Isolation, characterization, and extra-embryonic secretion of the Xenopus laevis embryonic epidermal lectin, XEEL

Saburo Nagata

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


E-mail: s-nagata{at}fc.jwu.ac.jp

Received on September 6, 2004; revised on October 26, 2004; accepted on October 27, 2004


    Abstract
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
The Xenopus laevis embryonic epidermal lectin (XEEL) is a novel member of a group of lectins including mammalian intelectins, frog oocyte cortical granule lectins, and plasma lectins in lower vertebrates and ascidians. We isolated the XEEL protein from the extract of tailbud embryos by affinity chromatography on a galactose-Sepharose column. The XEEL protein is a homohexamer of 43-kDa N-glycosylated peptide subunits linked by disulfide bonds. It requires Ca2+ for saccharide binding and shows a higher affinity to pentoses than hexoses and disaccharides. HEK-293T cells transfected with an expression vector containing the XEEL cDNA secrete into the culture medium the recombinant XEEL (rXEEL) that is similar to the purified XEEL in its molecular nature and saccharide-binding properties. Substitution of Asn-192 to Gln removed the N-linked carbohydrate and inhibited secretion of rXEEL but did not abolish the activity to bind to galactose-Sepharose. The embryo's XEEL content, as estimated by western blot analyses, increases during neurula/tailbud stages and declines after 1 week postfertilization. Immunofluorescence and immuno-electron microscopic analyses showed localization of the XEEL protein in a typical secretory granule pathway of nonciliated epidermal cells. When tailbud embryos were cultured in the standard medium, XEEL was accumulated in the medium, indicating secretion of XEEL into the environmental water. The rate of XEEL secretion greatly increased at around the hatching stage and stayed at a high level during the first week after hatching. XEEL may have a role in innate immunity to protect embryos and larvae against pathogenic microorganisms in the environmental water.

Key words: epidermal secretion / GPI anchor / innate immunity / intelectin / N-glycosylation


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Vertebrate lectins mediate a wide spectrum of specific biological functions, including intracellular protein trafficking, cell adhesion and communication, and innate immunity (see reviews, Dodd and Drickamer, 2001Go; Fujita, 2002Go; Schrag et al., 2003Go). Xenopus laevis cortical granule lectin (XCGL) was discovered ~30 years ago as a major protein content of oocyte cortical granules that is, on fertilization, secreted by exocytosis to the extracellular space (Wyrick et al., 1974Go). It binds to carbohydrate components of the oocyte extracellular matrix and vitelline coat and mediates its conversion to a sperm-impenetrable fertilization layer (Greve and Hedrick, 1978Go; Lee et al., 1997Go; Mozingo and Hedrick, 1996Go; Nishihara et al., 1986Go; Quill and Hedrick, 1994Go, 1996Go; Wyrick et al., 1974Go). In addition, it has been argued that XCGL, now known to be present as two closely related proteins, designated XCGL1 and XCGL2 (An et al., 2003Go; Lee et al., 1997Go), remains present on the surface of blastula/gastrula stage embryos and mediates adhesion of the blastomeres (Nomura et al., 1998Go; Outenreath et al., 1988Go; Roberson and Barondes, 1982Go, 1983Go).

Several independent lines of study have recently identified molecules structurally similar to XCGL in ascidian plasma (Abe et al., 1999Go), Xenopus embryonic epidermis (Nagata et al., 2003Go), mouse intestine (Komiya et al., 1998Go), and several human tissues (Lee et al., 2001Go; Suzuki et al., 2001Go; Tsuji et al., 2001Go). The DNA database also contains cDNA sequences encoding similar proteins in lamprey and Xenopus (Klein et al., 2002Go; DDBJ accession numbers AB055981, AB01238, and AB01239). Precursor proteins of XCGL and these XCGL-like lectins have an N-terminal signal peptide for secretion. The mature lectins consist of N-glycosylated peptide subunits of ~300–330 amino acid residues that contain a highly conserved fibrinogen-like motif found in the Ficolin/Opsonin p35 that probably serves as a carbohydrate-recognition domain (Sugimoto et al., 1998Go). They also have a rather divergent N-terminal and a relatively conserved C-terminal segments. Some of the lectins of this family are shown to be large oligomeric proteins of 120–500 kDa in size consisting of 3–12 subunits linked by disulfide bonds (Chamow and Hedrick, 1986Go; Suzuki et al., 2001Go; Tsuji et al., 2001Go). The lectins examined so far require Ca2+ for carbohydrate binding, but they do not have structural motifs characteristic to carbohydrate recognition domains of a class of Ca2+-dependent lectins, namely C-type lectins (Lee et al., 2001Go; Nishihara et al., 1986Go; Tsuji et al., 2001Go; Yokosawa et al., 1982Go). Thus XCGL and the XCGL-like molecules seem to fall into a unique group of lectins evolutionally conserved among chordates, designated the XCGL (Nagata et al., 2003Go) or the eglectin family (Chang et al., 2004Go).

Despite similarities of the XCGL-like lectins to XCGL in their molecular structures and carbohydrate-binding properties, little is known about their biological function. Human intelectin is thought to be a secretory protein that binds to a carbohydrate chain characteristic to the cell wall of certain class of bacteria, suggesting its role in antibacterial innate immunity of intestinal mucosa (Tsuji et al., 2001Go). However, the same protein has been isolated and characterized as a lactoferrin receptor, a glycosylphosphatidyl inositol (GPI)–anchored iron transporter–binding protein on the membrane of the human intestinal brush border, suggesting a role in iron uptake from milk (Suzuki et al., 2001Go). Furthermore, other researchers reported that the same human lectin, termed HL-1, is specifically expressed in the vascular endothelium and that a closely related lectin, HL-2, is expressed specifically in the intestine (Lee et al., 2001Go). Although possible roles in innate immunity were suggested for lower vertebrate lectins found in the plasma or serum (Abe et al., 1999Go), no substantial evidence to support this possibility has so far been available.

