Secretion of a cytoplasmic lectin from Xenopus laevis skin

The skin of Xenopus laevis contains a soluble beta-galactoside-binding lectin with a approximately 16,000-mol-wt subunit. It resembles similar lectins purified from a variety of tissues from other vertebrates, and differs from two other soluble X. laevis lectins from oocytes and serum that bind alpha-galactosides. The skin lectin is concentrated in the cytoplasm of granular gland and mucous gland cells, as demonstrated by immunohistochemistry with the electron microscope. Upon injection with epinephrine, there is massive secretion of the cytoplasmic lectin from the granular gland cells.     

Isolation and expression of a gene encoding L-14-II, a new human soluble lactose-binding lectin.

In the course of screening a human hepatoma cDNA library with antibody raised against a mammalian lectin with subunit molecular weight of about 14,000, we detected a partial cDNA encoding a related but distinct protein that was possibly a homologous lectin (Gitt and Barondes, 1986). We here report the isolation and sequencing of a full-length cDNA for this protein from a HepG2 cDNA library. The cDNA encodes a protein with subunit molecular weight of 14,650. Expression of the coding sequence in Escherichia coli yields a product that binds to a lactose affinity column and is specifically eluted with lactose, confirming that this new protein is a lectin. Like its well studied relative, here called L-14-I, the new lectin, L-14-II, exists as a homodimer in solution. The two related human lectins have 43% amino acid sequence identity. The genomic DNA encoding L-14-II (LGALS2) contains four exons with similar intron placement to L-14-I (LGALS1); but the genomic upstream region, which contains several sequences characteristic of regulatory elements, differs significantly from L-14-I.     

Isolation and characterization of a lectin from the cortical granules of Xenopus laevis eggs

A cortical granule lectin was isolated from eggs of the South African clawed toad Xenopus laevis. The lectin was released from the cortical granules by activation of dejellied eggs with the Ca2+ ionophore A23187. The lectin was purified by affinity chromatography with its natural ligand, the egg jelly coat, chemically coupled to a Sepharose matrix. The purified lectin was homogeneous by the criteria of isoelectric focusing (pI = 4.6), immunodiffusion, and immunoelectrophoresis but existed in two different molecular weight isomers as determined by sedimentation velocity ultracentrifugation and disc gel electrophoresis. Molecular weights of the isomers were determined by ultracentrifugation, disc gel electrophoresis, and gel filtration and found to be 539,000 and 655,000. Chemically, the lectin was a metalloglycoprotein, composed of 84.0% protein, 15.8% carbohydrate, and 0.19% calcium. No unusual types or amounts of amino acids were present. The carbohydrate moiety was composed of fucose, mannose, galactose, glucosamine, galactosamine, and sialic acid. The monosaccharide specificity of the lectin was investigated with the sugar inhibition of the precipitin reaction in gels. The lectin was specific for D-galactosyl sugars with the configuration at carbon atoms 2-4 of primary importance.     

Galactoside-binding serum lectin of Xenopus laevis. Estrogen-dependent hepatocyte synthesis and relationship to oocyte lectin

Xenopus laevis serum contains a lectin which binds alpha- and beta-galactosides. It was purified to homogeneity by affinity chromatography and consists of a single subunit with Mr approximately 69,000, associated in a multimer. The lectin is synthesized and secreted by hepatic parenchymal cells, and its synthesis is increased about 2-fold by estrogen treatment, both in vivo and in primary cell cultures. The serum lectin has the same carbohydrate binding properties as an oocyte lectin from X. laevis described previously, is immunologically cross-reactive, and shows similarities in its peptide map. However, marked differences in amino acid composition preclude the possibility that the serum lectin is a precursor of the oocyte lectin.     

Distribution of lectin binding sites in Xenopus laevis egg jelly.

