Complete amino acid sequence of 14 kDa beta-galactoside-binding lectin of chick embryo

The complete amino acid sequence of a soluble beta-galactoside-binding lectin (subunit MW 14,500) of chick embryo was determined. The protein consists of 134 amino acids beginning with serine and ending with glutamic acid, and its N-terminal was blocked with acetate. The agreement of the present result with that obtained from nucleotide sequence analysis (Y. Ohyama et al. (1986) Biochem. Biophys. Res. Commun. 134, 51-56) indicates the lack of a cleavable leader sequence. Internal homologies were observed in several regions along the polypeptide chain. The highest homology (55% identity) was found between residues 42-58 and residues 112-128. This suggests that chick 14 kDa lectin may have evolved via several gene duplications.     

Complete amino acid sequence of a beta-galactoside-binding lectin from human placenta

The complete amino acid sequence of a beta-galactoside-binding lectin from human placenta was determined at protein level. The lectin consists of 134 amino acids and its N-terminal alanine is blocked with acetate. The lectin shows about 50% similarity with chick 14K lectin, which was the first vertebrate beta-galactoside-binding lectin completely sequenced. Only 14 residues proved to be different from those of rat lung lectin, the sole mammalian lectin of which the complete sequence has been reported.     

Structure of chicken 16-kDa beta-galactoside-binding lectin. Complete amino acid sequence, cloning of cDNA, and production of recombinant lectin

The complete primary structure of chicken 16-kDa beta-galactoside-binding lectin (C-16) was determined. It was composed of 134 amino acid residues and has an acetylated NH2 terminus. A cDNA was also cloned, but no signal sequence was found in the initiator region. The initiator methionine remained as the NH2 terminus of the mature lectin. Although C-16 is distinct from chicken 14-kDa beta-galactoside-binding lectin (C-14), it proved to be a member of the vertebrate 14-kDa-type lectin family. Comparison of the primary structures between the vertebrate 14-kDa-type lectins suggests that C-14 and C-16 were produced by gene duplication of an ancestral lectin gene at a time close to the divergence of birds and mammals. Northern and Southern blot analysis indicated that these isolectins are encoded by individual genes which are differently regulated during the development of the embryo. A recombinant C-16 lectin was produced in Escherichia coli. The product was indistinguishable from the authentic C-16 lectin except that the NH2 terminus of the former was found to begin with free methionine.     

Evidence that Caenorhabditis elegans 32-kDa beta-galactoside-binding protein is homologous to vertebrate beta-galactoside-binding lectins. cDNA cloning and deduced amino acid sequence.

We have cloned a full-length cDNA for a beta-galactoside-binding protein with a relative molecular mass of 32 kDa (32-kDa GBP), recently purified from a nematode, Caenorhabditis elegans (Hirabayashi, J., Satoh, M., Ohyama, Y., and Kasai, K. (1992) J. Biochem. 111, 553-555). The clone contained a single open reading frame encoding 279 amino acids, including the initiator methionine. Significant sequence homology to metal-independent beta-galactoside-binding lectins (25-30% identities), which had previously been found only in vertebrates, was observed. Moreover, the nematode 32-kDa GBP proved to have a unique polypeptide architecture; that is, it is composed of two tandemly repeated homologous domains, each consisting of about 140 amino acids. The internal homology was about 32%. Thus, this protein is constructed with a duplicated fundamental unit which is similar to the subunit of vertebrate 14-kDa lectins. In spite of the extreme phylogenic distance between nematodes and vertebrates (divergence greater than 6 x 10(8) years ago), both of the two repeated domains of the nematode 32-kDa GBP retained most of the amino acid residues conserved in vertebrate lectins. This means that members of the metal-independent animal lectin family are distributed much more widely than had been believed: from nematodes to vertebrates. The implication is that proteins belonging to this family have fundamental roles which are not restricted to vertebrates but are common to almost all animals.     

