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Stephen V Evans - One of the best experts on this subject based on the ideXlab platform.

  • Sequence-dependent effects of cryoprotectAnts on the Active sites of the humAn ABO(H) Blood Group A And B glycosyltrAnsferAses.
    Acta Crystallographica Section D-biological Crystallography, 2012
    Co-Authors: A.r. Johal, B. Schuman, Javier A. Alfaro, S.n. Borisova, Nina O.l. Seto, Stephen V Evans
    Abstract:

    : The humAn ABO(H) A And B Blood Group glycosyltrAnsferAses GTA And GTB differ by only four Amino Acids, yet this smAll dissimilArity is responsible for significAnt differences in biosynthesis, kinetics And structure. Like other glycosyltrAnsferAses, these two enzymes hAve been shown to recognize substrAtes through drAmAtic conformAtionAl chAnges in mobile polypeptide loops surrounding the Active site. Structures of GTA, GTB And severAl chimerAs determined by single-crystAl X-rAy diffrAction demonstrAte A rAnge of susceptibility to the choice of cryoprotectAnt, in which the mobile polypeptide loops cAn be induced by glycerol to form the ordered closed conformAtion AssociAted with substrAte recognition And by MPD [hexylene glycol, (±)-2-methyl-2,4-pentAnediol] to hinder binding of substrAte in the Active site owing to chelAtion of the Mn²⁺ cofActor And thereby Adopt the disordered open stAte. Glycerol is often Avoided As A cryoprotectAnt when determining the structures of cArbohydrAte-Active enzymes As it mAy Act As A competitive inhibitor for monosAcchAride ligAnds. Here, it is shown thAt the use of glycerol As A cryoprotectAnt cAn AdditionAlly induce significAnt chAnges in secondAry structure, A phenomenon thAt could Apply to Any clAss of protein.

  • donor substrAte specificity of recombinAnt humAn Blood Group A b And hybrid A b glycosyltrAnsferAses expressed in escherichiA coli
    FEBS Journal, 2001
    Co-Authors: Nina O.l. Seto, Stephen V Evans, Catherine A Compston, David R Bundle, Saran A Narang, Monica M Palcic
    Abstract:

    The humAn Blood Group A And B glycosyltrAnsferAses cAtAlyze the trAnsfer of GAlNAc And GAl, to the (O)H-precursor structure Fucα(1–2)GAlβ-OR to form the Blood Group A And B Antigens, respectively. ChAnging four Amino Acids (176, 235, 266 And 268) Alters the specificity from An A to A B glycosyltrAnsferAse. A series of hybrid Blood Group A/B glycosyltrAnsferAses were produced by interchAnging these four Amino Acids in synthetic genes coding for soluble forms of the enzymes And expressed in EscherichiA coli. The purified hybrid glycosyltrAnsferAses were chArActerized by two-substrAte enzyme kinetic AnAlysis using both UDP-GAlNAc And UDP-GAl donor substrAtes. The A And B glycosyltrAnsferAses were screened with other donor substrAtes And found to Also utilize the unnAturAl donors UDP-GlcNAc And UDP-Glc, respectively. The kinetic dAtA demonstrAte the importAnce of A single Amino Acid (266) in determining the A vs. B donor specificity.

  • production crystAllizAtion And diffrAction to Atomic resolution of An Antibody fv specific for the Blood Group A oligosAcchAride Antigen
    Acta Crystallographica Section D-biological Crystallography, 1998
    Co-Authors: Sonia I Patenaude, C R Mackenzie, D Bilous, S E Ryan, N M Young, Stephen V Evans
    Abstract:

    The histoBlood-Group ABO cArbohydrAte Antigens Are well known As importAnt fActors in Blood trAnsfusions, but they cAn Also Act As receptors for infectious Agents And hAve been implicAted in susceptibility to certAin cArcinomAs. A single-chAin vAriAble-domAin Antigen-binding frAgment (scFv) gene bAsed on the known sequence of An Anti-Blood-Group-A monoclonAl Antibody (AC1001) hAs been synthesized And expressed in EscherichiA coli. The purified scFv prepArAtion existed primArily in the monomeric form but Also contAined lArge Amounts of dimeric And higher oligomeric forms. The corresponding vAriAble-domAin Antigen-binding frAgment (Fv) wAs generAted by cleAving the VL–VH linker with subtilisin, And its Activity wAs demonstrAted by surfAce plAsmon resonAnce with An immobilized bovine serum Albumin A–trisAcchAride conjugAte (KD = 290 µM). AC1001 Fv crystAls grown in the presence of N-AcetylgAlActosAmine diffrActed to 0.93 A resolution. This is the first reported exAmple of A crystAl of An Antibody Antigen-binding frAgment diffrActing to Atomic resolution.

Monica M Palcic - One of the best experts on this subject based on the ideXlab platform.

  • Abo h Blood Group A And b glycosyltrAnsferAses recognize substrAte viA specific conformAtionAl chAnges
    Journal of Biological Chemistry, 2008
    Co-Authors: Javier A. Alfaro, Nina O.l. Seto, Ruixiang Blake Zheng, Mattias Persson, James A Letts, Robert Polakowski, Yu Bai, Svetlana N Borisova, Todd L Lowary, Monica M Palcic
    Abstract:

    AbstrAct The finAl step in the enzymAtic synthesis of the ABO(H) Blood Group A And B Antigens is cAtAlyzed by two closely relAted glycosyltrAnsferAses, An α-(1→3)-N-AcetylgAlActosAminyltrAnsferAse (GTA) And An α-(1→3)-gAlActosyltrAnsferAse (GTB). Of their 354 Amino Acid residues, GTA And GTB differ by only four “criticAl” residues. High resolution structures for GTB And the GTA/GTB chimeric enzymes GTB/G176R And GTB/G176R/G235S bound to A pAnel of donor And Acceptor AnAlog substrAtes reveAl “open,” “semi-closed,” And “closed” conformAtions As the enzymes go from the unligAnded to the ligAnded stAtes. In the open form the internAl polypeptide loop (Amino Acid residues 177-195) AdjAcent to the Active site in the unligAnded or H Antigen-bound enzymes is composed of two α-helices spAnning Arg180-Met186 And Arg188-Asp194, respectively. The semi-closed And closed forms of the enzymes Are generAted by binding of UDP or of UDP And H Antigen AnAlogs, respectively, And show thAt these helices merge to form A single distorted helicAl structure with AlternAting α-310-α chArActer thAt pArtiAlly occludes the Active site. The closed form is distinguished from the semi-closed form by the ordering of the finAl nine C-terminAl residues through the formAtion of hydrogen bonds to both UDP And H Antigen AnAlogs. The semi-closed forms for vArious mutAnts generAlly show significAntly more disorder thAn the open forms, whereAs the closed forms displAy little or no disorder depending strongly on the identity of residue 176. FinAlly, the use of synthetic AnAlogs reveAls how H Antigen Acceptor binding cAn be criticAl in stAbilizing the closed conformAtion. These structures demonstrAte A delicAtely bAlAnced substrAte recognition mechAnism And give insight on criticAl Aspects of donor And Acceptor specificity, on the order of substrAte binding, And on the requirements for cAtAlysis.

  • donor substrAte specificity of recombinAnt humAn Blood Group A b And hybrid A b glycosyltrAnsferAses expressed in escherichiA coli
    FEBS Journal, 2001
    Co-Authors: Nina O.l. Seto, Stephen V Evans, Catherine A Compston, David R Bundle, Saran A Narang, Monica M Palcic
    Abstract:

    The humAn Blood Group A And B glycosyltrAnsferAses cAtAlyze the trAnsfer of GAlNAc And GAl, to the (O)H-precursor structure Fucα(1–2)GAlβ-OR to form the Blood Group A And B Antigens, respectively. ChAnging four Amino Acids (176, 235, 266 And 268) Alters the specificity from An A to A B glycosyltrAnsferAse. A series of hybrid Blood Group A/B glycosyltrAnsferAses were produced by interchAnging these four Amino Acids in synthetic genes coding for soluble forms of the enzymes And expressed in EscherichiA coli. The purified hybrid glycosyltrAnsferAses were chArActerized by two-substrAte enzyme kinetic AnAlysis using both UDP-GAlNAc And UDP-GAl donor substrAtes. The A And B glycosyltrAnsferAses were screened with other donor substrAtes And found to Also utilize the unnAturAl donors UDP-GlcNAc And UDP-Glc, respectively. The kinetic dAtA demonstrAte the importAnce of A single Amino Acid (266) in determining the A vs. B donor specificity.

Jacques Le Pendu - One of the best experts on this subject based on the ideXlab platform.

  • influence of the combined Abo fut2 And fut3 polymorphism on susceptibility to norwAlk virus AttAchment
    The Journal of Infectious Diseases, 2005
    Co-Authors: Severine Marionneau, Jacques Le Pendu, Nicolai V Bovin, Fabrice Airaud, Nathalie Ruvoenclouet
    Abstract:

    4 ShemyAkin Institute of BioorgAnic Chemistry, Moscow, RussiA The binding of NorwAlk virus (NV) recombinAnt cApsids wAs tested in A pAnel of sAlivA sAmples collected from 96 donors with different ABO, secretor, And Lewis phenotypes. As previously reported, binding occurred specificAlly to sAlivA from secretors, regArdless of their Lewis phenotype stAtus. Blood Group B sAlivA wAs poorly recognized, whereAs binding to Blood Group O sAlivA wAs higher And binding to Blood Group A sAlivA wAs highest. TrAnsfection of either Blood Group A or B enzyme into H epitope-expressing cells showed thAt mAsking of H epitopes by the A And B Antigens blocked the AttAchment of NV cApsids. The high level of binding to Blood Group A secretor sAlivA could be explAined by An optimAl H type 1 ligAnd density, which wAs lower thAn thAt in Blood Group O sAlivA And much higher thAn thAt in Blood Group B sAlivA. Indeed, despite A higher ligAnd density, sAlivA from homozygotes with 2 functionAl FUT2 Alleles wAs less strongly recognized thAn sAlivA from heterozygotes with 1 functionAl And 1 inActivAted FUT2 Allele. PArtiAl fucosidAse treAtment of duodenAl tissue sections And binding to A synthetic probe with vArying densities of H type 1 trisAcchAride indicAted thAt optimAl AttAchment occurred At medium ligAnd density.

  • Cloning of A rAt gene encoding the histo-Blood Group A enzyme. Tissue expression of the gene And of the A And B Antigens.
    European journal of biochemistry, 2002
    Co-Authors: Anne Cailleau-thomas, Béatrice Le Moullac-vaidye, Jézabel Rocher, Claude Szpirer, Danièle Bouhours, Jacques Le Pendu
    Abstract:

    The complete coding sequence of A BDIX rAt gene homologous to the humAn ABO gene wAs determined. IdentificAtion of the exon-intron boundAries, obtAined by compArison of the coding sequence with rAt genomic sequences from dAtA bAnks, reveAled thAt the rAt gene structure is identicAl to thAt of the humAn ABO gene. It locAlizes to rAt chromosome 3 (q11-q12), A region homologous to humAn 9q34. Phylogenetic AnAlysis of A set of sequences AvAilAble for the vArious members of the sAme gene fAmily confirmed thAt the rAt sequence belongs to the ABO gene cluster. The cDNA wAs trAnsfected in CHO cells AlreAdy stAbly trAnsfected with An AlphA1,2fucosyltrAnsferAse in order to express H oligosAcchAride Acceptors. AnAlysis of the trAnsfectAnts by flow cytometry indicAted thAt A but not B epitopes were synthesized. Direct AssAy of the enzyme Activity using 2' fucosyllActose As Acceptor confirmed the strong UDP-GAlNAc:FucAlphA1,2GAlAlphAGAlNAc trAnsferAse (AtrAnsferAse) Activity of the enzyme product And Allowed detection of A smAll UDP-GAl:FucAlphA1,2GAlAlphAGAl trAnsferAse (B trAnsferAse) Activity. The presence of the mRNA And of the A And B Antigens wAs seArched in vArious BDIX rAt tissues. There wAs A generAl good concordAnce between the presence of the mRNA And thAt of the A Antigen. Tissue distributions of the A And B Antigens in the homozygous BDIX rAt strAin were lArgely different, indicAting thAt these Antigens cAnnot be synthesized by Alleles of the sAme gene in this rAt inbred strAin.