We have recently isolated a cDNA clone that encodes a novel XCGL family lectin, XEEL, expressed exclusively in the X. laevis embryonic epidermis (Nagata et al., 2003Go). To gain insights into roles of XEEL in embryonic development, we isolated XEEL from embryos and produced recombinant XEEL (rXEEL) to characterize its molecular nature and carbohydrate-binding properties. We also demonstrated that XEEL is produced by a major subset of embryonic epidermal cells and secreted into the environmental water in a developmentally regulated manner. To our knowledge, XEEL is the first XCGL family lectin secreted from the embryonic epidermis.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Structure of XEEL protein
In the preliminary western blot analysis of embryo extracts, we found that the amount of XEEL content is highest at around the hatching stage. Therefore, we collected stage 35–37 embryos shortly after hatching and used their extracts for XEEL purification. First, the extract was loaded onto the galactose-Sepharose column in the presence of 10 mM Ca2+, and the bound proteins were eluted with Tris-buffered saline (TBS) containing 10 mM ethylenediamine tetra-acetic acid (EDTA). Combined sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and western blot analyses of the eluate showed a large amount of contaminating Sepharose-binding protein of ~30 kDa in size. Usage of TBS containing 20 mM D-xylose plus 10 mM CaCl2 in place of EDTA eliminated the contaminant protein from the eluate, resulting in purification of a 43-kDa protein as revealed by SDS–PAGE under reducing conditions (Figure 1A, left). SDS–PAGE of the purified sample under non-educing conditions resolved a single major protein band at 260 kDa (Figure 1B, left). Western blot analyses of the same sample demonstrated that a monoclonal anti-XEEL antibody 5G7 recognizes these major bands detected in reducing and nonreducing SDS–PAGE (Figure 1A, 1B, right). Although 5G7 antibody was found to cross-react to 40-kDa liver protein (see later discussion), we failed to find such a protein the purified XEEL fraction.



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Fig. 1. Characterization of XEEL and rXEEL proteins. (A) The crude embryo extract (Ext) and the affinity-purified XEEL (XEEL) were fractionated by SDS–PAGE on 12.5% polyacrylamide gels under reducing conditions. Proteins in the gels were visualized by staining with Coomasie brilliant blue (left), or XEEL was detected by western blotting using the monoclonal antibody 5G7 (right). (B) XEEL and rXEEL were fractionated by SDS–PAGE on 7.5% polyacrylamide gels under nonreducing conditions and visualized by protein staining (left) or western blotting (right). (C) XEEL and rXEEL were incubated with (+) or without (–) N-glycosidase, fractionated by SDS–PAGE on 12.5% polyacrylamide gel under reducing conditions and detected by western blotting.

 
The XEEL protein predicted from the cDNA sequence has a molecular mass of ~35 kDa (Nagata et al., 2003Go). It contains two possible N-glycosylation sites at Asn-183 and Asn-192 that are estimated from the amino acid sequence (consensus, Asn-Xaa-Ser/Thr where Xaa is not Pro). To determine whether or not the XEEL protein is N-glycosylated, we treated the affinity-purified protein with N-glycosidase and analyzed by western blotting. Figure 1C shows that the treatment reduces the molecular mass of the subunit from 43 kDa to ~37 kDa. These results indicate that the XEEL protein is a homohexamer of N-glycosylated 43-kDa peptide subunits linked by disulfide bonds.

Structure and secretion of rXEEL
HEK-293T cells were transfected with the pCEP-XEEL vector, and the transient expression of the rXEEL protein was examined. Western blotting of the extract and the culture supernatant showed single protein bands at a molecular mass of 43 kDa, indicating production and secretion of the rXEEL by the transfected cells (Figure 2A). These bands were absent in the cell extract and the culture supernatant of control cells transfected with vacant pCEP4 vector. To purify the rXEEL protein, we established a cell line stably secreting rXEEL by hygromycin selection and applied the supernatants of the large-scale cultures to a galactose-Sepharose column. SDS–PAGE analysis of the fractions eluted with TBS containing 10 mM EDTA resolved a major 260-kDa protein under nonreducing conditions (Figure 1B) and a single 43-kDa protein under reducing conditions (data not shown). These protein bands were recognized by the anti-XEEL monoclonal antibody 5G7 in western blotting (Figure 1B and 1C). Similar to XEEL isolated from embryos, rXEEL was reduced in protein size from 43 kDa to 37 kDa by N-glycosidase treatment (Figure 1C). Therefore, the basic structure of rXEEL protein seems identical to that of the XEEL protein isolated from embryos.



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Fig. 2. N-linked carbohydrate of rXEEL is required for secretion. (A) HEK-293T cells were transfected with recombinant expression vectors, and transient expression and secretion of wild-type or mutant rXEEL proteins were examined by western blotting. The cell extracts and culture supernatants were analyzed along with those transfected with the vacant vector (vector) and intact and N-glycosidase-digested (Ngase) XEEL. N/N, rXEEL; Q/N, rXEEL(N183Q); N/Q, rXEEL(N192Q); Q/Q, rXEEL(N183Q/N192Q). (B) HEK-293T cells were cotransfected with pCEP-AP vector and one of the vectors shown in A, and the cell extracts and culture supernatants were examined for transient expression and secretion of the recombinant proteins. The wild-type and mutant rXEEL proteins were detected by western blotting (upper panel), and the alkaline phosphatase activities were determined by enzyme assay (lower panel). (C) Carbohydrate-binding activities of the wild-type and mutant rXEEL proteins were examined using extracts of the transfected cells. The extracts were mixed with galactose-Sepharose matrix and incubated in the presence (+) or absence (–) of 10 mM Ca2+. rXEEL proteins bound to the matrix were detected by western blotting.

