Structure Of Lectin Research Articles

Molecular dynamics and binding energy analysis of Vatairea guianensis lectin: a new tool for cancer studies.

The Tn antigen is an epitope containing N-acetyl-D-galactosamine present in the extracellular matrix of some carcinoma cells in humans, and it is often used as a biomarker. Lectins are proteins capable of binding to carbohydrates and can be used as a molecular tool to recognize antigens and to differentiate cancer cells from normal cells. In this context, the present work aimed to characterize the interaction of Vatairea guianensis seed lectin with N-acetyl-D-galactosamine and the Tn antigen by molecular dynamics and molecular mechanics/Poisson-Boltzmann solvent-accessible surface area analysis. This study revealed new interacting residues not previously identified in static analysis of the three-dimensional structures of Vatairea lectins, as well as the configuration taken by the carbohydrate recognition domain, as it interacts with each ligand. During the molecular dynamics simulations, Vatairea guianensis lectin was able to bind stably to Tn antigen, which, as seen previously for other lectins, enables its use in cancer research, diagnosis, and therapy. This work further demonstrates the efficiency of bioinformatics in lectinology.

Glycan Binding Profiling of Jacalin-Related Lectins from the Pteria Penguin Pearl Shell.

We determined the primary structures of jacalin-related lectins termed PPL3s (PPL3A, 3B, and 3C, which are dimers consisting of sequence variants α + α, α + β, β + β, respectively) and PPL4, which is heterodimer consisting of α + β subunits, isolated from mantle secretory fluid of Pteria penguin (Mabe) pearl shell. Their carbohydrate-binding properties were analyzed, in addition to that of PPL2A, which was previously reported as a matrix protein. PPL3s and PPL4 shared only 35–50% homology to PPL2A, respectively; they exhibited significantly different carbohydrate-binding specificities based on the multiple glycan binding profiling data sets from frontal affinity chromatography analysis. The carbohydrate-binding specificity of PPL3s was similar to that of PPL2A, except only for Man3Fuc1Xyl1GlcNAc2 oligosaccharide, while PPL4 showed different carbohydrate-binding specificity compared with PPL2A and PPL3s. PPL2A and PPL3s mainly recognize agalactosylated- and galactosylated-type glycans. On the other hand, PPL4 binds to high-mannose-and hybrid-type N-linked glycans but not agalactosylated- and galactosylated-type glycans.

In silico analysis of bacteriophage tail tubular proteins suggests a putative sugar binding site and a catalytic mechanism

Bacteriophage base tailplate proteins were recently discovered to have hydrolytic activity towards disaccharides. The putative assignment of sugar binding sites was based on known lectin structures and identified residues a.a. 40–120 as the potential binding region for disaccharides [1]. To help verify the prediction, an in silico analysis was performed on the structure of a base tailplate protein gp31 from Klebsiella pneumoniae bacteriophage KP32 (PDB: 5MU4) which shows activity towards maltose but not trehalose [1]. Based on the information, a full surface docking was performed for both sugars which identified 2 regions different than originally predicted. The first region clearly favored maltose during the docking phase while the second one allowed for the energetically-equivalent binding of trehalose. To verify the assignment, a molecular dynamics simulation was performed to assess the stability of the docked substrates. MD simulations suggested that the first site included residues D131, D133, and E134, and was also superior for maltose binding while clearly disfavoring trehalose. Analysis of the putative catalytic mechanism suggested residues D131, D133 and E134 as critical for substrate binding. The residue D133 did participate in a stable substrate binding and was positioned near the scissile bond, potentially making it a catalytic residue. Catalytic residues were most likely D131 and D133, one of the two options proposed by Pyra et al. [1]. A comparison with known hydrolase mechanisms suggested that the enzyme most likely retains configuration during hydrolysis of maltose. The findings are discussed for other bacteriophage proteins regarding their potential specificities and catalytic mechanisms.

