Embryonic Epidermal Lectins in Three Amphibian Species, Rana ornativentris, Bufo japonicus formosus, and Cynops pyrrhogaster.

Intelectins (Itlns) are secretory lectins found in several chordate species that recognize carbohydrates on the bacterial cell surface depending on Ca2 + . In newly hatched larvae of Rana ornativentris (R. orn), Bufo japonicus formosus (B. jpn), and Cynops pyrrhogaster (C. pyr), an anti-Itln monoclonal antibody (mAb) labeled a subset of epidermal cells in whole-mount immunocytochemical assays. In western blot analyses, the mAb identified protein bands at approximately 33-37 kDa in the larval extracts and concentrated larval culture media. Using RT-PCR and RACE techniques, we isolated cDNAs from newly hatched larvae that encoded proteins of 343 (R. orn), 336 (B. jpn), and 337 (C. pyr) amino acids having 70%, 71%, and 60% identities with that of the Xenopus laevis embryonic epidermal lectin (XEEL), respectively. The proteins, designated REEL, BEEL, and CEEL, showed characteristics conserved among reported Itln proteins, and their amino acid sequences following the signal peptides were identical to those of the N-terminal peptides determined on Itln proteins in the respective larval extracts. Recombinant REEL (rREEL), rBEEL, and rCEEL proteins produced by HEK-293T cells were homo-oligomers of 34-37 kDa subunit peptides, which were similar to the Itlns found in the newly hatched larvae. The rEELs showed carbohydrate-binding specificities similar to that of XEEL and agglutinated Escherichia coli and Staphylococcus aureus cells depending on Ca2 + . These results suggest that REEL, BEEL, and CEEL are Itlns produced and secreted by epidermal cells of R. orn, B. jpn, and C. pyr larvae, respectively, and that Itlns have a conserved role as pathogen recognition molecules in the larval innate immune system.

Xenopus laevis macrophage-like cells produce XCL-1, an intelectin family serum lectin that recognizes bacteria.

Xenopus laevis Ca2+ -dependent lectin-1 (XCL-1) is an intelectin family serum lectin that selectively recognizes carbohydrate chains on the bacterial cell surface. Immunofluorescence examination of control spleen tissues from normal X. laevis revealed cells producing XCL-1 (XCL-1+ cells) exclusively in red pulps. Intraperitoneal injection of Escherichia coli lipopolysaccharide (LPS) caused a marked increase in the number of XCL-1+ cells in red pulps on day 3, followed by a rapid decrease to near control levels by day 7. XCL-1+ cells were also detected in peripheral blood leukocytes (PBLs) and peritoneal exudate cells (PECs), and their numbers increased upon LPS injection until day 7. The XCL-1+ cells exhibited the morphological characteristics of macrophages, with a large oval or lobulated nucleus and abundant cytoplasm with vacuoles and dendritic projections. Western blot analyses revealed concurrent increases in XCL-1 levels in the spleen, PBLs, and PECs. When LPS-stimulated frogs were intraperitoneally injected with paraformaldehyde-fixed, green fluorescent protein-labeled E. coli cells (GFP-Eco), these were phagocytosed by XCL-1+ PECs. The purified XCL-1 protein agglutinated GFP-Eco in a Ca2+ -dependent manner, which was blocked effectively by xylose and partly by LPS and Staphylococcus aureus peptidoglycan, but not by sucrose. These results indicate that X. laevis macrophage-like cells produce XCL-1 and suggest that XCL-1 promotes the clearance of invaded bacteria by facilitating phagocytosis.

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

The intelectin (Intl) family is a group of secretory lectins in chordates that serve multiple functions, including innate immunity, through Ca(2+)-dependent recognition of carbohydrate chains. Although six Intl family lectins have so far been reported in Xenopus laevis, none have been identified in the intestine. Using a monoclonal antibody to the Xenopus embryonic epidermal lectin (XEEL or Intl-1), I identified cross-reactive proteins in the intestines. The proteins were purified by affinity chromatography on a 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, identified two novel Intls (designated Intl-3 and Intl-4) that showed 59-79% amino acid identities with known Xenopus Intl family proteins. From the amino acid sequence, immunoreactivity, and properties of the recombinant protein, Intl-3 was considered the intestinal lectin identified by the anti-XEEL antibody. The purified Intl-3 protein could potentially bind to Escherichia coli and its lipopolysaccharides (LPS), and to Staphylococcus aureus and its peptidoglycans, depending on Ca(2+). In addition, the Intl-3 protein agglutinated E. coli cells in the presence of Ca(2+). 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.

