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.     

Lectins from bulbs of the Chinese daffodil Narcissus tazetta (family Amaryllidaceae).

The isolation of three lectins with similar N-terminal amino acid sequences from the bulbs of the Chinese daffodil Narcissus tazetta was achieved. The isolation protocol involved ion exchange chromatography on DEAE-cellulose, affinity chromatography on mannose-agarose, and fast protein liquid chromatography-gel filtration on Superose 12. The lectins were all adsorbed on mannose-agarose and demonstrated a single band with a molecular weight of 13 kDa in SDS-polyacrylamide gel electrophoresis and a single 26 kDa peak in gel filtration, indicating that they were mannose-binding, dimeric proteins. The lectins differed in hemagglutinating activity, with the magnitude of the activity correlating with the ionic strength of the buffer required to elute the lectin from the DEAE-cellulose column. The bulb lectin did not exert potent cytotoxicity against cancer cell lines or fetal bovine lung cells but inhibited syncytium formation in, and reinstated viability of, fetal bovine lung cells infected with bovine immunodeficiency virus.     

Isolation and characterization of a cDNA clone encoding the lima bean lectin

The complete amino acid sequence of the lima bean (Phaseolus lunatus) lectin was deduced from the nucleotide sequence of a cDNA clone. The lectin appears to be synthesized as a prepeptide consisting of a signal sequence of 21 residues and a mature protein of 241 amino acids. Comparison of the lima bean lectin sequence to the sequences of other leguminous seed lectins indicates regions of extensive homology. Northern blot analysis showed absence of lectin mRNA in the leaves, roots, or stems of 16-day-old lima bean plants.     

Purification and characterization of a lectin,BPL-3, from the marine green alga <Emphasis Type="Italic">Bryopsis plumosa</Emphasis>

A novel lectin was isolated and characterized from Bryopsis plumosa (Hudson) Agardh and named BPL-3. This lectin showed specificity to N-acetyl-d-galactosamine as well as N-acetyl-d-glucosamine and agglutinated human erythrocytes of all blood types, showing slight preference to the type A. SDS-PAGE and MALDI-TOF MS data showed that BPL-3 was a monomeric protein with molecular weight of 11.5 kDa. BPL-3 was a non-glycoprotein with pI value of ∼7.0. It was stable in high temperatures up to 70°C and exhibited optimum activity in pH 5.5–10. The N-terminal and internal amino acid sequences of the lectin were determined by Edman degradation and enzymatic digestion, which showed no sequence homology to any other reported proteins. The full sequence of the cDNA encoding this lectin was obtained from PCR using cDNA library, and the degenerate primers were designed from the N-terminal amino acid sequence. The size of the cDNA was 622 bp containing single ORF encoding the lectin precursor. This lectin showed the same sugar specificity to previously reported lectin, Bryohealin, involved in protoplast regeneration of B. plumosa. However, the amino acid sequences of the two lectins were completely different. The homology analysis of the full cDNA sequence of BPL-3 showed that it might belong to H lectin group, which was originally isolated from Roman snails.     

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.     

Sequence of a full-length cDNA for rat lung beta-galactoside-binding protein: primary and secondary structure of the lectin

A full-length cDNA for rat lung beta-galactoside lectin (subunit Mr approximately 14,000, lectin 14K) was cloned and the nucleotide sequence determined. The deduced amino acid sequence agrees with the amino acid composition and direct amino acid sequence analysis of purified rat lung lectin peptides. We found that the amino-terminal alanine is blocked with an acetyl group. Comparison of the amino acid sequence with other proteins shows a high degree of homology only with other vertebrate lectin sequences, supporting the suggestion that these lectins may constitute a unique class of vertebrate proteins. The amino acid composition and sequence of lectin peptides, the sequence of lectin cDNA, and isoelectric focusing of purified lectin indicate that rat lung lectin 14K is composed predominantly of a single protein. In addition, rat uterus lectin 14K was found to be the same protein as that present in lung. We characterized the secondary and tertiary structure of rat lung lectin 14K by circular dichroism, by analytical ultracentrifugation, and by computer analysis of its primary structure. Results of these experiments suggest that lectin 14K is primarily a hydrophilic protein with an asymmetric, elongated structure consisting of approximately equal amounts of alpha helix, beta sheet, beta turn, and random coil. We found that Cys-2 and Cys-130 react most rapidly with iodoacetamide; one or both of these residues may be primarily responsible for the thiol requirement of lectin activity.     