Isolation and characterization of the chick 14K beta-galactoside-binding lectin gene

Vertebrate endogenous lectins have been implicated in cellular interactions that contribute to embryonic development. We have isolated a cloned segment of the gene for chick 14K type beta-galactoside-binding lectin from a genomic DNA library. Analysis of the structure of the cloned gene as well as the results of genomic Southern blot hybridization revealed that the gene is unique and that the mRNA for the lectin is encoded by four exons separated by three introns. The whole sequence spans 3.1 kilobases in the gene. The first exon encodes only two amino acid residues of the N-terminus of the mature protein and the other three exons encode, respectively, one of the three repeating sequences found in this lectin. These facts strongly support the idea that gene duplications have occurred during the evolution of this lectin. The previous study (Y. Ohyama et al. (1986) Biochem. Biophys. Res. Commun. 134, 51-56) suggested that this lectin is not synthesized as a precursor molecule with a cleavable signal sequence at its amino terminus, although it is known to be secreted into the extracellular matrix. Sequence determination of the upstream region of the mRNA indicated that the ATG located just before the codon for the N-terminal amino acid of the mature protein is the actual translation initiator. Thus it was proved that this lectin is synthesized without an N-terminal cleavable signal sequence, as suggested before.     

Improved insulin stability through amino acid substitution.

Insulin analogs designed to decrease self-association and increase absorption rates from subcutaneous tissue were found to have altered stability. Replacement of HB10 with aspartic acid increased stability while substitutions at B28 and/or B29 were either comparable to insulin or had decreased stability. The principal chemical degradation product of accelerated storage conditions was a disulfide-linked multimer that was formed through a disulfide interchange reaction which resulted from beta-elimination of the disulfides. The maintenance of the native state of insulin was shown to be important in protecting the disulfides from reduction by dithiothreitol and implicitly from the disulfide interchange reaction that occurs during storage. To understand how these amino acid changes alter chemical stability, the intramolecular conformational equilibria of each analog was assessed by equilibrium denaturation. The Gibbs free energy of unfolding was compared with the chemical stability during storage for over 20 analogs. A significant positive correlation (R2 = 0.8 and P less than 0.0005) exists between the conformational stability and chemical stability of these analogs, indicating that the chemical stability of insulin's disulfides is under the thermodynamic control of the conformational equilibria.     

Cloning and nucleotide sequence of a full-length cDNA for human 14 kDa beta-galactoside-binding lectin

A full-length cDNA for a 14K-type human lung beta-galactoside-binding lectin was cloned. The cDNA includes a 405 bp open reading frame coding 135 amino acids including the initiator methionine, and having a single internal EcoRI site and a polyadenylation signal. The deduced amino-acid sequence agreed completely with the sequence of a human placenta lectin determined by direct amino-acid sequence analysis (Hirabayashi, J. and Kasai, K. (1988) J. Biochem. 104, 1-4). It showed extensive sequence similarity with other vertebrate 14K-type lectins and a 35K-type lectin (carbohydrate-binding protein 35) of mouse 3T3 cell. Search of a Genbank sequence data base revealed significant sequence similarity between the beta-galactoside-binding lectins and the carboxyl-terminal half of an IgE-binding protein, the cDNA of which has been cloned from rat basophilic leukemia cells. Thus, 14K-type lectin, 35K-type lectin and IgE-binding protein appeared to form a superfamily of proteins. Almost all invariant residues are located in the central region of the 14K-type lectins, so this region may constitute an essential part of the lectins, such as the sugar-binding domain.     

