the action of Intl-1 is dependent on carbohydrate recog- gene expression during tail regeneration and development. J. Biochem. 141, nition. Other studies have reported that human Intl-1, a designated 479e488. lactoferrin receptor and GPI-anchored membrane protein, could Kerr, S.C., Carrington, S.D., Oscarson, S., Gallagher, M.E., Solon, M., Yuan, S., Ahn, J.N., Dougherty, R.H., Finkbeiner, W.E., Peters, M.C., Fahy, J.V., 2014. Intelectin-1 is a bind and transport lactoferrin across the cell membrane prominent protein constituent of pathologic mucus associated with eosino- (Wrackmeyer et al., 2006; Suzuki et al., 2008; Akiyama et al., 2013). philic airway inflammation in asthma. Am. J. Respir. Crit. Care Med. 189, e These studies have also not been able to correlate lactoferrin 1005 1007. Komiya, T., Tanigawa, Y., Hirohashi, S., 1998. Cloning of the novel gene intelectin, transportation by Intl-1 with carbohydrate recognition activity. which is expressed in intestinal paneth cells in mice. Biochem. Biophys. Res. Further studies should focus on the molecular mechanisms Commun. 251, 759e762. involved in the diverse physiological functions of Intls, particularly Kralisch, S., Klein, J., Bluher, M., Paschke, R., Stumvoll, M., Fasshauer, M., 2005. Therapeutic perspectives of adipocytokines. Expert Opin. Pharmacother. 6, the putative roles and characteristics of carbohydrate recognition. 863e872. Xenopus Intl-3 is secreted from intestinal epithelia and is Lee, J.K., Schnee, J., Pang, M., Wolfert, M., Baum, L.G., Moremen, K.W., Pierce, M., possibly assimilated into the overlaying mucosa, whereas XCL-1 is 2001. Human homologs of the Xenopus oocyte cortical granule lectin XL35. Glycobiology 11, 65e73. secreted into the circulation. In contrast, human Intl-1 is present in Lee, J.K., Baum, L.G., Moremen, K., Pierce, M., 2004. The X-lectins: a new family with both intestinal secretions and blood plasma (Tsuji et al., 2009), homology to the Xenopus laevis oocyte lectin XL-35. Glycoconj. J. 21, 443e450. suggesting that the physiological roles of human Intl-1 may be Maruyama, S., Shibata, R., Kikuchi, R., Izumiya, Y., Rokutanda, T., Araki, S., Kataoka, Y., Ohashi, K., Daida, H., Kihara, S., Ogawa, H., Murohara, T., Ouchi, N., similar to those of both Intl-3 and XCL-1 in Xenopus. In addition, as 2012. Fat-derived factor omentin stimulates endothelial cell function and reported thus far, XCGL and XEEL are the lectins unique to Xenopus, ischemia-induced revascularization via endothelial nitric oxide synthase- suggesting that they might have evolved through functional dependent mechanism. J. Biol. Chem. 287, 408e417. fi divergence of the Intl family, for adaptation to the environment in Mogal, A.P., van der Meer, R., Crooke, P.S., Abdulkadir, S.A., 2007. Haploinsuf cient prostate tumor suppression by Nkx3.1: a role for chromatin accessibility in which fertilization and development of the frog occurs. These dosage-sensitive gene regulation. J. Biol. 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1 AGTACATGGGGCCGATTGACGTGGGTGGAGCAAAAGGTCTATACTATATGGGATATCACAGATTAATTTCTCTGCATCGGTGAACCCATCTGAACTGATG 100

101 CAAACACAAGTCAAATTCATCCGGATCCAATTAACTAACCAACAATATGTTTTTGCACAGTCTGGTGGTTCTACCCTTGCTCATTATTGAGGGAAGCTCT 200 M F L H S L V V L P L L I I E G S S

201 GCTGGGTGTGACCATAGCTCCCTTCATCAAAAGAAATTAAAGATCCTCAGTCTTCTGGCAGGTTGGGAAGATGATAAATCATACGTTGCTAATCCCTTGT 300 A G C D H S S L H Q K K L K I L S L L A G W E D D K S Y V A N P L W

