Comparative cross-linking activities of lactose-specific plant and animal lectins and a natural lactose-binding immunoglobulin G fraction from human serum with asialofetuin

Plant and animal lectins bind and cross-link certain multiantennaryoligosaccharides, glycopeptides, and glycoproteins. This canlead to the formation of homogeneous cross-linked complexes,which may differ in their stoichiometry depending on the natureof the sugar receptor involved. As a precisely defined ligand,we have employed bovine asialofetuin (ASF), a glycoprotein thatpossesses three asparagine-linked triantennary complex carbohydratechains with terminal LacNAc residues. In the present study,we have compared the carbohydrate cross-linking properties oftwo Lac-specific plant lectins, an animal lectin and a naturallyoccurring Lac-binding polyclonal iminunoglobulin G subfractionfrom human serum with the ligand. Quantitative precipitationstudies of the Lac-specific plant lectins, Viscum album agglutininand Ricinus communis agglutinin, and the Lac-specific 16 kDadimenc galectin from chicken liver demonstrate that these lectinsform specific, stoichiometric cross-linked complexes with ASF.At low concentrations of ASF, 1:9 ASF/lectin (monomer) complexesformed with both plant lectins and the chicken lectin. Withincreasing concentrations of ASF, 1:3 ASF/lectin (monomer) complexesformed with the lectins irrespective of their source or size.The naturally occurring polyclonal antibodies, however, revealeda different cross-linking behavior. They show the formationof 1:3 ASF/antibody (per Fab moiety) cross-linked complexesat all concentrations of ASF. These studies demonstrate thatLac-specific plant and animal lectins as well as the Lac-bindingimmunoglobulin subfraction form specific stoichiometric cross-linkedcomplexes with ASF. These results are discussed in terms ofthe structure-function properties of multivalent lectins andantibodies. asialofetuin Lac-specific lectins immunoglobulin subfraction     

Detection and characterization of a mannan-binding lectin from the mosquito, Anopheles stephensi (Liston).

Two lectins from the serum of the mosquito, Anopheles stephensi (Liston), with distinct characteristics, were detected by agglutination of various animal erythrocytes. The lectins were developmental stage-specific and/or sex-related. One adult female-specific lectin was identified as mannan-specific, and named mosquito mannan-binding lectin (MBL). MBL cross-reacted immunologically with antibodies against a previously characterized cockroach lectin, Blaberus discoidalis lectin (BDL1), and its activity was almost completely blocked by the antibodies. Mosquito MBL agglutinated erythrocytes from human, sheep, goat and rabbit, but not chicken or mouse, and agglutination was inhibited by mannan and nitrophenol-modified sugar derivatives, but not by simple sugars. Using affinity chromatography with immobilized mannan on Sepharose 6B, the mosquito MBL was partially purified. Purified mosquito MBL shared biochemical properties with BDL1, containing two subunits of molecular mass of 28 and 30 kDa under reducing conditions in SDS/PAGE. Its activity is dependent on Ca(2+), and it is stable at pH 7-9 and at temperatures less than 30 degrees C.     

Comparative FISH mapping in river buffalo and sheep chromosomes: assignment of forty autosomal type I loci from sixteen human chromosomes.

Forty autosomal type I loci earlier mapped in goat were comparatively FISH mapped on river buffalo (BBU) and sheep (OAR) chromosomes, noticeably extending the physical map in these two economically important bovids. All loci map on homoeologous chromosomes and chromosome bands, with the exception of COL9A1 mapping on BBU10 (homoeologous to cattle/goat chromosome 9) and OAR9 (homoeologous to cattle/goat chromosome 14). A FISH mapping control with COL9A1 on both cattle and goat chromosomes gave the same results as those obtained in river buffalo and sheep, respectively. Direct G- and R-banding comparisons between Bovinae (cattle and river buffalo) and Caprinae (sheep and goat) chromosomes 9 and 14 confirmed that a simple translocation of a small pericentromeric region occurred between the two chromosomes. Comparisons between physical maps obtained in river buffalo and sheep with those reported in sixteen human chromosomes revealed complex chromosome rearrangements (mainly translocations and inversions) differentiating bovids (Artiodactyls) from humans (Primates).     