Characterization of an S-type lectin purified from porcine heart

We have purified a carbohydrate-binding protein from porcine heart by affinity chromatography on asialofetuin-Sepharose and have characterized this protein with respect to its size, amino acid composition, partial amino acid sequence, and carbohydrate-binding specificity. Porcine heart lectin (PHL) has a subunit molecular mass of 14,700 and is immunologically cross-reactive with a polyclonal antibody raised against a lectin isolated from calf heart. The amino acid composition of PHL is similar to that of lectins that have been isolated from calf heart, bovine brain, and rat lung. Moreover, the primary sequences of four tryptic fragments (52 amino acids total) derived from PHL are closely related to sequences previously determined for 10 other vertebrate-derived lectins. The ability of PHL to agglutinate rabbit erythrocytes was inhibited only by oligosaccharides containing terminal beta-galactosyl residues. These data indicate that PHL is a vertebrate "S-type" lectin and provide further evidence that the structures and carbohydrate-binding specificities of these lectins are highly conserved across diverse vertebrate genera.     

Purification,characterization, molecular cloning,and expression of novel members of jacalin-related lectins from rhizomes of the true fern Phlebodium aureum (L) J. Smith (Polypodiaceae)

A lectin was purified from rhizomes of the fern Phlebodium aureum by affinity chromatography on mannose-Sepharose. The lectin, designated P. aureum lectin (PAL), is composed of two identical subunits of approximately 15 kDa associated by noncovalent bonds. From a cDNA library and synthetic oligonucleotide probes based on a partial amino acid sequence, 5'- and 3'-rapid amplification of cDNA ends allowed the generation of two similar full-length cDNAs, termed PALa and PALb, each of which had an open reading frame of 438 bp encoding 146 amino acid residues. The two proteins share 88% sequence identity and showed structural similarity to jacalin-related lectins. PALa contained peptide sequences exactly matching those found in the isolated lectin. PALa and PALb were expressed in Escherichia coli using pET-22b(+) vector and purified by one-step affinity chromatography. Native and recombinant forms of PAL agglutinated rabbit erythrocytes and precipitated with yeast mannan, dextran, and the high mannose-containing glycoprotein invertase. The detailed carbohydrate-binding properties of the native and recombinant lectins were elucidated by agglutination inhibition assay, and native lectin was also studied by isothermal titration calorimetry. Based on the results of these assays, we conclude that this primitive vascular plant, like many higher plants, contains significant quantities of a mannose/glucose-binding protein in its storage tissue, whose binding specificity differs in detail from either legume mannose/glucose-binding lectins or monocot mannose-specific lectins. The identification of a jacalin-related lectin in a true fern reveals for the first time the widespread distribution and molecular evolution of this lectin family in the plant kingdom.     

Isolation,characterization and molecular cloning of the mannose-binding lectins from leaves and roots of garlic (Allium sativum L.)

