Synthesis, isolation, and characterization of endogenous beta-galactoside-binding lectins in human leukocytes

Galaptin, a beta-galactoside-binding lectin, was isolated from human buffy coat cells (peripheral leukocytes) and spleen by affinity chromatography. The molecular weight (32K) of the native buffy coat galaptin was similar to that for splenic galaptin. Their subunit molecular weight (14.5K), pI (4.60-4.85), and amino acid composition were identical. Both galaptins showed the presence of a single polypeptide when subjected to reversed-phase HPLC. Monospecific rabbit polyclonal antiserum raised against the 14.5-kDa subunit of splenic galaptin reacted with a 14.5-kDa polypeptide present in buffy coat cells, Epstein-Barr virus-immortalized B lymphoblastoid cells, and HL-60 promyelocytic leukemia cells. However, galaptin was not synthesized in vitro by buffy coat cells. Rather, a monomeric beta-galactoside-binding protein of Mr 15.5-16.5K that is immunologically distinct from galaptin was synthesized. This galactoside-binding protein was separable from galaptin by polyacrylamide gel electrophoresis and by anion-exchange chromatography. In contrast, immunoprecipitation experiments confirmed that galaptin was synthesized by the B lymphoblastoid cells. cDNA corresponding to the B lymphoblastoid cell mRNA encoding galaptin was amplified by the polymerase chain reaction. The amplified product was partially sequenced, and 299 nucleotides were identified. The derived amino acids corresponded to residues 6-65, 84-114, and 118-126 found to be present in human splenic galaptin. Immunohistochemical analyses revealed that galaptin was distributed throughout the cytoplasm of B lymphoblastoid cells rather than being localized to the cell surface.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human splenic galaptin: physicochemical characterization

Mammalian spleens were previously reported to contain beta-galactoside-binding lectins [Allen, H. J., Cywinski, M., Palmberg, R., & DiCioccio, R. (1987) Arch. Biochem. Biophys. 256, 523-533]. The aim of the present investigation was to determine the relationship of human splenic galaptin to other beta-galactoside-binding lectins identified in other human and animal tissues. Galaptin of subunit molecular mass 14.5 kDa was the only lectin of this type found in human spleen as assessed by SDS-PAGE, RP-HPLC, and Western blot analyses. Three polypeptides of pI 4.60, 4.80, and 4.85 were detected by isoelectric focusing of purified galaptin, with the major band having pI 4.85. UV spectral analysis indicated the absence of prosthetic groups and gave A1%(1cm), 280 = 5.5. Circular dichroic analysis suggested the presence of 40% beta structure, considerable random coil, and 10% alpha helix structure. The amino acid composition was very similar to that for human placental galaptin. Amino acid sequence analyses were carried out on V8 protease, CNBr, and iodosobenzoic acid digestion fragments. A total of 94 residues were identified. All sequences determined could be aligned with placental galaptin sequences. We conclude that human splenic galaptin is identical with human placental galaptin. A related polypeptide of molecular mass approximately 14.5 kDa was found to be present in several different mammalian spleens as assessed by Western blot analysis using a monospecific polyclonal anti-human splenic galaptin antiserum.     

Lymphoblastoid cell adhesion mediated by a dimeric and polymeric endogenous beta-galactoside-binding lectin (galaptin).