Functional Characterization of OXYL, A SghC1qDC LacNAc-specific Lectin from The Crinoid Feather Star Anneissia Japonica.

We identified a lectin (carbohydrate-binding protein) belonging to the complement 1q(C1q) family in the feather star Anneissia japonica (a crinoid pertaining to the phylum Echinodermata). The combination of Edman degradation and bioinformatics sequence analysis characterized the primary structure of this novel lectin, named OXYL, as a secreted 158 amino acid-long globular head (sgh)C1q domain containing (C1qDC) protein. Comparative genomics analyses revealed that OXYL pertains to a family of intronless genes found with several paralogous copies in different crinoid species. Immunohistochemistry assays identified the tissues surrounding coelomic cavities and the arms as the main sites of production of OXYL. Glycan array confirmed that this lectin could quantitatively bind to type-2 N-acetyllactosamine (LacNAc: Galβ1-4GlcNAc), but not to type-1 LacNAc (Galβ1-3GlcNAc). Although OXYL displayed agglutinating activity towards Pseudomonas aeruginosa, it had no effect on bacterial growth. On the other hand, it showed a significant anti-biofilm activity. We provide evidence that OXYL can adhere to the surface of human cancer cell lines BT-474, MCF-7, and T47D, with no cytotoxic effect. In BT-474 cells, OXYL led to a moderate activation of the p38 kinase in the MAPK signaling pathway, without affecting the activity of caspase-3. Bacterial agglutination, anti-biofilm activity, cell adhesion, and p38 activation were all suppressed by co-presence of LacNAc. This is the first report on a type-2 LacNAc-specific lectin characterized by a C1q structural fold.

Sensing Glycans as Biochemical Messages by Tissue Lectins: The Sugar Code at Work in Vascular Biology.

Although a plethora of players has already been revealed to be engaged in the haemostatic system, a fundamental consideration of the molecular nature of information coding can give further explorations of the mechanisms of blood clotting, platelet functionality and vascular trafficking direction. By any measures, looking at ranges of occurrence and of potential for structural versatility, at strategic positioning to influence protein and cell sociology as well as at dynamics of processing and restructuring for phenotypic variability, using sugars as an alphabet of life for generating the glycan part of glycoconjugates is a success story. The handiwork by the complex system for glycan biosynthesis renders biochemical messages of exceptionally high coding capacity available. They are read and translated into cellular effects by receptors termed lectins. The different levels of regulation on both sides, that is, glycan and lectin, establish an intriguingly fine-tuned capacity for functional pairing. The emerging insights into the highly branched routes of glycosylation, into lectin structures up to complete characterization in solution and the shape of lectin networks, first obtained for the three selectins, now extended to considering many other C-type lectins, galectins and siglecs, as well as into intra- and inter-family cross-talk and cooperations are sure to push boundaries in our understanding of the molecular basis of haemostasis.

Structural analysis, molecular docking and molecular dynamics of an edematogenic lectin from Centrolobium microchaete seeds

Lectins represent a class of proteins or glycoproteins capable of reversibly binding to carbohydrates. Seed lectins from the Dalbergieae tribe (Leguminosae) have structural variability, carbohydrate specificity, and biological effects, such as inflammation, vasorelaxation and cancer antigen binding. To comprehensively address these factors, the present work aimed to establish and characterize the three-dimensional structure of Centrolobium microchaete lectin (CML) by homology modeling, investigate protein-carbohydrate interactions and evaluate its inflammatory effect on mice. Molecular docking was performed to analyze interactions of the lectin with monosaccharides, disaccharides and N-glycans. Two dimannosides, methyl mannose-1,3-α-D-mannose (MDM) and mannose-1,3-α-D-mannose (M13), were used in molecular dynamics (MD) simulations to study the behavior of the carbohydrate-recognition domain (CRD) over time. Results showed an expanded domain within which hydrophobic interactions with the methyl group in the MDM molecule were established, thus revealing novel interactions for mannose-specific Dalbergieae lectins. To examine its biological activities, CML was purified in a single step by affinity chromatography on Sepharose-mannose matrix. The lectin demonstrated inflammatory response in the paw edema model and stimulated leukocyte migration to the animal peritoneal cavities, an effect elicited by CRD. For the first time, this work reports the molecular dynamics of a lectin from the Dalbergieae tribe.

