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Arrays in rays: terminal addition in echinoderms and its correlation with gene expression.

The echinoderms are deuterostomes that superimpose radial symmetry upon bilateral larval morphology. Consequently, they are not the first animals that come to mind when the concepts of segmentation and terminal addition are being discussed. However, it has long been recognized that echinoderms have serial elements along their radii formed in accordance with the ocular plate rule (OPR). The OPR is a special case of terminal growth, forming elements of the ambulacra that define the rays in echinoderms. New elements are added at the terminus of the ray, which may or may not be marked by a calcified element called the terminal plate (the "ocular" of sea urchins). The OPR operates in every echinoderm, from the occasionally bizarre fossils of the Cambrian to the most familiar extant taxa. Using the OPR and other criteria of recognition, echinoderm body wall can be divided into two main regions: extraxial components are associated with the somatocoels, axial components (formed in accordance with the OPR) with the hydrocoel. We compare patterns of development in axial regions of echinoderms with those found in the anterior-posterior axes of the earliest echinoderms as well as other invertebrates. Although axial and extraxial skeletons appear to be composed of the same biomineral matrix, the genes involved in patterning these two skeletal components are likely distinct. During development of the axial skeleton, for instance, the genes engrailed and orthodenticle are expressed in spatial and temporal patterns consistent with the OPR. Other genes such as distal-less seem to demarcate early ontogenetic boundaries between the axial rudiment and the extraxial larval body. There is a complex and pervasive reorganization of gene expression domains to produce the highly divergent morphologies seen in the Echinodermata. We integrate morphological and genetic information, particularly with respect to the origins of radial symmetry in the rudiment, and the concomitant development of the rays.

Animals↗

PubMed2005

DrugScore(CSD)-knowledge-based scoring function derived from small molecule crystal data with superior recognition rate of near-native ligand poses and better affinity prediction.

Following the formalism used for the development of the knowledge-based scoring function DrugScore, new distance-dependent pair potentials are obtained from nonbonded interactions in small organic molecule crystal packings. Compared to potentials derived from protein-ligand complexes, the better resolved small molecule structures provide relevant contact data in a more balanced distribution of atom types and produce potentials of superior statistical significance and more detailed shape. Applied to recognizing binding geometries of ligands docked into proteins, this new scoring function (DrugScore(CSD)) ranks the crystal structures of 100 protein-ligand complexes best among up to 100 generated decoy geometries in 77% of all cases. Accepting root-mean-square deviations (rmsd) of up to 2 angstroms from the native pose as well-docked solutions, a correct binding mode is found in 87% of the cases. This translates into an improvement of the new scoring function of 57% with respect to the retrieval of the crystal structure and 20% with respect to the identification of a well-docked ligand pose compared to the original Protein Data Bank-based DrugScore. In the analysis of decoy geometries of cross-docking studies, DrugScore(CSD) shows equivalent or increased performance compared to the original PDB-based DrugScore. Furthermore, DrugScore(CSD) predicts binding affinities convincingly. Reducing the set of docking solutions to examples that deviate increasingly from the native pose results in a loss of performance of DrugScore(CSD). This indicates that a necessary prerequisite to successfully resolving the scoring problem with a more discriminative scoring function is the generation of highly accurate ligand poses, which approximate the native pose to below 1 angstroms rmsd, in a docking run.

Crystallography, X-Ray↗

PubMed1996

Recognition of subsets of the mammalian A/B-type core heterogeneous nuclear ribonucleoprotein polypeptides by novel autoantibodies.

