Contribution of tryptophan residues to the combining site of a monoclonal anti dinitrophenyl spin-label antibody

Two Fab fragments of the monoclonal anti dinitrophenyl (DNP) spin-label antibody AN02 were prepared by recombination of specifically deuterated heavy and light chains. In the recombinant H(I)L(II) all the tyrosines and phenylalanines were perdeuterated as were the tryptophan residues of the heavy chain. In the recombinant H(II)L(I) all the tyrosines and phenylalanines were perdeuterated as were the tryptophan residues of the light chain. Saturation of three resonances of H(I)L(II), assigned to tryptophan protons of the light chain, resulted in magnetization transfer to the aromatic proton at position 6 of the DNP ring and to the CH2 protons of the glycines linked to the DNP in a diamagnetic hapten (DNP-DG). Saturation of three resonances of H(II)L(I) assigned to tryptophan protons of the heavy chain resulted in magnetization transfer to the CH2 protons of the glycines in DNP-DG. From the dependence of the magnetization transfer on the irradiation time, the cross relaxation rates between the involved protons were estimated. The inferred distances between these protons of the hapten and certain tryptophan protons are 3-4 A. It is concluded that in the combining site of AN02 there is one tryptophan from the light chain and one tryptophan from the heavy chain that are very near the hapten. When all tyrosines and phenylalanines were perdeuterated and all tryptophan aromatic protons were deuterated except for the protons at positions 2 and 5, titration of the Fab fragments with variable amounts of paramagnetic hapten showed that one proton from the light chain tryptophan is near (less than 7 A) the unpaired electron and that three other protons are significantly closer than 15 A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformational multiplicity of the antibody combining site of a monoclonal antibody specific for a (6-4) photoproduct.

The antigen binding site of monoclonal antibody 64M5, which possesses a high degree of affinity for DNA containing pyrimidine (6-4) pyrimidone photoproducts, were investigated by use of stable-isotope-assisted NMR spectroscopy. A variety of 64M5 Fab fragments specifically labeled with 13C and 15N at backbone amide groups were prepared. Extensive assignments of amide resonances originating from the variable region of 64M5 were made by using 2D-HN(CO) measurements along with recombination of the heavy and light chains of 64M5. On the basis of chemical shift changes of the amide resonances caused upon addition of d(T[6-4]T) and d(GTAT[6-4]TATG), the binding sites of 64M5 Fab for the (6-4) photodimer and for the oligodeoxynucleotides flanking it were identified. It was revealed that the L1 and L3 segments, which are responsible for the binding to (6-4) photodimer, exhibit conformational multiplicities in the absence of antigens, and take different conformations between the d(T[6-4]T) and d(GTAT[6-4]TATG)-bound forms. On the basis of spectral comparison with another Fab fragment with a similarity in the amino acid sequence of the VL domain of 64M5, we suggest that the conformational multiplicities observed in the present study is caused by a substitution of an amino acid residue at the position of a key residue in L3 canonical structure, which leads to a preferable effect on the antigen binding, and by a specific combination of L1 and L3 canonical structures.     

Use of conformationally constrained peptides for a topographical analysis of the combining site of a monoclonal anti-substance P antibody

The topography of the binding site of a monoclonal anti-substance P antibody directed toward the C-terminal pentapeptide of substance P, Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH₂, was analyzed further using a wide range of constrained analogues of substance P. Results obtained in the present study show the following: (a) The binding subsites of Phe⁷ and Phe⁸ are large and deep, accommodating various side chains, including nonaromatic amino acids. (b) In contrast, the binding pockets for Gly-Leu-Met-NH₂ appear more restrictive. Consequently, five residues in the peptide are necessary for the high binding affinity to the antibody, the C-terminal tripeptide determining the binding specificity. These data, which appear to contradict those previously published, illustrate the limits of conclusions drawn from studies generally carried out using exclusively Ala-substituted peptides. In addition, the present results indicate that the topography of the binding site of this monoclonal antibody differs from that of the specific substance P neurokinin-1 receptor, in agreement with the differences observed in the fine specificities of these two substance P binding macromolecules. © 1996 John Wiley & Sons, Inc.     

Dissection of the combining site in a humanized anti-Tac antibody.

The genetically engineered "humanized" anti-Tac antibody (HAT) has been shown to bind the p55 chain of the human IL-2R with an affinity close to that of the murine anti-Tac. Although the HAT molecule contains all six mouse CDR, it was not known which, and to what extent, each of the CDR contributes to Ag binding. These questions were addressed by constructing a series of variant HAT antibodies, each substituting a single HAT CDR with a heterologous CDR. The association constants of the variant HAT antibodies to p55 were determined by competitive binding analysis. We find that CDR 1 and 3 of the H chain and CDR 3 of the L chain are essential for maintaining binding. The remaining three CDR appear to be involved to a lesser degree.     