The trimeric solution structure and fucose-binding mechanism of the core fucosylation-specific lectin PhoSL

The core α1–6 fucosylation-specific lectin from a mushroom Pholiota squarrosa (PhoSL) is a potential tool for precise diagnosis of cancers. This lectin consists of only 40 amino acids and can be chemically synthesized. We showed here that a synthesized PhoSL peptide formed a trimer by gel filtration and chemical cross-linking assays, and determined a structure of the PhoSL trimer by NMR. The structure possesses a β-prism motif with a three-fold rotational symmetry, where three antiparallel β-sheets are tightly connected by swapping of β-strands. A triad of Trp residues comprises the structural core, forming NH–π electrostatic interactions among the indole rings. NMR analysis with an excess amount of fucose revealed the structural basis for the molecular recognition. Namely, fucose deeply enters a pocket formed at a junction of β-sheet edges, with the methyl group placed at the bottom. It forms a number of hydrophobic and hydrogen-bonding interactions with PhoSL residues. In spite of partial similarities to the structures of other functionally related lectins, the arrangement of the antiparallel β-sheets in the PhoSL trimer is novel as a structural scaffold, and thus defines a novel type of lectin structure.

Exploring functional pairing between surface glycoconjugates and human galectins using programmable glycodendrimersomes

Precise translation of glycan-encoded information into cellular activity depends critically on highly specific functional pairing between glycans and their human lectin counter receptors. Sulfoglycolipids, such as sulfatides, are important glycolipid components of the biological membranes found in the nervous and immune systems. The optimal molecular and spatial design aspects of sulfated and nonsulfated glycans with high specificity for lectin-mediated bridging are unknown. To elucidate how different molecular and spatial aspects combine to ensure the high specificity of lectin-mediated bridging, a bottom-up toolbox is devised. To this end, negatively surface-charged glycodendrimersomes (GDSs), of different nanoscale dimensions, containing sulfo-lactose groups are self-assembled in buffer from a synthetic sulfatide mimic: Janus glycodendrimer (JGD) containing a 3'-O-sulfo-lactose headgroup. Also prepared for comparative analysis are GDSs with nonsulfated lactose, a common epitope of human membranes. These self-assembled GDSs are employed in aggregation assays with 15 galectins, comprising disease-related human galectins, and other natural and engineered variants from four families, having homodimeric, heterodimeric, and chimera architectures. There are pronounced differences in aggregation capacity between human homodimeric and heterodimeric galectins, and also with respect to their responsiveness to the charge of carbohydrate-derived ligand. Assays reveal strong differential impact of ligand surface charge and density, as well as lectin concentration and structure, on the extent of surface cross-linking. These findings demonstrate how synthetic JGD-headgroup tailoring teamed with protein engineering and network assays can help explain how molecular matchmaking operates in the cellular context of glycan and lectin complexity.

Molecular basis for intestinal mucin recognition by galectin-3 and C-type lectins.