Glutaraldehyde-polymerized human splenic galaptin, a beta-galactoside-binding lectin, was demonstrated to have enhanced hemagglutinating and asialofetuin binding activity relative to native dimeric galaptin when these lectins were present in solution. The polymerized lectin consisted primarily of 2-, 4- and 12-membered species after reductive alkylation. Both forms of galaptin bound, at 4 degrees C, to saturable B lymphoblastoid cell surface receptors. Estimates obtained by Scatchard analyses, with the binding data expressed in terms of 14.5 kDa subunit molarity, were 5 x 10(7) binding sites/cell with affinity constant Ka = 2.2 x 10(5) M for dimeric galaptin and 17 x 10(7) binding sites/cell with Ka = 3.4 x 10(5) M-1 for polymeric galaptin. Both forms of galaptin adsorbed to polystyrene with high efficiency; however, only plastic-adsorbed polymeric galaptin mediated adhesion of lymphoblastoid cells. Cell adhesion was inhibited by lactose. Plastic-adsorbed polymeric galaptin bound asialofetuin more efficiently than dimeric galaptin. Asialofetuin binding was inhibited 65% and 30-50% by lactose for plastic-adsorbed polymeric and dimeric galaptin, respectively. Native fetuin bound to the adsorbed dimeric galaptin in a lactose-insensitive manner. These data indicate that cell surface receptor-galaptin interaction is carbohydrate specific whereas polystyrene-adsorbed galaptin may demonstrate protein-protein interactions with soluble ligands.     

Soluble bovine galactose-binding lectin. cDNA cloning reveals the complete amino acid sequence and an antigenic relationship with the major encephalitogenic domain of myelin basic protein.

A full-length cDNA clone for the 13-14 kDa soluble beta-galactoside-binding lectin was isolated from a bovine fibroblast cDNA library. The derived amino acid sequence shows eight differences from a preliminary partial amino acid sequence given previously for the bovine heart lectin. This observation led to a re-examination of the data and correction of the heart lectin protein sequence. Except for a possible polymorphism of the heart lectin at position 57, the fibroblast and heart lectin sequences are considered identical. The epitope recognized by two monoclonal anti-(bovine lectin) antibodies, 36/8 and 9/5, was identified as the tetrapeptide sequence W-G-A/S-E/D by the isolation of several different cDNA clones from a human intestine cDNA library. A similar tetrapeptide is present in all of the soluble beta-galactoside-binding animal lectins sequenced thus far. It is also found in myelin basic protein, which we show is antigenically cross-reactive with the lectin. In myelin basic protein the tetrapeptide is a part of the major domain previously shown to be responsible for the induction of experimental allergic encephalomyelitis.     

Isolation and characterization of lactose-binding lectins from the venoms of the snakes Lachesis muta and Dendroaspis jamesonii

Two lectins have been isolated: one from the venom of Lachesis muta (bushmaster lectin) and one from Dendroaspis jamesonii venom (Jameson's mamba lectin). The lectin from bushmaster venom (BML) is similar to the lactose-binding lectins previously isolated from snake venoms (Gartner et al. (1980) FEBS Lett. 117, 13-16; Gartner & Ogilvie (1984) Biochem. J. 224, 301-307) in that it is calcium-dependent, lactose inhibitable, and is a dimer of molecular weight 28,000. In contrast, the lactose-blockable lectin from Jameson's mamba venom (JML) has an apparent molecular weight of 26,000 and agglutinates erythrocytes in the presence of EDTA. The absorption spectra of BML were affected by the binding of calcium, or calcium and lactose to the lectin. However, JML spectra were not affected by these conditions. While the hemagglutination activity of each of the previously described lactose-binding snake venom lectins is inhibited by reducing agent, the activities of BML and JML are not affected by reducing agent. Antiserum against bushmaster lectin cross-reacts with thrombolectin, cottonmouth lectin (CML), rattlesnake lectin (RSL), and copperhead lectin (CuHL) but not lectin from Jameson's mamba venom. This evidence plus a comparison of atomic absorption spectra, isoelectric points and amino acid analyses of the lectins demonstrate that JML and BML are different from thrombolectin, CML, RSL, and CuHL.     

Description of a monomeric prototype galectin from the lizard Podarcis hispanica

Galectins are a continuously expanding family of beta-galactoside-binding lectins present in a variety of evolutionarily divergent animal species. Here we report, for the first time, that expression of galectins extends to the reptilia lineage of lizards. Up to five lactose-binding proteins were isolated from the lizard Podarcis hispanica by affinity chromatography on asialofetuin-Sepharose. The main component, which is most abundantly expressed in skin, was purified from this tissue and further characterized. Under native conditions the protein behaved as a monomer with a molecular mass of 14,500 Da and an isoelectric point of 6.3. Based on sequence homology of the 58 N-terminal amino acid residues with galectins, and on its demonstrated galactoside-binding activity, this lectin we named LG-14 (from Lizard Galectin and 14 kDa) is classified as a new member of the galectin family. LG-14 falls into and strengthen the still thinly populated category of monomeric prototype galectins.     