Sponge aggregation factor: identification of the specific collagen-binding site by means of a monoclonal antibody

The aggregation factor (AF) from the sponge Geodia cydonium is known to be a complex proteinaceous particle, composed of a series of different (glyco)proteins (Mr lower than 150,000) around a 90S sunburst-like core structure. One of the low-Mr proteins is the 47-KD cell binding fragment. We describe a new monoclonal antibody (mAb), III1E6, raised against purified AF particles, which recognizes in tissue slices structures present both on the plasma membrane and in a network-like manner in the extracellular space. By applying immunoelectron microscopical, immunoblotting, and immunoaffinity chromatographical techniques, the mAb III1E6 was shown to recognize the core structure of the AF particle. Cell adhesion studies revealed that the mAb does not inhibit AF mediated cell-cell adhesion but abolishes AF-caused attachment of cells to collagen. Electron microscopic data show that III1E6 prevents association of AF particles with collagen fibrils. By applying the techniques of immunoblotting and of protein-protein recognition on the solid phase in vitro, we could formulate the following series of events: the AF particle recognizes, with its 47-KD cell binding fragment, the aggregation receptor protein in the plasma membrane and with its core structure the collagen fibrils. These fibrils interact optionally, either via the same route or via the collagen assembly factor, with an adjacent cell surface. These findings demonstrate that the AF particle is not only the key molecule for cell-cell adhesion but also a component of cell-matrix interactions.     

A monoclonal antibody specific for the amino terminal cleavage site of procollagen type I

A monoclonal mouse IgG1 antibody was produced against the aminopropeptide of dermatosparactic sheep procollagen type I by using the hybridoma technique. Radioimmunoassays demonstrated an apparent affinity constant of 10(8) l X mol-1. The antibody reacted with a 19-amino-acid-long sequence spanning the procollagen N-proteinase cleavage site with stronger binding to structures contributed by the aminopropeptide. The antibody showed strong cross-reactions with similar antigens of bovine, human or chick origin but failed to react with the aminopropeptide of procollagen type III. Incubation of chick or sheep procollagen type I with stoichiometric amounts of antibody blocked the release from procollagen molecules of the aminopropeptide by procollagen N-proteinase. Thus, this antibody seems useful for studying various biological problems encountered in the conversion of procollagen.     

Production of a monoclonal antibody specific for a Flavobacterium species isolated from soil

Abstract Hybridomas secreting monoclonal antibodies (MABs) specific for a soil Flavobacterium species (P25) were isolated. The MAB (D10) was used to target P25 using an enzyme-linked immunosorbant assay (ELISA) and indirect immunofluorescence. Cross-reactivity of the MAB with other Gram-negative bacteria (including Flavobacterium spp.) and a number of Gram-positive bacteria was investigated but none were found. Cross-reactivity with other orange/yellow pigmented Gram-negative rods ( Pseudomonas/Flavobacterium type) isolated from the soil into which P25 has been introduced in field experiments was also assessed using a modified colony blotting procedure. None of the indigenous species tested were recognised by the monoclonal antibody, thereby allowing unambiguous identification of P25 in soil. The MAB D10 was shown to recognise P25 growth under low-nutrient or stored under starvation conditions, suggesting that the antigen is a constitutive component of the cell and that the microorganism should be detected in oligotrophic environments such as soil. The pattern of fluorescence of P25 gave a clear indication of the localisation of the antigen in the outer membrane/cell wall region, and this was confirmed by immunogold labelling. Preliminary studies on the limits of detection of P25 using immunofluorescence suggest that densities as low as 20 bacteria g⁻¹ soil can be enumerated.     

Construction of recombinant monoclonal antibodies from a chicken hybridoma line secreting specific antibody

The chicken is a useful animal for the development of the specificantibodies against the mammalian conserved proteins. We generated twotypes of recombinant chicken monoclonal antibodies (mAbs), using a phagedisplay technique from a chicken hybridoma HUC2-13 which secreted themAb to the N-terminal of the mammalian prion protein (PrP). Althoughthe mAb HUC2-13 is a useful antibody for the prion research, thehybridoma produces a low level of antibody production. In order to producea large amount of the mAb, we have constructed a single chain fragmentvariable region (scF_V) mAb by using the variable heavy(V_H) and light (V_L)genes which were amplified by using the two primer pairs and theflexible linker. The two phage display mAbs (HUC2p3 and HUC2p5)expressed on a M13 filamentous phage and their soluble type mAbs(HUC2s3 and HUC2s5) were reacted with the PrP peptide antigen in theELISA. In the Western blot analysis, the mAbs HUC2p3 and HUC2s3 wereas reactive to PrP^c from mouse brains as the mAb HUC2-13 was. The nucleotide sequences of V_H and V_L genes from HUC2-13 and the two cloneswere identical except for only one residue. These results indicate that themethods presented here provide an effective tool for the improvement ofthe low levels of antibody production in the chicken hybridoma system.     