 
To examine whether the N-linked carbohydrate is required for secretion or carbohydrate-binding activity of XEEL, we create the expression vectors modified to produce mutant rXEEL proteins with amino acid substitution at Asn-183 and Asn-192 to Gln. HEK-293T cells were transfected with one of the vectors, pCEP-XEEL(N183Q), pCEP-XEEL(N192Q), and pCEP-XEEL(N183Q/N192Q), and the extracts and the lysates were examined by western blotting using 5G7 antibody. The transiently expressed rXEEL(N183Q) protein was similar to rXEEL in size and found in both the cell lysate and the culture supernatant (Figure 2A). In contrast, the rXEEL(N192Q) and rXEEL(N183Q/N192Q) proteins resembled deglycosylated XEEL in size and were detectable in the cells but not in the culture supernatant. Similar results were obtained using the cell lines producing mutant XEEL proteins established by long-term hygromycin selection (data not shown). When the pCEP-AP vector was cotransfected, the cells transiently producing the wild-type or mutant rXEEL proteins secreted alkaline phosphatase activities into the medium (Figure 2B), although the amounts of secreted activities relative to those in the cells are smaller in the cells producing rXEEL(N192Q) or rXEEL(N183Q/N192Q). The results indicate that production of these mutant rXEEL proteins does not completely block the general secretory pathways in the cells, suggesting that N-glycosylation at Asn-192 is required for secretion of the rXEEL protein.

Next, we examined the effects of removal of N-linked carbohydrate on the carbohydrate-binding activity of rXEEL. The extracts were prepared from cells transiently expressing wild-type or mutant rXEEL proteins, incubated with galactose-Sepharose in the presence or absence of 10 mM CaCl2, and the bound proteins were examined by western blotting. Figure 2C demonstrates that the wild-type rXEEL and the mutant rXEEL(N183Q) bind similarly to galactose-Sepharose in the presence of Ca2+. The mutant rXEEL(N192Q) and rXEEL(N183Q/N192Q) also bind to galactose-Sepharose, although the relative amount of the bound proteins in the extracts is less than those of rXEEL and rXEEL(N183Q).

Carbohydrate-binding properties of XEEL and rXEEL
The purified XEEL and rXEEL were adsorbed onto galactose-Sepharose and eluted with TBS containing either EDTA or 100 mM of various saccharides in the presence of 10 mM CaCl2. The XEEL immunoreactivities in the eluates were detected by western blotting using 5G7 antibody and quantified by densitometry of the blots. XEEL bound to galactose-Sepharose was completely eluted by TBS containing 10 mM EDTA (Figure 3A). Pentoses released 70% (D-ribose) –95% (D-xylose) XEEL from galactose-Sepharose, whereas hexoses were less effective in releasing XEEL, with elutions of 5% (D-glucose) to 50% (D-fructose) of the galactose-bound XEEL (Figure 3B). Disaccharides (maltose, melibiose, and lactose) were completely ineffective in releasing the bound XEEL. These results indicate that among the saccharides used in the present study, pentoses have a higher affinity to XEEL than hexoses and disaccharides. rXEEL and rXEEL(N183Q) showed similar carbohydrate specificity profiles to XEEL, exhibiting a higher affinity to pentoses than to hexoses and disaccharides (Figure 3A, 3B; data for rXEEL(N183Q) not shown).



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Fig. 3. Elution of XEEL and rXEEL adsorbed to the galactose-Sepharose with various saccharides. (A) XEEL (upper panel) or rXEEL (lower panel) was adsorbed to galactose-Sepharose matrix and incubated in TBS, TBS containing 10 mM EDTA (EDTA), or 10 mM CaCl2 plus 100 mM saccharides, as indicated. XEEL or rXEEL in the eluates was detected by western blotting. (B) Western blots of the eluates were quantitatively analyzed by densitometry. The results are presented as percentages of the XEEL (closed column) and rXEEL (open column) eluted from the gel, with those eluted by EDTA regarded as 100%. Each column shows the mean ± SD (bar) of the data obtained from three separate experiments using independently purified samples.

 
Tissue distribution and developmental expression of XEEL
Extracts of various adult tissues were examined by western blotting with two monoclonal antibodies raised against recombinant XEEL peptide (Figure 4A). Both 3E4 and 5G7 antibodies react to the 43-kDa XEEL protein in embryo extract. In addition, 3E4 also recognizes a smeared protein band between 30–50 kDa in ovary extract and 5G7 a 40-kDa protein in liver. However, none of the adult tissues examined contains XEEL detected by both of these antibodies. Because the XEEL peptide used to immunize mice includes amino acid sequences conserved among XCGL family lectins (Nagata et al., 2003Go), the monoclonal antibodies may recognize the determinants shared by XEEL and XCGL (ovary) or the Xenopus serum lectin (liver).



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Fig. 4. Western blot analyses of the XEEL protein in adult tissues and embryos. (A) Extracts of various adult tissues and sera were analyzed using monoclonal antibodies to XEEL (5G7 and 3E4) or actin. (B) Whole embryo extracts at various developmental stages were examined similarly. The sample loaded in each lane contained amounts of proteins from 1/2 embryo (left, stages 6–45) or 1/10 embryo (right, stages 45–50).