The structurally related A/B-type core heterogeneous nuclear ribonucleoprotein (hnRNP) polypeptides of 34-39 kDa (A1, A2, B1 and B2) belong to a family of RNA-binding proteins that are major components of 40 S hnRNP complexes. By two-dimensional gel electrophoresis and peptide mapping analysis we compared each member of the A/B-type core proteins in the human and rat liver cells. This comparison revealed the unique presence in rat cells of major protein species, referred to as mBx polypeptides, that appeared as three charge isoforms at a position corresponding to the minor HeLa B1b protein spot. In addition, clear differences in the ratios of the A1 polypeptide to the A1b isoform were observed. The detection, in sera of patients with rheumatic autoimmune diseases, of two novel autoantibody specificities, one recognizing solely B2 protein and the second both the B2 and mBx polypeptides, helped to identify mBx proteins as new A/B-type hnRNP components, immunologically related to B2 protein. A common immunoreactive V8 protease peptide of approx. 17 kDa has been identified in B2 and mBx hnRNP polypeptides. mBx protein species are identified in cells of murine origin, and have a ubiquitous tissue distribution and developmental appearance.

Animals↗

PubMed2001

Synthesis and molecular recognition of novel oligo(ethylenediamino) bridged bis(beta-cyclodextrin)s and their copper(II) complexes: enhanced molecular binding ability and selectivity by multiple recognition.

Four bridged bis(beta-cyclodextrin)s tethered by different lengths of oligo(ethylenediamine)s have been synthesized and their inclusion complexation behavior with selected substrates elucidated by circular dichroism spectroscopy and fluorescence decay. In order to study their binding ability quantitatively, inclusion complexation stability constants with four dye guests, that is, brilliant green (BG), methyl orange (MO), ammonium 8-anilino-1-naphthalenesulfonic acid (ANS), and sodium 6-(p-toluidino)-2-naphthalenesulfonate (TNS), have been determined in aqueous solution at 25 degrees C with spectrophotometric, spectropolarimetric, or spectrofluorometric titrations. The results obtained indicate that the two tethered cyclodextrin units might cooperatively bind to a guest, and the molecular binding ability toward model substrates, especially linear guests such as TNS and MO, could be extended. The tether length plays a crucial role in the molecular recognition, the binding constants for ANS and TNS decrease linearly with an increase in the tether length of dimeric cyclodextrin. The Gibbs free energy changes (-deltaGo) for the unit increment per ethylene are 0.99 kJ mol(-1) for ANS and 0.44 kJmol(-1) for TNS, respectively. On the other hand, the presence of a copper(II) ion in metallobis(beta-cyclodextrin)s oligo(ethylenediamino) tethers enhances not only the original binding ability, but also the molecular selectivity through triple or multiple recognition, as compared with the parent bis(beta-cyclodextrin)s.

Journal Article↗

PubMed1988

The origin of conjugative IncP plasmid transfer: interaction with plasmid-encoded products and the nucleotide sequence at the relaxation site.

To characterize protein-DNA interactions involved in the initiation of conjugative transfer replication we isolated and dissected the transfer origins (oriT) of the promiscuous IncP plasmids RP4 and R751. Essential features of oriT are conserved: symmetric sequence repeats, the nic site and a pair of potential promoter sites that allow for divergent transcription of two tra operons. The relaxation nick and the end of a 19 bp inverted repeat are interspaced by eight basepairs. The 5'-terminal nucleotide at the nick is modified by an alkali-resistant residue and the 3'-nucleotide is accessible to extension by DNA polymerase I. Transfer gene products essential for the formation of the initiation complex (relaxosome) of conjugative DNA synthesis map adjacent to oriT. Two of these products, TraJ and TraK confer specificity to their homologous oriT exclusively. Proteins TraJ and TraK are the only components of the RP4 and R751 transfer machinery which cannot be interchanged. TraJ and at least two additional plasmid-encoded products are necessary for specific relaxation. The purified TraJ protein of RP4 possesses oriT-binding ability. The recognition sequence contains a palindromic sequence located within the right arm of the 19 bp inverted repeat. The TraJ binding site and the nic site are located on one side of the DNA double helix. We presume that this nucleoprotein structure is the initial complex in the pathway to the assembly of functional relaxosomes.

Bacterial Proteins↗

PubMed1998

Glycyl-tRNA synthetase from Thermus thermophilus--wide structural divergence with other prokaryotic glycyl-tRNA synthetases and functional inter-relation with prokaryotic and eukaryotic glycylation systems.