301 GGTCCCAAAACCCCAGAGGAGACAATGGCATATACAGGAGCTGCATGGAAATCAGGAAATTGAACAATAATGCTGAAGATGGAATCTACACACTGAGAAC 400 S Q N P R G D N G I Y R S C M E I R K L N N N A E D G I Y T L R T

401 TGGAGGTGGGATCTACTACCAGACATTCTGTGACATGACAACTGATGGTGGAGGCTGGACCTTAGTGGCCAGTGTCCATGAGAACAACATGTTTGGCAAG 500 G G G I Y Y Q T F C D M T T D G G G W T L V A S V H E N N M F G K

501 TGCACAGTTGGAGACCGATGGACCAGTCAACAGGGAAACGTACCCAACTACCCAGCAGGAGATGGTAACTGGGCAAACGATGCCACGTTTGGGTTACCTG 600 C T V G D R W T S Q Q G N V P N Y P A G D G N W A N D A T F G L P D

601 ATGGAGCTACCAGTGATGACTACAAGAATCCAGGCTATTACGATATCATTTCCAGTAATTTGGGATTGTGGCATGTGCCAAACAACACACCATTTTCCCA 700 G A T S D D Y K N P G Y Y D I I S S N L G L W H V P N N T P F S H

701 CTGGAGGAATAACTCACTGCTAAGGTATCACACACAGAATGGCTTCTTTTCTGCTGAAGGTGGGAATCTCTTCCATCTGTACCAGAAATATCCACTAAAG 800 W R N N S L L R Y H T Q N G F F S A E G G N L F H L Y Q K Y P L K

801 TTTGGTGCTGGAACCTGCCCAAAAGACAACGGACCTGCTGTGCCAGTTGCGTATGATTTGGGAAATCCTGACATAGCCACTGGTTACTTCTCTCCAGATG 900 F G A G T C P K D N G P A V P V A Y D L G N P D I A T G Y F S P D A

901 CTAGAGGTATGTTTTCTGCAGGATTTGTTCAGTTCCGAGTGTTTAATACTGAAACAGCAGCACTGGCTCTCTGTCCAGGAGTAAAGATGACTACCTGCAA 1000 R G M F S A G F V Q F R V F N T E T A A L A L C P G V K M T T C N

1001 CTCAGAACATTTCTGCATAGGAGGTGGGGGCTATTTTCCTGAAGGAAATCCCAGACAGTGTGGAGATTTTACAGCCTTTGATTGGGATGGTTATGGGGAA 1100 S E H F C I G G G G Y F P E G N P R Q C G D F T A F D W D G Y G E

1101 CATAAAGGTGTGAGTATGAGTAAGGAAATGACAGAGGCTGCCGTGCTGCTCTTCTACCGCTGATTTATTTTCAAGGCACAACAACTTACAGATCTCTAGC 1200 H K G V S M S K E M T E A A V L L F Y R *

1201 ATAAAGCTGCTTTCATTTATATACAAACTCGACTGTTGGATCTAGGGTGGGTTTAACATAACATACTAATTTCCAATATTATATGGATATCTTATATATA 1300 1301 TCTGTACATGGTCATGTAATATGTAATTTAAAGAGACAATGTACCTATATGTCAAAAATACTGTACCGTATAGGATCTGTTATCCAGAAACCCATTATCC 1400 1401 AGAAAGCTCTGAATTACAGGAAGGCCATCTCCCAAAGACTCCATTTTAATCAAATAATGCAGATTTTTAAAAATGATTTCCTATTTCTCTATAATAATAA 1500 1501 AACAGTACCTTTTACTTGATCCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 1552

Supplementary Fig. S1. Translation of XCL-3a cDNA. The underline, rectangular frame and N in

black box represent a signal sequence, fibrinogen-like domain and possible N-glycosylation residue,

respectively. The shaded box represents the nucleotide and amino acid sequences that are spliced out

in the XCL-3b transcript and translate.