 
To assess the developmental changes of the XEEL content in embryos, we analyzed whole embryo extracts by western blotting using the 5G7 antibody (Figure 4B). The XEEL protein becomes detectable in early tailbud embryos at stage 20. Its amount increases during the tailbud stages up to stage 40 and decreases after stage 45 to a barely detectable level at stage 50. However, we failed to detect a 40-kDa liver protein to which 5G7 antibody cross-reacts in any of these samples.

We have shown by in situ hybridization analyses that XEEL mRNA localizes in nonciliated epidermal cells of neurula and tailbud embryos (Nagata et al., 2003Go). To visualize distribution of the XEEL protein in embryos, tailbud embryos at stage 34 were whole-mount stained using the monoclonal antibody 5G7. Confocal fluorescence microscopy of the stained embryos demonstrated that the XEEL immunoreactivities were localized in a subset of epidermal cells and that no other internal tissues were immunolabeled. Strong immunoreactivities were found in patches in the perinuclear cytoplasm and irregular arrays of fine dots that may have been discernible as Golgi apparatus, cytoplasmic vesicles, and endoplasmic reticulum (Figure 5A). In the apical cytoplasm, the majority of the immunoreactivities were localized in round or oval granules of about 0.6–1.2 µm in diameter (Figure 5B). When embryos were incubated with either control mouse IgG or 5G7 antibody preabsorbed with rXEEL, no immunoreactivity was found in these structures of the epidermal cells (Figure 5C).



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Fig. 5. Distribution of XEEL in embryonic epidermis. (A) A stage 34 embryo was whole mount immunolabeled using the monoclonal antibody 5G7 and the mid-basal level of the epidermal cell sheet was viewed with a confocal fluorescent microscope. (B) The same field of the epidermal sheet shown in A was viewed so as to focus on the apical levels. (C) A confocal fluorescence micrograph of the epidermis of the control embryo that was incubated with the 5G7 antibody preabsorbed with the purified rXEEL protein. (D) Immuno-electron micrographs of a cross-section of the embryonic epidermis, showing gold particles on the cytoplasmic granules (arrowheads) of nonciliated epidermal cells. The inset is an enlarged view of the apical cytoplasm of a nonciliated cell, showing that the contents of the secretory granules are heavily labeled. Asterisks in A and B, nonimmunolabeled cells; CEC, ciliated epidermal cells; NCEC, nonciliated epidermal cells; YG, yolk granule; scale bars in AC, 20 µm, and D, 1 µm.

 
On immuno-electron microscopy, the XEEL immunoreactivities were found in the content of granules adjacent to the apical plasma membrane as well as in cytoplasmic vesicles of nonciliated epidermal cells (Figure 5D). The 5G7-immunoreactive cell types are identical with the cells expressing XEEL mRNA (Nagata et al., 2003Go). In addition, in situ hybridization analyses using specific probes failed to demonstrate epidermal expression of XSL and XSL2 in tail bud embryos (unpublished data). These strongly support that the present immunofluorescence and immuno-electron microscopic analyses using 5G7 antibody could visualize XEEL distribution instead of possible cross-reactivity to other lectins or related proteins. Thus XEEL is likely to be produced, processed in secretory pathways, and accumulated actively in the apical secretory granules at this stage of embryonic development.

Extra-embryonic secretion of XEEL
To examine whether XEEL is secreted into the environmental water, we incubated the embryo-conditioned medium with galactose-Sepharose and examined the adsorbed proteins by western blotting using 5G7 antibody. Figure 6A shows accumulation of the XEEL protein in the conditioned medium during the cultivation of embryos for 6 h, demonstrating extra-embryonic secretion of XEEL. Next we cultured denuded or hatched embryos at various developmental stages in 1/10 Steinberg's solution for 3 h, and the embryo-conditioned media were analyzed by slot blotting. Figure 6B shows immunological detection of XEEL on the slot blot (inset) and developmental changes in the rate of embryonic XEEL secretion estimated by densitometry of the blot (histogram). XEEL secretion was undetectable or at low levels before hatching (stages 26 and 32) and then quickly increased to reach the highest level at stage 42, stayed at a high level during the first week after hatching (stages 36–48), and declined to an undetectable level by the second week (stage 50). Similar results were obtained using double-distilled water for embryo culture.



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Fig. 6. Secretion of the XEEL from embryos. (A) Embryos at stage 34 were cultured for 3 h or 6 h. The XEEL in the culture medium was adsorbed to galactose-Sepharose and was detected by western blotting. (B) Embryo-conditioned media was harvested from cultures of embryos at various developmental stages and dot-blotted onto a nitrocellulose membrane. The XEEL on the blots was detected with anti-XEEL antibody (inset), and quantified by densitometry (histogram). A similar result was obtained from an additional experiment using separately prepared embryo-conditioned media.

 

    Discussion
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Analyses of XEEL isolated from Xenopus tailbud embryos revealed that the protein is a 260-kDa homohexamer consisting of N-glycosylated 43-kDa peptide subunits linked by disulfide bonds. This is consistent with the finding that the human cell line transfected with the expression vector containing a single XEEL cDNA produces rXEEL similar to XEEL in structural and carbohydrate-binding properties. The XCGL1 protein purified from Xenopus oocyte extract has been shown to be a ~540-kDa glycoprotein consisting of 12 subunit peptides (Chamow and Hedrick, 1986Go), whereas the human intelectin/lactoferrin receptor is reportedly a 120-kDa or 136-kDa protein consisting of 3 or 4 subunits (Suzuki et al., 2001Go; Tsuji et al., 2001Go). In addition, the ascidian plasma lectin is present as multiple forms including dimers, tetramers, and hexamers of 41-kDa subunits (Abe et al., 1999Go). Thus, the homo-oligomeric structure seems to be an additional property common to the XCGL family lectins, although the numbers of constituent subunits differ among the lectins. Six Cys residues perfectly conserved among the XCGL family lectins may contribute to link subunits by disulfide bonds to form the multimeric structure.