Bacterial lipopolysaccharides stimulate production of XCL1, a calcium-dependent lipopolysaccharide-binding serum lectin, in Xenopus laevis.

Xenopus laevis serum lectin XCL1 is a newly identified molecule of the XCGL (or X-lectin) family, a unique group of Ca(2+)-dependent lectins that have a fibrinogen-like domain. The XCL1 protein was purified from lipopolysaccharide (LPS)-stimulated frog sera by sequential affinity chromatography on heparin-acrylic beads and galactose-Sepharose. XCL1 comprises multiple oligomeric proteins consisting of 37-kDa subunit polypeptides, as revealed by sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) and Western blot analyses using the monoclonal antibody (mAb) produced against the recombinant XCL1 polypeptide. In the presence of Ca(2+), the protein bound to Escherichia coli, Staphylococcus aureus, LPS and galactose and the bound XCL1 was competitively eluted using ribose and xylose, and the elution was as efficient as that using EDTA, whereas elution using hexoses, GalNAc or GlcNAc was less effective. In reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analyses, XCL1 expression was ubiquitously detected in frog tissues, with relatively high levels in hematopoietic tissues including the spleen, liver and kidney. Intraperitoneal injection of E. coli, S. aureus or 100-300µg S-type LPS from various bacteria induced several-fold increases in serum XCL1 concentrations on day 3, and the elevated levels retained up to day 12. It also caused a remarkable increase of the splenic XCL1 expression on day 3, followed by a rapid decline to nearly nonstimulated control levels by day 7. The R-type LPS with shortened polysaccharide chains was less effective in inducing the serum XCL1 response, indicating that the sugar chains of LPS were important, if not essential, for the stimulation of XCL1 production. These results suggest that XCL1 is a pathogen recognition molecule involved in antimicrobial innate immunity in Xenopus.

Structure and expression of myelin basic protein gene products in Xenopus laevis.

To study roles of the myelin basic protein (mbp) gene products in nervous system development, cDNA cloning and expression analyses were performed in Xenopus laevis. We cloned cDNAs for XMBP.1 and XMBP.2 encoded by xmbp.1 and xmbp.2 genes, respectively. We also identified xmbp.1 gene transcripts encoding three XGolli (X.laevis gene of the oligodendrocyte lineage) proteins, XBG21.1, XJ37.1, and XTP8.1, which are homologues of mouse BG21, J37, and TP8, respectively. In reverse transcription-polymerase chain reaction (RT-PCR) analyses, the XMBP, XJ37, and XTP8 mRNAs were expressed in brain, ovaries, testes, and/or thymus in frogs and in larvae after hatching. In contrast, the XBG21 mRNA was found fairly ubiquitously in adult tissues, unfertilized eggs and embryos throughout the developmental stages examined. Western blot analyses using three different monoclonal antibodies (mAbs) showed that the central and peripheral myelin contained 20kDa and18.5 kDa XMBP variants. In addition, XMBP was found in thymus by Western blotting and in thymocyte cytoplasm immunocytochemically. However, the XGolli protein, most provably XBG21, was detectable only in testes. The results indicate that the structure of xmbp gene products seems highly conserved among amphibians and mammals, although their expression patterns and thus physiological roles may partially differ. This is the first report that systematically describes the mbp gene products in nonmammalian vertebrates.

Contactin 1 knockdown in the hindbrain induces abnormal development of the trigeminal sensory nerve in Xenopus embryos.

In Xenopus tailbud embryos, the mandibular branch of trigeminal sensory nerve has a transient pathway innervating the cement gland. This pathway is settled by pioneer neurons in the trigeminal ganglion and along which extend later-growing axons from the trigeminal ganglion and the hindbrain. Axons in this branch express a neuronal recognition molecule, Contactin 1, from the initial stage of its outgrowth in early tailbud embryos and form a tightly joined, strongly Contactin 1-positive fascicle in the later stages. When the expression vector encoding the enhanced green fluorescent protein was electrotransfected into the brain neurons of early tailbud embryos, the fluorescence was detected in the hindbrain and the trigeminal nerve at late tailbud stages. Cotransfection of antisense vector caused knockdown of Contactin 1 concurrent with defasciculation and misguidance of the sensory axons in the trigeminal mandibular branch. The results suggest that Contactin 1 is required for the growing axon of hindbrain sensory neurons to recognize and follow the pathway settled by the pioneer neurons.