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.     

Stimulation of vascular cell proliferation by beta-galactoside specific lectins

An investigation was conducted to assess the effects of various beta-galactoside specific lectins on the growth of vascular cells in vitro. The plant lectins from peanut (Arachis hypogaea), mushroom (Agaricus bisporus), and coral tree (Erythrina corallodendron) were used in these studies with the ultimate purpose of comparing those findings with data derived with the lectin isolated from rat lung. Peanut lectin was added to confluent and subconfluent cultures of smooth muscle cells (SMC), pulmonary arterial (PEC), and aortic endothelial cells (BAEC) at concentrations of 2, 3.5, and 7.0 micrograms/ml. There was a dose-dependent increase in cell proliferation for both confluent and subconfluent SMC, with maximal stimulation noted between 3.5 and 7 micrograms/ml of peanut lectin. A dose-dependent stimulation of PEC proliferation was also found with maximal stimulation between 3.5 and 7.0 micrograms/ml. Peanut lectin did not stimulate BAEC to multiply. The stimulation of PEC and SMC by peanut lectin could be prevented by the addition of 50 mM lactose. Peanut and mushroom lectin stimulated the proliferation of sparse cultures of SMC in a dose-dependent fashion in both standard (10% fetal bovine serum, or FBS) or low (0.5% FBS) serum to about the same degree. Coral tree lectin did not have a significant stimulation of proliferation under either serum conditions. The incorporation of [3H]thymidine into the DNA of PEC was increased 30 and 150% by peanut lectin and lung galaptin, respectively, under standard serum conditions. However, under low serum conditions, both lectins increased incorporation by about the same extent (93 and 78% for peanut lectin and galaptin, respectively). Both lectins produced a 30% increase in DNA synthesis by SMC under standard serum conditions, and about a 200% increase under low serum conditions. These studies indicate that beta-galactoside specific lectins such as lung galaptin have mitogenic activity toward vascular cells.     

Carbohydrate-Binding Proteins in the Leech: I. Isolation and Characterization of Lactose-Binding Proteins

Abstract: Three lactose-binding proteins with apparent molecular masses of 16, 35, and 63 kDa [leech lectin 16, 35, and 63 (LL16, LL35, and LL63, respectively)] were isolated from leech membranes. Polyclonal antibodies raised against LL35 cross-reacted with LL16 and LL63, indicating that all three lectins were immunologically related. These leech lectins, however, can be subdivided into two groups based on their tissue distributions and binding affinities for galactose derivatives. LL16 and LL35 are endogenous to the leech's CNS, whereas LL63 is only present in peripheral organs. LL16 and LL35, found in the CNS, bind both the α and β anomers of methylgalactose, whereas the peripheral lectin LL63 binds only the β form. LL35 and LL63 also differ in their binding affinities for galactosamine and N -acetylgalactosamine. The binding activity of LL35 was calcium independent and active over a wide pH range. Triton X-100 and 2-mercaptoethanol were necessary to recover LL35 binding activity during extraction. These characteristics strongly suggest that LL35 is another member of the calcium-independent galactose/lactose-specific lectins previously described in vertebrates and recently demonstrated in sponges and nematodes. Because a single leech expresses up to 100 µg of LL35, this leech lectin is readily amenable to structural and functional analysis.     