Eggs from the anuran Xenopus laevis are surrounded by a thick jelly coat that is required during fertilization. The jelly coat contains three morphologically distinct layers, designated J1, J2, and J3. We examined the lectin binding properties of the individual jelly coat layers as a step in identifying jelly glycoproteins that may be essential in fertilization. The reactivity of 31 lectins with isolated jelly coat layers was examined with enzyme-linked lectin-assays (ELLAs). Using ELLA we found that most of the lectins tested showed some reactivity to all three jelly layers; however, two lectins showed jelly layer selectivity. The lectin Maackia amurensis (MAA) reacted only with J1 and J2, while the lectin Trichosanthes kirilowii (TKA) reacted only with J2 and J3. Some lectins were localized in the jelly coat using confocal microscopy, which revealed substantial heterogeneity in lectin binding site distribution among and within jelly coat layers. Wheat germ agglutinin (WGA) bound only to the outermost region of J3 and produced a thin, but very intense, band of fluorescence at the J1/J2 interface while the remainder of J2 stained lightly. The lectin MAA produced an intense fluorescence-staining pattern only at the J1/J2 interface. Several lectins were also tested for the ability to inhibit fertilization. WGA, MAA, and concanavalin A significantly inhibited fertilization and WGA was found to block fertilization by preventing sperm from penetrating the jelly. Using Western blotting, we identified high-molecular-weight components in J1 and J2 that may be important in fertilization.     

Hemagglutinating activity and conformation of a lactose-binding lectin from mushroom Agrocybe cylindracea

A lactose-binding lectin (Agrocybe cylindracea Lectin, ACL) purified from fruiting bodies of the mushroom A. cylindracea was investigated to determine the hemagglutinating activity and conformation changes after chemical modification, removal of metal ion and treatment at different temperatures and pH. ACL agglutinated both rabbit and human erythrocytes and its hemagglutinating activity could be inhibited by lactose. This lectin was stable in the pH range of 6-9 and temperature up to 60 degrees C. Fluorescence quenching and modification of tryptophan residues indicated that there were about two tryptophan residues in ACL molecule and one of them might be located on the surface, while the other was buried in the hydrophobic shallow groove near the surface. Chemical modification of serine/threonine and histidine showed that the partial necessity of these residues for the hemagglutinating activity of ACL. However, modifications of arginine, tyrosine and cysteine residues had no effect on its agglutinating activity.     

Sequence analysis of 28S ribosomal DNA from the amphibian Xenopus laevis.

We have determined the complete nucleotide sequence of Xenopus laevis 28S rDNA (4110 bp). In order to locate evolutionarily conserved regions within rDNA, we compared the Xenopus 28S sequence to homologous rDNA sequences from yeast, Physarum, and E. coli. Numerous regions of sequence homology are dispersed throughout the entire length of rDNA from all four organisms. These conserved regions have a higher A + T base composition than the remainder of the rDNA. The Xenopus 28S rDNA has nine major areas of sequence inserted when compared to E. coli 23S rDNA. The total base composition of these inserts in Xenopus is 83% G + C, and is generally responsible for the high (66%) G + C content of Xenopus 28S rDNA as a whole. Although the length of the inserted sequences varies, the inserts are found in the same relative positions in yeast 26S, Physarum 26S, and Xenopus 28S rDNAs. In one insert there are 25 bases completely conserved between the various eukaryotes, suggesting that this area is important for eukaryotic ribosomes. The other inserts differ in sequence between species and may or may not play a functional role.     

Novel short antimicrobial peptide isolated from Xenopus laevis skin

A rich source of bioactive peptides, including a large number of antimicrobial peptides, has been found in amphibian skin. In this study, a novel short antimicrobial peptide was purified from Xenopus laevis skin and characterised through reversed‐phase high‐performance liquid chromatography, Edman degradation and matrix‐assisted laser desorption/ionisation time‐of‐flight mass spectrometry. The peptide was composed of six amino acids with a sequence of DEDLDE and thus named X. laevis antibacterial peptide‐P2 (XLAsp‐P2). Transmission electron microscopy revealed that this peptide showed potential antimicrobial abilities against bacteria by damaging the bacterial cell membrane. XLAsp‐P2 maybe inhibit bacterial growth by binding to the microbial genomic DNA. The peptide also exhibited a weak haemolytic activity against rabbit red blood cells. Therefore, XLAsp‐P2 is a novel short anionic antibacterial peptide with broad activities. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Toxoplasma gondii micronemal protein MIC1 is a lactose-binding lectin.