The tRNA glycylation system is amongst the most complex aminoacylation systems since neither the oligomeric structure of the enzymes nor the discriminator base in tRNAs are conserved in the phylae. To understand better this structural diversity and its functional consequences, the prokaryotic glycylation system from Thermus thermophilus, an extreme thermophile, was investigated and its structural and functional inter-relations with those of other origins analyzed. Alignments of the protein sequence of the dimeric thermophilic glycyl-tRNA synthetase (Gly-tRNA synthetase) derived from its gene with sequences of other dimeric Gly-tRNA synthetases revealed an atypical character of motif 1 in all these class 2 synthetases. Interestingly, the sequence of the prokaryotic thermophilic enzyme resembles eukaryotic and archaebacterial Gly-tRNA synthetases, which are all dimeric, and diverges drastically from the tetrameric enzymes from other prokaryotes. Cross aminoacylations with tRNAs and synthetases of different origins provided information about functional interrelations between the glycylation systems. Efficient glycylations involving partners from T. thermophilus and Escherichia coli showed conservation of the recognition process in prokaryotes despite strong structural variations of the synthetases. However, Gly-tRNA synthetase from T. thermophilus acylates eukaryotic tRNA(Gly) while the charging ability of the E. coli enzyme is restricted to prokaryotic tRNA(Gly). A similar behaviour is found in eukaryotic systems where the restricted species specificity for tRNA glycylation of mammalian Gly-tRNA synthetase contrasts with the relaxed specificity of the yeast enzyme. The consensus sequence of the tRNAs charged by the various Gly-tRNA synthetases reveals conservation of only G1-C72 in the acceptor arm, C35 and C36 in the anticodon, and the (G10-Y25)-G45 triplet involved in tRNA folding. Conservation of these nucleotides indicates their key role in glycylation and suggests that they were part of the ancestral glycine identity set. These features are discussed in the context of the phylogenic connections between prokaryotes, eukaryotes, and archaebacteria, and of the particular place of T. thermophilus in this phylogeny.

Amino Acid Sequence↗

PubMed1996

Origin of carbohydrate recognition specificity of human lysozyme revealed by affinity labeling.

In order to reveal the origin of carbohydrate recognition specificity of human lysozyme by clarifying the difference in the binding mode of ligands in the active site, the inactivation of human lysozyme by 2',3'-epoxypropyl beta-glycoside derivatives of the disaccharides, N,N'-diacetylchitobiose [GlcNAc-beta-(1-->4)-GlcNAc] and N-acetyllactosamine [Gal-beta-(1-->4)-GlcNAc], was investigated and the three-dimensional structures of the affinity-labeled enzymes were determined by X-ray crystallography at 1.7 A resolution. Under the conditions comprising 2.0 x 10(-3) M labeling reagent and 1.0 x 10(-5) M human lysozyme at pH 5.4, 37 degrees C, the reaction time required to reduce the lytic activity against Micrococcus luteus cells to 50% of its initial activity was lengthened by 3.7 times through the substitution of the nonreducing end sugar residue, GlcNAc to Gal. The refined structure of human lysozyme labeled by 2',3'-epoxypropyl beta-glycoside derivatives of N,N'-diacetylchitobiose (HL/NAG-NAG-EPO complex) indicated that the interaction mode of the N,N'-diacetylchitobiose moiety in substites B and C in this study was essentially the same as in the case of the complex of human lysozyme with the free ligand. On the other hand, the hydrogen-bonding pattern and the stacking interaction at subsite B were remarkably different between the HL/NAG-NAG-EPO complex and human lysozyme labeled by the 2',3'-epoxypropyl beta-glycoside of N-acetyllactosamine (HL/GAL-NAG-EPO complex). The reduced number of possible hydrogen bonds as well as the less favorable stacking between the side chain of Tyr63 in human lysozyme and the galactose residue in the HL/GAL-NAG-EPO complex reasonably explained the less efficient ability of the 2',3'-epoxypropyl beta-glycoside of N-acetyllactosamine as compared to that of N,N'-diacetylchitobiose as an affinity labeling reagent toward human lysozyme.