Immunological cross-reactivity between beta-galactoside-binding lectins of vertebrates. Possible relationship of antigenicity to oligomeric structure

Structural relationships among five beta-galactoside-binding lectins isolated from human, mouse and chick were studied using immunochemical methods. The lectins examined were human placenta lectin with a 14-kDa subunit (human 14K lectin), two types of mouse lectin (mouse 15K and mouse 16K lectin), and two types of chick lectin (chick 14K and chick 16K lectin). Five polyclonal antibodies raised against these lectins were used. Antibody to human 14K lectin cross-reacted with mouse 15K and chick 14K lectins. Antibodies to both mouse 15K and chick 14K lectins cross-reacted with human 14K and chick 16K lectins. Antibody to chick 16K lectin cross-reacted with mouse 15K lectin. An immunological relationship was not found between human 14K and chick 16K lectins, or between mouse 15K and chick 14K lectins. Mouse 16K lectin did not show any immunological relationship with any of the other lectins. A monoclonal antibody raised against chick 14K lectin cross-reacted with chick 16K lectin. These results cannot be explained simply in terms of phylogenic distance but suggest that vertebrate beta-galactoside-binding lectins can be classified into two structural groups on the basis of their antigenicities. One group, which is characterized as a monomer type, includes human 14K and chick 14K lectins. The other group, which is characterized as a dimer type, includes mouse 15K and chick 16K lectins.     

Sequence and specificity of a soluble lactose-binding lectin from Xenopus laevis skin.

A 16-kDa lactose-binding lectin comprises 5% or more of the soluble protein in Xenopus laevis skin. This lectin is mainly localized in the cytoplasm of granular gland cells. In response to stress, the lectin along with a variety of toxic and antibiotic peptides are released onto the skin surface by holocrine secretion. We have purified the lectin, sequenced tryptic peptides using tandem mass spectrometry and Edman degradation, and isolated full-length cDNA using a deduced oligonucleotide. Comparison of the cDNA and peptide sequences revealed expression of at least two isolectins, which differ in sequence at only two or three amino acids. Comparison of cDNA with complementary message by ribonuclease protection confirmed expression in approximately equal abundance of two nearly identical messages. The major soluble lactose-binding lectin expressed in Xenopus muscle is composed of these same isolectins, but at 100-fold lower levels. Similarities and distinctions in sequence and carbohydrate-binding specificity indicate that this lectin is a novel member of a family of soluble lactose-binding lectins expressed in a wide range of vertebrate tissues.     

Comparative analysis of galactoside-binding lectins isolated from mammalian spleens

Galactoside-inhibitable lectins have been isolated from rabbit, rat, mouse, pig, lamb, calf, and human spleens. Native molecular mass, subunit structure, pI, and hemagglutinating activity have been compared for these lectins. The yields of lectin varied from 1.8 mg/kg for rabbit spleen to 79 mg/kg for lamb spleen. Pig, lamb, calf, and human spleen lectins yielded single protein peaks when subjected to Superose 12 fast-protein liquid chromatography. The apparent molecular mass for these lectins was 33-34 kDa. In contrast, rat and mouse spleen lectin preparations were separated into three components ranging from 8.4 to 34 kDa. Superose 12 chromatography of rabbit spleen lectin revealed the presence of at least six components. Gradient slab gel sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the presence of single polypeptides for pig, calf, lamb, and human lectins corresponding to a molecular mass of 14-14.5 kDa. Multiple polypeptides were detected for the mouse, rat, and rabbit lectins. The molecular mass of the major polypeptides were 15, 15, and 17 kDa for rat, mouse, and rabbit, respectively. The presence of isolectins in all preparations was shown by isoelectric focusing. The major isolectins were acidic proteins with pI 4.38-4.80. Hemagglutination and hemagglutination inhibition assays demonstrated similarities as well as differences among the lectin preparations. Hemagglutinating activity could not be demonstrated in rabbit spleen extracts nor for isolated putative lectin. Human buffy coat cells were reversibly agglutinated by calf and human spleen lectins, demonstrating the presence of leucocyte cell surface lectin receptors.     

Isolation of lactose-binding lectins from axolotl (Ambystoma mexicanum).

