The carbohydrate-binding sequences in lectins of the clovers Trifolium repens, T. pratense, and T. trichocephalum

The carbohydrate-binding sequences (CBS) in the lectin genes of Trifolium repens, T. pratense, and T. trichocephalum were sequenced. The gene regions encoding lectin CBS of T. pratense and T. repens displayed a considerable similarity; however, the CBS of these species differed essentially. Moreover, T. repens formed a compact cluster with Melilotus albus and M. officinalis in the phylogenetic trees constructed according to the nucleotide sequences and the corresponding CBS of legume lectins. T. trichocephalum does not fall into the group of the tribe Trifolieae members according to both the amino acid sequence of lectin carbohydrate-binding region and the nucleotide sequence of lectin gene.     

Carbohydrate-binding sequences of the lectin genes in leguminous plants from the Galegeae and Hedysareae Tribes

The carbohydrate-binding sequences (CBS) of the lectin genes from legume plants of the genera Astragalus Lam., Oxytropis DC., and Hedysarum L. were determined. Computer-assisted analysis of nucleotide and predicted amino acid sequences of the lectin gene fragments examined revealed a high homology level between their CBS. At the same time, the CBS of Astragalus and Oxytropis were considerably different from the lectin gene CBS in the earlier examined representatives of the tribe Galegeae, Caragana frutex and C. arborescens. This fact probably points to the differences in the carbohydrate-binding specificity of the proteins examined, which can eventually affect their functional activity.     

Polymorphism of lectin genes in Lathyrus plants

The carbohydrate-binding sequences of the lectin genes from spring vetchling Lathyrus vernus (L.) Bernh., marsh vetchling L. palustris (L.), and Gmelin's vetchling L. gmelinii (Fitsch) (Fabaceae) were determined. Computer-aided analysis revealed substantial differences between nucleotide and predicted amino acid sequences of the lectin gene regions examined in each of the three vetchling species tested. In the phylogenetic trees based on sequence similarity of carbohydrate-biding regions of legume lectins, the sequences examined formed a compact cluster with the lectin genes of the plants belonging to the tribe Fabeae. In each plant, L. vernus, L. palustris, and L. gmelinii, three different lectin-encoding genes were detected. Most of the substitutions were identified within the gene sequence responsible for coding the carbohydrate-binding protein regions. This finding may explain different affinity of these lectins to different carbohydrates, and as a consequence, can affect the plant host specificity upon development of symbiosis with rhizobium bacteria.     

Polymorphism of lectin genes in <Emphasis Type="Italic">Lathyrus</Emphasis> plants

The carbohydrate-binding sequences of the lectin genes from spring vetchling Lathyrus vernus (L.) Bernh., marsh vetchling L. palustris (L.), and Gmelin’s vetchling L. gmelinii (Fitsch) (Fabaceae) were determined. Computer-aided analysis revealed substantial differences between nucleotide and predicted amino acid sequences of the lectin gene regions examined in each of the three vetchling species tested. In the phylogenetic trees based on sequence similarity of carbohydrate-biding regions of legume lectins, the sequences examined formed a compact cluster with the lectin genes of the plants belonging to the tribe Fabeae. In each plant, L. vernus, L. palustris, and L. gmelinii, three different lectin-encoding genes were detected. Most of the substitutions were identified within the gene sequence responsible for coding the carbohydrate-binding protein regions. This finding may explain different affinity of these lectins to different carbohydrates, and as a consequence, can affect the plant host specificity upon development of symbiosis with rhizobium bacteria.     