Affinity Labels↗

PubMed2004

Effects of monofunctional adducts of platinum(II) complexes on thermodynamic stability and energetics of DNA duplexes.

Effects of adducts of [PtCl(NH3)3]Cl or chlorodiethylenetriamineplatinum(II) on DNA stability were studied with emphasis on thermodynamic origins of that stability. Oligodeoxyribonucleotide duplexes (15-bp) containing the single, site-specific monofunctional adduct at G-residues of the central sequences TGT/ACA or 5'-AGT/5'-ACT were prepared and analyzed by differential scanning calorimetry, temperature-dependent ultraviolet absorption and circular dichroism. The unfolding of the platinated duplexes was accompanied by relatively small unfavorable free energy terms. This destabilization was enthalpic in origin. On the other hand, a relatively large reduction of melting temperature (T(m)) was observed as a consequence of the monofunctional adduct in the TGT sequence, whereas T(m) due to the adduct in the AGT sequence was reduced only slightly. We also examined the efficiency of the mammalian nucleotide excision repair system to remove from DNA the monofunctional adducts and found that these lesions were not recognized by this repair system. Thus, rather thermodynamic than thermal characterization of DNA adducts of monofunctional platinum compounds is a property implicated in the modulation of downstream effects such as protein recognition and repair.

DNA↗

PubMed2001

Self and nonself recognition of chiral catalysts: the origin of nonlinear effects in the amino-alcohol catalyzed asymmetric addition of diorganozincs to aldehydes.

Asymmetric addition of dialkylzincs to aldehydes in the presence of (2S)-3-exo-(dimethylamino)isoborneol [(S)-DAIB] exhibits various nonclassical phenomena. The enantiomeric excess (ee) of the alkylation product, obtained with partially resolved DAIB, is much higher than that of the chiral amino alcohol, while the rate decreases considerably as the ee of DAIB is lowered. The asymmetric amplification effects reflect the relative turnover numbers of two enantiomorphic catalytic cycles, where an essential feature is the reversible homochiral and heterochiral dimerization of the coexisting enantiomeric DAIB-based Zn catalysts. The interplay between the thermodynamics of the monomer/dimer equilibration and the kinetics of alkylation reaction strongly affect the overall profile of asymmetric catalysis. The self and nonself recognition of the chiral Zn catalysts is a general phenomenon when (S)-DAIB is mixed with its enantiomer, diastereomer, or even an achiral beta-amino alcohol. The degree of nonlinearity is highly affected not only by the structures and purity of catalysts but also by various reaction parameters. The salient features have been clarified on the basis of molecular weight measurements, NMR and X-ray crystallographic studies of organozinc complexes, and kinetic experiments, as well as computer-aided quantitative analysis.

Journal Article↗

PubMed2002

Recognition of IgG-derived peptides by a human IgM with an unusual combining site.

A monoclonal immunoglobulin (Ig)M cryoglobulin (Mez) with interesting binding behaviour was isolated from a Waldenström's macroglobulinemia (WM) patient. It demonstrated very strong binding to peptides derived from the sequences of human IgG. However, when tested for binding to intact IgG, this antibody (Ab) did not show any rheumatoid factor (RF) activity. We propose several nonexclusive structural interpretations of the Mez-binding propensities, based on the orientations and solvent accessibilities of ligand residues and the nature of the Ab-binding site. To further characterize the structural features of Mez-peptide binding, IgG-derived octapeptides were docked into the Mez fragment variable (Fv)-binding site, revealing additional reasons for Mez-binding selectivity based on the interactions of the docked peptides with the Mez Fv. The problem was also approached from an immunological perspective. Comparisons of Mez variable region of the light chain (VL)/variable region of the heavy chain (VH) sequences with those of human germlines and known IgM RFs allowed us to provide a possible outline tracing the structural and functional origins of the Mez IgM. Coupled with examinations of interactions in docked complexes, this analysis led us to propose that the potential for RF activity, demonstrated through Mez binding to IgG-derived peptides, was owing to the inherent sequence and structure of the Mez IgM, rather than to somatic mutations. Thus, Mez IgM may occupy an intermediate niche between IgMs with and without RF activity.

