Identifying residues in antigenic determinants by chemical modification.
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Enzyme-Linked Immunosorbent Assay↗
Biomedical subjects
N M Young
Publications and source records attributed to N M Young.
Early diagnosis of Usher syndrome in infants and children.
The electroretinogram (ERG) is the only test currently available that has the potential to identify patients with Usher syndrome before onset of fundoscopic and visual abnormalities. The ERG is a noninvasive technique that can diagnose retinitis pigmentosa via measurement of corneal-retinal potentials. Forty-seven children with bilateral severe to profound sensorineural hearing loss were evaluated. Usher syndrome was diagnosed in five children, all of whom subsequently underwent cochlear implantation. We have found that accurate diagnosis of Usher syndrome in infants and young children is possible via ERG. Benefits of early diagnosis include timely counseling regarding genetic issues, educational and vocational placement, and cochlear implantation. For this reason we recommend that all newly identified children with bilateral severe to profound sensorineural hearing loss undergo ERG testing.
Child↗
Thermal stabilization of a single-chain Fv antibody fragment by introduction of a disulphide bond.
A disulphide bond was introduced into a single-chain Fv form of the anticarbohydrate antibody, Se155-4 by replacing Ala-L57 of the light chain and Asp-H106 of the heavy chain with cysteines, by site-directed mutagenesis. To maintain the salt-bridge from the latter residue to Arg-H98, Tyr-107 was also altered to Asp. The resulting ds-scFv was shown to retain full antigen-binding activity, by enzyme immunoassay and surface plasmon resonance analysis of binding kinetics. Compared with the parent scFv, the disulphide bonded form was shown to have enhanced thermal stability, by Fourier transform IR spectroscopy. The Tm was raised from 60 degrees C to 69 degrees C. The ds-scFv form thus combines the stable monomeric form of the disulphide form with the expression advantages of the scFv.
Binding Sites↗
Basis for selection of improved carbohydrate-binding single-chain antibodies from synthetic gene libraries.
A technique is described for the simultaneous and controlled random mutation of all three heavy or light chain complementarity-determining regions (CDRs) in a single-chain Fv specific for the O polysaccharide of Salmonella serogroup B. Sense oligonucleotides were synthesized such that the central bases encoding a CDR were randomized by equimolar spiking with A, G, C, and T at a level of 10% while the antisense strands contained inosine in the spiked regions. Phage display of libraries assembled from the spiked oligonucleotides by a synthetic ligase chain reaction demonstrated a bias for selection of mutants that formed dimers and higher oligomers. Kinetic analyses showed that oligomerization increased association rates in addition to slowing dissociation rates. In combination with some contribution from reduced steric clashes with residues in heavy-chain CDR2, oligomerization resulted in functional affinities that were much higher than that of the monomeric form of the wild-type single-chain Fv.
Amino Acid Sequence↗
C-terminal post-translational proteolysis of plant lectins and their recombinant forms expressed in Escherichia coli. Characterization of "ragged ends" by mass spectrometry.
Electrospray mass spectrometry was used to accurately measure the molecular masses of single chain lectins from legume seeds and also of three recombinant lectins, expressed in Escherichia coli. The five single chain lectins, Erythrina corallodendron lectin, soybean and peanut agglutinins, Dolichos biflorus lectin, and Phaseolus vulgaris hemagglutinin E, all showed evidence of C-terminal proteolytic processing, in some cases to "ragged" ends, when their masses were compared to those expected from their cDNA sequences and their known carbohydrate chains. Recombinant forms of the lectins from E. corallodendron, soybean, and peanut also showed C-terminal trimming, but not to the same points as the natural forms. Discrepancies between the protein and cDNA sequences of the E. corallodendron lectin were resolved by combined liquid chromatography-mass spectrometry peptide mapping and protein sequencing experiments, and the presence of a second glycosylation site was demonstrated. Our data show that all of these lectins undergo C-terminal proteolytic processing of a readily attacked peptide segment. This trimming is frequently imprecise, and the resulting heterogeneity may be a major contributor to the appearance of isolectin forms of these proteins.