Rhizobium strains that nodulate Trifolium semipilosum Fres. are phylogenetically distinct

The genetic relationships of Rhizobium isolated from temperate and tropical perennial Trifolium species were investigated using PCR-based nucleotide sequence analysis of 16S and 23S rDNA regions. Comparative analysis of partial 23S rDNA sequences clustered Rhizobium isolates effective with T. semipilosum, T. repens, T. pratense, T. hybridum and T. fragiferum into two distinct groups. These groups were consistent with the pattern of symbiotic effectiveness observed in cross-inoculation experiments. Our data suggested that strains from T. semipilosum were more closely related phylogenetically to R. etli, indicating that these strains do not belong in the R. leguminosarum bv trifolii group. Further differentiation of Rhizobium strains effective on T. semipilosum was reflected in the broader metabolic profile observed using the BIOLOG MicroPlate ^TM system to evaluate carbon utilization. This revised version was published online in August 2006 with corrections to the Cover Date.     

Sequence of the T4 recombination gene, uvsX, and its comparison with that of the recA gene of Escherichia coli.

We have determined the nucleotide sequence of the uvsX gene of bacteriophage T4 which is involved in DNA recombination and damage repair, and whose product catalyzes in vitro reactions related to recombination process in analogous manners to E. coli recA gene product. The coding region consisted of 1170 nucleotides directing the synthesis of a polypeptide of 390 amino acids in length with a calculated molecular weight of 43,760. Amino acid composition, the sequence of seven NH2-terminal amino acids and molecular weight of the protein deduced from the nucleotide sequence were consistent with the data from the analysis of the purified uvsX protein. The nucleotide sequence and the deduced amino acid sequence were compared with those of the recA gene. Although a significant homology was not found in the nucleotide sequences, the amino acid sequences included 23% of identical and 15% of conservatively substituted residues.     

Structure and evolutionary origin of the gene encoding a human serum mannose-binding protein.

The N-terminal sequence of the major human serum mannose-binding protein (MBP1) was shown to be identical at all positions determined with the amino acid sequence predicted from a cDNA clone of a human liver MBP mRNA. An oligonucleotide corresponding to part of the sequence of this cDNA clone was used to isolate a cosmid genomic clone containing a homologous gene. The intron/exon structure of this gene was found to closely resemble that of the gene encoding a rat liver MBP (MBP A). The nucleotide sequence of the exons differed in several places from that of the human cDNA clone published by Ezekowitz, Day & Herman [(1988) J. Exp. Med. 167, 1034-1046]. The MBP molecule comprises a signal peptide, a cysteine-rich domain, a collagen-like domain, a 'neck' region and a carbohydrate-binding domain. Each domain is encoded by a separate exon. This genomic organization lends support to the hypothesis that the gene arose during evolution by a process of exon shuffling. Several consensus sequences that may be involved in controlling the expression of human serum MBP have been identified in the promoter region of the gene. The consensus sequences are consistent with the suggestion that this mammalian serum lectin is regulated as an acute-phase protein synthesized by the liver.     

Carbohydrate-binding specificities of mouse ficolin A, a splicing variant of ficolin A and ficolin B and their complex formation with MASP-2 and sMAP

Ficolins are a group of proteins mainly consisting of collagen-like and fibrinogen-like domains and are thought to play a role in innate immunity via their carbohydrate-binding activities. Two types of ficolins have been identified in mice, ficolin A, and ficolin B. However, their structure and function are not fully understood. In this study, we isolated the cDNA encoding a novel variant of ficolin A having a shorter collagen-like domain and a longer gap sequence, which was generated from the ficolin A gene by alternative splicing. We delineated the structure and function of mouse ficolins, including this splicing variant, by preparing the respective recombinants. Recombinant ficolin A, its splicing variant, and ficolin B showed multimeric structures and revealed binding to both N-acetylglucosamine and N-acetylgalactosamine. Interestingly, ficolin B specifically recognized sialic acid residues. Ficolin A and its variant, but not ficolin B, bound to mannose-binding lectin (MBL)-associated serine protease-2 (Masp-2) and small MBL-associated protein (smap), and the resulting complexes showed a potent complement activating capacity. In addition, smap competed with Masp-2 in association with ficolin A and its variant, and inhibited the complement activation by the ficolin A (or ficolin A variant)/MASP-2 complex, indicating its regulatory role in the lectin pathway. These results suggest that ficolin A and its variant function as recognition molecules of the lectin pathway, and ficolin B plays a distinct role through its unique carbohydrate-binding specificity. The nucleotide sequences reported in this paper have been submitted to the DDBJ, EMBL, and GenBank nucleotide sequence databases and have been assigned accession numbers AB222271 for ficolin A variant cDNA.     