Amino Acid Sequence↗

PubMed1987

Structural elements highly preserved during the evolution of the D-loop-containing region in vertebrate mitochondrial DNA.

A detailed comparative study of the regions surrounding the origin of replication in vertebrate mitochondrial DNA (mtDNA) has revealed a number of interesting properties. This region, called the D-loop-containing region, can be divided into three domains. The left (L) and right (R) domains, which have a low G content and contain the 5' and the 3' D-loop ends, respectively, are highly variable for both base sequence and length. They, however, contain thermodynamically stable secondary structures which include the conserved sequence blocks called CSB-1 and TAS which are associated with the start and stop sites, respectively, for D-loop strand synthesis. We have found that a "mirror symmetry" exists between the CSB-1 and TAS elements, which suggests that they can act as specific recognition sites for regulatory, probably dimeric, proteins. Long, statistically significant repeats are found in the L and R domains. Between the L and R domains we observed in all mtDNA sequences a region with a higher G content which was apparently free of complex secondary structure. This central domain, well preserved in mammals, contains an open reading frame of variable length in the organisms considered. The identification of common features well preserved in evolution despite the high primary structural divergence of the D-loop-containing region of vertebrate mtDNA suggests that these properties are of prime importance for the mitochondrial processes that occur in this region and may be useful for singling out the sites on which one should operate experimentally in order to discover functionally important elements.

Amino Acid Sequence↗

PubMed1988

Complement deficiency. Predisposing factor to autoimmune syndromes.

The recognition of the association between complete and partial complement (C) deficiencies and immune complex-mediated diseases is of clinical and etiopathologic interest. From studies of sera deficient in C1, C4, C2, or C3, the crucial role of these complement components in promoting the solubility and clearance of immune complexes has been elucidated. Moreover, partial C4 deficiency appears to be a common risk factor for the development of systemic lupus erythematosus, with complete C4A deficiency (C4A null) being present in 10 to 15 percent and heterozygous C4A deficiency present in 50 to 80 percent of patients with systemic lupus erythematosus. Most importantly, there is an obvious pathophysiologic relationship between the function of complement relative to immune complex processing and the disease that results from their deficiency. Systemic lupus erythematosus is characterized by excessive quantities of inappropriately deposited immune complexes. More subtle complement component and receptor deficiencies are likely to be predisposing factors for autoimmune disease. The complement deficiencies provide us with a unique opportunity to investigate the origin and development of immune complex excess syndromes.

Antigen-Antibody Complex↗

PubMed2005

Sequential information processing using time-delay connections in ontogenic CALM networks.

In this paper, a variant of the categorization-and-learning-module (CALM) network is presented that is not only capable of categorizing sequential information with feedback, but can adapt its resources to the current training set. In other words, the modules of the network may grow or shrink depending on the complexity of the presented sequence-set. In the original CALM algorithm, modules did not have access to activations from earlier stimulus presentations. To bypass this limitation, we introduced time-delay connections in CALM. These connections allow for a delayed propagation of activation, such that information at a given time will be available to a module at a later timestep. In addition, modules can autonomously add and remove resources depending on the structure and complexity of the task domain. The performance of this ontogenic CALM network with time-delay connections is demonstrated and analyzed using a sample set of overlapping sequences from an existing problem domain.

Algorithms↗

PubMed1990

Preferential usage of V alpha 4 and V beta 10 T cell receptor genes by lymphocytic choriomeningitis virus glycoprotein-specific H-2Db-restricted cytotoxic T cells.

