Molecular characterization of monovalent and multivalent hapten-protein conjugates for analysis of the antigen--antibody interaction

We prepared a hapten-protein conjugate using (4-hydroxy-3-nitrophenyl)acetyl (NP) hapten and hen egg lysozyme (HEL) or bovine serum albumin (BSA) and defined hapten modification sites on the former protein based on results of reverse-phase high-performance liquid chromatography (HPLC) and mass spectrometric analyses performed after enzymatic digestion. The most reactive residue for aminoacetylation in HEL was found to be Lys33, and the second was Lys96 or Lys97. The homogeneous NP-HEL conjugates were purified by HPLC and used for examining the effect of hapten valence on the antigen-antibody interaction. We also examined the molecular nature of NP conjugates of BSA. Analysis using mass spectroscopy showed that the mass distribution of NP-BSA conjugates was limited, although it became broader with an increase in NP valence. Surface plasmon resonance biosensor measurements were employed in measuring antigen-antibody interactions. The results showed that the apparent binding avidity depends on hapten valence, hapten density, size of carrier proteins, and intrinsic binding affinity of the antibody.     

Quantitative analysis of alachlor protein adducts by gas chromatography-mass spectrometry

This study examined the potential use of hemoglobin (Hb)- and serum-protein adducts of alachlor as potential biomarkers of alachlor exposure, a genotoxic and carcinogenic herbicide. The method developed was based on the observation that cleavage of S-cysteinyl alachlor-protein adducts by methanesulfonic acid gave the rearrangement product 3-(2',6'-diethylphenyl)-1, 3-thiazolidine-4-one (TZO). The structure of TZO was confirmed by mass spectroscopy, NMR spectroscopy, and independent synthesis. In the assay, treatment of alachlor-cysteinyl protein adducts by methanesulfonic acid was followed by extraction and analysis. TZO was detected and quantitated by electron-impact GC/MS in the single ion-monitoring mode. [ring-13C6]Alachlor-N-acetylcysteine was added as an internal standard prior to treatment and was converted to [ring-13C6]TZO, allowing response factors to be used to quantitate TZO concentrations. Incubations of alachlor (0-1000 microM) with human albumin and bovine serum albumin (BSA) resulted in linear adduct formation with both proteins. Maximal adduction levels of 613-1130 pmol alachlor-albumin adducts/mg protein were observed, with BSA binding close to twice that of human albumin. A linear concentration response of alachlor-Hb adducts was observed when whole blood from female CD rats was incubated with alachlor in vitro at concentrations up to 300 microM. Maximal binding was 1860 pmol alachlor-Hb adducts/mg globin. Male CD rats treated with alachlor at 150 mg/kg body wt/day ip for 0, 1, 2, and 3 days were sacrificed 4 days after final dosing. A maximal binding of 2250 pmol alachlor-Hb adducts/mg globin was observed. This assay provides a new approach for biomonitoring alachlor levels in experimental animals and has the potential for use in humans.     

Solid-state NMR studies of the structure and mechanisms of proteins

Magic-angle spinning solid-state NMR experiments are well suited to investigating the structures and mechanisms of important proteins that are inaccessible to X-ray crystallography and solution NMR spectroscopy, including membrane proteins and disease-related protein aggregates. Good progress has been made in the development of methods for the complete structure determination of small (<20 kDa) solid proteins using uniformly 13C, 15N-labeled samples. Studies of selectively labeled proteins focusing on labeled active sites have yielded insights into the mechanisms of enzymes and of membrane proteins involved in energy and signal transduction. Studies of selectively labeled synthetic peptides have yielded structural models for biomedically important systems, including amyloid fibrils and surface-associated peptides involved in biomineralization and cell adhesion. Novel NMR and biochemical methods are being developed to target solid-state NMR experiments within large proteins and whole cells. These approaches are being used to investigate mechanisms of transmembrane signaling by membrane receptors and to characterize binding interactions between antibiotics and bacterial cell walls. Thus, solid-state NMR is proving to be a valuable biophysical tool for probing structure and dynamics in a wide range of biomolecules.     