Thermodynamics of oligosaccharide binding to a monoclonal antibody specific for a Salmonella O-antigen point to hydrophobic interactions in the binding site.

The thermodynamic characteristics of oligosaccharide binding to an antibody binding site that is dominated by aromatic amino acids suggest that the hydrophobic effect contributes substantially to complex formation as well as hydrogen bonding and van der Waals interactions. A detailed titration microcalorimetric study on the temperature dependence of the binding of a trisaccharide, representing the epitope of a Salmonella O-antigen, showed that its maximum binding to the monoclonal antibody Se155-4 occurs just below room temperature and both enthalpy and entropy changes are strongly dependent on temperature in a mutually compensating manner. The heat capacity change also shows an unusually strong temperature dependence being large and negative above room temperature and positive below. van't Hoff analysis of the temperature dependence of the binding constant yielded a biphasic curve with two apparent intrinsic enthalpy estimations (approximately -100 kJ mol-1 above 18 degrees C and approximately +100 kJ mol-1 below), each very different from the calorimetrically determined enthalpies (ranging from about -60 kJ mol-1 to -20 kJ mol-1). This was interpreted as being due to large enthalpy contributions from concomitant reactions, most notably changes in solvation. Linear plots, -delta H0 versus -T delta S0, observed for temperature-dependent measurements mirror the behavior seen for a series of functional group replacements, suggesting that the molecular and physical origin of these phenomena are closely related and linked to the role of water in complex formation. The thermodynamic results are compared to the mode of binding determined from a 2.05-A resolution structure of the Fab-oligosaccharide complex, and with literature data for the heat capacities of sugars in aqueous solution and for the thermodynamics of carbohydrate binding to transport proteins and lectins.     

Site-directed mutants of charged residues in the active site of tyrosine hydroxylase

The active site of tyrosine hydroxylase consists of a hydrophobic cleft with an iron atom near the bottom. Within the cleft are several charged residues which are conserved across the family of pterin-dependent hydroxylases. We have studied four of these residues, glutamates 326 and 332, aspartate 328, and arginine 316 in tyrosine hydroxylase, by site-directed substitution with alternate amino acid residues. Replacement of arginine 316 with lysine results in a protein with a Ktyr value that is at least 400-fold greater and a V/Ktyr value that is 4000-fold lower than those found in the wild-type enzyme; substitution with alanine, serine, or glutamine yields insoluble enzyme. Arginine 316 is therefore critical for the binding of tyrosine. Replacement of glutamate 326 with alanine has no effect on the KM value for tyrosine and results in a 2-fold increase in the KM value for tetrahydropterin. The Vmax for DOPA production is reduced 9-fold, and the Vmax for dihydropterin formation is reduced 4-fold. These data suggest that glutamate 326 is not directly involved in catalysis. Replacement of aspartate 328 with serine results in a 26-fold higher KM value for tyrosine, a 8-fold lower Vmax for dihydropterin formation, and a 13-fold lower Vmax for DOPA formation. These data suggest that aspartate 328 has a role in tyrosine binding. Replacement of glutamate 332 with alanine results in a 10-fold higher KM value for 6-methyltetrahydropterin with no change in the KM value for tyrosine, a 125-fold lower Vmax for DOPA formation, and an only 3.3-fold lower Vmax for tetrahydropterin oxidation. These data suggest that glutamate 332 is required for productive tetrahydropterin binding.     

Production of a monoclonal antibody specific for Salmonella spp.

A monoclonal antibody (MAb) specific for heat-treated Salmonella spp. which shows no cross-reactivity with other enteric bacteria when tested by an ELISA system, has been developed. The MAb isotype is IgG2a and has been successfully purified using protein A. Preliminary work suggests that the MAb recognizes a protein present in the Boivin lipopolysaccharide extracts of Salmonella spp.     

Interaction of smoking and atopy in producing specific IgE antibody against a hapten protein conjugate.

A survey was carried out of a population of workers exposed to tetrachlorophthalic anhydride, an acid anhydride epoxy resin curing agent known to cause asthma. Using a radioallergosorbent test with a tetrachlorophthalic anhydride human serum albumin conjugate, specific IgE antibody was detected in serum from 24 out of 300 factory floor workers exposed to tetrachlorophthalic anhydride. Of these 24, 20 (83.3%) were current smokers compared with 133 (48.2%) of 276 without antibody (p less than 0.01), and there was a weaker association with atopy, defined by skin tests with common allergens. Smoking and atopy interacted, the prevalence of antibody being 16.1% in atopic smokers, 11.7% in non-atopic smokers, 8.3% in atopic non-smokers, and nil in non-atopic non-smokers (p less than 0.025). Smoking may predispose to, and interact with atopy in, the production of specific IgE antibody to this hapten protein conjugate.     