Amino Acid Sequence↗
Mass spectrometric analysis of genetic and post-translational heterogeneity in the lectins Jacalin and Maclura pomifera agglutinin.
Jacalin and M. pomifera agglutinin are T-antigen specific lectins with alpha 4 beta 4 structures that show far greater micro-heterogeneity than plant lectins from other families, due to multiple genetic isoforms and post-translational processing. Electrospray mass spectrometry and combined liquid chromatography-electrospray mass spectrometry were used to characterize the various forms. For both lectins, the mass data were consistent with previous protein sequencing of the major alpha-chain species of 133 residues and three beta-chain species of 20 or 21 residues. In addition, for jacalin the mass of one minor alpha-chain species was consistent with a second of the four reported gene sequences. However, the glycopeptide alpha-chain form and one beta-chain form did not match any of the genes, suggesting a fifth gene remains to be found. For M. pomifera agglutinin, three more beta-chain forms were found, but all six could arise from only two genes, with additional post-translational proteolysis and post-translational substitution with an unidentified component of 106 Da creating the set of six forms. Only two alpha-chain forms were found also, with no glycosylation.
Amino Acid Sequence↗
Bifunctional fusion proteins consisting of a single-chain antibody and an engineered lanthanide-binding protein.
The combination of an antibody fragment with a lanthanide chelating protein has desirable characteristics for fluorescence-based immunoassays and tumor radioimmunotherapy. As a model for this design, a fusion protein consisting of a single-chain antibody linked to an engineered version of oncomodulin, a protein with two Ca(2+)-binding motifs (the CD and EF loops), was produced by secretion from Escherichia coli in good yield. The single-chain antibody was specific for a Salmonella O-polysaccharide. The CD loop of oncomodulin had been redesigned to bind lanthanide ions with high affinity. The fusion protein was shown to have antigen-binding activity that was comparable to that of the unfused single-chain antibody, to bind Tb3+ with very high affinity and to give strong, sensitized Tb3+ luminescence via excitation of the tryptophan residue in the CD loop. A second fusion protein containing a 30-residue helix-loop-helix motif as the lanthanide-binding component was also prepared, but showed considerably lower solubility. Competition for Tb3+ binding by a series of metal chelators indicated that the affinities of the oncomodulin and 30 residue fusions for Tb3+ were approximately 10(11) M-1 and 10(7) M-1, respectively. Time-resolved lanthanide luminescence photography of electrophoresis gels demonstrated that the helix-loop-helix Ca(2+)-binding could be used to specifically visualize the scFv fragment.
Amino Acid Sequence↗
Location of the active site of the bean alpha-amylase inhibitor and involvement of a Trp, Arg, Tyr triad.
Seeds of the common bean contain three homologous proteins: phytohaemagglutinin E, phytohaemagglutinin L and the lectin-like protein alpha-amylase inhibitor (alpha AI). Whereas the active site of lectins has been studied in great detail, there is no information on the active site of the related protein alpha AI, which exerts its biological activity by making a 1:1 complex with alpha-amylase. alpha-amylase inhibitor is synthesized as a 30 kDa precursor glycoprotein that needs to be processed at Asn77 to form an active molecule. Comparison of the amino acid sequence of the bean alpha AI with that of the bacterial amylase inhibitor, tendamistat, suggested that a region around Trp188 might be involved in the inhibitory site. When a three-dimensional model of the bean alpha AI was constructed based on its homology to the legume lectins, this Trp region was alongside Asn77. To test this site hypothesis, mutants of alpha AI were created by site-directed mutagenesis of the cDNA and expressed in transgenic tobacco. The mutant proteins R74N and WSY188-190GNV, as well as the double mutant, were inactive as inhibitors. These findings suggest that the active site of alpha AI consists of W188, R74 and Y190, in analogy to the Trp-Arg-Tyr motif of tendamistat, and that the processing of the polypeptide at Asn77 may be necessary to bring these residues in close proximity.
Amino Acid Sequence↗
Cochlear implants in young children with Usher's syndrome.