Structural and functional characterization of the mutant Escherichia coli glutaredoxin (C14----S) and its mixed disulfide with glutathione.

Glutaredoxin is essential for the glutathione (GSH)-dependent synthesis of deoxyribonucleotides by ribonucleotide reductase, and in addition, it displays a general GSH disulfide oxidoreductase activity. In Escherichia coli glutaredoxin, the active site contains a redox-active disulfide/dithiol of the sequence Cys11-Pro12-Tyr13-Cys14. In this paper, we have prepared and characterized the Cys14----Ser mutant of E. coli glutaredoxin and its mixed disulfide with glutathione. The Cys14----Ser mutant of glutaredoxin is shown to retain 38% of the GSH disulfide oxidoreductase activity of the wild-type protein with hydroxyethyl disulfide as substrate but to be completely inactive with ribonucleotide reductase, demonstrating that dithiol glutaredoxin is the hydrogen donor for ribonucleotide reductase. The covalent structure of the mixed disulfide of glutaredoxin(C14S) with GSH prepared with 15N-labeling of the protein was confirmed with nuclear magnetic resonance (NMR) spectroscopy, establishing a basis for NMR structural studies of the glutathione binding site on glutaredoxin.     

Further evidence for the specificity of anti-5-HT (serotonin)-like antisera in immunocytochemistry. Existence of cyclic secondary amino groups in the immunogen

The serotonin antigen (5-HT-BSA formaldehyde conjugate) used for obtaining anti-5-HT antibodies was studied to obtain additional data concerning the nature of its immunogen. Dialysis against 0.1 M acetic acid and then against distilled water proved to be the best way of removing 5-HT condensation products not bound to BSA. The hapten has the configuration of a tetrahydro-beta-carboline (THBC) ring structure that is coupled to protein most probably via the carbon(s) ortho to the phenolic hydroxyl group and the indole nitrogen. The cyclic secondary amine of the THBC remained unsubstituted and was not involved in the bridging to BSA. This functional group was effectively blocked by acetylation and was unreactive to glutaraldehyde. On the other hand, in 5-HT conjugates synthesized using glutaraldehyde as the coupling agent, no cyclization to THBC occurred, and the amino groups were blocked. The chemical reactivity of the secondary amino group of the hapten in the synthesized conjugates was compared to the immunoreactivity of 5-HT conjugates formed in tissues. Immunostaining of formaldehyde-fixed serotoninergic neurons of the raphe of rats was suppressed by acetylation and the use of glutaraldehyde as the primary fixative, but the staining was unaffected when glutaraldehyde was reacted with formaldehyde-fixed 5-HT neurons. It is concluded that the cyclic secondary amine of the THBC structure is not conjugated to protein and forms part of the 5-HT-antibody-binding site in immunogens formed in vitro and in tissues.     

The influence of epitope density on the immunological properties of hapten-protein conjugates. I. Characteristics of the immune response to hapten-coupled albumen with varying epitope density

The relationship between the epitope density of hapten-protein conjugates (DNP· BSA), and their immunogenicity in mice has been investigated. As others have found, lightly substituted protein (DNP₅BSA) elicited primary and secondary antibody responses which were mainly IgG. In contrast, DNP₅₀BSA induced a primary IgM response with relatively little IgG, and little or no immunological memory. The transition in immunogenic behaviour from “low” to “high” occurred with a hapten: protein molar ratio around 30. DNP₅₀BSA does not contain any serologically detectable native BSA determinants or neodeterminants resulting from dinitrophenylation. Although this antigen elicits a mainly IgM response as do thymus-independent antigens, antibody production to both DNP₅BSA and DNP₅₀BSA is highly thymus dependent. The possible reasons for the thymus dependence of immune responsiveness to highepitope-density hapten-protein conjugates are discussed.     