A chimeric lectin formed from Bauhinia purpurea lectin and Lens culinaris lectin recognizes a unique carbohydrate structure

Lectins are carbohydrate-binding proteins widely used in biochemical, immunochemical, and histochemical studies. Bauhinia purpurea lectin (BPA) is a leguminous lectin with an affinity for galactose and lactose. Nine amino acids, DTWPNTEWS, corresponding to the amino acid sequence from aspartic acid-135 to serine-143 in the primary structure of BPA were replaced with the corresponding amino acid residues from the mannose-binding Lens culinaris lectin (LCA), and the chimeric lectin obtained was expressed in Escherichia coli cells. The carbohydrate-binding specificity of the recombinant chimeric lectin was investigated in detail by comparing the elution profiles of various glycopeptides and oligosaccharides with defined carbohydate structures from immobilized lectin columns. Glycopeptides carrying three constitutive carbohydrate sequences of Galbeta1-3GalNAc-Ser/Thr and a complex-type biantennary glycopeptide, which show a high affinity for BPA or LCA, were shown to have no affinity for the chimeric lectin. In contrast, hybrid-type and high mannose-type glycopeptides with a Manalpha1-6(Manalpha1-3)Manalpha1-6Man sequence were found to have a moderate affinity for the chimeric lectin. This result demonstrates that a novel type of lectin with a unique carbohydrate-binding specificity can be constructed from BPA by substituting several amino acid residues in its metal-binding region with other amino acid residues. Additional lectin(s) with distinctly different carbohydrate-binding specificities will provide a powerful tool for many studies.     

Evolutionary relationships between the former species Saccharomyces uvarum and the hybrids Saccharomyces bayanus and Saccharomyces pastorianus; reinstatement of Saccharomyces uvarum (Beijerinck) as a distinct species

Analysis of the nucleotide sequence of the GDH1 homologues from Saccharomyces bayanus strain CBS 380T and S. pastorianus strains showed that they share an almost identical sequence, SuGDH1*, which is a diverged form of the SuGDH1 from the type strain of the former species S. uvarum, considered as synonym of S. bayanus. SuGDH1* is close to but differs from SuGDH1 by the accumulation of a high number of neutral substitutions designated as Multiple Neutral Mutations Accumulation (MNMA). Further analysis carried out with three other markers, BAP2, HO and MET2 showed that they have also diverged from their S. uvarum counterparts by MNMA. S. bayanus CBS 380T is placed between S. uvarum and S. pastorianus sharing MET2, CDC91 sequences with the former and BAP2, GDH1, HO sequences with the latter. S. bayanus CBS 380T has been proposed to be a S. uvarum/S. cerevisiae hybrid and this proposal is confirmed by the presence in its genome a S. cerevisiae SUC4 gene. Strain S. bayanus CBS 380T, with a composite genome, is genetically isolated from strains of the former S. uvarum species, thus justifying the reinstatement of S. uvarum as a distinct species.     

Multiplicity of carbohydrate-binding sites in beta-prism fold lectins: occurrence and possible evolutionary implications