1 CAGTTATTAGATTATTAATTATTATATGAGCATATGACTAATTTCTAACATTTCAATGCAATTTAGAAATACTTTCTACTTTATCTTTCAGAGCAAGATG 100 M

101 TTGGCCTACGCACTAATTATTTTTGTCTTAGTATTACCAAGGAGCAATGCTAGAGAATGCGACCAAGCATCAGTTTCTGAAAAGAAGCAGAGAATTCTGA 200 L A Y A L I I F V L V L P R S N A R E C D Q A S V S E K K Q R I L N

201 ACCTTTTGGCATGCTGGACAGAGGATATGGCAGACAGCAGCAGCCCTGGCAGTTCAGGTGGCTTTCCAACTGGCGGCTATGGCTATGGATACAGGAGCTG 300 L L A C W T E D M A D S S S P G S S G G F P T G G Y G Y G Y R S C

301 CATGGAGATCAAAAAGTCAGACTCAGCAGCCAAAGATGGCATATACTCGTTGACCACTGAACAAGGTGAGGCCTATCAAACTTTCTGTGACATGACAACT 400 M E I K K S D S A A K D G I Y S L T T E Q G E A Y Q T F C D M T T

401 AACGGGGGAGGATGGACTTTGGTGGCCAGTGTCCACGAGAATAACCTGTTTGGGAAGTGCACTGAGGGTGATCGCTGGTCCTCACAGCAAGGAAATAATA 500 N G G G W T L V A S V H E N N L F G K C T E G D R W S S Q Q G N N I

501 TACAAAATCCAGGAGGTGATGGCAACTGGGCAAACTATGCCACCTTTGGTTTACCTGAAGGAGCCACAAGTGATGACTATAAGAATCCTGGCTATTATGA 600 Q N P G G D G N W A N Y A T F G L P E G A T S D D Y K N P G Y Y D

601 CATTGAGGCCAAGAATGTGGCTGTTTGGCATGTGCCCAATAAGACACCAATGGTTATGTGGAGGAATTCCTCAATTCTGAGATATCGTACACAGAACGGC 700 I E A K N V A V W H V P N K T P M V M W R N S S I L R Y R T Q N G

701 TTCCTTACTGAAGAAGGAGGAAATCTATTTCAACTTTACAAGAAATATCCTGTGAAATATGGTGTTGGAAAATGCTTAGGAGACAATGGACCTGCTGAGC 800 F L T E E G G N L F Q L Y K K Y P V K Y G V G K C L G D N G P A E P

801 CAATAGTCTATGACGTTGGCAACACCGAGAAGACAGCATCTCTGTACTCATCATTTGGAAGCAATGAATTTACCCCCGGTTTTATTCAGTTCCGAGCTAT 900 I V Y D V G N T E K T A S L Y S S F G S N E F T P G F I Q F R A I

901 TAATACTGAGGGAGCTGCACTTGCTCTTTGCTCAGGAGTGAAGGTGAAAGGCTGCAATGTGGAGCATCACTGCATTGGAGGAGGTGGTTACATCCCTGAA 1000 N T E G A A L A L C S G V K V K G C N V E H H C I G G G G Y I P E

1001 GGAAACCCCAGACAGTGTGGAGACTTTGCATCAATGGACTGGGATGGGTATGGGACACGCACAGGATGGAGCTCAAGCAAGGAGATGATTGAAGCAGCAG 1100 G N P R Q C G D F A S M D W D G Y G T R T G W S S S K E M I E A A V

1101 TAATGTTGTTCTACCGTTAATCTAGAGTTCTGCTACATTGCTCAACAAGAACAGTCATTCCATCGTTTATGCATTTTTGACCTCCATGACTATACCACCC 1200 M L F Y R *

1201 ACAAGTAGCATGTAAAAATCAGAATGCACAACTGTTCTTCTGAAAAGCCTAAGCACCTAAGAGCTCTGGAATGAAAAGAAAAATCATATATTTGTAACTA 1300

1301 TATAACTG 1308

Supplementary Fig. S2. Translation of Intl-3 cDNA. The underline, rectangular frame and N in

black box represent a signal sequence, fibrinogen-like domain and possible N-glycosylation residue,

respectively.