Antibodies against neuroactive amino acids and neuropeptides. I. A new two-step procedure for their conjugation to carrier proteins and the production of an anti-Met-enkephalin antibody reactive with glutaraldehyde-fixed tissues.

We developed a new two-step procedure to couple haptens to bovine serum albumin (BSA) via glutaraldehyde (GA). After activation of BSA with excess GA and removal of unreacted GA, the hapten was bound to the activated protein in a second step. This two-step procedure is easy to use, the desired molecular ratio of coupled hapten to protein is conveniently adjusted, and no visible precipitation of the conjugate is detected. Using a low peptide concentration, nearly 50% of the inserted haptens are bound to the protein, and unbound expensive peptide can be recovered after Sephadex chromatography. Antisera to neuroactive amino acids (GABA, glycine, and glutamate) and neuropeptides (Met-enkephalin) were prepared by immunization of rabbits with these conjugates. Immunological analysis of immune sera by dot-blot and ELISA techniques and subsequent removal of crossreactivities by solid-phase adsorption yielded monospecific antibodies, which were further purified by affinity chromatography. The immunocytochemical specificities of these purified antibodies were verified in adjacent sections of GA-fixed rat spinal cord. Pre-embedding staining with anti-Met-enkephalin in combination with post-embedding staining for amino acids such as GABA allowed double staining of the two antigens in a single semi-thin section.     

Further evidence for the specificity of anti-5-HT (serotonin)-like antisera in immunocytochemistry

Summary The serotonin antigen (5-HT-BSA formaldehyde conjugate) used for obtaining anti-5-HT antibodies was studied to obtain additional data concerning the nature of its immunogen. Dialysis against 0.1 M acetic acid and then against distilled water proved to be the best way of removing 5-HT condensation products not bound to BSA. The hapten has the configuration of a tetrahydro--carboline (THBC) ring structure that is coupled to protein most probably via the carbon(s) ortho to the phenolic hydroxyl group and the indole nitrogen. The cyclic secondary amine of the THBC remained unsubstituted and was not involved in the bridging to BSA. This functional group was effectively blocked by acetylation and was unreactive to glutaraldehyde. On the other hand, in 5-HT conjugates synthesized using glutaraldehyde as the coupling agent, no cyclization to THBC occurred, and the amino groups were blocked. The chemical reactivity of the secondary amino group of the hapten in the synthesized conjugates was compared to the immunoreactivity of 5-HT conjugates formed in tissues. Immunostaining of formaldehyde-fixed serotoninergic neurons of the raphe of rats was suppressed by acetylation and the use of glutaraldehyde as the primary fixative, but the staining was unaffected when glutaraldehyde was reacted with formaldehyde-fixed 5-HT neurons. It is concluded that the cyclic secondary amine of the THBC structure is not conjugated to protein and forms part of the 5-HT-antibody-binding site in immunogens formed in vitro and in tissues.     

Use of polystyrene-supported 2-isobutoxy-1-isobutoxycarbonyl-1,2-dihydroquinoline for the preparation of a hapten–protein conjugate for antibody development

Polystyrene-supported 2-isobutoxy-1-isobutoxycarbonyl-1,2-dihydroquinoline (PS-IIDQ), a polymer-supported covalent coupling reagent, was successfully employed for the first time in the bioconjugation of an example hapten (phytanic acid derivative) to a carrier protein (bovine serum albumin (BSA)) within the context of immunogen preparation for antibody development. The ability of the prepared example phytanic acid derivative–BSA conjugate to bind an anti-phytanic acid antibody was confirmed using an enzyme-linked immunosorbent assay (ELISA).     