Characterization of a mouse monoclonal antibody specific for O-linked N-acetylglucosamine

beta-O-linked N-acetylglucosamine (O-GlcNAc) is an abundant posttranslational modification of resident nuclear and cytoplasmic proteins in eukaryotes. Increasing evidence suggests that O-GlcNAc plays a regulatory role in numerous cellular processes. Here we report on the production and characterization of a highly specific mouse monoclonal antibody, MAb CTD110.6, that specifically reacts with O-GlcNAc. The antibody recognizes O-GlcNAc in beta-O-glycosidic linkage to both serine and threonine. We could detect no cross-reactivity with alpha-linked Ser/Thr-O-GlcNAc, alpha-linked Ser-O-linked N-acetylgalactosamine (O-GalNAc), or N-linked oligosaccharides on ovalbumin and immunoglobulin G. The monosaccharide GlcNAc, but not GalNAc, abolishes immunoreactivity, further demonstrating specificity toward O-GlcNAc. Furthermore, galactose capping of O-GlcNAc sites also inhibits CTD110.6 immunoreactivity. Enrichment of GlcNAc-containing glycoproteins using the lectin wheat germ agglutinin dramatically enriches for CTD110.6-reactive proteins. The antibody reacts with a large number of proteins from cytoplasmic and nuclear extracts and readily detects in vivo changes in O-GlcNAc modification. These studies demonstrate that CTD110.6 is highly specific toward O-GlcNAc, with no cross-reactivity toward similar carbohydrate antigens or toward peptide determinants.     

Proteolytic dissection of a hapten binding site

IgG Gar, a human myeloma protein that binds riboflavin with a high affinity, was used to derive variable region fragments from the heavy chain and the light chain. Riboflavin binding ability of the active site generated by V(H) and light chain and the active site generated by V(H) and V(L) was compared to riboflavin binding by the F(ab) fragment. The riboflavin binding ability of the F(ab) fragment is the same as the intact molecule, while the binding ability of the active site formed by V(H) and light chain is lowered by two to three orders of magnitude, indicating that the removal of C(H1) domain decreases the interaction between riboflavin and the amino acids that is important in tight binding of riboflavin. Removal of the third hypervariable region and the constant region domain from the light chain further lowers the binding constant by one order of magnitude. The results indicate that the V(H) and V(L) segments of IgG Gar can reconstitute a riboflavin binding site. The decrease in affinity probably reflects a decrease in the rigidity with which the hypervariable loops are held together to place the contact amino acid residues in optimal contact with the hapten.     

Recognition of specific Physarum alpha-tubulin isotypes by a monoclonal antibody. Sequence heterogeneity around the acetylation site at lysine 40

The monoclonal antibody 6-11B-1 recognises specifically the acetylated form of alpha-tubulin. The acetylation event occurs on a unique lysine residue, lysine 40. Using 6-11B-1, acetylated alpha-tubulin was detected in myxamoebae but not plasmodia of Physarum polycephalum. Following chemical acetylation plasmodial alpha-tubulin was detected by 6-11B-1. The monoclonal antibody KMP-1 recognises certain Physarum alpha-tubulin isotypes but only in non-acetylated form. Whilst recognising all the non-acetylated fraction of myxamoebal alpha-tubulin only a proportion of plasmodial alpha-tubulin was recognised by KMP-1. Peptides were synthesised corresponding to the acetylation domains (containing lysine 40) of myxamoebal alpha-tubulin and the inferred acetylation domains of two plasmodial-specific alpha-tubulin isotypes. The only difference between the two peptides was at a single residue corresponding to amino acid 44 in the polypeptide. Tyrosine was present in myxamoebal alpha-tubulin and glycine was present in the plasmodial specific peptides; the peptides are referred to as the Tyr44 and Gly44 peptides respectively. Both peptides in acetylated form blocked 6-11B-1 reactivity towards acetylated myxamoebal alpha-tubulin. The Tyr44 but not the Gly44 peptide blocked KMP-1 reactivity towards non-acetylated myxamoebal alpha-tubulin. Tyrosine at position 44 is not found in any other known alpha-tubulin. Thus a unique antigenic determinant exists in certain Physarum alpha-tubulin isotypes, close to the acetylation site at lysine 40. This antigenic determinant forms part of the KMP-1 recognition epitope and explains the unique isotype selectivity of this monoclonal antibody.