Assuntos
Dextranos/genética , Epitopos/genética , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Diversidade de Anticorpos/genética , Diversidade de Anticorpos/imunologia , Sequência de Bases , Sítios de Ligação de Anticorpos/genética , Clonagem Molecular , Dextranos/imunologia , Epitopos/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Região de Junção de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Região Variável de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/genética , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Homologia de Sequência do Ácido NucleicoRESUMO
Nine groove-type mAb to alpha(1----6)dextran were cloned and sequenced. Together with previous reports from this laboratory, the VH and VL of 34 mAb have been sequenced, in which 10 VH19.1.2 and 11 VH9.14.7 combined with the V kappa-Ox1 gene to form two major families of anti-alpha(1----6)dextrans. The same D minigene (DFL16) was used by all VH19.1.2 and VH9.14.7 mAb; however, the patterns of JH and J kappa usage are quite different. VH19.1.2 mAb used only JH3 and J kappa 2, whereas VH9.14.7 mAb used three JH (JH1, JH2, and JH3) and all four active J kappa (J kappa 1, J kappa 2, J kappa 4, and J kappa 5). Relative uniformity in the lengths of VH CDR3 and the junctional sequences is seen in both families. Some mAb from different mouse strains share common structural features. The differences in idiotypic specificities and in the amino acid sequences suggest that VH19.1.2 and VH9.14.7 may differ in the conformation of CDR1 and CDR2. Combining with V kappa-Ox1 gene to generate groove-type combining sites to the single site-filling epitope of alpha(1----6)dextran, the two VH chains may require certain conformations of CDR3. Whether such conformational requirements influence the choice of J minigenes, the selection of the length of VH CDR3 and the sequences at junctions, are discussed.
Assuntos
Anticorpos Monoclonais/imunologia , Dextranos/imunologia , Rearranjo Gênico de Cadeia Pesada de Linfócito B , Rearranjo Gênico de Cadeia Leve de Linfócito B , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA/genética , Genes , Camundongos , Camundongos Endogâmicos , Dados de Sequência MolecularRESUMO
An idiotype defined by mAb and polyclonal antibodies to 10.16.1, an anti-alpha(1----6) dextran was previously reported to be expressed on most BALB/c anti-alpha(1----6)dextrans with groove-type sites and to involved CDR3 and probably CDR2. By comparing amino acid sequences of VH and VL derived from cDNA of idiotype+ and idiotype- anti-alpha(1----6)dextran hybridoma proteins, an idiotope was assigned to VH CDR2. Substitution of phenylalanine for leucine at residue 52 in CDR2 coupled with amino acid changes at either residue 58 or residues 57 and 60 abolished expression of this idiotype without affecting Ag binding.
Assuntos
Anticorpos Monoclonais , Sítios de Ligação de Anticorpos , Dextranos/imunologia , Cadeias Pesadas de Imunoglobulinas , Idiótipos de Imunoglobulinas , Região Variável de Imunoglobulina , Relação Estrutura-Atividade , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/isolamento & purificação , Sequência de Bases , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/isolamento & purificação , Idiótipos de Imunoglobulinas/genética , Idiótipos de Imunoglobulinas/isolamento & purificação , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/isolamento & purificação , Cadeias kappa de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/isolamento & purificação , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Dados de Sequência MolecularRESUMO
The combining site of Ricinus communis agglutinin (RCA1) was studied by quantitative precipitin and precipitin inhibition assays. Of 31 complex carbohydrates tested, all except active and inactive antifreeze glycoproteins, Streptococcus group C polysaccharide, and native rat salivary glycoprotein, reacted strongly, and 22 completely precipitated the lectin, indicating that RCA1 has both a broad range of affinity and a low solubility of its carbohydrate-bound complex. Of the monosaccharides and glycosides tested for inhibition of precipitation, p-nitrophenyl beta-D-galactopyranoside was the best. It was about 6.4 times better than methyl beta-D-galactopyranoside. The beta anomer of glycosides of D-galactose was much more potent than the corresponding alpha anomer. Among the oligosaccharides tested, beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----6)-D-Gal was the best inhibitor, which was approximately 2/3 as active as p-nitrophenyl beta-D-galactopyranoside. It was approximately 1.4 times as active as beta-D-Gal-(1----4)-D-GlcNAc (N-acetyllactosamine), twice as active as beta-D-Gal-(1----3)-D-GlcNAc, and 4.5 times more active than lacto-N-tetraose. From the results, it can be concluded that; (a) hydrophobic interaction is important for binding; (b) the combining site of this lectin is at least as large as a trisaccharide; and (c) of the compounds studied, the trisaccharide beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----6)-D-Gal was the most complementary to the human blood group I Ma determinant beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----6)-D-Gal.
Assuntos
Amino Açúcares/metabolismo , Lectinas/metabolismo , Lectinas de Plantas , Trissacarídeos/metabolismo , Animais , Bovinos , Feminino , Cavalos , Humanos , Imuno-Histoquímica , Especificidade por Substrato , SuínosRESUMO
Monoclonal antibodies to alpha(1----6)dextrans produced in mice immunized with the T-independent antigens alpha(1----6)dextran or the stearylisomaltosyl oligosaccharides have been characterized immunochemically. To correlate the immunochemical properties of these monoclonal antibodies with their primary structure, we have sequenced the variable (V) regions of the light (L) and heavy (H) chains. Three V kappa germ-line genes belonging to two major gene families were used; differential J usages also contribute to diversity. Five different VH germ-line genes belonging to three major VH families were used. The VH genes were further modified by junctional diversity and differential J usage and possibly by somatic mutations. The effects of these modifications on the fine specificities of anti-alpha(1----6)dextrans are discussed. Thus far, six different combinations of VLJL-VH(D)JH chains that form groove-type combining sites specific for alpha(1----6)dextran have been found. We conclude that entirely different VL and VH can form combining sites specific for the internal linear sequence of alpha(1----6)dextran.