Polyclonal Antibodies against a Structure Mimicking the Covalent Linkage Unit between Picornavirus RNA and VPg: An Immunochemical Study

We propose that therapy of patients with anticancer drugs that poison DNA topoisomerases induces formation of covalent complexes of cellular RNAs and DNA topoisomerases. The appearance of these complexes can be detected with antibodies against a synthetic hapten mimicking the covalent linkage unit Tyr-pU(p) of picornavirus RNA and VPg. We synthesized hapten [N(Ac),CO(NH2)]Tyr-(5 P --> O)Up-O-(CH2)6NH2, conjugated it with BSA, and immunized rabbits with the antigen obtained. The raised polyclonal antibodies were purified by successive affinity chromatography on BSA-Sepharose and hapten-Sepharose columns. Target antibodies recognized hapten and encephalomyocarditis virus RNA-VPg complex specifically as found using the dot-immunogold method. We believe that these antibodies might be useful to study mechanism of picorna and similar virus RNA synthesis. The discovery and qualitative determination of the cellular RNA-DNA topoisomerases covalent complexes with these antibodies might be useful to monitor therapy efficacy by drugs "freezing" dead-end complexes of DNA topoisomerases and nucleic acids and to understand the mechanism of DNA topoisomerase poisoning in situ.     

Structure of an elastin-mimetic polypeptide by solid-state NMR chemical shift analysis

The conformation of an elastin-mimetic recombinant protein, [(VPGVG)4(VPGKG)]39, is investigated using solid-state NMR spectroscopy. The protein is extensively labeled with 13C and 15N, and two-dimensional 13C-13C and 15N-13C correlation experiments were carried out to resolve and assign the isotropic chemical shifts of the various sites. The Pro 15N, 13Calpha, and 13Cbeta isotropic shifts, and the Gly-3 Calpha isotropic and anisotropic chemical shifts support the predominance of type-II beta-turn structure at the Pro-Gly pair but reject a type-I beta-turn. The Val-1 preceding Pro adopts mostly beta-sheet torsion angles, while the Val-4 chemical shifts are intermediate between those of helix and sheet. The protein exhibits a significant conformational distribution, shown by the broad line widths of the 15N and 13C spectra. The average chemical shifts of the solid protein are similar to the values in solution, suggesting that the low-hydration polypeptide maintains the same conformation as in solution. The ability to measure these conformational restraints by solid-state NMR opens the possibility of determining the detailed structure of this class of fibrous proteins through torsion angles and distances.     

Recent developments in solid-state magic-angle spinning, nuclear magnetic resonance of fully and significantly isotopically labelled peptides and proteins

In recent years, a large number of solid-state nuclear magnetic resonance (NMR) techniques have been developed and applied to the study of fully or significantly isotopically labelled ((13)C, (15)N or (13)C/(15)N) biomolecules. In the past few years, the first structures of (13)C/(15)N-labelled peptides, Gly-Ile and Met-Leu-Phe, and a protein, Src-homology 3 domain, were solved using magic-angle spinning NMR, without recourse to any structural information obtained from other methods. This progress has been made possible by the development of NMR experiments to assign solid-state spectra and experiments to extract distance and orientational information. Another key aspect to the success of solid-state NMR is the advances made in sample preparation. These improvements will be reviewed in this contribution. Future prospects for the application of solid-state NMR to interesting biological questions will also briefly be discussed.     

Maleimide-mediated protein conjugates of a nucleoside triphosphate gamma-S and an internucleotide phosphorothioate diester.

The purpose of this study was to determine whether the gamma-S of nucleoside thiotriphosphates and the non-bridging sulfur of internucleotide phosphorothioate diesters possess sufficient thiol character to form adducts with maleimides. Adenosine triphosphate gamma-S (ATPS) and thymidyl-PS-thymidine (TPST) were each reacted with the reporter molecule N-1 pyrene maleimide (PM) and the fluorescence intensity was recorded. The observed reactivity of the phosphorothioate nucleotides towards maleimide was used as a basis for preparing covalent protein-nucleotide conjugates of ATPS and of the internucleotide phosphorothioate diester, deoxyadenylyl-PS-deoxy-adenylyl-PS-deoxyadenosine (dA3(PS)2). The absorbance spectra of bovine serum albumin (BSA) conjugates of ATPS and of dA3(PS)2 showed the formation of protein-nucleotide conjugates, with absorbance maxima near 260 nm. The degree of conjugation was 1.69 nucleotides (nt)/BSA molecule for ATPS and 0.44 nt/BSA molecule for dA3(PS)2. The extent of conjugation of the gamma-S of the nucleoside thiotriphosphate and of the non-bridging sulfur of the internucleotide phosphorothioate diester with maleimide-derivatized protein agreed with their relative reactivity towards PM. Both the gamma-S of the nucleoside thiotriphosphate and the internucleotide phosphorothioate diester were found to possess sufficient thiol character to permit formation of maleimide-mediated protein conjugates.     