The beta-prism II fold lectins of known structure, all from monocots, invariably have three carbohydrate-binding sites in each subunit/domain. Until recently, beta-prism I fold lectins of known structure were all from dicots and they exhibited one carbohydrate-binding site per subunit/domain. However, the recently determined structure of the beta-prism fold I lectin from banana, a monocot, has two very similar carbohydrate-binding sites. This prompted a detailed analysis of all the sequences appropriate for two-lectin folds and which carry one or more relevant carbohydrate-binding motifs. The very recent observation of a beta-prism I fold lectin, griffthsin, with three binding sites in each domain further confirmed the need for such an analysis. The analysis demonstrates substantial diversity in the number of binding sites unrelated to the taxonomical position of the plant source. However, the number of binding sites and the symmetry within the sequence exhibit reasonable correlation. The distribution of the two families of beta-prism fold lectins among plants and the number of binding sites in them, appear to suggest that both of them arose through successive gene duplication, fusion and divergent evolution of the same primitive carbohydrate-binding motif involving a Greek key. Analysis with sequences in individual Greek keys as independent units lends further support to this conclusion.It would seem that the preponderance of three carbohydrate-binding sites per domain in monocot lectins, particularly those with the beta-prism II fold, is related to the role of plant lectins in defence.     

The (QxW)3 domain: a flexible lectin scaffold.

Lectins form a class of proteins that have evolved a specialized carbohydrate-binding function. Based on amino acid sequence analysis, several lectin families have been described and a lectin domain, the (QxW)3 domain, was discussed recently based on 11 family members. In this paper, the (QxW)3 domain family is extended to 45 sequences, several of which have very low sequence identity with the previously known members of the family. A hidden Markov model was used to identify the most divergent members of the family. The expanded set of sequences gives us a more complete appreciation of the conserved features, and the lack thereof, in this lectin family. This, in turn, provides new insights in the structural and functional properties of the individual family members.     

The lectin from leaves of Japanese cycad, Cycas revoluta Thunb. (gymnosperm) is a member of the jacalin-related family.

A novel lectin was isolated from leaves of the Japanese cycad, Cycas revoluta Thunb. (gymnosperm), and its characteristics including amino acid composition, molecular mass, carbohydrate binding specificity and partial amino acid sequences were examined. The inhibition analysis of hemagglutinating activity with various sugars showed that the lectin has a carbohydrate-binding specificity similar to those of mannose recognizing, jacalin-related lectins. Partial amino acid sequences of the lysylendopeptic peptides shows that the lectin might have a repeating structure and belong to the jacalin-related lectin family.     

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 and cloning of a ricin B-like lectin from mushroom Clitocybe nebularis with antiproliferative activity against human leukemic T cells

Background

Lectins are a diverse group of carbohydrate-binding proteins exhibiting numerous biological activities and functions.

Methods

Two-step serial carbohydrate affinity chromatography was used to isolate a lectin from the edible mushroom clouded agaric (Clitocybe nebularis). It was characterized biochemically, its gene and cDNA cloned and the deduced amino acid sequence analyzed. Its activity was tested by hemagglutination assay and carbohydrate-binding specificity determined by glycan microarray analysis. Its effect on proliferation of several human cell lines was determined by MTS assay.

Results

A homodimeric lectin with 15.9-kDa subunits agglutinates human group A, followed by B, O, and bovine erythrocytes. Hemagglutination was inhibited by glycoprotein asialofetuin and lactose. Glycan microarray analysis revealed that the lectin recognizes human blood group A determinant GalNAcα1–3(Fucα1–2)Galβ-containing carbohydrates, and GalNAcβ1–4GlcNAc (N,N'-diacetyllactosediamine). The lectin exerts antiproliferative activity specific to human leukemic T cells.

Conclusions

The protein belongs to the ricin B-like lectin superfamily, and has been designated as C. nebularis lectin (CNL). Its antiproliferative effect appears to be elicited by binding to carbohydrate receptors on human leukemic T cells.

General significance

CNL is one of the few mushroom ricin B-like lectins that have been identified and the only one so far shown to possess immunomodulatory properties.     

Nucleotide sequence of a type II DNA topoisomerase gene. Bacteriophage T4 gene 39.