Host cell invasion by Toxoplasma gondii is a multistep process with one of the first steps being the apical release of micronemal proteins that interact with host receptors. We demonstrate here that micronemal protein 1 (MIC1) is a lactose-binding lectin. MIC1 and MIC4 were recovered in the lactose-eluted (Lac(+)) fraction on affinity chromatography on immobilized lactose of the soluble antigen fraction from tachyzoites of the virulent RH strain. MIC1 and MIC4 were both identified by N-terminal microsequencing. MIC4 was also identified by sequencing cDNA clones isolated from an expression library following screening with mouse polyclonal anti-60/70 kDa (Lac(+) proteins) serum. This antiserum localized the Lac(+) proteins on the apical region of T. gondii tachyzoites by confocal microscopy. The Lac(+) fraction induced hemagglutination (mainly type A human erythrocytes), which was inhibited by beta-galactosides (3 mM lactose and 12 mM galactose) but not by up to 100 mM melibiose (alpha-galactoside), fucose, mannose, or glucose or 0.2 mg/ml heparin. The lectin activity of the Lac(+) preparation was attributed to MIC1, because blotted MIC1, but not native MIC4, bound human erythrocyte type A and fetuin. The copurification of MIC1 and MIC4 may have been due to their association, as reported by others. These data suggest that MIC1 may act through its lectin activity during T. gondii infection.     

Sequence of C region of L chains from Xenopus laevis Ig

A cDNA expression library, prepared from Xenopus laevis splenocytes, was screened with antibodies to Xenopus Ig. One clone, lambda XIg23, reacted with antibodies to IgY and to IgM; the insert hybridized to approximately 1.3-kb RNA from spleen, the approximate size expected for L chain mRNA. An additional clone, lambda XIg31, was identified by cross-hybridization. The inserts of lambda XIg23 and lambda XIg31 begin in the third framework region of the V region and extend through the C region to the poly(A) tail. Except for a single nucleotide difference, the two C region sequences are identical. The amino acid sequence of the C region was compared with the sequences of a variety of C kappa and C lambda, as well as to C region sequences of L chains from Rana catesbeiana and from two species of shark. The Xenopus C region resembles mouse and human C kappa slightly more than C lambda. The similarity of the Xenopus and Rana C regions to each other is approximately the same as that of either amphibian sequence to mammalian CL. The data are discussed in terms of the evolution of kappa and lambda C regions.     

FGF3 from Xenopus laevis.

Fibroblast growth factor 3 (FGF3) was first identified as the product of a cellular oncogene activated by mouse mammary tumour virus but its normal role appears to be in the developing embryo. To gain further insights into its function, we have isolated sequences encoding the FGF3 homologue in Xenopus laevis, XFGF3. COS-1 cells transfected with XFGF3 cDNA express a 31 kDa product, p31, generated by signal peptide cleavage and Asn-linked glycosylation at the single consensus site. This product is secreted and becomes associated with the cell surface and extracellular matrix. Proteolytic cleavage of p31 in the extracellular compartment results in an amino-terminally truncated product, p27, that is also glycosylated. Both p31 and p27 bind quantitatively to heparin-Sepharose and can be displaced from the cell surface and extracellular matrix by soluble heparin. Conditioned medium containing these two proteins is capable of inducing transient morphological transformation of NIH3T3 cells and of stimulating DNA synthesis in quiescent C57MG and BALB/MK cells which express different isoforms of FGF receptors 1 and 2. Since XFGF3 behaves very differently from its mouse counterpart, we constructed chimeras in which amino-terminal sequences from XFGF3 were fused with carboxy-terminal sequences from mouse FGF3. Increasing the contribution from mouse FGF3 led to a more restricted host range for the chimeric ligand.     