Intestinal mucins trigger immune responses upon recognition by dendritic cells via protein–carbohydrate interactions. We used a combination of structural, biochemical, biophysical, and cell-based approaches to decipher the specificity of the interaction between mucin glycans and mammalian lectins expressed in the gut, including galectin (Gal)-3 and C-type lectin receptors. Gal-3 differentially recognized intestinal mucins with different O-glycosylation profiles, as determined by mass spectrometry (MS). Modification of mucin glycosylation, via chemical treatment leading to a loss of terminal glycans, promoted the interaction of Gal-3 to poly-N-acetyllactosamine. Specific interactions were observed between mucins and mouse dendritic cell-associated lectin (mDectin)-2 or specific intercellular adhesion molecule–grabbing nonintegrin-related-1 (SIGN-R1), but not mDectin-1, using a cell-reporter assay, as also confirmed by atomic force spectroscopy. We characterized the N-glycosylation profile of mouse colonic mucin (Muc)-2 by MS and showed that the interaction with mDectin-2 was mediated by high-mannose N-glycans. Furthermore, we observed Gal-3 binding to the 3 C-type lectins by force spectroscopy. We showed that mDectin-1, mDectin-2, and SIGN-R1 are decorated by N-glycan structures that can be recognized by the carbohydrate recognition domain of Gal-3. These findings provide a structural basis for the role of mucins in mediating immune responses and new insights into the structure and function of major mammalian lectins.—Leclaire, C., Lecointe, K., Gunning, P. A., Tribolo, S., Kavanaugh, D. W., Wittmann, A., Latousakis, D., MacKenzie, D. A., Kawasaki, N., Juge, N. Molecular basis for intestinal mucin recognition by galectin-3 and C-type lectins.

Influence of Trp flipping on carbohydrate binding in lectins. An example on Aleuria aurantia lectin AAL.

Protein–carbohydrate interactions are very often mediated by the stacking CH–π interactions involving the side chains of aromatic amino acids such as tryptophan (Trp), tyrosine (Tyr) or phenylalanine (Phe). Especially suitable for stacking is the Trp residue. Analysis of the PDB database shows Trp stacking for 265 carbohydrate or carbohydrate like ligands in 5 208 Trp containing motives. An appropriate model system to study such an interaction is the AAL lectin family where the stacking interactions play a crucial role and are thought to be a driving force for carbohydrate binding. In this study we present data showing a novel finding in the stacking interaction of the AAL Trp side chain with the carbohydrate. High resolution X-ray structure of the AAL lectin from Aleuria aurantia with α-methyl-l-fucoside ligand shows two possible Trp side chain conformations with the same occupation in electron density. The in silico data shows that the conformation of the Trp side chain does not influence the interaction energy despite the fact that each conformation creates interactions with different carbohydrate CH groups. Moreover, the PDB data search shows that the conformations are almost equally distributed across all Trp–carbohydrate complexes, which would suggest no substantial preference for one conformation over another.

Structure and function analysis of Polygonatum cyrtonema lectin by site-directed mutagenesis.

The crystal structure of mature Polygonatum cyrtonema lectin (PCL) showed three similar carbohydrate-binding sites (CBS I, CBS II, and CBS III). The Gln58 and Asp60 residues of CBS II are substituted with His58 and Asn60. To establish the relationship between the key amino acid residues and structure or activity of PCL, we constructed four recombinant mutants in CBS I, CBS II, and CBS III. The experimental results indicate that CBS I, CBS III and the disulfide bond play vital roles in the binding with mannose. Furthermore, molecular dynamics simulations and binding free energy calculation illustrate that CBS I has a direct and strong relationship with the activity of PCL. CBS II does not play a critical role in the model for mannose binding by PCL. Although CBS III does not enhance the activity, it helps to maintain the activity and 3D structure. These results suggest that the carbohydrate-binding site of PCL may be in a hydrophilic environment, and Asn and Tyr are the key amino acids involved in its binding with sugar, but Gln and Asp are not necessary to maintain its activity.

Mannobiose Binding Induces Changes in Hydrogen Bonding and Protonation States of Acidic Residues in Concanavalin A As Revealed by Neutron Crystallography.

Plant lectins are carbohydrate-binding proteins with various biomedical applications. Concanavalin A (Con A) holds promise in treating cancerous tumors. To better understand the Con A carbohydrate binding specificity, we obtained a room-temperature neutron structure of this legume lectin in complex with a disaccharide Manα1-2Man, mannobiose. The neutron structure afforded direct visualization of the hydrogen bonding between the protein and ligand, showing that the ligand is able to alter both protonation states and interactions for residues located close to and distant from the binding site. An unprecedented low-barrier hydrogen bond was observed forming between the carboxylic side chains of Asp28 and Glu8, with the D atom positioned equidistant from the oxygen atoms having an O···D···O angle of 101.5°.