Two novel lectins were isolated from roots and leaves of garlic. Characterization of the purified proteins indicated that the leaf lectin ASAL is a dimer of two identical subunits of 12 kDa, which closely resembles the leaf lectins from onion, leek and shallot with respect to its molecular structure and agglutination activity. In contrast, the root lectin ASARI, which is a dimer of subunits of 15 kDa, strongly differs from the leaf lectin with respect to its agglutination activity. cDNA cloning of the leaf and root lectins revealed that the deduced amino acid sequences of ASAL and ASARI are virtually identical. Since both lectins have identical N-terminal sequences the larger Mr of the ASARI subunits implies that the root lectin has an extra sequence at its C-terminus. These results not only demonstrate that virtually identical precursor polypeptides are differently processed at their C-terminus in roots and leaves but also indicate that differential processing yields mature lectins with strongly different biological activities. Further screening of the cDNA library for garlic roots also yielded a cDNA clone encoding a protein composed of two tandemly arrayed lectin domains. Since the presumed two-domain root lectin has not been isolated yet, its possible relationship to the previously described two-domain bulb lectin could not be studied at the protein level.     

Isolation and characterization of a mannan-binding lectin from the freshwater cyanobacterium (blue-green algae) Microcystis viridis

Microcystis viridis NIES-102 strain, a unicellular freshwater bloom-forming cyanobacterium, showed transient hemagglutinating activity in laboratory culture during stationary phase under nonaeration conditions. However, the hemagglutinating activity which was inhibited with yeast mannan could not be observed during culture with aeration. A mannan-binding lectin named MVL was isolated with the assay of the hemagglutinating activity against rabbit erythrocytes from the cyanobacterium by successive hydrophobic and gel filtration chromatography. MVL was composed of a single polypeptide of 13 kDa. The gene (mvl) for MVL was cloned from a genomic DNA of NIES-102 strain as a template, and its sequence was determined. The deduced amino acid sequence showed that MVL consisted of 113 amino acid residues and was composed of two tandemly repeated homologous domains of 54 amino acid residues. MVL showed no sequence homology to any other lectins or proteins.     

The partial amino acid sequence of bovine cartilage proteoglycan, deduced from a cDNA clone, contains numerous Ser-Gly sequences arranged in homologous repeats.

We have determined the sequence of a partial cDNA clone encoding the C-terminal region of bovine cartilage aggregating proteoglycan core protein. The deduced amino acid sequence contains a cysteine-rich region which is homologous with chicken hepatic lectin. This lectin-homologous region has previously been identified in rat and chicken cartilage proteoglycan. The bovine sequence presented here is highly homologous with the rat and chicken amino acid sequences in this apparently globular region. A region containing clusters of Ser-Gly sequences is located N-terminal to the lectin homology domain. These Ser-Gly-rich segments are arranged in tandemly repeated, approx. 100-residue-long, homology domains. Each homology domain consists of an approx. 75-residue-long Ser-Gly-rich region separated by an approx. 25-residue-long segment lacking Ser-Gly dipeptides. These dipeptides are arranged in 10-residue-long segments in the 100-residue-long homology domains. The shorter homologous segments are tandemly repeated some six times in each 100-residue-long homology domain. Serine residues in these repeats are potential attachment sites for chondroitin sulphate chains.     

The sequence of human retinal S-antigen reveals similarities with alpha-transducin

The complete amino acid sequence of human retinal S-antigen (48 kDa protein), a retinal protein involved in the visual process has been determined by cDNA sequencing. The largest cDNA was 1590 base pairs (bp) and it contained an entire coding sequence. The similarity of nucleotide sequence between the human and bovine is approximately 80%. The predicted amino acid sequence indicates that human S-antigen has 405 residues and its molecular mass is 45050 Da. The amino acid sequence homology between human and bovine is 81%. There is no overall sequence similarity between S-antigen and other proteins listed in the National Biomedical Research Foundation (NBRF) protein data base. However, local regions of sequence homology with alpha-transducin (T alpha) are apparent including the putative rhodopsin binding and phosphoryl binding sites. In addition, human S-antigen has sequences identical to bovine uveitopathogenic sites, indicating that some types of human uveitis may in part be related to the animal model of experimental autoimmune uveitis (EAU).     