The results of N-glycosidase digestion of XEEL and of analyses of the mutant rXEEL proteins suggest that XEEL is glycosylated at Asn-192, although the latter is based on the transfected cultured mammalian cell line. Substitution of Asn-192 of rXEEL to Gln caused a decrease of the apparent molecular size to that of deglycosylated XEEL, inhibition of its secretion, and accumulation in the cells. However, it did not abolish the carbohydrate-binding activity. The N-linked carbohydrate is, therefore, probably required for extracellular secretion of XEEL but is not an essential component of the carbohydrate recognition domain. It has been shown that N-glycosylation is critical (if not essential) for trafficking of certain proteins along the secretory pathways (Simizu et al., 2004Go). Alternative or additional roles of N-glycan may also be possible; for example, stabilization of XEEL after synthesis in embryonic cells and interaction with other proteins after secretion.

Figure 7A shows alignment of partial amino acid sequences of XCGL family lectins (Abe et al., 1999Go; Komiya et al., 1998Go; Lee et al., 2001Go; Mozingo and Hedrick, 1996Go; Nagata et al., 2003Go; Tsuji et al., 2001Go; Suzuki et al., 2001Go; DDBJ accession numbers AB055981, AB01238, and AB01239) containing conserved N-glycosylation motifs. The first sites (Asn-183 in XEEL) are conserved among all the lectins, whereas the second sites (Asn-192) are present in a limited group of lectins including XEEL. The first sites are always associated with a Pro residue C-terminal to Ser/Thr, which is in many cases known to prevent N-glycosylation (Gave and von Heijne, 1990Go). Indeed, Asn-183 in rXEEL has a limited amount (if any at all) of linked carbohydrates with probably minor functional significance. Substitution of Asn-183 to Gln alone did not affect the apparent molecular size, carbohydrate-binding properties and secretion. However, both XCGL1 and XCGL2 have been shown to be nonglycosylated at the second sites but glycosylated at the first site (An et al., 2003Go). Thus it may be worthwhile to see whether N-glycosylation at one of these sites is required for secretion of the other XCGL family lectins.



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Fig. 7. Alignment of partial amino acid sequences of XCGL family lectins. (A) Sequences containing possible N-glycosylation sites are aligned. The shaded boxes indicate Asn residues at the predicted glycosylation site, and the open box the Pro residues C-terminal to the Ser/Thr. Dot, same amino acid as XEEL; bar, gap. (B) Analysis of C-terminal amino acid sequences for GPI anchor motifs. Underlined amino acids and the shaded boxes are predicted cleavage sites and hydrophobic amino acid sequences in GPI anchor motifs, respectively. The sequences are analyzed by DGPI version 2.04 software. GenBank accession numbers and references for the sequence data (parentheses) are as follows: XEEL (Nagata et al., 2003Go); XCGL1 (X82626); XSL1, X. laevis serum lectin 1 (AB01238); XSL2, X. laevis serum lectin 2 (AB01239); MIL, mouse intelectin (Komiya et al., 1998Go); HL1, human intelectin 1 (Tsuji et al., 2001Go); HL2, human intelectin 2 (Lee et al., 2001Go); LSL, lamprey serum lectin (AB055981); APL, ascidian plasma lectin (Abe et al., 1999Go).

 
A group of researchers has reported that XCGL is secreted into the extracellular space on fertilization (Greve and Hedrick, 1978Go; Mozingo and Hedrick, 1996Go; Nishihara et al., 1986Go; Quill and Hedrick, 1994Go, 1996Go; Wyrick et al., 1974Go), but other groups suggest that it is associated with the plasma membrane of early embryos, possibly by a GPI anchor (Nomura et al., 1998Go; Roberson and Barondes, 1983Go). Similarly, human intelectin is found to be a secretory protein (Tsuji et al., 2001Go), whereas the same molecule is identified as a lactoferrin receptor bound to the intestinal cell membrane via a GPI anchor (Suzuki et al., 2001Go). Other XCGL family lectins, including the ascidian plasma lectin (Abe et al., 1999Go), have been shown to occur in the plasma or the serum, suggesting that they are secretory proteins. To understand the biological function of these lectins, it is important to determine whether they are secreted or membrane-bound. The present study provides a direct evidence for extra-embryonic secretion of XEEL, suggesting its biological function in the embryo's environmental water.

Figure 7B aligns C-terminal amino acid sequences of the XCGL family lectins, showing possible GPI anchor motifs that are determined by analyses of the amino acid sequences using DGPI version 2.04 software (DGPI Web site: http://dgpi.pathbot.com/). Unexpectedly, it predicts that XEEL and XCGL can be GPI-anchored to the plasma membrane at the C-terminus. In our preliminary study, embryonic epidermal cells or HEK-293T cells producing the wild-type or the mutant rXEEL were treated with phosphatidyl inositol–specific phospholipase C, and the liberated proteins were examined by western blotting. This study has failed to demonstrate GPI-anchored XEEL or rXEEL. These observations as well as the findings that XCGL and the human intelectin/transferrin receptor can take both GPI-anchored and secretory forms (Greve and Hedrick, 1978Go; Nomura et al., 1998Go; Quill and Hedrick, 1994Go; Roberson and Barondes, 1983Go; Wyrick et al., 1974Go) indicate that the destination of lectins is not determined by the presence of a GPI anchor motif alone. This in turn suggests that the choice of these lectins to take secretory or GPI-anchored forms depends on their posttranslational modification, including N-glycosylation in different cell types or even in a single cell.