Usher's syndrome (US) is an autosomal recessive disorder characterized by congenital sensorineural hearing loss and retinitis pigmentosa. The majority of affected individuals have severe to profound sensorineural hearing loss and are therefore potential cochlear implant candidates. A goal of our otology program has been early identification and implantation of children with US. At our center, early diagnosis of US has been achieved by comprehensive ophthalmologic evaluation including electroretinography. Four of our 19 pediatric cochlear implant recipients with congenital sensorineural hearing loss were identified with US and implanted prior to the onset of signs or symptoms of visual loss. All children have received measurable benefit from cochlear implantation. In light of the dual sensory deficits present in US, the need for early diagnosis and cochlear implantation is of special importance in this population.
Child↗
Exploring the mimicry of polysaccharide antigens by anti-idiotypic antibodies. The crystallization, molecular replacement, and refinement to 2.8 A resolution of an idiotope-anti-idiotope Fab complex and of the unliganded anti-idiotope Fab.
The monoclonal antibody YsT9.1 is specific for the lipopolysaccharide A antigen of Brucella abortus. A complex formed between the Fab of YsT9.1 (Ab1) and the Fab of its antidiotopic monoclonal antibody T91AJ5 (Ab2) has been crystallized, and data have been collected to 2.8 A resolution. The space group is monoclinic P2(1), with one molecule per asymmetric unit. The structure was solved using a limited Patterson-correlation search over the asymmetric-unit of rotation space, and has been refined to an R-factor of 0.174. The complex is a head-to-head dimer, with the contact between the two Fabs almost completely restricted to their hypervariable loops. The interactions between the two Fabs in the complex are dominated by tyrosine residues, not only in the formation of hydrogen bonds, but in their participation in an aromatic ring network that spans the two Fv domains. The anti-idiotope was found to be unable to carry an internal image of the antigen and induce polysaccharide-specific "anti-anti-idiotopes" (Ab3) because the polysaccharide binding cleft on the Ab1 is too narrow and deep to allow comprehensive contact with the binding site of Ab2. The antibody T91AJ5 therefore is a class gamma anti-idiotope. The contact surfaces of the two Fabs are highly complementary; however, they are distinctly different in character. Each Fab has two separate binding surfaces of approximately equal size, but while the two binding surfaces of Ab1 are partitioned between the light and heavy chains, the two binding surfaces of Ab2 are shared between the chains, with the heavy chain responsible for about 60% of the total binding area. The structure of the unliganded Fab of Ab2 has also been solved by molecular replacement, and refined to an R-factor of 0.152 at 2.8 A resolution. This Fab crystallizes in the orthorhombic space group P2(1)2(1)2(1), with one molecule per asymmetric unit. The second hypervariable loop of the heavy chain of the Ab2 Fab is observed to undergo a significant and necessary conformational rearrangement in going from the unliganded to complexed form, and thus complex formation is an example of "induced fit" of antigen to antibody. The unliganded Ab1 Fab packs in the standard head-to-tail fashion observed for other Fabs. This mode of packing is precluded in the complex, by its head-to-head nature, and it is found to pack in tilted layers with most intermolecular contacts made between adjacent Ab2 Fab molecules.(ABSTRACT TRUNCATED AT 400 WORDS)
Antibodies, Anti-Idiotypic↗
Structure of a single-chain antibody variable domain (Fv) fragment complexed with a carbohydrate antigen at 1.7-A resolution.
We describe here the 1.7-A resolution structure of a single-chain antibody variable domain (scFv) molecule, based on the carbohydrate-binding antibody Se155-4, complexed with the trisaccharide ligand alpha-D-Gal(1-->2)[alpha-D-Abe(1-->3)]alpha-D-Manp1-->OMe, where Abe is abequose. The scFv expressed in Escherichia coli has the variable region light chain to heavy chain polarity with the domains connected by a 19-residue linker. Although the linker is partially disordered in the crystal, the packing of the molecules suggests a monomeric state of the scFv. The carbohydrate adopts a different conformation about the Man-Gal linkage than was observed previously in the Fab-trisaccharide complex. Instead of a direct hydrogen bond between O2Abe and O2Gal, these two atoms are bridged by a water molecule in the present complex.
Amino Acid Sequence↗
Molecular recognition of a Salmonella trisaccharide epitope by monoclonal antibody Se155-4.