Purification and characterisation of a jacalin-related, coleoptile specific lectin from Hordeum vulgare

A plant lectin was isolated from barley (Hordeum vulgare) coleoptiles using acidic extraction and different chromatographic methods. Sequencing of more than 50% of the protein sequence by Edman degradation confirmed a full-length cDNA clone. The subsequently identified open reading frame encodes for a 15 kDa protein which could be found in the soluble fraction of barley coleoptiles. This protein exhibited specificity towards mannose sugar and is therefore, accordingly named as Horcolin (Hordeum vulgare coleoptile lectin). Database searches performed with the Horcolin protein sequence revealed a sequence and structure homology to the lectin family of jacalin-related lectins. Together with its affinity towards mannose, Horcolin is now identified as a new member of the mannose specific subgroup of jacalin-related lectins in monocot species. Horcolin shares a high amino acid homology to the highly light-inducible protein HL#2 and, in addition to two methyl jasmonic acid-inducible proteins of 32.6 and 32.7 kDa where the jasmonic acid-inducible proteins are examples of bitopic chimerolectins containing a dirigent and jacalin-related domain. Immunoblot analysis with a cross-reactive anti-HL#2 antibody in combination with Northern blot analysis of the Horcolin cDNA revealed tissue specific expression of Horcolin in the coleoptiles. The function of Horcolin is discussed in the context of its particular expression in coleoptiles and is then compared to other lectins, which apparently share a related response to biotic or abiotic stress factors.     

Amino acid sequence homology between bovine and human platelet proteins

The complete amino acid sequence of bovine platelet factor 4 (PF-4) was determined. Comparison of the 88 residue bovine protein with its 70 residue human counterpart indicated 73% homology. There is 53% homology between this bovine protein and another human platelet protein, beta-thromboglobulin (beta-TG). These heparin binding proteins share greatest homology around a lysine-rich octa-peptide near the carboxy-terminus which is the putative heparin binding domain. Graphic comparison of these proteins suggests that a point mutation at position 55 (human PF-4 numbering) could cause a significant difference among the folding properties of these 3 proteins and might be critical for their different heparin binding properties.     

Lectins homologous to those of monocotyledonous plants in the skin mucus and intestine of pufferfish,Fugu rubripes

We have characterized pufflectin, a novel mannose-specific lectin, from the skin mucus of the pufferfish, Fugu rubripes. Molecular mass estimations by gel filtration and matrix-assisted laser desorption ionization time-of-flight mass spectrometry and the SDS-PAGE pattern suggest that pufflectin is a homodimer composed of non-covalently associated subunits of 13 kDa. The full-length pufflectin cDNA consists of 527 bp, with 116 amino acid residues deduced from the open reading frame. The amino acid sequence of pufflectin shows no homology with any known animal lectin. Surprisingly, pufflectin shares sequence homology with mannose-binding lectins of monocotyledonous plants and has conserved two of three carbohydrate recognition domains of these plant lectins. The pufflectin gene is expressed in gills, oral cavity wall, esophagus, and skin. In addition, an isoform occurs exclusively in the intestine. Pufflectin differs from mannose-binding lectins purified from the blood plasma of Fugu. Whereas pufflectin did not agglutinate five bacterial species tested, it was demonstrated to bind to the parasitic trematode, Heterobothrium okamotoi. This finding suggests that pufflectin contributes to the parasite-defense system in Fugu.     

Identifying glycoconjugate-binding domains. Building on the past.