Specific cleavage of beta-amyloid peptides by a metallopeptidase from Xenopus laevis skin secretions

Dactylysin (EC 3.5.24.60) is a metalloendopeptidase first isolated from the skin granular gland secretions of Xenopus laevis. This peptidase hydrolyzes bonds on the amino-terminus of singlets and between doublets of hydrophobic amino acids and was considered to play a role in the in vivo inactivation of biologically active regulatory peptides. Here, we show that dactylysin has also the ability to cleave human β[1-40]-amyloid peptide and related peptides. Cleavage of the wild type β[1-40]-amyloid peptide form, and to a lesser extent Flemish and Dutch mutants, occurred predominantly at the His¹⁴-Glu¹⁵ bond. We demonstrate that frog skin exudate contains a full-length amyloid protein precursor detected by immunochemical cross-reactivity with monoclonal antibody against C-terminal human amyloid protein precursor. The possibility that dactylysin, might be involved in normal catabolism of β amyloid peptide of Xenopus laevis is discussed.     

Properties of a cytostatic factor from Xenopus laevis eggs.

The cytoplasm of mature eggs of Xenopus laevis was found to contain a cytostatic factor (CSF) which induces cleavage arrest at metaphase when microinjected into one blastomere of a two-cell embryo of Xenopus laevis or Rana pipiens. The Rana CSF was found to be incapable of arresting mitosis in Xenopus embryos. Both Xenopus and Rana CSF were stabilized during the transfer procedure by Ca²⁺-chelation in the donor egg. The Xenopus CSF was not present in the germinal vesicle of immature oocytes, but arose in the cytoplasm at the time of germinal vesicle breakdown and subsequently disappeared at the time of fertilization or egg activation.     

Purification of a calcium dependent ribonuclease from Xenopus laevis.

We have purified a Ca2+ dependent ribonuclease from the oocytes of Xenopus leavis. Two properties of this ribonuclease set it apart from other known nucleases. First, Ca2+ was required for ribonuclease activity, and Mg2+ would not substitute. Second, the enzyme specifically degraded RNA and digestion of double or single stranded DNA was not observed. Ca2+ dependent ribonuclease activity of the purified 36-kDa protein was directly observed after renaturation of the protein following electrophoresis in an SDS-Laemmli gel. In addition, the enzyme was shown to have endoribonuclease activity at numerous sites. The Ca2+ dependence suggests that the ribonuclease activity may be modulated by changes in the level of intracellular Ca2+ and thereby provide a direct link to signal transduction systems.     

Levitide, a neurohormone-like peptide from the skin of Xenopus laevis. Peptide and peptide precursor cDNA sequences

A novel peptide, levitide, less than Glu-Gly-Met-Ile-Gly-Thr-Leu-Thr-Ser-Lys-Arg-Ile-Lys-Gln-NH2 has been isolated from skin secretions of the South African frog Xenopus laevis and sequenced by fast atom bombardment mass spectrometry. Synthetic oligonucleotides were used as probes to screen a X. laevis skin cDNA library for species coding for preprolevitide. Two such clones were detected and their sequences are reported here. Preprolevitide is 88 residues long, exhibits a putative signal sequence at the amino terminus, and contains the levitide peptide at the carboxyl terminus. The levitide precursor shows a striking nucleotide and amino acid (86%) sequence homology with the precursor of xenopsin, a biologically active octapeptide from Xenopus skin, and also encodes a 25-residue amphipathic peptide that is released by processing at a single arginine residue.     

Peptide C-terminal alpha-amidating enzyme purified to homogeneity from Xenopus laevis skin

The C-terminal alpha-amide formation of the peptides is one of the most important events of prohormone processing. In this study, we have developed a simple and sensitive assay for monitoring alpha-amidating activity by using radioiodinated Ac-Tyr-Phe-Gly as a substrate. By utilizing this assay, an alpha-amidating enzyme was first purified to homogeneity from Xenopus laevis skin. The purified enzyme has a single polypeptide chain with an apparent molecular weight of 39,000 and its N-terminal sequence was determined as Ser-Leu-Ser-. The enzyme converts several synthetic peptides with C-terminal glycine to the corresponding des-glycine peptide alpha-amides. The enzyme activity, with an optimal pH 6-7, was dependent on the copper ion and ascorbate. In the presence of 0.25 mM ascorbate, the enzyme exhibited a Km of 0.35 microM and a Vmax of 1.9 nmol/microgram/h for Ac-Tyr-Phe-Gly.