Purification and characterization of two types of Cytisus sessilifolius anti-H(O) lectins by affinity chromatography.

Two anti-H(O) lectins were separated from extracts of Cytisus sessilifolius seeds by successive affinity chromatographies on columns of di-N-acetylchitobiose- and galactose-Sepharose 4B. One was found to be inhibited most by di-N-acetylchitotriose or tri-N-acetylchitotriose [Cytisus-type anti-H(O) lectin designated as Cytisus sessilifolius lectin I (CSA-I)] and the other anti-H(O) lectin was inhibited by galactose or lactose and designated as Cytisus sessilifolius lectin II (CSA-II). These two anti-H(O) lectins were further purified by gel filtration on TSK-Gel G3000SW. These preparations were homogeneous as judged by polyacrylamide gel electrophoresis and gel filtration. The molecular masses of the purified lectins I and II were found to be 95,000 and 68,000 Da, respectively, by gel filtration on TSK-Gel G3000SW. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and 2-mercaptoethanol, both lectins gave a single component of molecular masses of 27,000 +/- 2,000 and 34,000 +/- 2,000 Da, respectively, suggesting that the lectins I and II were composed of four and two apparently identical subunits, respectively. Lectins I and II contain 38% and 13% carbohydrate, respectively, and only very small amounts of cysteine and methionine, but they are rich in aspartic acid, serine and glycine. The N-terminal amino-acid sequences of these two lectins were determined and compared with those of several lectins already published.     

Sequence analysis of UTR and coding region of kappa-casein gene of Indian riverine buffalo (Bubalus bubalis).

In this study, complete nucleotide as well as derived amino acid sequence characterization of water buffalo (Bubalus bubalis) kappa-casein gene has been presented. Kappa-casein cDNA clones were identified and isolated from a buffalo lactating mammary gland cDNA library. Sequence analysis of kappa-casein cDNA revealed 850 nucleotides with an open reading frame (ORF) of 573 nucleotides, encoding mature peptide of 169 amino acids. The 5' untranslated region (UTR) comprised 71 nucleotides, while 3' UTR was of 206 nucleotides. A total of 11 nucleotide and seven amino acid changes were observed in, buffalo (Bubalus bubalis) as compared to cattle (Bos taurus), sheep (Ovis aries) and goat (Capra hircus). Among these nucleotide changes, eight were unique in buffalo as they were fully conserved in cattle, sheep and goat. Majority of the nucleotide changes and all the amino acid changes; 14 (Asp-Glu), 19(Asp/Ser-Asn), 96(Ala-Thr), 126(Ala-Val), 128(Ala/Gly-Val), 156(Ala/Pro-Val) and 168(Ala/Glu-Val) were limited to exon IV. Three glycosylation sites, Thr 131, Thr 133 and Thr 142 reported in cattle and goat kappa-casein gene were also conserved in buffalo, however, in sheep Thr 142 was replaced by Ala. Chymosin hydrolysis site, between amino acids Phe 105 and Met 106, important for rennet coagulation process, were found to be conserved across four bovid species. Buffalo kappa-casein with the presence of amino acids Thr 136 and Ala 148 seems to be an intermediate of "A" and "B" variants of cattle. Comparison with other livestock species revealed buffalo kappa-casein sharing maximum nucleotide (95.5%) and amino acid (92.6%) similarity with cattle, whereas with pig it showed least sequence similarity of 76.0% and 53.2%, respectively. Phylogenetic analysis based on both nucleotide and amino acid sequence indicated buffalo kappa-casein grouping with cattle, while sheep and goat forming a separate cluster close to them. The non-ruminant species viz. camel, horse and pig were distantly placed, in separate lineages.     

Comparison of lectin receptor and membrane coat-associated glycoproteins of milk lipid globule membranes.