Correlations between the T cell receptor (TcR) V gene usage and the specificity of T cells have been primarily described for major histocompatibility complex (MHC) class II-restricted helper T cell responses. In the present study the TcR genes expressed by MHC class I-restricted murine cytotoxic T cells (CTL) specific for a major epitope of the lymphocytic choriomeningitis virus (LCMV), LCMV-GP2(275-289), were investigated. The TcR primary structure of an LCMV-GP2(275-289) specific H-2Db-restricted CTL clone has been determined. It uses a member of the V alpha 4 family joined to J alpha AN14.4 for the alpha chain and V beta 10 rearranged to D beta 2.1 and J beta 2.4 for its beta chain. Four other independent LCMV-GP2(275-289) specific H-2Db-restricted CTL clones also expressed V alpha 4 and V beta 10 gene elements. Furthermore, V alpha 4 and V beta 10 were preferentially expressed by polyclonal CTL of C57BL/6 origin specific for LCMV. These results suggest that both TcR V alpha and V beta regions are important for the recognition of the LCMV-GP2(275-289) epitope on H-2Db molecules.

Amino Acid Sequence↗

PubMed2000

A quantitative study of the in vitro binding of the C-terminal domain of p21 to PCNA: affinity, stoichiometry, and thermodynamics.

Proliferating cell nuclear antigen (PCNA) plays an essential role in DNA replication, repair, and control of cell proliferation, and its activity can be modulated by interaction with p21(Waf1/Cip1) [Cox, L. S., (1997) Trends Cell Biol. 7, 493-497]. This protein-protein interaction provides a particularly good model target for designing therapeutic agents to treat proliferative disorders such as cancer. In this study, the formation of complexes between PCNA and peptides derived from the C-terminus of p21 has been investigated at the molecular level and quantified using a competitive PCNA binding assay and isothermal titration calorimetry (ITC). The affinity constant for the interaction between p21 (141-160) peptide and PCNA has been determined to be 1.14 x 10(7) M(-)(1), corresponding to a K(d) of 87.7 nM. Measurement of the interaction of truncation and substitution analogues based on the p21 (141-160) sequence with PCNA revealed that the N-terminal part (residues 141-152) of the above peptide is the minimum recognition motif, required for PCNA binding. Truncation of the C-terminal region p21 (153-160), though, inhibited significantly the ability of the peptides to compete with the full-length p21 (141-160) for binding to PCNA. Alanine mutation of Met 147 or Asp 149 completely abolished or significantly decreased, respectively, the level of the PCNA binding and the inhibition of SV40 DNA replication. Comparison of the data obtained by the competitive PCNA binding assay and the ITC measurements demonstrated the usefulness of this assay for screening for compounds that could modulate the PCNA-p21 interaction. Using this assay, we have screened rationally designed peptides for binding to PCNA and interruption of the PCNA-p21 (141-160) complex. As a result of this screening, we have identified a 16-residue peptide (consensus motif 1 peptide) with the following sequence: SAVLQKKITDYFHPKK. Consensus motif 1 peptide and p21 (141-160) have similar affinities for binding PCNA and abilities to inhibit in vitro replication of DNA originated from SV40. Such peptides could prove useful in assessing p21-mimetic strategies for cancer treatment.

Amino Acid Sequence↗

PubMed1983

Immune-response gene-associated antigens (Ia/DR). Structure and function in immunologically related diseases.

Major histocompatibility complex-determined antigens were originally identified as a consequence of their ability to induce rejection of tissue grafts between organisms that are not genetically identical. Currently, much is known about their biochemical nature and intended biological functions. Major histocompatibility complex antigens are found on three types of glycoprotein molecules. One type (class I) is associated with beta 2-microglobulin in the cell-surface membranes of all body tissues and includes H-2K and D molecules in mice and HLA-A, B, and C molecules in humans. These antigens are the major cause of rejection of transplanted organs. The other two types of glycoproteins (class II) are noncovalently linked to each other, are found in the cell-surface membranes of a limited number of cell types, and include H-2-Ia molecules in mice and HLA-DR molecules in humans. They are noted for their ability to elicit graft-versus-host disease. Both class I and class II molecules are, however, important for the immune recognition of pathogens, although the types of responses they modulate are different. Class I molecules are important in the recognition of cell-surface antigens, whereas class II molecules control responsiveness to soluble antigens. Major histcompatibility complex-encoded molecules are also involved in certain autoimmune diseases. As our understanding of major histocompatibility complex-controlled immune responsiveness broadens and hybridoma and gene-cloning technology advances, specific enhancement of desired immune responses and suppression of deleterious ones will most likely become possible.