1 CACACGGGCAGCCAGTATAAGTGGAACCACTGGGGCTCCTTCCGAAAGCTGCCCACTAACTGCATAATTAAACAGACAACCCGTATTGGAGTTAAAAGTT 100

101 GGTATATACAGTAGATTCAGCTAAAATGCTCTCTCATTTATTGCTACTACTTGTCCTTGTAAAAGAATCTTCAGCATGTGATGAAATCTGCGGAAATGTT 200 M L S H L L L L L V L V K E S S A C D E I C G N V

201 CCCTTATCTGAGAAGAAGAAAAGAATTATGAACATTCTGGCTTGTTGGGATGAAAATAATTTTGAGGATCAGTTTAGGCCTCCAAGTACCTCAGGCTATA 300 P L S E K K K R I M N I L A C W D E N N F E D Q F R P P S T S G Y T

301 CATATGGAAGCTGTAAGGAGATCAAGCTGTATAACCAGAATGCCCAAGATGGCATTTATACACTGAGCACTAAGAATGGAATGACCTATCAAACATTCTG 400 Y G S C K E I K L Y N Q N A Q D G I Y T L S T K N G M T Y Q T F C

401 TGACATGAGCACGAATGGAGGAGGTTGGACGTTGGTGGCCAGTATTCATGAGAACTACATACAAGGCAAGTGCAAATCCGGAGATCGCTGGAGCAGTCAG 500 D M S T N G G G W T L V A S I H E N Y I Q G K C K S G D R W S S Q

501 TCAGGAAACAATGCAAACAACCCTGCAGGGGAGGGTAATTGGGCAAATTATGCCACATTTGGACTGCCTGAAGGAGCAACTAGTGATGACTACAAGAACC 600 S G N N A N N P A G E G N W A N Y A T F G L P E G A T S D D Y K N P

601 CTGGATATTATGACATCAAAGCCAAAAATGTAGCTGTGTGGCATGTGCCGAACAATACACCTCTGGAACTTTGGAAGAACTCTTCTTATTTAAGATATCG 700 G Y Y D I K A K N V A V W H V P N N T P L E L W K N S S Y L R Y R

701 AACAGACAATGACTTTCTAAAGGATGAAGGAGGGAACCTATTCAATTTGTATACAAAATACCCAGTGAAATACAATGCTGGAAGCTGCCCAAAGGACAAT 800 T D N D F L K D E G G N L F N L Y T K Y P V K Y N A G S C P K D N

801 GGACCAGCCATTCCTATTATTTATGACCTTGGAGACCCTGAGCAGACATCAAAGTTATACTCACCAAACGGAAGAAAGGAACTAATTCCTGGCTTTATCC 900 G P A I P I I Y D L G D P E Q T S K L Y S P N G R K E L I P G F I Q

901 AGTTCCGTGCAATGAACAATGAAAAAGCTGCCCTGGCTTTGTGTTCTGGGGTAAAAATGTCTGGCTGCAATGCAGAAGTGAACTGCATTGGAGGAGGAGG 1000 F R A M N N E K A A L A L C S G V K M S G C N A E V N C I G G G G

1001 ATATATCCCCGAGGGGAACCCTAGACAGTGTGGAGACTTTGCAGCAATGGACTGGGATGGGTACGGGACGCATGTTGGCTGGAGCTCCAGTAGACAACTG 1100 Y I P E G N P R Q C G D F A A M D W D G Y G T H V G W S S S R Q L

1101 ATAGAGTCTGCTGTTTTGTTATTTTACCATTAAGAATAAAAAGATTCCTTAACTGTT 1157 I E S A V L L F Y H *

Supplementary Fig. S3. Translation of Intl-4 cDNA. The underline, rectangular frame and N in

black box represent a signal sequence, fibrinogen-like domain and possible N-glycosylation residue,

respectively.