1. Glycoproteins of bovine (Bos taurus) and human (Homo sapiens) milk lipid globule membranes were characterized by ability to bind lectins after electrophoretic separation. 2. Seven lectin receptor glycoproteins were detected in bovine and five in human milk lipid globule membranes. Bovine and human globule membrane glycoproteins differed in ability to interact with certain lectins. 3. Two major nonionic detergent insoluble glycoproteins were present in bovine and human lipid globule membrane; these constituents had apparent molecular weights of 155,000 and 69,000. Detergent-insoluble polypeptides with similar or identical electrophoretic mobilities were found in milk lipid globule membranes from four other species, rat (Rattus norvegicus), sheep (Ovis aries), pig (Sus scrofa) and goat (Capra hircus). Tryptic peptide mapping revealed these polypeptides to be nonidentical among species.     

Isolation and properties of three lectins from mistletoe (Viscum album L.).

Three lectins have been isolated from an extract of mistletoe (Viscum album) by affinity chromatography on partially hydrolysed Sepharose and human immunoglobulin- Sepharose. The lectins differ in molecular weight and sugar specificity (lectin I, mol.wt. 11500, D-galactose-specific; lectin II, mol.wt. 60000, both D-galactose- and N-acetyl-D-galactosamine-specific; lectin III, mol. wt. 50000, N-acetyl-D-galactosamine-specific). All three lectins react with human erythrocytes without specificity for the A, B, and O blood groups. In contrast with abrin and ricin the mistletoe lectins cannot be divided into "toxins" and "haemagglutinins".     

Analysis of conserved microsatellite sequences suggests closer relationship between water buffalo Bubalus bubalis and sheep Ovis aries.

The distribution and evolutionary pattern of the conserved microsatellite repeat sequences (CA)n, (TGG)6, and (GGAT)4 were studied to determine the divergence time and phylogenetic position of the water buffalo, Bubalus bubalis. The mean allelic frequencies of these repeat loci showed a high level of heterozygosity among the euartiodactyls (buffalo, cattle, sheep, and goat). Genetic distances calculated from the allelic frequencies of these microsatellites were used to position Bubalus bubalis in the phylogenetic tree. The tree topology revealed a closer proximity of the Bubalus bubalis to the Ovis aries (sheep) genome than to other domestic species. The estimated time of divergence of the water buffalo genome relative to cattle, goat, sheep, pig, rabbit, and horse was found to be 21, 0.5, 0.7, 94, 20.3, and 408 million years (Myr), respectively. Although water buffaloes share morphological and biochemical similarities with cattle, our study using the microsatellite sequences places the bubaline species in an entirely new phylogenetic position. Our results also suggest that with respect to these repeat loci, the water buffalo genome shares a common ancestry with sheep and goat after the divergence of subfamily Bovinae (Bos taurus) from the family Bovidae.     

Cloning and characterization of alpha(s2)-casein gene of Riverine buffalo.

The present study was carried out to characterize the alpha(s2)-casein gene in Riverine buffalo. Total RNA was extracted from the mammary tissue of buffalo and alpha(s2)-casein cDNA were synthesized by RT-PCR, then cloned using pDRIVE-cloning vector and sequenced. The sequencing revealed that the size of alpha(s2)-casein was 669 bp with GC content of 41.11%. The gene encoded for 222 amino acid precursors and that it possessed 15 amino acids signal peptide. The similarity of buffalo alpha(s2)-casein mRNA sequence with that of cattle, sheep, goat, pig and camel were estimated as 97.9, 93.6, 93.4, 73.5 and 73.0%, respectively. In the phylogenetic trees, constructed from the data of the alpha(s2)-casein mRNA sequences as well as protein sequences, it has been observed that the cattle and buffalo were in the same group whereas sheep and goat formed another group. The camel and swine were placed in two separate groups.     