Molecular cloning, characterization, and expression of a human 14-kDa lectin

Full length cDNAs coding for a 14-kDa beta-galactoside binding lectin have been isolated from HL-60 cells and human placenta. Oligonucleotide probes based on a pentapeptide present in several partial sequences of homologous human lectins were used to screen a lambda GT10 HL-60 cDNA library. The HL-60 cDNA clones that were isolated were used to design a synthetic primer representing the 3'-untranslated region of the HL-60 lectin. This primer was then used to synthesize a lambda GT10 human placenta cDNA library, and restriction fragments of the HL-60 cDNA clones were used to screen the library. The cDNA clones for both HL-60 and placenta lectin had identical sequences with short 5'- and 3'-untranslated regions and coded for a 135-amino acid protein which lacks a hydrophobic signal peptide sequence. Biochemical data show that, despite the presence of a possible N-linked glycosylation site, the protein is not glycosylated. Northern and Southern blot analyses indicate that the 14-kDa lectin is encoded for by a single gene. The lectin cDNA was expressed in Escherichia coli and biologically active protein was purified from cell lysates by affinity chromatography.     

Identification of a 14-kDa laminin binding protein (HLBP14) in human melanoma cells that is identical to the 14-kDa galactoside binding lectin.

The carbohydrate moieties present on laminin play a crucial role in the multiple biological activities of this basement membrane glycoprotein. We report the identification of a human laminin binding protein with an apparent molecular mass of 14 kDa on sodium dodecyl sulfate-polyacrylamide gels that was found, after purification and amino acid microsequencing, to be identical to the previously described 14-kDa galactoside binding soluble L-14 lectin. We have designated this human laminin binding protein as HLBP14. HLBP14 was purified from human melanoma cells in culture by laminin affinity chromatography and gel electroelution. We demonstrate that HLBP14 binds specifically to the poly-N-acetyllactosamine residues of murine laminin and does not bind to other glycoproteins that do not contain such structures, such as fibronectin. HLBP14 was eluted from a murine laminin column by lactose, N-acetyllactosamine, and galactose but not by other control saccharides, including glucose, fucose, mannose, and melibiose. It did not bind to laminin treated with endo-beta-galactosidase. Lactose also eluted HLBP14 off a human laminin affinity column, implying that human laminin also contains poly-N-acetyllactosamine residues. On immunoblots, polyclonal antibodies raised against HLBP14 recognized HLBP14 as well as 31- and 67-kDa molecules that are also laminin binding proteins, indicating that these proteins share common epitopes. L-14, a dimeric lactose binding lectin, is expressed in a wide variety of tissues. Although the expression of this molecule has been linked to a variety of biological events, the elucidation of its specific functions has been elusive. The observation that HLBP14, a human cancer cell laminin binding protein, is identical to L-14 strongly suggests that the functions attributed to this lectin could be mediated, at least in part, through its ability to interact with the poly-N-acetyllactosamine residues of laminin. HLBP14 could potentially play a role during tumor invasion and metastasis by modulating the interactions between cancer cells and laminin.     

Purification and properties of lung lectin. Rat lung and human lung beta-galactoside-binding proteins.

Lung is one of the organs of the rat with a particular abundance of haemagglutinating activity that is inhibited by beta-galactosides. This lectin activity can be attributed to a single protein that has been purified from rat lung; a similar protein has been purified from human lung. The molecular weights and subunit structures were estimated from gel filtration and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis; the human lung lectin appeared to be composed to two identical subunits, mol.wt. 14500, whereas rat lung lectin was observed as both a dimer and a tetramer of one subunit type, mol.wt. 13500. Both lectins bind to disaccharides or oligosaccharides with terminal beta-linked galactose residues. The carbohydrate moiety may be free [lactose or D-galactopyranosyl-beta-(1 leads to 4)-thiogalactopyranoside], protein-bound (asialofetuin) or lipid-bound (cerebrosides). The molecular properties of the beta-galactoside-binding proteins of rat lung and human lung are closely similar to those of embryonic chick muscle lectin [Nowak, Kobiler, Roel & Barondes (1977) Proc. Natl. Acad. Sci. U.S.A. 73, 1383--1387] and calf heart lectin [De Waard, Hickman & Kornfeld (1976) J. Biol. Chem. 251, 7581--7587].     