It has long been known that the epidermis of various vertebrates produces and secretes lectins onto the body surface (Barondes, 1984Go; Tasumi et al., 2002Go). In Xenopus, the adult epidermis produces and secretes into the lumen of skin glands a 16-kDa lactose-binding lectin of unknown function (Bols et al., 1986Go; Marschal et al., 1992Go). In addition, one of a family of ß-galactoside-binding lectins, Xgalectin VIIa has been found in unfertilized eggs, embryos, larvae, and some adult tissues (Shoji et al., 2002Go, 2003Go). This lectin is ~34 kDa in size and abundant in embryonic and larval epidermis, although its secretion has not been confirmed. Other Xenopus tissues also produce several groups of lectins, including galactoside–binding lectins produced by adult liver (Roberson et al., 1985Go) or embryonic tissues (Frunchak et al., 1993Go; Harris and Zalik, 1985Go; Milos et al., 1998Go). All these lectins obviously differ from XEEL in protein size and structure, carbohydrate-binding properties, tissue distribution, patterns of developmental expression, and/or secretion. Although a gene encoding a possible ortholog of XEEL has been found in the Xenopus tropicalis genome (Klein et al., 2002Go), no information is available on the protein. Thus, to our knowledge, XEEL is the first XCGL family lectin that has been demonstrated in embryonic epidermal secretion.

The overall carbohydrate-binding properties of XEEL, including Ca2+ dependence and affinity profiles to various saccharides, are quite similar to those of human intelectin (Tsuji et al., 2001Go). Both XEEL and human intelectin exhibit high affinity to pentoses (D-arabinose, D-ribose, D-xylose), moderate affinity to hexoses (D-fructose, D-galactose, D-glucose, D-mannose), and undetectable affinity to disaccharides (lactose, maltose, melibiose) in similar competitive assays using galactose-Sepharose. Human intelectin has been shown to recognize galactofuranosyl residues of carbohydrates present in the cell walls of certain bacteria (Tsuji et al., 2001Go). It has also been reported that recombinant mouse intelectin produced by Escherichia coli has a toxic activity in the bacteria (Komiya et al., 1998Go). Based on these results, we estimated that mammalian intelectins act as antimicrobial effecter molecules in the innate immune system of intestinal mucosa (Tsuji et al., 2001Go). XEEL may also participate in a similar antimicrobial activity in the secretion of the outer (epidermal) surface of the body, instead of the inner (intestinal) surface.

Patterns of changes in the XEEL content of the embryo and the rate of extra-embryonic secretion indicate that XEEL production and secretion are developmentally regulated. Onset of XEEL gene expression occurs zygotically at early gastrula stages (Nagata et al., 2003Go), and the protein appears in the epidermis of early tailbud embryos. The secretion starts at around the hatching stage and stays at high levels during the first week after hatching (stages 36–48), followed by a decline to an undetectable level at stage 50. Acquired immunity in Xenopus, as judged by antibody production and allograft rejection, is not demonstrable before 2 weeks after fertilization (stage 49) (Du Pasquier et al., 1996Go). In addition, various antimicrobial peptides in skin secretions have so far been identified only in adult and late larval stages of X. laevis (Kreil, 1996Go). Combining these facts and the above-mentioned possible antimicrobial activity, we presume that XEEL may play a role to protect free-living embryos and early larvae from pathogenic microorganisms in the environmental water, although the antimicrobial activity of XEEL remains to be demonstrated.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Embryos and their conditioned medium
Mature X. laevis females and males were purchased from local dealers, and eggs were obtained by injecting females with human chorionic gonadotropin (Teikokuzoki, Tokyo). Fertilized eggs obtained by insemination were dejellied in 2.5% cysteine-HCl (pH 8.0) immediately following rotation and rinsed extensively in modified Steinberg's solution (58 mM NaCl, 0.67 mM KCl, 0.34 mM Ca(NO3)2, 0.83 mM MgSO4 and 5 mM HEPES, pH 7.4) (Peng, 1991Go). The embryos were cultured in aerated 1/10 Steinberg's solution at 23°C, and the developmental stages were determined according to the normal table of Nieuwkoop and Faber (1994)Go. To obtain an embryo-conditioned medium, we removed the fertilization membrane manually and cultured 200 denuded or hatched embryos in 3 ml of 1/10 Steinberg's solution at 23°C. The culture medium was harvested, centrifuged for 10 min at 11,000 x g at 4°C, and the supernatant was stored at –80°C.

Purification of XEEL
The galactose-Sepharose affinity gel was prepared by incubation of epoxy-activated Sepharose 6B (Pharmacia Biotech, Uppsala, Sweden) with D-galactose according to the manufacturer's instructions. Two to three thousand hatched embryos at stages 35–37 were homogenized in 10 ml TBS (20 mM Tris–HCl buffer [pH 7.5] containing 150 mM NaCl), supplemented with 10 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 5 mM iodoacetamide by passing them several times through a 20-gauge needle attached to a syringe barrel. The homogenate was centrifuged at 500 x g for 5 min at 4°C. The supernatant was transferred to a new tube and centrifuged again at 11,000 x g for 30 min. The pellet was washed twice by homogenization in 20 ml TBS, centrifuged at 11,000 x g for 30 min, and resuspended in 10 ml TBS containing 1 mM phenylmethylsulfonyl fluoride, 5 mM iodoacetamide, and 10 mM 3-[(3-chloramidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) (Sigma, St. Louis, MO). The suspension was kept overnight at 4°C with continuous rocking. It was then centrifuged at 11,000 x g for 30 min, supplemented with CaCl2 to a final concentration of 10 mM, and mixed with 3 ml of the galactose-Sepharose matrix. The mixture was incubated for 3 h at room temperature with continuous rocking.