Assuntos
Sítios de Ligação de Anticorpos , Dextranos/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Região Variável de Imunoglobulina/genética , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Sequência de Bases , Genes , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias J de Imunoglobulina/genética , Cadeias J de Imunoglobulina/imunologia , Cadeias Leves de Imunoglobulina/imunologia , Região Variável de Imunoglobulina/imunologia , Camundongos , Camundongos Endogâmicos BALB C/genética , Camundongos Endogâmicos BALB C/imunologia , Camundongos Endogâmicos C57BL/genética , Camundongos Endogâmicos C57BL/imunologiaRESUMO
The specificities of the combining sites of 19 mouse monoclonal antibodies to dextran B1355S have been characterized immunochemically by quantitative precipitin and precipitin inhibition assays; association constants for B1355S were determined by affinity gel electrophoresis. Cross-reactive and individual idiotypes related to the BALB/c B1355S-binding myeloma proteins MOPC104E [IdI(MOPC104E)] and J558 [IdI(J558)], determined by a radioimmunoassay, and heavy-chain variable-region sequences, are presented. Antibodies to B1355S are "alpha (1----3) alpha (1----6)-specific" as determined by precipitin and precipitin inhibition assays with dextrans and oligosaccharides, respectively, containing alternating alpha (1----3) alpha (1----6) linkages compared with oligosaccharides composed solely of alpha (1----3) or alpha (1----6) linkages; all antibodies have low association constants (less than or equal to 10(5) ml/g). However, there is also considerable diversity among the proteins as seen in the five groups of different patterns of reactivity with numerous dextrans having different structures, and the variability in affinity even among antibodies showing the same fine specificity by precipitin assay. There is little observable correlation of heavy-chain variable-region amino-acid sequence with specificity or affinity; however, all proteins having D-region amino acids Tyr,Asp at positions 96,97 express the MOPC104E individual idiotype and belong to precipitin specificity group 5, the group most cross-reactive with numerous dextrans, whereas those proteins having the J558 individual idiotype, Arg,Tyr or Asn,Tyr at 96,97 are found in all five precipitin groups.
Assuntos
Anticorpos Monoclonais/imunologia , Dextranos/imunologia , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Afinidade de Anticorpos , Especificidade de Anticorpos , Sítios de Ligação de Anticorpos , Idiótipos de Imunoglobulinas/análise , Imunoglobulina M/análise , Região Variável de Imunoglobulina/análise , Camundongos , Testes de PrecipitinaRESUMO
Treatment of blood group A active glycoprotein from human ovarian cyst fluid by one stage of Smith degradation followed by alkaline beta-elimination in the presence of NaB[ 3H4 ] (Carlson degradation) liberated tritiated oligosaccharide alditols. The carbohydrate mixture was fractionated by gel filtration, elution from charcoal, paper chromatography, and high pressure liquid chromatography. Structures were established based on sugar composition, periodate oxidation, methylation analysis, and analysis of oligosaccharide alditols as permethylated and N-trifluoroacetylated derivatives by gas-liquid chromatography-mass spectrometry. The following structures have been deduced: Gal beta 1----3GalNAc-ol, GlcNAc beta 1---- 6GalNAc -ol, Gal beta 1---- 3GlcNAc beta 1----6(3-deoxy)GalNAc-ol, Gal beta 1---- 3GlcNAc beta 1---- 6GalNAc -ol, Gal beta 1----4GlcNAc beta 1---- 6GalNAc -ol, GlcNAc beta 1----3Gal beta 1----3GalNAc-ol, Gal beta 1----3[GlcNAc beta 1----6]GalNAc-ol, Gal beta 1----3[Gal beta 1----4GlcNAc beta 1----6]GalNAc-ol, Gal beta 1---- 3GlcNAc beta 1----3Gal beta 1----3GalNAc-ol, GlcNAc beta 1----3Gal beta 1----4GlcNAc beta 1---- 6GalNAc -ol, GlcNAc beta 1----3Gal beta 1----3[Gal beta 1----4GlcNAc beta 1----6]GalNAc-ol, Gal beta 1---- 3GlcNAc beta 1----3Gal beta 1---- 3GlcNAc beta 1----3Gal beta 1----3Gal beta 1----3GalNAc-ol, Gal beta 1---- 3GlcNAc beta 1----3[Gal beta 1----4GlcNAc beta 1----6]Gal beta 1----3GalNAc-ol, Gal beta 1---- 3GlcNAc beta 1----3Gal beta 1----3[Gal beta 1----4GlcNAc beta 1----6]GalNAc-ol. The smaller structures represent pieces of the larger structures. Together they provide direct evidence for the core structure of the carbohydrate side chains in the blood group substances as proposed by K. O. Lloyd and E. A. Kabat [1968) Proc. Natl. Acad. Sci. U.S.A. 61, 1470-1477). Oligosaccharides previously isolated after Carlson degradation of intact human ovarian cyst fluid HLeb , Lea, and B substances and from human and horse B substances contained various alpha-linked L- fucopyranose and alpha-linked Gal substitutions on the composite structure.(ABSTRACT TRUNCATED AT 400 WORDS)