The potent anti-cancer activity of Dioclea lasiocarpa lectin

The lectin DLasiL was isolated from seeds of the Dioclea lasiocarpa collected from the northeast coast of Brazil and characterized for the first time by mass spectrometry, DNA sequencing, inductively coupled plasma-mass spectrometry, electron paramagnetic resonance, and fluorescence spectroscopy. The structure of DLasiL lectin obtained by homology modelling suggested strong conservation of the dinuclear Ca/Mn and sugar-binding sites, and dependence of the solvent accessibility of tryptophan-88 on the oligomerisation state of the protein. DLasiL showed highly potent (low nanomolar) antiproliferative activity against several human carcinoma cell lines including A2780 (ovarian), A549 (lung), MCF-7 (breast) and PC3 (prostate), and was as, or more, potent than the lectins ConBr (Canavalia brasiliensis), ConM (Canavalia maritima) and DSclerL (Dioclea sclerocarpa) against A2780 and PC3 cells. Interestingly, DLasiL lectin caused a G2/M arrest in A2780 cells after 24h exposure, activating caspase 9 and delaying the on-set of apoptosis. Confocal microscopy showed that fluorescently-labelled DLasiL localized around the nuclei of A2780 cells at lectin doses of 0.5–2× IC50 and gave rise to enlarged nuclei and spreading of the cells at high doses. These data reveal the interesting antiproliferative activity of DLasiL lectin, and suggest that further investigations to explore the potential of DLasiL as a new anticancer agent are warranted.

CDNA cloning, molecular modeling and docking calculations of L-type lectins from Swartzia simplex var. grandiflora (Leguminosae, Papilionoideae), a member of the tribe Swartzieae

The genus Swartzia is a member of the tribe Swartzieae, whose genera constitute the living descendants of one of the early branches of the papilionoid legumes. Legume lectins comprise one of the main families of structurally and evolutionarily related carbohydrate-binding proteins of plant origin. However, these proteins have been poorly investigated in Swartzia and to date, only the lectin from S. laevicarpa seeds (SLL) has been purified. Moreover, no sequence information is known from lectins of any member of the tribe Swartzieae. In the present study, partial cDNA sequences encoding L-type lectins were obtained from developing seeds of S. simplex var. grandiflora. The amino acid sequences of the S. simplex grandiflora lectins (SSGLs) were only averagely related to the known primary structures of legume lectins, with sequence identities not greater than 50–52%. The SSGL sequences were more related to amino acid sequences of papilionoid lectins from members of the tribes Sophoreae and Dalbergieae and from the Cladratis and Vataireoid clades, which constitute with other taxa, the first branching lineages of the subfamily Papilionoideae. The three-dimensional structures of 2 representative SSGLs (SSGL-A and SSGL-E) were predicted by homology modeling using templates that exhibit the characteristic β-sandwich fold of the L-type lectins. Molecular docking calculations predicted that SSGL-A is able to interact with D-galactose, N-acetyl-D-galactosamine and α-lactose, whereas SSGL-E is probably a non-functional lectin due to 2 mutations in the carbohydrate-binding site. Using molecular dynamics simulations followed by density functional theory calculations, the binding free energies of the interaction of SSGL-A with GalNAc and α-lactose were estimated as −31.7 and −47.5 kcal/mol, respectively. These findings gave insights about the carbohydrate-binding specificity of SLL, which binds to immobilized lactose but is not retained in a matrix containing D-GalNAc as ligand.

