The human ABO(H) blood group antigens are produced by specific glycosyltransferase enzymes. An N-acetylgalactosaminyltransferase (GTA) uses a UDP-GalNAc donor to convert the H-antigen acceptor to the A antigen, whereas a galactosyltransferase (GTB) uses a UDP-galactose donor to convert the H-antigen acceptor to the B antigen. GTA and GTB differ only in the identity of four critical amino acid residues. Crystal structures at 1.8-1.32 A resolution of the GTA and GTB enzymes both free and in complex with disaccharide H-antigen acceptor and UDP reveal the basis for donor and acceptor specificity and show that only two of the critical amino acid residues are positioned to contact donor or acceptor substrates. Given the need for stringent stereo- and regioselectivity in this biosynthesis, these structures further demonstrate that the ability of the two enzymes to distinguish between the A and B donors is largely determined by a single amino acid residue.
The specificity of antibody recognition of the ABO blood group trisaccharide antigens has been explored by crystal structure analysis and mutation methods. The crystal structure of the Fv corresponding to the antiblood group A antibody AC1001 has been determined to 2.2-Å resolution and reveals a binding pocket that is complementary to the blood group A-trisaccharide antigen. The effect of mutating specific residues lining this pocket on binding to the A and B blood group oligosaccharide antigens was investigated through a panel of single point mutations and through a phage library of mutations in complementarity determining region H3. Both approaches gave several mutants with improved affinity for antigen. Surface plasmon resonance indicated up to 8-fold enhancement in affinity for the Apentasaccharide with no observable binding to the blood group B antigen. This is the first example of single point mutations in a carbohydrate-binding antibody resulting in significant increases in binding affinity without loss of specificity.The affinity of anti-carbohydrate antibodies for their antigens is commonly observed to be 3-5 orders of magnitude lower than affinities of anti-protein or anti-peptide antibodies for their antigens, yet there is no clear mechanism to explain this phenomenon. One means of exploring this question is to attempt to generate mutant anti-carbohydrate antibodies with higher affinities, either by design of site-directed mutants or by randomizing selected codons in a phage library. However, the production of antibodies with improved affinities that maintain antigen specificity has proven challenging (1-4). For example, in an exhaustive site-directed mutagenesis study of CDR 1 H3 of an anti-Salmonella Fab, Brummell et al. (1) found that all of the 90 mutant Fabs produced showed similar or decreased binding affinity for the O-antigen. Mutants of the anti-Lewis Y antibody BR96 were obtained as phage-displayed scFv; although the mutant scFv binding affinity had increased by up to 6-fold versus the native antibody, its specificity was altered (2, 3). More recently, higher affinity antibodies against levan, a model for the polysaccharide capsules of bacteria, were obtained by mutational analysis though their fine specificity varied from that of the parent antibody (5). In contrast, significant enhancements have been achieved with antibodies to protein antigens. For example, a mutant scFv of an anti-c-erbB-2 tumor antigen obtained by chain shuffling and phage display had a 5-6-fold increase in affinity (4). In a later study by the same group (6) sequential mutation of light and heavy chains of the scFv yielded 16-and 9-fold increases in affinity for the protein antigen. Yang et al. (7) obtained mutant anti-HIV-1 Fab by phage-display techniques with a 96-fold increase in binding, even though the native antibody fragments already possessed high affinity for the protein antigen. Though phage-display techniques have been reported to yield higher binding mutant scFvs, the resulting antibodies often contain mult...
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 microM). 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.
Meningococcal meningitis is a severe childhood disease which often results in significant disability or death. Two major etiological agents of meningitis are the group B meningococci and capsular type K1 E. coli. The virulence of these organisms is attributable to structural mimicry between their common alpha(2-8)-polysialic acid capsular polysaccharide and human tissue antigens, which allows the bacteria to evade immune surveillance. There is currently no effective vaccine to protect against this infection. It has been demonstrated that the capsular polysaccharide of the bacteria can adopt a unique 'antigenic conformation'. This antigenic conformation has formed the basis for the development of an N-propionylated polysialic acid vaccine. Immunization trials in mice with this vaccine show the production of two groups of antibodies, of which only N-propionylated polysialic acid-specific were protective. Knowledge of the structure of the antigen-binding site which recognizes the protective epitope is essential to determining the antigenic conformation of the polysaccharides, and is a critical aspect in understanding and improving the action of potential vaccines. The antigen-binding fragments (Fab) of one protective (13D9) and one non-protective (6B9) monoclonal antibody specific for the capsular polysaccharides of group B meningococci have been crystallized and have undergone preliminary X-ray diffraction analysis. Both crystals are observed to scatter X-rays to approximately 1.7 A resolution at the A1 station at the Cornell High-Energy Synchrotron Source. 13D9 has an orthorhombic unit cell with a = 41.8, b = 102.3, c = 134.7 A, with space group P212121. Fab 6B9 has an orthorhombic unit cell with a = 89.6, b = 132.0 and c = 36.9 A, with space group P21212.
The protozoan parasites Trypanosoma brucei and 7: cruzi are the causative agents of African Sleeping Sickness and Chagas' Disease, respectively. Their surface molecules play a crucial role in protection and infectivity. Galactose residues are thought to play an important role in parasite survival: 7: brucei bloodstream forms are covered by a layer of variant surface glycoprotein (VSG) attached to the cell membrane via a glycosylphosphatidylinositol (GPI) anchor which is modified by a branched a-Gal side chain. In 7: cruzi p-galactopyranoside residues of m u c k are the acceptor sites for the trans-sialylation reaction which is essential for parasite survival. The oxidoreductase UDP-glucose 4' epimerase interconverts glucose and galactose. In mammals this enzyme is important for converting Gal to Glc because excess Gal can be toxic. In trypanosomes, the reverse reaction (Glc to Gal) is probably more important since Glc is freely available from blood whereas they cannot transport Gal. We have cloned the epimerase from both 7: brucei and 7: cruzi using partial sequences found by database searches. The parasite epimerase genes were cloned into pUCl8; these plasmids successfully rescued an E. coliepimerase mutant (galE-) for growth on McConkey agar with galactose as the sole carbon source. Rescued cells had the epimerase gene in reading frame +1 and grew as large red colonies, whereas their mutant counterparts formed smaller, paler colonies. We have also expressed the parasite epimerases from pET15b and pQE-30. Overexpressed protein will be used for enzyme assays and crystallisation trials. We will also attempt to make epimerase knockouts in I: brucei and 7: cruzi to assess the role of Gal in these parasites. MFIS: a high throughput technology for studying the ligandreceptor interactionThe Multi-parameter Fluorescence Immunosensor System (MFIS) is designed to study the specificity and cross-reactivity of the ligand-receptor interaction on a large scale. Using a micro-spotting device, many thousands of ligands or receptors can be patterned on a micro-glass slide. A solution containing specific ligands or receptors with distinct fluorescent tags can then be applied on the slide and their binding monitored by fluorescence signals. To investigate the feasibility of this method, we have taken advantage of a wellestablished antigen-antibody system, the dextrans and anti-dextran antibodies. A panel of purified dextran preparations with different linkage compositions and ratios of terminal/internal epitopes were immobilized on a surface-treated glass slide and than incubated with anti-dextran antibodies of defined specificities, either cavitytype or groove-type. The former is specific for the terminal nonreducing end structure of alpha(l,6)dextran; the latter recognizes the internal linear chain of the polysaccharide. When a cavity-type mAb, 16.4.12E, was applied on the glass slide, it bound to the immobilized alpha( 1,6)dextran preparations having branches but not those with an internal linear chain structure. By contrast, a...
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