Reaction of hen egg white lysozyme with Fischer-type metallocarbene complexes. Characterization of the conjugates and determination of the metal complex binding sites.

The introduction of heavy atoms into protein crystals is sometimes rendered difficult and tedious because of the poor specificity of the available reagents for particular target residues. On the other hand, transition organometallic chemistry offers an almost untouched field for this purpose. In particular, Fischer-type metallocarbene complexes of the general formula (CO)5W=C(OR1)R2 may be attractive reagents because they contain the heavy element tungsten and specifically target amino groups to form stable, covalent aminocarbene adducts. With a small protein such as hen egg white lysozyme (HEWL) with a limited number of potential binding sites, it was possible to form protein-aminocarbene conjugates that have an average of one aminocarbene moiety per protein molecule. RP-HPLC combined with matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS analysis of the conjugates revealed that they were mixtures of the native protein, monoaminocarbenes and diaminocarbenes. Tryptic proteolysis experiments performed on the protein conjugates combined with MALDI-TOF-MS analysis of the aminocarbenic peptides allowed us to determine that lysines 13, 33, 97 and 116 were involved in the reaction of HEWL with (CO)5W=C(OMe)Me.     

Protein–nucleotide contacts in motor proteins detected by DNP-enhanced solid-state NMR

DNP (dynamic nuclear polarization)-enhanced solid-state NMR is employed to directly detect protein–DNA and protein–ATP interactions and identify the residue type establishing the intermolecular contacts. While conventional solid-state NMR can detect protein–DNA interactions in large oligomeric protein assemblies in favorable cases, it typically suffers from low signal-to-noise ratios. We show here, for the oligomeric DnaB helicase from Helicobacter pylori complexed with ADP and single-stranded DNA, that this limitation can be overcome by using DNP-enhanced spectroscopy. Interactions are established by DNP-enhanced ³¹P–¹³C polarization-transfer experiments followed by the recording of a 2D ¹³C–¹³C correlation experiment. The NMR spectra were obtained in less than 2 days and allowed the identification of residues of the motor protein involved in nucleotide binding.     

Influence of the site of conjugation on the specificity of antibodies to progesterone

In order to determine how the site on the molecule used for conjugation influences the specificty of the resulting antiserum, progesterone was conjugated to bovine serum albumin (BSA) through substituents on the A(C3), B(C6), C(C11), and D(C20) rings for use as a hapten to elicit antibody formation in rabbits. Specificty of the antisera was determined by testing the ability of 24 representative steroids to displace radioactive progesterone in a radioimmunoassay procedure. Progesterone-tyrosine methyl ester (TME) conjugates were radioiodinated and used as the radioactive form of the hormone and radioactivity bound to antibody was separated from free radioactivity by a double antibody procedure. Immunization with progesterone conjugated at C20 resulted in the formation of antibodies which could not distinguish between progesterone and other Δ⁴-3-ketosteroids with structures similar in the A, B, and C ring (namely 17-hydroxyprogesterone, 20α and 20β-hydroxy-4-pregnen-20-one, deoxycorticosterne and testosterone). Immunization with progesterone-3-BSA resulted in the formation of antisera which were fairly specific for progesterone while immunization with progesterone conjugated at the 11 or 6 positions resulted in antisera which were very specific for progesterone. It was concluded that steroid hormones should be conjugated to protein at sites on the B or C ring of the molecule for the production of specific antisera.     