Kluyveromyces bulgaricus yeast lectins. Isolation of N-acetylglucosamine and galactose-specific lectins: their relation with flocculation

Kluyveromyces bulgaricus is a yeast which, upon culture in a calcium-enriched glucose-peptone medium, flocculates. Its flocculation can be reversed by the addition of galactose. In this paper, it is shown that two lectins can be isolated either from the concentrated culture broth or from the supernatant of deflocculated cells suspended in galactose solution. The N-acetylglucosamine-specific lectin, at pH 7.4, agglutinates untreated sheep red blood cells, but agglutinates neither untreated rabbit red blood cells nor glutaraldehyde-fixed sheep or rabbit red blood cells. Conversely, at pH 4.5, this lectin agglutinates glutaraldehyde-fixed sheep red blood cells. The galactose-specific lectin, at pH 7.4, agglutinates both untreated and glutaraldehyde-fixed rabbit red blood cells but does not agglutinate untreated or glutaraldehyde-fixed sheep red blood cells. At pH 4.5, this lectin agglutinates both glutaraldehyde-fixed sheep and rabbit red blood cells and induces flocculation of deflocculated K. bulgaricus cells. In all cases, the agglutination and the flocculation induced by one of these two lectins were inhibited by free or conjugated N-acetyl-D-glucosamine or by free or conjugated D-galactose, respectively. No glycosylhydrolase activity could be detected in the purified lectins.     

Physicochemical and biological properties of glycoproteins isolated from the plasma of different species of animals

1. Glycoproteins were isolated from the plasma of sheep, goat, cow, buffalo and monkey. They were homogeneous by electrophoresis; on ultracentrifugation, a faster-sedimenting fraction, to an extent of 5–8% only, was observed in each case. 2. Similar physical properties were exhibited by these glycoproteins and they each have a molecular weight of about 105000. 3. In chemical composition, differences have been observed and the glycoproteins can be classified into three groups: (a) sheep and goat glycoproteins; (b) cow and buffalo glycoproteins; (c) monkey glycoprotein. Glucose, galactosamine and N-terminal amino acid were absent from these proteins. 4. These glycoproteins were trypsin inhibitors and prolonged the clotting time of plasma.     

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.     

Amino acid sequence of beta-galactoside-binding bovine heart lectin. Member of a novel class of vertebrate proteins

A variety of animal tissues contain beta-galactoside-binding lectins with molecular masses in the range 13-17 kDa. There is evidence that these lectins may constitute a new protein family although their function in vivo is not yet clear. In this work the major part of the amino acid sequence of the 13 kDa lectin from bovine heart muscle has been determined. Comparison of this sequence with the cDNA-deduced sequence published for the chick embryo skin lectin showed 58% homology. Comparison of the bovine lectin sequence with partial sequences from two cDNA clones from a human hepatoma library and partial amino acid sequences of human lung lectin showed 70, 40 and 85% homology, respectively. The sequences of these vertebrate lectins are thus clearly related, supporting earlier results of immunological cross-reactivity within this group of proteins. Computer searching of protein sequence databases did not detect significant homologies between the bovine lectin sequence and other known proteins.     

Physicochemical Properties and Oxidative Inactivation of Soluble Lectin from Water Buffalo (<Emphasis Type="Italic">Bubalus bubalis</Emphasis>) Brain