Animals↗

PubMed2006

Understanding the bacterial polysaccharide antigenicity of Streptococcus agalactiae versus Streptococcus pneumoniae.

Bacterial surface capsular polysaccharides (CPS) that are similar in carbohydrate sequence may differ markedly in immunogenicity and antigenicity. The structural origin of these phenomena is poorly understood. Such a case is presented by the Gram-positive bacteria Streptococcus agalactiae (Group B Streptococcus; GBS) type III (GBSIII) and Streptococcus pneumoniae (Pn) type 14 (Pn14), which share closely related CPS sequences. Nevertheless, antibodies (Abs) against GBSIII rarely cross-react with the CPS from Pn14. To establish the origin for the variation in CPS antigenicity, models for the immune complexes of CPS fragments from GBSIII and Pn14, with the variable fragment (Fv) of a GBS-specific mAb (mAb 1B1), are presented. The complexes are generated through a combination of comparative Ab modeling and automated ligand docking, followed by explicitly solvated 10-ns molecular dynamics simulations. The relationship between carbohydrate sequence and antigenicity is further quantified through the computation of interaction energies using the Molecular Mechanics-Generalized Born Surface Area (MM-GBSA) method, augmented by conformational entropy estimates. Despite the electrostatic differences between Pn14 and GBSIII CPS, analysis indicates that entropic penalties are primarily responsible for the loss of affinity of the highly flexible Pn14 CPS for mAb 1B1. The similarity of the solution conformation of the relatively rigid GBSIII CPS with that in the immune complex characterizes the previously undescribed 3D structure of the conformational epitope. The analysis provides a comprehensive interpretation for a large body of biochemical and immunological data related to Ab recognition of bacterial polysaccharides and should be applicable to other Ab-carbohydrate interactions.

Amino Acid Sequence↗

PubMed1985

Antibody-nucleic acid interactions. Monoclonal antibodies define different antigenic domains in 2',5'-oligoadenylates.

To define the epitopes involved in binding anti-oligonucleotide antibodies, several hybridomas producing monoclonal antibodies directed against 2',5'-oligoadenylate were established. A solid-phase enzyme-linked immunoassay that employed microtiter wells coated with Ficoll-2',5'-oligoadenylate conjugates proved useful in screening and characterizing hybridoma supernatants. Control experiments demonstrated that the conjugates were irreversibly adsorbed to polystyrene wells under the conditions employed in the assay. Reactivity of monoclonal antibodies with numerous analogues of 2',5'-oligoadenylate was measured by using a competition assay. Several monoclonal antibodies originating from different mice immunized with the same or different immunogens possessed distinctive fine specificities. At least one 2',5'-phosphodiester bond was important in forming each epitope, suggesting that the ribose phosphate backbone is a critical element in defining an antigenic domain of an oligonucleotide. The purine bases were also important, and modification of the bases had varied effects on the extent of antibody recognition. The length of the oligonucleotide and the nature of the termini were also of some importance. In several instances the modification created by linkage of 2',5'-oligoadenylate to carrier protein also contributed to the determinant. The monoclonal antibody most specific for 2',5'-oligoadenylates was relatively insensitive to ionic strength. In contrast, a monoclonal antibody with a 2',5'-oligopurine specificity appeared to bind 2',5'-oligoadenylate through one ion pair, whereas the binding of a monoclonal antibody with a low degree of base specificity appeared to bind through two ion pairs. The results demonstrated that 2',5'-linked oligoadenylate-protein complexes possess at least three distinct oligonucleotide-related antigenic surfaces that can be recognized with high apparent affinity by monoclonal antibodies. A model for the three epitopes is presented.

Adenine Nucleotides↗