1. Lactose-inhibitable hemagglutination activity was identified in extracts of axolotl (Ambystoma mexicanum) larvae. 2. Two types of lectin were isolated from extracts by affinity chromatography on lactose-Sepharose. 3. A thiol-independent lectin of subunit mol. wt 15 kDa and a thiol-dependent lectin of subunit mol. wt 18 kDa were identified. 4. The 15 kDa and a 18 kDa polypeptides were weakly reactive with polyclonal anti-human galaptin serum.     

The major tuber storage protein of araceae species is a lectin. Characterization and molecular cloning of the lectin from Arum maculatum L.

A new lectin was purified from tubers of Arum maculatum L. by affinity chromatography on immobilized asialofetuin. Although this lectin is also retained on mannose-Sepharose 4B, under the appropriate conditions free mannose is a poor inhibitor of its agglutination activity. Pure preparations of the Arum lectin apparently yielded a single polypeptide band of approximately 12 kD upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, N-terminal sequencing of the purified protein combined with molecular cloning of the lectin have shown that the lectin is composed of two different 12-kD lectin subunits that are synthesized on a single large precursor translated from an mRNA of approximately 1400 nucleotides. Lectins with similar properties were also isolated from the Araceae species Colocasia esculenta (L.) Schott, Xanthosoma sagittifolium (L.) Schott, and Dieffenbachia sequina Schott. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration of the different Araceae lectins have shown that they are tetrameric proteins composed of lectin subunits of 12 to 14 kD. Interestingly, these lectins are the most prominent proteins in the tuber tissue. Evidence is presented that a previously described major storage protein of Colocasia tubers corresponds to the lectin.     

Soluble lactose-binding vertebrate lectins: a growing family

Extracts of rat intestine contain nine soluble lactose-binding lectins with subunit molecular weights ranging from 14,500 to 19,000 that were purified by affinity chromatography and ion-exchange chromatography. Two of them are either identical with or closely related to other known rat lectins. A third appears to be the isolated carbohydrate-binding C-terminal domain of a known lectin but lacks the N-terminal domain presumed to mediate a different function. The others have not been described previously. Among them, the major rat intestinal lectin, RI-H, and a related protein, RI-G, have N-terminal amino acid sequences with similarities to sequences found in other known rat lectins. Therefore, these results introduce new members of a growing family of these structurally homologous soluble lactose-binding proteins.     

The sequence of the mouse 14 kDa beta-galactoside-binding lectin and evidence for its synthesis on free cytoplasmic ribosomes.

The partial amino acid sequence of the mouse 14 kDa beta-galactoside-binding lectin has been deduced from cDNA clones corresponding to 86% of the coding sequence and extending to the polyadenylation signal. The deduced amino acid sequence for the murine lectin shows 94% identity with the rat, 89% with human, 86% with bovine and 46% with the chicken 14 kDa lectins. A cDNA probe has been used to analyse genomic DNA and identify a single mRNA of approx. 570 bp in 3T3 fibroblasts, murine erythroleukaemia cells and the murine basement-membrane-secreting Engelbreth-Holm-Swarm tumour. Analysis of free and bound polyribosomes has shown that the lectin message is translated on free cytoplasmic ribosomes.     

Isolation and partial characterisation of galactose-specific lectins from African yam beans, Sphenostyles stenocarpa Harms.

A new galactose-specific lectin was isolated from African yam bean (Sphenostyles stenocarpa Harms) by affinity chromatography on galactose-Sepharose 4B. SDS-PAGE analysis resulted in four polypeptide bands of approximately 27, 29, 32 and 34 kDa, respectively. Based on the analysis of carbohydrate content and native PAGE, it is likely that the Sphenostyles lectin is a tetrameric glycoprotein with M(r) of approximately 122 kDa. N-terminal protein sequencing of purified lectins from four different Sphenostyles accessions shows that the four polypeptides have largely identical amino acid sequences. The sequences contain the conserved consensus sequence F-F-LILG characteristic of legume lectins, as well as Phaseolus vulgaris proteins in the arcelin-alpha-amylase inhibitor gene family. The lectin agglutinates both rabbit and human erythrocytes, but with a preference for blood types A and O. Using Western blotting, the lectin was shown to accumulate rapidly during seed development, but levels dropped slightly as seeds attained maturity. This is the first time a lectin has been purified from the genus Sphenostyles. The new lectin was assigned the abbreviation LECp.SphSte.se.Hga1.     