T4 DNA topoisomerase is a type II enzyme and is thought to be required for normal T4 DNA replication T4 gene 39 codes for the largest of the three subunits of T4 DNA topoisomerase. I have determined the nucleotide sequence of a region of 2568 nucleotides of T4 DNA which includes gene 39. The location of the gene was established by the identification of the first fifteen amino acids in the large open reading frame in the DNA sequence as those found at the amino-terminus of the purified 39-protein. The coding region of gene 39 has 1560 bases, and it is followed by two in-frame stop codons. The gene is preceded by a typical Shine-Dalgarno sequence as well as possible promoter sequences for E. coli RNA polymerase. T4 39-protein consists of 520 amino acids, and it has a calculated molecular weight of 58,478. By comparing the amino acid sequences, T4 39-protein is found to share homology with the gyrB subunit of DNA gyrase. This suggests that these topoisomerase subunits may be equivalent functionally. Some of the characteristics of the 39-protein and its structural features predicted from the DNA sequence data are discussed.     

Nucleotide sequence and complementation studies of the gene 10 region of bacteriophage T3

The nucleotide sequence of bacteriophage T3 gene 10 and surrounding regulatory elements has been determined and compared to the analogous region of bacteriophage T7. T3 genes 9, 10 and 11 have been shown to complement T7 mutants. The DNA sequences of T3 and T7 gene 10A are homologous, as are the amino acid sequences of the respective products. The translational shift to the -1 frame is predicted to occur at the same position in gene 10 of T3 and T7, though different nucleotide sequences are probably responsible. The resulting gp10B products have completely different C termini.     

The taxonomic position of Saccharomyces boulardii as evaluated by sequence analysis of the D1/D2 domain of 26S rDNA, the ITS1-5.8S rDNA-ITS2 region and the mitochondrial cytochrome-c oxidase II gene

A taxonomic study was carried out on eight strains of Saccharomyces boulardii. Morphological and physiological characteristics were consistent with those of Saccharomyces cerevisiae. Sequences of the D1/D2 domain of the 26S rDNA were identical for all strains examined and had a similarity value of 100% compared to sequences of the type strain of S. cerevisiae (CBS 1171T) and strain S288c. For all S. boulardii isolates was found the exact same ITS1-5.8S rDNA-ITS2 sequence, which displayed a close resemblance with the sequences published for S288c (99.9%), CBS 1171(T) (99.3%) and other S. cerevisiae strains. Sequence analysis of the mitochondrial cytochrome-c oxidase II gene (COX2) also resulted in identical sequences for the S. boulardii isolates and comparisons with available nucleotide sequences revealed close relatedness to strains of S. cerevisiae including S288c (99.5%) and CBS 1171(T) (96.6%). The electrophoretic karyotypes of the S. boulardii strains appeared quite uniform and although very typical of S. cerevisiae, they formed a cluster separate from strains of this species. The results of the present study strongly indicate a close relatedness of S. boulardii to S. cerevisiae and thereby support the recognition of S. boulardii as a member of S. cerevisiae and not as a separate species.     

Candida ruelliae sp. nov., a novel yeast species isolated from flowers of Ruellia sp. (Acanthaceae)

Two novel yeast strains designated as 16Q1 and 16Q3 were isolated from flowers of the Ruellia species of the Acanthaceae family. The D1/D2 domain and ITS sequences of these two strains were identical. Sequence analysis of the D1/D2 domain of large-subunit rRNA gene indicated their relationship to species of the Candida haemulonii cluster. However, they differ from C. haemulonii by 14% nucleotide sequence divergence, from Candida pseudohaemulonii by 16.1% and from C. haemulonii type II by 16.5%. These strains also differ in 18 physiological tests from the type strain of C. haemulonii, and 12 and 16 tests, respectively, from C. pseudohaemulonii and C. haemulonii type II. They also differ from C. haemulonii and other related species by more than 13% sequence divergence in the internal transcribed spacer region. In the SSU rRNA gene sequences, strain 16Q1 differs by 1.7% nucleotide divergence from C. haemulonii. Sporulation was not observed in pure or mixed cultures on several media examined. All these data support the assignment of these strains to a novel species; we have named them as Candida ruelliae sp. nov., and designate strain 16Q1(T)=MTCC 7739(T)=CBS10815(T) as type strain of the novel species.