Lectins are carbohydrate-binding proteins present in a wide variety of plants and animals, which serve various important physiological functions. A soluble β-galactoside binding lectin has been isolated and purified to homogeneity from buffalo brain using ammonium sulphate precipitation (40–70%) and gel permeation chromatography on Sephadex G_50–80 column. The molecular weight of buffalo brain lectin (BBL) as determined by SDS-PAGE under reducing and non-reducing conditions was 14.2 kDa, however, with gel filtration it was 28.5 kDa, revealing the dimeric form of protein. The neutral sugar content of the soluble lectin was estimated to be 3.3%. The BBL showed highest affinity for lactose and other sugar moieties in glycosidic form, suggesting it to be a β-galactoside binding lectin. The association constant for lactose binding as evidenced by Scatchard analysis was 6.6 × 10³ M⁻¹ showing two carbohydrate binding sites per lectin molecule. A total inhibition of lectin activity was observed by denaturants like guanidine HCl, thiourea and urea at 6 M concentration. The treatment of BBL with oxidizing agent destroyed its agglutination activity, abolished its fluorescence, and shifted its UV absorption maxima from 282 to 250 nm. The effect of H₂O₂ was greatly prevented by lactose indicating that BBL is more stable in the presence of its specific ligand. The purified lectin was investigated for its brain cell aggregation properties by testing its ability to agglutinate cells isolated from buffalo and goat brains. Rate of aggregation of buffalo brain cells by purified protein was more than the goat brain cells. The data from above study suggests that the isolated lectin may belong to the galectin-1 family but is glycosylated unlike those purified till date.     

Cross-linking activity of the 14-kilodalton beta-galactoside-specific vertebrate lectin with asialofetuin: comparison with several galactose-specific plant lectins.

We have previously shown that plant lectins with a wide range of carbohydrate binding specificities can bind and cross-link (precipitate) specific multiantennary oligosaccharides and glycopeptides [cf. Bhattacharyya, L., Fant, J., Lonn, H., & Brewer, C. F. (1990) Biochemistry 29, 7523-7530]. This leads to a new source of binding specificity: namely, the formation of homogeneous cross-linked lattices between lectins and carbohydrates. Recently, we have demonstrated the existence of highly ordered cross-linked lattices that form between the D-Man/D-Glc-specific plant lectin concanavalin A and the soybean agglutinin which is a tetrameric glycoprotein possessing a single Man9 oligomannose chain per monomer [Khan, M. I., Mandal, D. K., & Brewer, C. F. (1991) Carbohydr. Res. 213, 69-77]. In the present study, we have compared the ability of the 14-kDa beta-galactoside-specific lectin from calf spleen, a dimeric S-type animal lectin, and several galactose-specific plant lectins from Erythrina indica, Erythrina cristagalli, and Glycine max (soybean agglutinin) to form specific cross-linked complexes with asialofetuin (ASF), a 48-kDa monomeric glycoprotein, using quantitative precipitation analyses. The results show the formation of 1:9 and 1:3 stoichiometric cross-linked complexes (per monomer) of ASF to the 14-kDa lectin, depending on their relative ratio in solution. Evidence indicates that the three triantennary N-linked complex-type oligosaccharide chains of ASF mediate the cross-linking interactions and that each chain expresses either trivalency in the 1:9 cross-linked complex or univalency in the 1:3 complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular cloning and characterization of buffalo alpha(s1)-casein gene.

Buffaloes in Indian subcontinent play an important role as the producer of milk and milk products. The alpha(s1)-casein constitutes 38% of the total milk proteins. The present study was carried out to characterize the gene in Murrah breed of Riverine buffalo. Buffalo alpha(s1)-casein cDNA was synthesized by RT-PCR, then cloned using pDRIVE-cloning vector and sequenced. The sequencing revealed that the size of alpha(s1)-casein cDNA was of 645 bp with GC content of 45.58%. The alpha(s1)-casein gene coded 214 amino acids precursor with a signal peptide of 15 amino acid residues. The similarity of buffalo alpha(s1)-casein mRNA sequence with that of cattle, goat, sheep, pig, camel, equine and human were estimated as 97.2, 93, 92.3, 57.2, 59.5, 55.9 and 46.6%, respectively. A similar trend was observed when compared amino acid sequences of these species. In the phylogenetic trees, constructed from the data of the alpha(s1)-casein mRNA as well as protein sequences, it has been observed that buffalo, cattle, goat and sheep formed a cluster with a closer relationship between cattle and buffalo followed by goat and sheep.     