The gel in the mixture was washed twice in buffer and loaded onto a small plastic column with a diameter of 1 cm. The column was washed with 20 ml TBS containing 10 mM CHAPS and 10 mM CaCl2 at a flow rate of 0.3 ml/min, and the galactose-binding proteins were eluted with TBS containing 10 mM CaCl2 and 20 mM D-xylose. The eluate was collected as 0.2-ml fractions, and XEEL was detected by western blotting. On some occasions, the fractions containing XEEL were pooled, dialyzed in TBS, and concentrated by centrifugation in a Centricut Mini tube (Biofield, Tokyo) before storing at –80°C.

Production and analyses of rXEEL
The cDNA fragment encoding the whole coding region of XEEL was amplified by polymerase chain reaction (PCR) using XEEL cDNA (Nagata et al., 2003Go) as a template. The amplified product was cloned into the pCEP4 plasmid vector (Invitrogen, Carlsbad, CA) to create pCEP-XEEL. The plasmid DNA was purified using a QIAGEN column (QIAGEN, Tokyo) and used to transfect human epithelial kidney cell line HEK-293T. The cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 100 µg/ml streptomycin sulfate, and 100 U/ml penicillin in a CO2 incubator. The cells were plated in 3 ml medium at 5 x 105 cells per well of six-well plates (Corning, Corning, NY) and transfected 24 h later with the plasmid DNA by the calcium phosphate method (Sambrook and Russel, 2001Go). The medium was changed the next day, and the cells and the culture supernatants were harvested 48 h later to examine transient expression and secretion of the recombinant protein.

To establish cell lines producing the recombinant protein, we selected transfected cells by cultivation in medium containing 100 µg/ml hygromycin B (Sigma). To purify rXEEL from the large-scale culture, we harvested the culture supernatant by centrifugation at 10,500 x g for 20 min; supplemented it with iodoacetamide (5 mM at a final concentration), CHAPS (10 mM), and CaCl2 (10 mM); and loaded it onto the galactose-Sepharose column as described previously. After washing, rXEEL was eluted with TBS containing 10 mM EDTA and detected by western blotting. The fractions containing the rXEEL were pooled, dialyzed in TBS, and concentrated by centrifugation in a Centricut Mini tube.

XEEL has two putative N-glycosylation sites at Asn-183 and Asn-192 (consensus, Asn-Xaa-Ser/Thr where Xaa is not Pro). We generated mutant cDNAs encoding rXEEL with amino acid substitution of one or both of the Asn residues to Gln residues by PCR-mediated, site-directed mutagenesis using the overlap extension technique (Ho et al., 1989Go). The sequences of antisense mutagenesis primers with nucleotide substitutions (underscored) are 5'-GTGTTTTCTGGGGCACATGC-3', 5'-GATGACTGCCTCCACATAAC-3', and 5'-GACTGCCTCCACATAACCATTGGTGTTTTCTGGGGCAC-3'. The mutant cDNAs were amplified using KOD-Plus-DNA polymerase (Toyobo, Tokyo). The sequences were confirmed, and the restriction fragments containing the mutated site were introduced into pCEP-XEEL to create pCEP-XEEL(N183Q), pCEP-XEEL(N192Q), and pCEP-XEEL(N183Q/N192Q), respectively. The cDNA encoding heat-stable human alkaline phosphatase was excised from APtag-4 vector (GenHunter, Nashville, TN) and inserted into the pCEP4 vector to create pCEP-AP. HEK-293T cells were transfected with the vectors, and the cells and the culture supernatants were harvested 2 days after transfection to examine properties of mutant rXEEL proteins. Alkaline phosphatase activities in extracts and culture supernatants of the transfected cells were assayed using AP Activity Assay Reagent A (GenHunter) according to the manufacturer's instructions.

Monoclonal antibodies
To obtain antibodies to XEEL, we produced a bacterial rXEEL peptide and used it as an antigen. The peptide (amino acid 43 to 142 of the conceptual translate of the cDNA clone) contains the XEEL-specific segment and a part of the fibrinogen-like motif that is highly conserved in the XCGL family (Nagata et al., 2003Go). A fragment of the XEEL cDNA encoding the peptide was amplified by PCR and cloned into the pET32c plasmid (Novagen, Madison, WI). BL21 strain E. coli bacteria were transformed with the recombinant vector, and production of the recombinant fusion protein with a thioredoxin tag was induced with 1 mM ispropylthiogalactoside (Ambion, Austin, TX). Soluble bacterial extracts were fractionated by SDS–PAGE, and the recombinant protein was isolated from the gel using an Electroeluter apparatus (Bio-Rad Laboratories, Hercules, CA). Balb/c mice were immunized with the purified recombinant fusion protein, and two hybridoma clones secreting IgG1({kappa}) antibodies, 3E4 and 5G7, were established by standard procedures (Harlow and Lane, 1988Go). The hybridoma cells were injected intraperitoneally into Balb/c mice, and the ascites containing high titer monoclonal antibody was harvested and stored at –80°C. A monoclonal antibody to chicken gizzard actin was purchased from Chemicon (Temecula, CA).