Molecular cloning and expression analysis of two lectin genes in Sophora flavescens

Plant lectins are greatly introduced in modern medical diagnostics, which play a key role to diagnose the degrees of malignant tumor and used to inhibit different types of cancer cells. To further characterize the structure and function of plant lectins, we cloned, expressed and analyzed two lectin genes from a Chinese medicinal plant Sophora flavescens (sfl1, sfl2). The two genes were successfully cloned by reverse transcription-polymerase chain reaction (RT-PCR). The full-length open reading frame (ORF) of the sfl1 contained 864bp encoding SFL1 protein with 287 amino acids, whereas the sfl2 contained 891bp encoding SFL2 protein of 296 amino acids. The two genes were subcloned to pET-22b(+) vector and the recombinant plasmids pET-22b(+)-sfl1 and pET-22b(+)-sfl2 were transformed into E. coli BL21 for protein expression. The two expressed proteins were detected by Western-blotting. The physicochemical properties, homologous proteins and evolution, secondary structures, and three dimensional (3D) structures of the SFL1 and SFL2 proteins were analyzed by bioinformatic tools. Our results provide a foundation for exploring plant lectins as anti-cancer drugs.

Change in the structure and function of lectin by photodissociation of NO.

We have shown here that the structure and sugar-binding activity of lectin can be changed by the photodissociation of NO. Intramolecular S-S bonds are photogenerated from SNO in the protein, which can be used to photo-control the structure and function of proteins.

Crystal Structure of Colocasia esculenta Tuber Agglutinin at 1.74 Å Resolution and Its Quaternary Interactions

Many members of the monocot mannose-binding lectin family, characterized by specificity towards mannose have been characterized and cloned. A majority of these lectins molecules contain 1-4 polypeptides of about 110 residues each. From the previously solved crystal structures of a few such lectins, mostly from non-edible plants, these lectins are thought to possess a common β-prism II fold structure. The major tuber storage protein of Colocasia esculenta is a monocot mannose-binding, widely used, dietary lectin. This tuber agglutinin contains two polypeptides of 12.0 and 12.4 kDa by matrix assisted laser desorption ionisation time-of-flight mass spectrometry. By gel filtration at pH 7.2, the purified lectin has a α2β2 form of apparent molecular mass of 48.2 kDa in solution but at pH 3, it has the heterodimeric αβ form. Lectin crystals were obtained by hanging-drop, vapor-diffusion method at room temperature and high-resolution X-ray diffraction data were collected using a home X-ray source. Among previously solved crystal structures of this family are garlic, Solomon’s seal, snowdrop, daffodil and Spanish blue-bell lectins, but the protein sequence of the Colocasia esculenta tuber agglutinin was found to be closest to that of the Remusatia vivipara lectin having no simple mannose-binding property. Using the previously solved 2.4A crystal structure of the Remusatia vivipara lectin, that of Colocasia esculenta has been solved by molecular replacement and subsequent crystallographic refinement and root mean square deviations between various lectins are tabulated and rationalized. The asymmetric unit in our lectin crystal structure contains four β-prism II domains or two αβ heterodimers, each forming a α2β2 heterotetramer with a symmetry related unit. The tetrameric interface obtained from our crystal structure is used to explain the conversion to dimers in acidic pH. Five ordered magnesium ions were located in the asymmetric unit and the presence of magnesium verified by atomic absorption spectroscopy.

Structure prediction and functional analysis of a non-permutated lectin from Dioclea grandiflora.

Legume lectins have been widely studied and applied for many purposes in the last few decades, but many of their physiological aspects remain elusive. The Diocleinae legume subtribe, which includes intensively explored lectins, such as ConA, presents an unusual and extensive post-translational process which results in minor alterations in protein structure, in turn making its function elusive. Despite previous reports about Diocleinae precursor activity, no structural or functional analyses have ever been carried out to understand the impacts of post-translational processing relative to lectin structure and binding specificity. Here we analyzed the functionality of a non glycosylated, recombinantly expressed lectin precursor from Dioclea grandiflora through inhibition assays, corroborating the experimental data with structural information generated by molecular modeling, docking calculations and molecular dynamics simulations. We demonstrate that Diocleinae precursors are active and share the same carbohydrate specificity as mature lectins. At the same time, however, subtle structural alterations were detected and mostly result in an "incomplete" functionality of the precursor, as consequence of an immature binding site and an unstructured tetramer interface, affecting carbohydrate binding and oligomer formation, respectively.