Supplementary Fig. S4. Alignment of Intl-3 and Intl-4 with the other Xenopus intelectin family proteins. Amino acid sequences of Intl-3, Intl-4 were deduced from isolated cDNAs. The underlines and shaded boxes show signal peptides and fibrinogen-like domains, respectively. Identical amino acids, gaps, and potential N-glycosylation residues are shown as dots, bars and reverse color, respectively. The amino acids important to carbohydrate recognition are boxed.

Supplementary Table S1. Degenerate primers for amplification of intelectin cDNA fragments

Tm No. of Product Name Sequence (˚C) cycles size (bp) XIntl-S1 TGGACBYTRGTSGCYAGTGTTC 54.0 30 179 Xintl-As CCHGGRTTCTTGTAGTCATC Xintl-S2 TGGACBYTRGTSGCYAGTGTCC 54.0 30 179 Xintl-As CCHGGRTTCTTGTAGTCATC

Supplementary Table S2. Primers for RACE of Intl-3 and Intl-4

Tm No. of Product Name Sequence (˚C) cycles size (bp) Universal primer GATTACGCCAAGCTTGGACCTTAGTGGCCAGTGTCCATG 72.0 30 _ Xintl-3S CCTTTGGTTTACCTGAAGGAGCCACAAG Xintl-3As GTGAGGACCAGCGATCACCCTCAGTGC 72.0 30 _ Xintl-4S GGACTGCCTGAAGGAGCAACTAGTGATGAC Xintl-4As CTGCTCCAGCGATCTCCGGATTTGC

Supplementary Table S3. Primers for RT-PCR

Tm No. of Product Name Sequence (˚C) cycles size (bp) XIntl-3-641S TGGCTATTATGACATTGAGG 56.0 27-30 230 XIntl-3-870As CAACGTCATAGACTATTGGC XIntl-4-699S ATACACCTCTGGAACTTTGG 56.0 27-30 274 XIntl-4-972As GACATTGGTAAATGCCTAGC XEEL-708S GACATTGGTAAATGCCTAGC 56.0 27-30 313 XEEL-1020As TCAATGATCTGTTTGCTTGC XCL-3-691S AATCCTGACATAGCCACTGG 58.0 27-30 291 XCL-3-891As TCCACACTGTCTGGGATTTC XCL-1-23S AGGATTTAGTTCACTATGGG 52.0 27-30 270 XCL-1-292As GTTCCTGTACATTTTACTGG XCL-2-696S CTCTCCAAATGGCAGAGGAG 60.0 27-30 173 XCL-2-868As CACATTGGACTGGGTTTCCT ODC-1204S TGAAGAAGATGCTGCCAATG 60.0 25-27 192 ODC-1395As TCTACGATACGATCCAGCCC

Supplementary Table S4. Primers for expression vectors Tm No. of Product Name Sequence (˚C) cycles size (bp) Xintl-3-154S CTCGAGCCATGGTGGCCTACGCACTAATTATTTTT 58.0 30 1019 Xintl-3-1172As GGATCCTTAACGGTAGAACAACATTACTGC Xintl-4-174S CTCGAGCCATGGTCTCTCATTTATTGCTACTACTTGTCC 56.0 30 1005 Xintl-4-1178As GGATCCTTAACGGTAGAACAACATTACTGC

CTCGAGCATGGGCAAGATGTTGTCAT XEEL-19S 56.0 30 1025 XEEL-1044As GGATCCGCATTTTACCACATTAAC CTCGAGCCATGGTTGTGCACAGTCTGGTTC XCL-1-90S 58.0 30 1005 XCL-1-1094As GGATCCTCAGCGGTAAAAGAGGAGTAC CTCGAGGACAAGATGGTGCCCCACAG XCL-2-16S 60.0 30 979 XCL-2-994As GGATCCAGGATCAGCGGTACATCAGG XCL-3−25S CTCGAGCCATGGTTTTGCACAGTCTGGTGG 60.0 30 960 XCL-3-984As GGATCCTTGAAAATAAATCAGCGG Restriction sites for cloning are underlined.