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.     

Destabilization of pea lectin by substitution of a single amino acid in a surface loop

Legume lectins are considered to be antinutritional factors (ANF) in the animal feeding industry. Inactivation of ANF is an important element in processing of food. In our study on the stability ofPisum sativum L. lectin (PSL), a conserved hydrophobic amino acid (Val¹⁰³) in a surface loop was replaced with alanine. The mutant lectin, PSL V103A, showed a decrease in unfolding temperature (T _m ) by some 10 °C in comparison with wild-type (wt) PSL, and the denaturation energy (H) is only about 55% of that of wt PSL. Replacement of an adjacent amino acid (Phe¹⁰⁴) with alanine did not result in a significant difference in stability in comparison with wt PSL. Both mutations did not change the sugarbinding properties of the lectin, as compared with wt PSL and with PSL from pea seeds, at ambient temperatures. The double mutant, PSL V103A/F104A, was produced inEscherichia coli, but could not be isolated in an active (i.e. sugar-binding) form. Interestingly, the mutation in PSL V103A reversibly affected sugar-binding at 37 °C, as judged from haemagglutination assays. These results open the possibility of production of lectins that are activein planta at ambient temperatures, but are inactive and possibly non-toxic at 37 °C in the intestines of mammals.     

Identification and synthesis of a novel 15 kDa beta-galactoside-binding lectin in human leukocytes.

Knowledge of the identity, synthesis and secretion of beta-galactoside-binding lectins by leukocytes is of importance because lactosaminoglycans present at the leukocyte cell surface may be physiologically significant lectin receptors that could mediate autocrine or paracrine functions and/or cell adhesion. This paper presents data that show that a previously identified 15.5-16.5 kDa lactose-binding protein synthesized in vitro by human peripheral leukocytes is actually comprised of three different polypeptides. One of these is related to a novel 15 kDa lectin isolated from human spleen and which is synthesized by B lymphoblastoid cells. Spleen contains at least six lactose-binding polypeptides for which the carbohydrate-binding activity is independent of the presence of divalent cations and mercaptoethanol. The splenic 15 kDa polypeptide does not appear to be immunologically related to previously characterized beta-galactoside-binding lectins. It is separable from galaptin, another galactoside-binding lectin (subunit mol. wt 14.5 kDa) by chromatography on DEAE-Sephacel. Western blot analyses and immunoprecipitation/fluorography experiments with metabolically labelled cells showed the presence of the 15 kDa lectin in peripheral leukocytes and in Epstein-Barr virus-immortalized B lymphoblastoid cells. The 15 kDa lectin yielded polypeptide fragments of approximately 6.2 and approximately 8.6 kDa after cyanogen bromide (CNBr) degradation. These fragments were partially sequenced and 12 residues/fragment were identified. A similarity search of the SWISS PROT protein data base did not reveal a relationship of the 15 kDa polypeptide to known lectins, including galaptin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural basis of carbohydrate recognition by the lectin LecB from Pseudomonas aeruginosa

The crystal structure of Pseudomonas aeruginosa fucose-specific lectin LecB was determined in its metal-bound and metal-free state as well as in complex with fucose, mannose and fructopyranose. All three monosaccharides bind isosterically via direct interactions with two calcium ions as well as direct hydrogen bonds with several side-chains. The higher affinity for fucose is explained by the details of the binding site around C6 and O1 of fucose. In the mannose and fructose complexes, a carboxylate oxygen atom and one or two hydroxyl groups are partly shielded from solvent upon sugar binding, preventing them from completely fulfilling their hydrogen bonding potential. In the fucose complex, no such defects are observed. Instead, C6 makes favourable interactions with a small hydrophobic patch. Upon demetallization, the C terminus as well as the otherwise rigid metal-binding loop become more mobile and adopt multiple conformations.     

