Subunit structure of Vicia graminea anti-(blood-group N) lectin.
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ABSTRACT:
SUBMITTER: Prigent MJ
PROVIDER: S-EPMC1161931 | biostudies-other | 1980 Jul
REPOSITORIES: biostudies-other
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Project description:The lectin from Moluccella laevis seeds agglutinates specifically blood-type-A and -N erythrocytes, and both activities are inhibited by micromolar concentrations of N-acetyl-D-galactosamine. The lectin consists of three subunits: a 67 kDa heterodimer, made up of two S-S-linked polypeptides of 28 and 46 kDa, and two non-covalently linked moieties of 26 and 42 kDa, the latter migrating after reduction with an apparent molecular mass of 46 kDa. Here we demonstrate that affinity chromatography of a crude protein fraction from M. laevis seeds on immobilized D-galactose in the presence of 8 M-urea affords a fully active lectin practically devoid of the 42 kDa subunit. We also present data showing that the 26 kDa subunit is devoid of cysteine residues, that the 28 kDa subunit contains two cysteine residues engaged in S-S bonds with the 46 kDa subunit, and that the latter has, in addition, two intramolecular cystine residues. Gel filtration on Sephadex G-150 in 8 M-urea/0.2 M-D-galactose of the lectin, affinity-purified in the presence of urea, afforded a pure 26 kDa subunit which exhibited both anti-A and anti-N activity, as well as high specificity for N-acetyl-D-galactosamine. In addition to demonstrating that the lectin is unusually stable and retains its carbohydrate-binding activity in 8 M-urea, our findings also show that the activity for different blood groups resides in the same subunit.
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Project description:Zonal isoelectric precipitation was shown to be an effective method for the preparation of legumin which was homogeneous as judged by ultracentrifugation and polyacrylamide-gel electrophoresis. The subunit structure of legumin was investigated by preparative sodium dodecyl sulphate-polyacrylamide-gel electrophoresis and ion-exchange chromatography in urea. Five distinct subunits, of which two were acidic (alpha) and had a molecular weight of 37000, and three were basic (beta) with molecular weights of 20100, 20900 and 23800, were identified. The alpha and beta subunits were present in equimolar amounts in the legumin molecule and, in view of this and molecular-weight considerations, an alpha(6)beta(6) subunit model was proposed for legumin.
| S-EPMC1168094 | biostudies-other
Project description:Toxoplasma gondii maintains its intracellular life cycle using an extraordinary arsenal of parasite-specific organelles including the inner membrane complex (IMC), rhoptries, micronemes, and dense granules. While these unique compartments play critical roles in pathogenesis, many of their protein constituents have yet to be identified. We exploited the Vicia villosa lectin (VVL) to identify new glycosylated proteins that are present in these organelles. Purification of VVL-binding proteins by lectin affinity chromatography yielded a number of novel proteins that were subjected to further study, resulting in the identification of proteins from the dense granules, micronemes, rhoptries and IMC. We then chose to focus on three proteins identified by this approach, the SAG1 repeat containing protein SRS44, the rhoptry neck protein RON11 as well as a novel IMC protein we named IMC25. To assess function, we disrupted their genes by homologous recombination or CRISPR/Cas9. The knockouts were all successful, demonstrating that these proteins are not essential for invasion or intracellular survival. We also show that IMC25 undergoes substantial proteolytic processing that separates the C-terminal domain from the predicted glycosylation site. Together, we have demonstrated that lectin affinity chromatography is an efficient method of identifying new glycosylated parasite-specific proteins.
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Project description:Vicia faba lectin contained 40-57% beta-conformation, 4-23% alpha-conformation along with random coil at pH 7.2 depending upon the analytical methods used. The percentage of beta-conformation increased with the addition of N-acetyl-D-glucosamine or methyl alpha-D-mannopyranoside. The structural transitions of V. faba lectin were affected by alkali at pH 9.6 and 10.6. Binding constants and free energy changes for the interaction between V. faba lectin and N-acetyl-D-glucosamine and methyl alpha-D-mannopyranoside were estimated at pH 7.2 using the c.d. and fluorescence methods.
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Project description:Lectins mediate adhesion of pathogens to host tissues, filling in a key role in the first steps of infection. Belonging to the opportunistic pathogen Burkholderiacenocepacia, BC2L-C is a superlectin with dual carbohydrate specificity, believed to mediate cross-linking between bacteria and host cells. Its C-terminal domain binds to bacterial mannosides while its N-terminal domain (BCL2-CN) recognizes fucosylated human epitopes. BC2L-CN presents a tumor necrosis factor alpha (TNF-) fold previously unseen in lectins with a novel fucose binding mode. We report, here, the production of a novel recombinant form of BC2L-CN (rBC2L-CN2), which allowed better protein stability and unprecedented co-crystallization with oligosaccharides. Isothermal calorimetry measurements showed no detrimental effect on ligand binding and data were obtained on the binding of Globo H hexasaccharide and l-galactose. Crystal structures of rBC2L-CN2 were solved in complex with two blood group antigens: H-type 1 and H-type 3 (Globo H) by X-ray crystallography. They provide new structural information on the binding site, of importance for the structural-based design of glycodrugs as new antimicrobials with antiadhesive properties.