P6012 Use of targeted next generation re-sequencing in the identification of polymorphisms in the bovine collagenous lectin gene family

Collagenous lectins bind carbohydrate motifs on pathogens, leading either to the activation of the lectin complement pathway or to the opsonization or agglutination of pathogens. They play an important role in innate immunity against a variety of bacteria, viruses, and fungi. Functionally altered proteins resulting from genetic mutations in collagenous lectins have been shown in other species to predispose animals to infectious disease. This study aimed to 1) identify genetic variation in the bovine collagenous lectin genes; and 2) determine whether polymorphisms were associated with an increased susceptibility to infectious disease. We used pooled, targeted next generation re-sequencing to identify variants in the bovine collagenous lectin genes. Cattle submitted for post-mortem examination at the University of Guelph were classified as normal (n = 40) or diseased (n = 80) based on the presence or absence of infectious disease. Cattle were placed into groups of 5 based on the similarity of diagnosis, and an equal amount of DNA from each animal was pooled. The collagenous lectin genes (including three unique to bovids) along with 3 kb of downstream DNA and up to 50 kb of upstream DNA were targeted for re-sequencing. The sequencing library was prepared with a Roche Nimblegen EZ Developer kit and sequenced on an Illumina MiSeq. In total, 4.6 Gb of usable sequence data was obtained with an average read depth of 42x/cow over the target region. Following application of quality control filters, 6525 single nucleotide variants (SNVs) were found, including 510 not reported in dbSNP. This included 3948 upstream region SNVs, 2672 downstream region SNVs, and 411 intronic SNVs. Within exons, 107 SNVs, including 54 missense mutations, were identified. In silico analysis of the missense mutations identified 16 SNVs with significant potentially disruptive effects on protein structure. A mutation in MBL2, resulting in a P42Q change in the collagen-like domain, holds particular interest, as similar mutations in orthologous genes have been shown to have impact on susceptibility to disease. Allele frequencies between the normal and diseased populations were compared and the potential impact of promoter SNVs investigated. This study demonstrates that pooled, targeted re-sequencing is a cost effective method of polymorphism identification and discovery in cattle. We identified 510 previously unreported SNVs, as well as 16 mutations potentially affecting collagenous lectin structure or function. These SNVs will help in our understanding of the genetics of disease susceptibility, and represent potential candidates for genetic selection.

Coagulant Activity of Water-Soluble Moringa oleifera Lectin Is Linked to Lowering of Electrical Resistance and Inhibited by Monosaccharides and Magnesium Ions.

Moringa oleifera seeds contain a water-soluble lectin [water-soluble M. oleifera lectin (WSMoL)] that has shown coagulant activity. Magnesium ions are able to interfere with the ability of this lectin to bind carbohydrates. In this study, we performed structural characterization of WSMoL and analyzed its effect on the electrical resistance of a kaolin clay suspension in both presence and absence of monosaccharides (N-acetylglucosamine, glucose, or fructose) and magnesium ions. The coagulant activity of WSMoL was monitored by measuring optical density and electrical resistance over a period of 60min. Native WSMoL had a molecular mass of 60kDa and exhibited anionic nature (pI 5.5). In sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), it appeared as three polypeptide bands of 30, 20, and 10kDa. WSMoL reduced the optical density and electrical resistance of the kaolin suspension, which suggests that suspended particles are destabilized and that this is followed by formation of complexes. The coagulant activity of lectin decreased in the presence of Mg2+ ions and carbohydrates at concentrations that also inhibited hemagglutinating activity. This was most likely due to conformational changes in lectin structure. Our findings suggest that the coagulant activity of WSMoL is enhanced by lowering of electrical resistance of the medium and is impaired by lectin-carbohydrate and lectin-Mg2+ interactions.