The binding site of monoclonal antibody Se155-4, which has been the object of successful crystallographic and antibody-engineering studies, is shown by solid-phase immunoassays to be complementary to a branched trisaccharide, alpha-D-Galp(1-->2) [alpha-D-Abep(1-->3)]-alpha-D-Manp(1, rather than to the tetrasaccharide repeating unit alpha-D-Galp(1-->2) [alpha-D-Abep(1-->3)]-alpha-D-Manp(1-->4) alpha-L-Rhap(1- of the bacterial antigen. Specificity for the 3,6-dideoxy-D-xylo-hexose (3,6-dideoxy-D-galactose) epitope present in Salmonella paratyphi B O-antigens was ensured by screening hybridoma experiments with glycoconjugates derived from synthetic oligosaccharides. Detailed epitope mapping of the molecular recognition by modified and monodeoxy oligosaccharide derivatives showed that complementary surfaces and three antibody-saccharide hydrogen bonds are essential for full binding activity. Both hydroxyl groups of the 3,6-dideoxy-D-galactose residue were obligatory for binding and consistent with the directional nature of their involvement in carbohydrate-protein hydrogen bonds; related tetrasaccharides built from the isomeric 3,6-dideoxyhexoses, 3,6-dideoxy-D-glucose, paratose, and 3,6-dideoxy-D-mannose, tyvelose were not bound by the antibody. Titration microcalorimetry measurements were consistent with the hydrogen-bonding map inferred from the crystal structure and suggest that the displacement of water molecules from the binding site accounts for the favorable entropy that accompanies binding of the native trisaccharide determinant. The protein sequences determined for the antibody VL and VH domains reveal somatic mutation of the VL germ line gene, implying that this antibody-binding site results from a mature antibody response.
Amino Acid Sequence↗
Selection of antibody single-chain variable fragments with improved carbohydrate binding by phage display.
A single-chain variable fragment (Fv) version of a murine monoclonal antibody, Se155-4, specific for Salmonella serogroup B O-polysaccharide, was used as a model system for testing monovalent phage display as a route for enhancing the relatively low affinities that typify anti-carbohydrate antibodies. Random single-chain Fv mutant libraries generated by chemical and error-prone polymerase chain reaction methods were panned against the serogroup B lipopolysaccharide. Panning of a randomly mutated heavy chain variable domain library indicated selection for improved serogroup B binders and yielded six mutants, five of which showed wild type activity by enzyme immunoassay. Two of these were apparently selected on the basis of better functional single-chain Fv yield in Escherichia coli. A heavy chain mutation (Ile77-->Thr) in one mutant, 3B1, appeared to have a particularly dramatic effect, resulting in yields of approximately 120 mg/liter of functional periplasmic product. The sixth mutant, 4B2, had complementarity determining region 1 (CDR1) and CDR2 mutations and demonstrated 10-fold improved binding, by enzyme immunoassay, relative to the wild type. Extensive analysis of antigen-antibody interactions indicated that the improved binding properties of 4B2 were attributable to a higher association rate constant and interaction with an epitope that is larger than the trisaccharide recognized by the wild type. None of the mutations involved known trisaccharide contact residues; this was consistent with analysis of wild type and mutant single-chain Fvs by titration microcalorimetry. Examination of the structure indicated that two mutations in the heavy chain CDR2 provided improved surface complementarity between the protein and the extended epitope encompassing 2 additional hexose residues. However, introduction of only the CDR2 mutations into the wild type structure failed to confer the improved binding properties of 4B2, indicating an indirect effect by the more distant mutations. Panning of randomly mutated light chain variable domain and full-length single-chain Fv mutant libraries did not yield mutants with improved assembly or binding properties.
Antibodies, Monoclonal↗
Cochlear implants in children.
Children with such profound deafness that they are not helped by hearing aids are now candidates for cochlear implantation. This technology permits us to provide these children with a significant degree of useful hearing. The degree of improvement in speech perception and spoken language in pediatric cochlear implant recipients varies. However, the younger the children and the less time they have been completely deprived of auditory stimuli, the more likely they are to make significant progress. The evaluation of the deaf child for implantation is best done by a multidisciplinary team who understands the needs of hearing-impaired children and who can work with the family, the child, and classroom teachers, as well as other school professionals. The decision to proceed with cochlear implantation in a child is one that requires long-term commitment on the part of the family and the cochlear implant team.