Repulsive guidance of axons of spinal sensory neurons in Xenopus laevis embryos: roles of Contactin and notochord-derived chondroitin sulfate proteoglycans.

An immunoglobulin superfamily neuronal adhesion molecule, Contactin, has been implicated in axon guidance of spinal sensory neurons in Xenopus embryos. To identify the guidance signaling molecules that Contactin recognizes in tailbud embryos, an in situ binding assay was performed using recombinant Contactin-alkaline phosphatase fusion protein (Contactin-AP) as a probe. In the assay of whole-mount or sectioned embryos, Contactin-AP specifically bound to the notochord and its proximal regions. This binding was completely blocked by either digestion of embryo sections with chondroitinase ABC or pretreatment of Contactin-AP with chondroitin sulfate A. When the spinal cord and the notochord explants were co-cultured in collagen gel, growing Contactin-positive spinal axons were repelled by notochord-derived repulsive activity. This repulsive activity was abolished by the addition of either a monoclonal anti-Contactin antibody, chondroitin sulfate A or chondroitinase ABC to the culture medium. An antibody that recognizes chondroitin sulfate A and C labeled immunohistochemically the notochord in embryo sections and the collagen gel matrix around the cultured notochord explant. Addition of chondroitinase ABC into the culture eliminated the immunoreactivity in the gel matrix. These results suggest that the notochord-derived chondroitin sulfate proteoglycan acts as a repulsive signaling molecule that is recognized by Contactin on spinal sensory axons.

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

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 Ca(2+) 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.

Overexpression of receptor-type protein tyrosine phosphatase beta causes abnormal development of the cranial nerve in Xenopus embryos.

Roles of receptor-type protein tyrosine phosphatase beta (RPTPbeta, also called PTPzeta) were investigated in the nervous system development of Xenopus embryos. We previously showed that Xenopus embryos express mRNAs for 11 receptor-type (XRPTPbeta.1-XRPTPbeta.11) and two secretory (sXRPTPbeta.1 and sXRPTPbeta.2) variants generated by alternative RNA splicing. Whole-mount in situ hybridization analyzes demonstrated central nervous system-specific gene transcription in tailbud embryos. Distributions of mRNAs for receptor-type and secretory variants partially differ in the hindbrain. Overexpression of XRPTPbeta.4 or sXRPTPbeta.2, which was brought about by microinjection of the recombinant plasmid vectors, caused abnormal development of the cranial nerve X. Deletion of the cytoplasmic segment from XRPTPbeta.4 did not affect the ability to cause the abnormality, but deletion of the extracellular segment abolished it. These results suggest that normal development of the cranial nerve X requires regulated expression of the XRPTPbeta gene products.

Developmental expression of XEEL, a novel molecule of the Xenopus oocyte cortical granule lectin family.

We have isolated cDNA clones from a Xenopus laevis embryo library that encode a predicted translation product of 342 amino acids containing a signal sequence for secretion. The predicted protein has 62-70% amino acid identity with the Xenopus oocyte cortical granule lectin (XCGL), the mouse intelectin, the human HL-1/intelectin and HL-2. Onset of gene expression occurs by gastrulation, and the transcripts localize in non-ciliated epidermal cells all over the tailbud embryos. The results suggest that the molecule, designated XEEL ( Xenopus embryonic epidermal lectin), is a novel XCGL family molecule secreted from the embryonic epidermis.

Development of Prolactin and Growth Hormone Production in the Fetal Rat Pituitary: An Immunochemical Study: (hormone production/ontogeny/fetal rat pituitary/immunochemistry).

Western immunoblot analysis was performed to monitor contents of prolactin (PRL), growth hormone (GH) and Pit-1, a transcription factor required for both GH and PRL gene expression, in developing fetal rat pituitaries. Both PRL and GH were first detected in pituitary extracts at 18 days of fetal age and their contents increased during the rest of the fetal period. Pit-1 was detectable from 16 days on. Quantitative densitometry scanning analysis of Western immunoblots showed that the PRL and GH contents were less than 10 ng per pituitary at 18 days. The PRL content increased to about 19 ng at 21 days (the last day of gestation), whereas the GH content showed much larger increase in the same period to reach about 1.2 µg per pituitary. In primary culture, dispersed pituitary cells from 19 day-old fetuses incorporated [35 S]-amino acids into immunoprecipitable GH. The incorporation into PRL was detectable after stimulation with 1 nM 17ß-estradiol. These results suggests that the rat pituitary starts to produce both PRL and GH at around 18 days of fetal age.