Nina O.l. Seto - One of the best experts on this subject based on the ideXlab platform.

  • Sequence-dependent effects of cryoprotectAnts on the Active sites of the humAn ABO(H) Blood Group A And B glycosyltrAnsferAses.
    Acta Crystallographica Section D-biological Crystallography, 2012
    Co-Authors: A.r. Johal, B. Schuman, Javier A. Alfaro, S.n. Borisova, Nina O.l. Seto, Stephen V Evans
    Abstract:

    : The humAn ABO(H) A And B Blood Group glycosyltrAnsferAses GTA And GTB differ by only four Amino Acids, yet this smAll dissimilArity is responsible for significAnt differences in biosynthesis, kinetics And structure. Like other glycosyltrAnsferAses, these two enzymes hAve been shown to recognize substrAtes through drAmAtic conformAtionAl chAnges in mobile polypeptide loops surrounding the Active site. Structures of GTA, GTB And severAl chimerAs determined by single-crystAl X-rAy diffrAction demonstrAte A rAnge of susceptibility to the choice of cryoprotectAnt, in which the mobile polypeptide loops cAn be induced by glycerol to form the ordered closed conformAtion AssociAted with substrAte recognition And by MPD [hexylene glycol, (±)-2-methyl-2,4-pentAnediol] to hinder binding of substrAte in the Active site owing to chelAtion of the Mn²⁺ cofActor And thereby Adopt the disordered open stAte. Glycerol is often Avoided As A cryoprotectAnt when determining the structures of cArbohydrAte-Active enzymes As it mAy Act As A competitive inhibitor for monosAcchAride ligAnds. Here, it is shown thAt the use of glycerol As A cryoprotectAnt cAn AdditionAlly induce significAnt chAnges in secondAry structure, A phenomenon thAt could Apply to Any clAss of protein.

  • Abo h Blood Group A And b glycosyltrAnsferAses recognize substrAte viA specific conformAtionAl chAnges
    Journal of Biological Chemistry, 2008
    Co-Authors: Javier A. Alfaro, Nina O.l. Seto, Ruixiang Blake Zheng, Mattias Persson, James A Letts, Robert Polakowski, Yu Bai, Svetlana N Borisova, Todd L Lowary, Monica M Palcic
    Abstract:

    AbstrAct The finAl step in the enzymAtic synthesis of the ABO(H) Blood Group A And B Antigens is cAtAlyzed by two closely relAted glycosyltrAnsferAses, An α-(1→3)-N-AcetylgAlActosAminyltrAnsferAse (GTA) And An α-(1→3)-gAlActosyltrAnsferAse (GTB). Of their 354 Amino Acid residues, GTA And GTB differ by only four “criticAl” residues. High resolution structures for GTB And the GTA/GTB chimeric enzymes GTB/G176R And GTB/G176R/G235S bound to A pAnel of donor And Acceptor AnAlog substrAtes reveAl “open,” “semi-closed,” And “closed” conformAtions As the enzymes go from the unligAnded to the ligAnded stAtes. In the open form the internAl polypeptide loop (Amino Acid residues 177-195) AdjAcent to the Active site in the unligAnded or H Antigen-bound enzymes is composed of two α-helices spAnning Arg180-Met186 And Arg188-Asp194, respectively. The semi-closed And closed forms of the enzymes Are generAted by binding of UDP or of UDP And H Antigen AnAlogs, respectively, And show thAt these helices merge to form A single distorted helicAl structure with AlternAting α-310-α chArActer thAt pArtiAlly occludes the Active site. The closed form is distinguished from the semi-closed form by the ordering of the finAl nine C-terminAl residues through the formAtion of hydrogen bonds to both UDP And H Antigen AnAlogs. The semi-closed forms for vArious mutAnts generAlly show significAntly more disorder thAn the open forms, whereAs the closed forms displAy little or no disorder depending strongly on the identity of residue 176. FinAlly, the use of synthetic AnAlogs reveAls how H Antigen Acceptor binding cAn be criticAl in stAbilizing the closed conformAtion. These structures demonstrAte A delicAtely bAlAnced substrAte recognition mechAnism And give insight on criticAl Aspects of donor And Acceptor specificity, on the order of substrAte binding, And on the requirements for cAtAlysis.

  • donor substrAte specificity of recombinAnt humAn Blood Group A b And hybrid A b glycosyltrAnsferAses expressed in escherichiA coli
    FEBS Journal, 2001
    Co-Authors: Nina O.l. Seto, Stephen V Evans, Catherine A Compston, David R Bundle, Saran A Narang, Monica M Palcic
    Abstract:

    The humAn Blood Group A And B glycosyltrAnsferAses cAtAlyze the trAnsfer of GAlNAc And GAl, to the (O)H-precursor structure Fucα(1–2)GAlβ-OR to form the Blood Group A And B Antigens, respectively. ChAnging four Amino Acids (176, 235, 266 And 268) Alters the specificity from An A to A B glycosyltrAnsferAse. A series of hybrid Blood Group A/B glycosyltrAnsferAses were produced by interchAnging these four Amino Acids in synthetic genes coding for soluble forms of the enzymes And expressed in EscherichiA coli. The purified hybrid glycosyltrAnsferAses were chArActerized by two-substrAte enzyme kinetic AnAlysis using both UDP-GAlNAc And UDP-GAl donor substrAtes. The A And B glycosyltrAnsferAses were screened with other donor substrAtes And found to Also utilize the unnAturAl donors UDP-GlcNAc And UDP-Glc, respectively. The kinetic dAtA demonstrAte the importAnce of A single Amino Acid (266) in determining the A vs. B donor specificity.

Robert J Woods - One of the best experts on this subject based on the ideXlab platform.

  • predicting the origins of Anti Blood Group Antibody specificity A cAse study of the Abo A And b Antigens
    Frontiers in Immunology, 2014
    Co-Authors: Spandana Makeneni, David C Watson, Martin N Young, Robert J Woods
    Abstract:

    The ABO Blood Group system is the most importAnt Blood type system in humAn trAnsfusion medicine. Here, we explore the specificity of Antibody recognition towArds ABO Blood Group Antigens using computAtionAl modeling And biolAyer interferometry. AutomAted docking And moleculAr dynAmics (MD) simulAtions were used to explore the origin of the specificity of An Anti-Blood Group A Antibody vAriAble frAgment (Fv AC1001). The AnAlysis predicts A number of Fv-Antigen interActions thAt contribute to Affinity, including A hydrogen bond between A HisL49 And the cArbonyl moiety of the GAlNAc in Antigen A. This interAction wAs consistent with the dependence of Affinity on pH, As meAsured experimentAlly; At lower pH there is An increAse in binding Affinity. Binding energy cAlculAtions provide unique insight into the origin of interAction energies At A per-residue level in both the scFv And the trisAcchAride Antigen. The cAlculAtions indicAte thAt while the Antibody cAn AccommodAte both Blood Group A And B Antigens in its combining site, the A Antigen is preferred by ApproximAtely 4 kcAl/mol, consistent with the lAck of binding observed for the B Antigen.