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Project description:The lectin of the seeds of hairy vetch (Vicia villosa Roth), which selectively binds murine cytotoxic T-lymphocytes, was purified by simple affinity-chromatographic procedures on two different N-acetyl-alpha-D-galactosaminyl-carriers. The lectin thus obtained is homogeneous on polyacrylamide-gel electrophoresis both in acid and alkaline media and has a mol. wt. of approx. 120000. The lectin molecule appears to comprise four subunits of equal electrophoretic mobility, contains 4.3% of covalently bound neutral sugar and 0.72 Mn and 0.94 Zn atoms respectively. The anti-(blood-group A1) specific erythroagglutinating activity of the lectin can be detected at a limit concentration of 15 microgram/ml and is inhibitable most effectively by N-acetyl-D-galactosamine.
| S-EPMC1162933 | biostudies-other
Project description:The Kell blood group is a major antigenic system in human erythrocytes. Kell antigens reside on a 93-kDa membrane glycoprotein that is surface-exposed and associated with the underlying cytoskeleton. We isolated tryptic peptides and, based on the amino acid sequence of one of the peptides and by using the PCR, prepared a specific oligonucleotide to screen a lambda gt10 human bone-marrow cDNA library. Four clones were isolated, one containing cDNA with an open reading frame for an 83-kDa protein. All known Kell amino acid sequences were present in the deduced sequence; moreover, rabbit antibody to a 30-amino acid peptide, prepared from this sequence, reacted on an immunoblot with authentic Kell protein. The Kell cDNA sequence predicts a 732-amino acid protein. Hydropathy analysis indicates a single membrane-spanning region, suggesting that Kell protein is oriented with 47 of its N-terminal amino acids in the cell cytoplasm, and a 665-amino acid segment, which contains six possible N-glycosylation sites, is located extracellularly. Computer-based search showed that Kell has structural and sequence homology to a family of zinc metalloglycoproteins with neutral endopeptidase activity.
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Project description:MOA (Marasmius oreades agglutinin), a lectin isolated from fruiting bodies of the mushroom M. oreades, specifically binds nonreducing terminal Galalpha(1,3)Gal carbohydrates, such as that which occurs in the xenotransplantation epitope Galalpha(1,3)Galbeta(1,4)GlcNAc and the branched blood group B determinant Galalpha(1,3)[Fucalpha(1,2)]Gal. Here, we present the crystal structure of MOA in complex with the blood group B trisaccharide solved at 1.8 A resolution. To our knowledge, this is the first blood-group-B-specific structure reported in complex with a blood group B determinant. The carbohydrate ligand binds to all three binding sites of the N-terminal beta-trefoil domain. Also, in this work, Ca(2+) was included in the crystals, and binding of Ca(2+) to the MOA homodimer altered the conformation of the C-terminal domain by opening up the cleft containing a putative catalytic site.
| S-EPMC2721272 | biostudies-literature
Project description:cDNA clones encoding a human blood group Rh polypeptide were isolated from a human bone marrow cDNA library by using a polymerase chain reaction-amplified DNA fragment encoding the known common N-terminal region of the Rh proteins. The entire primary structure of the Rh polypeptide has been deduced from the nucleotide sequence of a 1384-base-pair-long cDNA clone. Translation of the open reading frame indicates that the Rh protein is composed of 417 amino acids, including the initiator methionine, which is removed in the mature protein, lacks a cleavable N-terminal sequence, and has no consensus site for potential N-glycosylation. The predicted molecular mass of the protein is 45,500, while that estimated for the Rh protein analyzed in NaDodSO4/polyacrylamide gels is in the range of 30,000-32,000. These findings suggest either that the hydrophobic Rh protein behaves abnormally on NaDodSO4 gels or that the Rh mRNA may encode a precursor protein, which is further matured by a proteolytic cleavage of the C-terminal region of the polypeptide. Hydropathy analysis and secondary structure predictions suggest the presence of 13 membrane-spanning domains, indicating that the Rh polypeptide is highly hydrophobic and deeply buried within the phospholipid bilayer. In RNA blot-hybridization (Northern) analysis, the Rh cDNA probe detects a major 1.7-kilobase and a minor 3.5-kilobase mRNA species in adult erythroblasts, fetal liver, and erythroid (K562, HEL) and megakaryocytic (MEG01) leukemic cell lines, but not in adult liver and kidney tissues or lymphoid (Jurkat) and promyelocytic (HL60) cell lines. These results suggest that the expression of the Rh gene(s) might be restricted to tissues or cell lines expressing erythroid characters.
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Project description:The RH (rhesus) blood group locus from RhD-positive donors is composed of two homologous structural genes, one of which encodes the Cc and Ee polypeptides, whereas the other, which is missing in the RhD-negative condition, encodes the D protein that carries the major antigen of the RH system. Recently, different splicing isoforms transcribed from the CcEe gene were isolated. We report now the characterization of two other Rh clones, RhII and RhXIII, generated by alternative choices for poly(A) addition sites that were identified as the RhD gene transcripts. That these cDNAs represented the RhD messenger and that the previously described Rh clones were derived from the CcEe gene was demonstrated by amplification of RhII/XIII sequences only from D-positive genomes and by cloning and sequencing of D- and CcEe-specific gene fragments. The predicted translation product of the RhD mRNA is a 417-amino acid protein (M(r) = 45,500) that exhibited a similar membrane organization with 13 bilayer-spanning domains compared with the polypeptide encoded by the CcEe gene. The D and Cc/Ee polypeptides differ by 36 amino acid substitutions (8.4% divergence), but the NH2- and COOH-terminal regions of the two proteins are well conserved. Similarly, five of the six cysteine residues of the Cc/Ee proteins were conserved in the D protein, including the unique exofacial cysteine, which is critical for antigenic reactivity. The sequence homology between the Cc/Ee and D proteins supports the concept that the genes encoding these polypeptides have evolved by duplication of a common ancestor gene.
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