Effect of site-specific DNA methylation and mutagenesis on recognition by methylated DNA-binding protein from human placenta.

Methylated DNA-binding protein (MDBP) from human placenta is the first protein shown to bind specifically to certain DNA sequences only when they are methylated at cytosine residues. Among the sites recognized by MDBP is pB site 1, a pBR322-derived sequence which has a high affinity for MDBP when methylated at all CpG positions. We have substituted pB site 1 with 5-methyl-cytosine (m5C) residues at one to three of its CpG dinucleotides on one strand by the use of m5C-containing oligonucleotides. MDBP binds best when all three CpG dinucleotides in the region 5'-ATCGTCACGGCGAT-3' are methylated. Even more binding is obtained when both strands are methylated. Alteration of various residues in this binding site by oligonucleotide-directed mutagenesis decreased the binding. However, two mutations which increased the dyad symmetry of part of the binding site yielded ligands with a higher affinity for MDBP.     

Galactose recognition by a tetrameric C-type lectin, CEL-IV, containing the EPN carbohydrate recognition motif

CEL-IV is a C-type lectin isolated from a sea cucumber, Cucumaria echinata. This lectin is composed of four identical C-type carbohydrate-recognition domains (CRDs). X-ray crystallographic analysis of CEL-IV revealed that its tetrameric structure was stabilized by multiple interchain disulfide bonds among the subunits. Although CEL-IV has the EPN motif in its carbohydrate-binding sites, which is known to be characteristic of mannose binding C-type CRDs, it showed preferential binding of galactose and N-acetylgalactosamine. Structural analyses of CEL-IV-melibiose and CEL-IV-raffinose complexes revealed that their galactose residues were recognized in an inverted orientation compared with mannose binding C-type CRDs containing the EPN motif, by the aid of a stacking interaction with the side chain of Trp-79. Changes in the environment of Trp-79 induced by binding to galactose were detected by changes in the intrinsic fluorescence and UV absorption spectra of WT CEL-IV and its site-directed mutants. The binding specificity of CEL-IV toward complex oligosaccharides was analyzed by frontal affinity chromatography using various pyridylamino sugars, and the results indicate preferential binding to oligosaccharides containing Galβ1-3/4(Fucα1-3/4)GlcNAc structures. These findings suggest that the specificity for oligosaccharides may be largely affected by interactions with amino acid residues in the binding site other than those determining the monosaccharide specificity.     

Differences in amino acid sequences of mistletoe lectin I and III B-subunits determining carbohydrate binding specificity

Toxic lectins of European mistletoe Viscum album L.--MLI (viscumin), MLII and MLIII--are present in water extracts of this plant. Earlier we have cloned the full-length gene of MLIII precursor [A.G. Tonevitsky, I.I. Agapov, I.B. Pevzner, N.V. Maluchenko, M.M. Mojsenovich, U. Pfueller, M.P. Kirpichnikov, (2004) Biochemistry (Mosc.), 69 (6), 790-800, in press]. Here for the first time we report the cloning and expression in Escherichia coli cells of MLIII gene fragment encoding the carbohydrate-binding subunit. We have proved with our panel of monoclonal antibodies against ML toxins that the cloned fragment encoded MLIII B-subunit. The immunochemical and sugar-binding activities of renatured recombinant MLIII B-subunit were demonstrated in ELISA and ELLA, respectively. The comparative analysis of amino acid sequences of the cloned rMLIIIB and the B-subunits of other type II RIPs--MLI, ricin, abrin and nigrin b--was performed, revealing the main differences in primary structure of MLI and MLIII B-chains, which could determine their sugar specificity. The antigenicity analysis of MLI and MLIII B-subunits showed one epitope 25RDDDFRDGNQ34 in MLIB that is absent in MLIIIB sequence. The role of the toxic lectins and their subunits in immunological properties of mistletoe extracts is discussed.