Purification and properties of a beta-galactoside-binding lectin from neonatal marmoset.

A beta-galactoside-binding lectin was extracted from whole neonatal marmoset homogenate with lactose solution and purified to homogeneity by ion-exchange chromatography on Q Sepharose Fast Flow and by affinity adsorption to trypsinized and glutaraldehyde-fixed ghosts of rabbit erythrocytes. The lectin has a dimeric structure composed of two 15K subunits. Its amino acid composition and partial amino acid sequences were quite similar to those of beta-galactoside-binding lectins from human placenta and lung.     

An intestinal galactose-specific lectin mediates the binding of murine IgE to mouse intestinal epithelial cells.

A number of lactose-binding lectins have recently been identified in the rat and mouse intestine, one of which corresponds to the C-terminal domain of IgE-binding proteins, originally identified in rat basophilic leukemia (RBL) cells and mouse 3T3 fibroblasts. In the present report, we describe the affinity purification of a rat intestinal lactose-specific lectin which binds murine IgE antibodies. This binding most likely occurs via the immunoglobulin carbohydrate chains, as it is inhibited by lactose. This intestinal lectin molecule is also immunologically related to the previously described IgE-binding protein (epsilon BP) isolated from RBL cells, since it is recognized by antibodies raised against recombinant epsilon BP. This intestinal form of epsilon BP has a molecular mass of 17.5 kDa, which is much lower than that of its RBL cell analogue (31 kDa). The attachment of IgE to the mouse intestinal epithelium was demonstrated by immunohistochemistry, along with the presence of a corresponding mouse intestinal epsilon BP. The carbohydrate-dependent nature of this attachment was established by demonstrating that IgE binding to mouse epithelium was specifically abolished by lactose (4 mM) and by a blood-group-A-active tetrasaccharide (0.2 mM), but not by mannose (10 mM). Finally, the association of IgE with the mouse intestinal epithelium was prevented by competition with the purified IgE-binding lectin isolated from rat intestine. Although the physiological function of this intestinal protein is still unknown, the finding that IgE binds to a lectin in the intestinal epithelium pinpoints a possible novel mechanism for the regulation of IgE-mediated disorders, such as food allergy.     

Different immunoreactivities of anti-soluble lactose lectin antisera to tissues from early chick embryos: a histochemical study

The location of soluble lactose-binding proteins (S-lac lectins) has been studied by immunohistochemical methods during morphogenesis of the chick embryo, when segregation and early differentiation of organ primordia was occurring. Using a panel of polyclonal antisera raised to various purified lectin preparations, we observed striking differences in the antigenic properties of these antisera, indicating that diverse versions of the lectins may be expressed during development. The antisera referred to as anti-L-16, anti-M-16, anti-S-14 and anti-I-14 were respectively raised to native or denatured 16 kDa lectins from adult liver and embryonic muscle and to 14 kDa lectins from embryonic skin and adult intestine. Having determined the optimal immunohistochemical conditions in the preparation of embryo sections (fixation, embedding, sectioning) we show that anti-L-16, anti-S-14 and anti-I-14 mostly bind the lectins expressed at the cell surface, in the extracellular matrix and in some released secretion. As previously shown, anti-L-16 and anti-S-14 are also able to recognize the cytoplasmic form of some migrative lectin-rich cells (primitive streak, neural crest cells, germ cells). Anti-M-16 was bound exclusively to the cytoplasmic form of the 16 kDa lectin in the same cell lines as above and also in some others, such as in the notochord, the myotomal part of the somites, the pharyngeal endoderm and the cardiac muscle. These different antigenic properties may be applied to the accurate mapping of various lectin isoforms and evaluation of the respective contribution of their intra-and extracellular variants during development and differentiation.