A novel mitogenic and antiproliferative lectin from a wild cobra lily, Arisaema flavum

A novel lectin having specificity towards a complex glycoprotein asialofetuin was purified from tubers of Arisaema flavum (Schott.) by affinity chromatography on asialofetuin-linked amino-activated silica beads. A. flavum gave a single peak on HPLC size exclusion and a single band on non-denatured PAGE at pH 4.5. The molecular mass of the lectin, as determined by gel filtration chromatography, was 56 kDa. In SDS-PAGE, pH 8.3, the lectin migrated as a single band of 13.5 kDa, under reducing and non-reducing conditions, indicating the homotetrameric nature. A. flavum lectin (AFL) readily agglutinated rabbit, rat, sheep, goat, and guinea pig erythrocytes but not human ABO blood group erythrocytes even after neuraminidase treatment. This lectin is stable up to 55 degrees C and does not require metal ions for its hemagglutination activity. AFL was completely devoid of sulphur containing amino acids and was rich in aspartic acid and glycine. In Oucterlony's double immunodiffusion, the antisera raised against A. flavum lectin showed distinct lines of identity with those of other araceous lectins. AFL showed potent mitogenic activity towards BALB/c splenocytes and human lymphocytes in comparison to Con A, a well-known plant mitogen. AFL also showed significant in vitro antiproliferative activity towards J774 and P388D1 murine cancer cell lines.     

Detection of polymorphism and sequence characterization of toll-like receptor 7 gene of Indian goat revealing close relationship between ruminant species

In this study, approximately 3.4?kb nucleotide sequence of caprine TLR7 (Toll-like receptor 7) gene was generated from twelve different Indian goat breeds belonging to different geographical regions. Goat TLR7 gene ORF (Open Reading Frame) was found to be 3141 nucleotides long coding for 1046?amino acids similar to sheep. The sequence analysis at nucleotide level revealed goat TLR7 having 99.5% homology with sheep, followed by other livestock species. Simple Modular Architecture Research Tool (SMART) was used for the structural analysis of goat TLR7 that showed the presence of 22 leucine rich repeats (LRRs) along with single Toll/interleukin-1 receptor (TIR) domains. TIR domain, when compared, was found to be similar in ruminant species, goat, sheep, cattle, and buffalo. The phylogenetic analysis also revealed grouping of all ruminant species together, goat being closer to sheep followed by cattle and buffalo. A total of 22 polymorphic sites were observed in TLR7 gene of 24 goats representing 12 different breeds, out of which 19 were present within the coding region and three in 3'UTR. Out of the seven nonsynonymous SNPs, two were in ectodomains and one in TIR domain. Overall our results indicate substantial variation within goat TLR7 gene, which could be exploited for association with disease susceptibility.     

Detection of Polymorphism and Sequence Characterization of Toll-Like Receptor 7 Gene of Indian Goat Revealing Close Relationship Between Ruminant Species

In this study, approximately 3.4 kb nucleotide sequence of caprine TLR7 (Toll-like receptor 7) gene was generated from twelve different Indian goat breeds belonging to different geographical regions. Goat TLR7 gene ORF (Open Reading Frame) was found to be 3141 nucleotides long coding for 1046 amino acids similar to sheep. The sequence analysis at nucleotide level revealed goat TLR7 having 99.5% homology with sheep, followed by other livestock species. Simple Modular Architecture Research Tool (SMART) was used for the structural analysis of goat TLR7 that showed the presence of 22 leucine rich repeats (LRRs) along with single Toll/interleukin-1 receptor (TIR) domains. TIR domain, when compared, was found to be similar in ruminant species, goat, sheep, cattle, and buffalo. The phylogenetic analysis also revealed grouping of all ruminant species together, goat being closer to sheep followed by cattle and buffalo. A total of 22 polymorphic sites were observed in TLR7 gene of 24 goats representing 12 different breeds, out of which 19 were present within the coding region and three in 3'UTR. Out of the seven nonsynonymous SNPs, two were in ectodomains and one in TIR domain. Overall our results indicate substantial variation within goat TLR7 gene, which could be exploited for association with disease susceptibility.