Western and slot blotting
Freshly dissected adult tissues were homogenized in 19 volumes of SDS-sample buffer (62.5 mM Tris-HCl, 2.3% SDS, 5% ß-mercaptoethanol, 10% glycerol, pH 6.8) using a Polytron homogenizer (Kinematica, Littau, Switzerland). Embryos and cultured cells were homogenized in buffer at 50 embryos/ml or 5 x 106 cells/ml. The homogenates were centrifuged at 11,000 x g for 10 min, and the supernatant was used as samples. Embryo conditioned media was concentrated to 0.5 ml by centrifugation in Centricut Mini tubes, mixed with 25 µl 20x concentrated TBS and 5 µl 1 M CaCl2, and incubated for 1 h at room temperature with 50 µl 50% galactose-Sepharose suspension in TBS. The cultured cells were homogenized in SDS-sample buffer at 2 x 106 cells/ml, and the culture supernatant was mixed with the same volume of 2x SDS sample buffer.

Alkaline phosphatase activities in extracts and culture supernatants of the transfected cells were assayed using AP Activity Assay Reagent A (GenHunter) according to the manufacturer's instructions. The affinity beads were washed in TBS containing 10 mM CaCl2, and the bound proteins were extracted with 30 µl SDS–PAGE sample buffer. Proteins in the sample were heat denatured for 3 min at 100°C, fractionated by SDS–PAGE, and transferred to a polyvinylidene difluoride membrane. The embryo-conditioned medium was slot blotted onto a nitrocellulose membrane using a Bio-Dot apparatus (Bio-Rad) according to the manufacturer's instructions. The membranes were blocked with 5% skim milk in TBS-T and incubated sequentially with a mouse monoclonal antibody and an anti-mouse IgG goat antibody conjugated with horseradish peroxidase (Biosource, Camarillo, CA). The blots were developed with ECL-Plus western blot detection reagent (Amersham Biosciences, Tokyo), and the chemiluminescent signals were captured and analyzed with a VersaDoc 5000 image analysis system and Quantity One software (Bio-Rad).

N-glycosidase treatment
The purified XEEL or rXEEL fractions containing 50 ng protein were dissolved in 20 µl 200 mM Tris–HCl buffer (pH 8.0) containing 0.1% SDS, 50 mM ß-mercaptoethanol, and 50 mM EDTA and denatured at 100°C for 5 min. The denatured samples were mixed with the same volume of 2.5% Nonidet P-40 and incubated with 1 µl of either 0.5 U of N-glycosidase (Boehringer, Mannheim, Germany) or TBS at 37°C for 16 h. The treated samples were analyzed by western blotting using an anti-XEEL monoclonal antibody 5G7.

Saccharide-binding assay
To examine activities of XEEL or rXEEL to bind various saccharides, we mixed aliquots of 200 µl of the purified samples containing 10 mM CaCl2 with 20 µl galactose-Sepharose and incubated for 1 h at room temperature with continuous shaking. The gels were washed four times with TBS containing 10 mM CaCl2 and then resuspended in TBS containing either 10 mM EDTA or 10 mM CaCl2 plus 100 mM of one of the following saccharides: pentoses D-arabinose, D-ribose, D-xylose; hexoses D-fructose, D-galactose, D-glucose, D-mannose; or disaccharides lactose, maltose, melibiose. After the mixtures were agitated for 10 min at room temperature, they were centrifuged, and the supernatants were subjected to western blotting.

Immunofluorescence staining and immuno-electron microscopy
For immunofluorescence staining, tailbud embryos at stage 34 were fixed overnight with 4% paraformaldehyde in phosphate buffered saline, bleached, and permeabilized with 10% H2O2 and 5% dimethyl sulfoxide in methanol for 2–3 days. The embryos were washed in phosphate buffered saline, blocked in 1% normal goat sera, and then incubated sequentially with monoclonal anti-XEEL antibody 5G7 (diluted 1:2000) and tetramethylrhodamine isothiocyanate–labeled anti-mouse IgG goat antibody (Biosource). The embryos were whole mounted, or the epidermis was peeled off of the stained embryo under the dissecting microscope and then mounted on a glass slide. The preparations were observed and the images were captured with a confocal laser beam microscope (Leica Microsystems, Wetzlar, Germany).

For immuno-electron microscopy, embryos were fixed in 0.1 M Na-cacodylate buffer containing 0.5% glutaraldehyde and 4% paraformaldehyde for 2 h and embedded in LR White (London Resin, Reading, U.K.). Thin sections were successively incubated with 5G7 monoclonal antibody and anti-mouse IgG-colloidal gold conjugates (Aurion, Wageningen, Netherlands), followed by treatment with a silver enhancement kit (Aurion). Immunolabeled sections were examined and photographed with an electron microscope JEM1200 EXS (JEOL, Tokyo).


    Acknowledgements
 
We thank Dr. M. Sato of Laboratory of Electron Microscopy and Ms. N. Fujita of the Department of Chemical and Biological Sciences, Japan Women's University, for immuno-electron microscopy and confocal fluorescence microscopy, respectively.


    Abbreviations
 
CHAPS, 3-[(3-chloramidopropyl)-dimethylammonio]-1-propanesulfonate; EDTA, ethylenediamine tetra-acetic acid; GPI, glycosylphosphatidyl inositol; PCR, polymerase chain reaction; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; TBS, Tris-buffered saline; XCGL, Xenopus laevis oocyte cortical granule lectin; XEEL, Xenopus laevis embryonic epidermal lectin


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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