Age Factors↗
Effect of C lambda-C kappa domain switching on Fab activity and yield in Escherichia coli: synthesis and expression of genes encoding two anti-carbohydrate Fabs.
We have used a strategy of hybrid gene synthesis and constant domain shuffling to construct and functionally express in Escherichia coli genes encoding two anti-carbohydrate Fabs, one specific for a Brucella cell-surface polysaccharide and the second for the human blood group A determinant. Very similar VL amino acid sequences made possible the simultaneous synthesis of the two corresponding genes. A class switching approach was used in Fd and light chain gene assembly. The two independently synthesized VH genes were fused to a previously made sequence encoding the C(gamma 1)1 domain as an alternative to synthesis of the natural C gamma 2b 1 and C mu 1 sequences. The VL genes were initially coupled to a synthetic C kappa gene. When these light chain and the above Fd genes, each preceded by the ompA signal sequence, were expressed from two-cistron DNA, yields of functional periplasmic Fab were low and, in each instance, limited by light chain availability. Replacement of the C kappa domains with a C lambda 1 domain resulted in a significant increase in the amount of soluble periplasmic light chain and functional Fab for both the Brucella and blood group A antibodies. The C kappa and C lambda 1 forms of each of the Brucella and blood group A Fabs, with His5 fusions at the C-termini of the Fd chains, were purified by immobilized metal affinity chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Inhibition of serine palmitoyl-transferase activity by lipoxamycin.
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Acyltransferases↗
Characterisation of residues in antibody binding sites by chemical modification of surface-adsorbed protein combined with enzyme immunoassay.
Specific functional group modification of an antibody adsorbed to microtitre plates has been used to probe the binding site residues that determine antigen specificity. Chemical modification of adsorbed protein in tandem with enzyme immunoassay (termed CMAP-EIA) consumes only modest amounts of antibody, while allowing a variety of reagents to be rapidly screened in situ. Modification of tyrosine and arginine residues with 1-fluoro-2,4-dinitrobenzene, and p-hydroxyphenylglyoxal resulted in reduced binding of polysaccharide antigen from Yersinia enterocolitica O-polysaccharide to its homologous monoclonal antibody, YsT9-1. Modification with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide under various conditions indicated that carboxylate groups may also be involved. Parallel experiments with diethylpyrocarbonate and acetic anhydride were used to rule out the involvement of histidine and lysine residues respectively. In all cases, binding of an anti-idiotypic antibody, AJ5, could only be reduced at concentrations of modifying reagent substantially higher than those required to reduce polysaccharide antigen binding to YsT9-1. The results are discussed with regard to the structure of the combining site of YsT9-1 as determined by X ray crystallography and by modelling, and the role of particular residues in complex formation with antigen and in the idiotope.
Acetic Anhydrides↗
Crystal structure to 2.45 A resolution of a monoclonal Fab specific for the Brucella A cell wall polysaccharide antigen.
The atomic structure of an antibody antigen-binding fragment (Fab) at 2.45 A resolution shows that polysaccharide antigen conformation and Fab structure dictated by combinatorial diversity and domain association are responsible for the fine specificity of the Brucella-specific antibody, YsT9.1. It discriminates the Brucella abortus A antigen from the nearly identical Brucella melitensis M antigen by forming a groove-type binding site, lined with tyrosine residues, that accommodates the rodlike A antigen but excludes the kinked structure of the M antigen, as envisioned by a model of the antigen built into the combining site. The variable-heavy (VH) and variable-light (VL) domains are derived from genes closely related to two used in previously solved structures, M603 and R19.9, respectively. These genes combine in YsT9.1 to form an antibody of totally different specificity. Comparison of this X-ray structure with a previously built model of the YsT9.1 combining site based on these homologies highlights the importance of VL:VH association as a determinant of specificity and suggests that small changes at the VL:VH interface, unanticipated in modeling, may cause significant modulation of binding-site properties.
Antibody Specificity↗