The molecular details of how glycoconjugate-binding proteins interact with their ligands have been revealed by a variety of techniques. For example, proteases, chemical-modifying reagents and antibodies have served as effective probes of lectin functional domains. Protein crystallography has providing insight into how lectins are structured, and aided in determining which amino acids in these proteins are positioned appropriately for bond formation with glycoconjugates. In addition, the characterization and sequencing of naturally occurring, non-functional lectin variants have led to the identification of amino acids which play critical roles in a lectin's glycoconjugate-binding domain. Similarly, studies of lectin mutants produced by site-directed mutagenesis, and of synthetic peptides that mimic lectin binding properties, have demonstrated the importance of particular amino acids for glycoconjugate binding. An alternate approach to understanding lectin functional domains has been to compare the primary sequences of these proteins to reveal common sequence elements which allow them to be organized into families. For example, the discovery of amino acid homologies dispersed over long segments of the primary sequences of several lectins has suggested that many of these proteins have a related three-dimensional organization. In addition, the identification of more highly focused regions of sequence homology has indicated that many structures within the lectin glycoconjugate-binding domains themselves may be conserved. Scanning protein data banks for sequences homologous to known lectins has led to the identification of several previously unrecognized lectins, and aided in determining what portions of these proteins function in their glycoconjugate-binding domains.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular cloning of mouse p47, a second group mammalian RuvB DNA helicase-like protein: homology with those from human and Saccharomyces cerevisiae

A 47k protein (p47) in a high-salt buffer extract of a rat liver nuclear matrix fraction was purified by means of a wheat germ agglutinin affinity column, reversed phase HPLC, and SDS-PAGE, and partial amino acid sequences were analyzed. Based on these sequences, the mouse cDNA of the protein was cloned and sequenced, and its amino acid sequence was deduced. Mouse p47 consists of 463 amino acid residues with a molecular weight of 51,112. The amino acid sequences of human and Saccharomyces cerevisiae p47s were also deduced from the nucleotide sequences of "expressed sequence tag" fragments and genomic DNA, respectively. These sequences contain helicase motifs and show homology to bacterial RuvB DNA helicases acting in homologous recombination. They also show homology with the putative mammalian helicases p50/TIP49 and RUVBL1. Comparison of the amino acid sequences of p47 group proteins and those of p50/TIP49 group proteins revealed the p47 group proteins to comprise a group distinct from the p50/TIP49 proteins. Ultracentrifugation and gel filtration analyses showed that p47 in the rat liver cytosol fraction exists as large complexes of 697k.     

cDNA cloning and expression of Bauhinia purpurea lectin.

Bauhinia purpurea lectin (BPA) was purified from seeds of B. purpurea alba. The purified lectin was digested with an endoproteinase, Asp-N, or trypsin and then the amino acid sequences of the resultant fragments were analyzed. Furthermore, a cDNA library for BPA was constructed using RNA isolated from germinated Bauhinia purpurea seeds. By gene cloning, the nucleotide sequence of BPA cDNA and its deduced amino acid sequence were analyzed. The cloned BPA cDNA comprised 1,152 nucleotides and the open reading frame of the cDNA encodes a polypeptide of 290 amino acids including a signal peptide composed of 28 amino acids. BPA expressed in Escherichia coli showed a relative molecular mass of 29 kDa on sodium dodecyl sulfate-polyacrylamide gel. On comparison of its sequence with those of other leguminous seed lectins, BPA showed high homology to the others.     

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.     

The X-lectins: A new family with homology to the Xenopus laevis oocyte lectin XL-35

The Xenopus laevis oocyte cortical granule lectin (XL35) has been studied in fertilization and embryonic development. Several nucleic acid sequences that predict proteins homologous to XL35 have since been reported in frog, human, mouse, lamprey, trout, ascidian worm. These proteins also showed high degrees of amino acid sequence homology to a common fibrinogen-like motif that may involve carbohydrate binding. Although their biological functions and carbohydrate binding specificities have not been studied in detail, this new family of lectins has common characteristics. Several independent studies on this new family of lectins strongly suggest that the lectins are expressed and stored in specialized vesicles that may be released upon the infection by pathogens. In addition, some family members have been shown to bind to oligosaccharides from bacterial pathogens. Therefore, this family of lectins likely participates in pathogen surveillance as part of the innate immune system. We propose the name X-lectin family for these homologs of XL35. Published in 2004.