Magnetic resonance studies of the binding site interactions between phosphorylcholine and specific mouse myeloma immunoglobulin

The interaction of phosphorycholine-binding mouse myeloma protein M603 and the isotopically substituted hapten phosphoryl[methyl-13C] choline has been investigated using 13C and 31P nuclear magnetic resonance (NMR) spectroscopy. Upon binding to antibody, upfield shifts of 0.7 and 1.5 ppm are observed for the hapten 13C and 31P resonances, respectively, and both spectra are in the "slow" exchange limit. Linewidth analysis indicates some immobilization of the phosphate group but essentially unrestricted methyl group rotation for the bound hapten. Hapten-antibody dissociation rate constants of 10 and 38 s-1 are calculated from 13C and 31P NMR spectra, respectively, suggesting the possibility of differential dissociation rates for the two opposing ends of the phosphorylcholine molecule. The NMR data are entirely consistent with the known x-ray structure of the M603 Fab'-phosporylcholine complex (Segal,D.M., Padlan, E.A., Cohen G.H., Rudikoff S., Potter,M., and Davies, D.R. (1974), Proc. Natl. Acad. Sci. U.S.A. 71, 4298).     

4D Non-uniformly sampled C,C-NOESY experiment for sequential assignment of 13C,15N-labeled RNAs

¹³C spin diluted protein samples can be produced using [1-¹³C] and [2-¹³C]-glucose (Glc) carbon sources in the bacterial growth medium. The ¹³C spin dilution results in favorable ¹³C spectral resolution and polarization transfer behavior. We recently reported the combined use of [1-¹³C]- and [2-¹³C]-Glc labeling to facilitate the structural analysis of insoluble and non-crystalline biological systems by solid-state NMR (ssNMR), including sequential assignment, detection of long-range contacts and structure determination of macromolecular assemblies. In solution NMR the beneficial properties of sparsely labeled samples using [2-¹³C]-glycerol (¹³C labeled Cα sites on a ¹²C diluted background) have recently been exploited to provide a bi-directional assignment method (Takeuchi et al. in J Biomol NMR 49(1):17–26, 2011 ). Inspired by this approach and our own recent results using [2-¹³C]-Glc as carbon sources for the simplification of ssNMR spectra, we present a strategy for a bi-directional sequential assignment of solid-state NMR resonances and additionally the detection of long-range contacts using the combination of ¹³C spin dilution and 3D NMR spectroscopy. We illustrate our results with the sequential assignment and the collection of distance restraints on an insoluble and non-crystalline supramolecular assembly, the Salmonella typhimurium type III secretion system needle.     

Uniform isotope labeling of a eukaryotic seven-transmembrane helical protein in yeast enables high-resolution solid-state NMR studies in the lipid environment

Overexpression of isotope-labeled multi-spanning eukaryotic membrane proteins for structural NMR studies is often challenging. On the one hand, difficulties with achieving proper folding, membrane insertion, and native-like post-translational modifications frequently disqualify bacterial expression systems. On the other hand, eukaryotic cell cultures can be prohibitively expensive. One of the viable alternatives, successfully used for producing proteins for solution NMR studies, is yeast expression systems, particularly Pichia pastoris. We report on successful implementation and optimization of isotope labeling protocols, previously used for soluble secreted proteins, to produce homogeneous samples of a eukaryotic seven-transmembrane helical protein, rhodopsin from Leptosphaeria maculans. Even in shake-flask cultures, yields exceeded 5 mg of purified uniformly ¹³C,¹⁵N-labeled protein per liter of culture. The protein was stable (at least several weeks at 5°C) and functionally active upon reconstitution into lipid membranes at high protein-to-lipid ratio required for solid-state NMR. The samples gave high-resolution ¹³C and ¹⁵N solid-state magic angle spinning NMR spectra, amenable to a detailed structural analysis. We believe that similar protocols can be adopted for challenging mammalian targets, which often resist characterization by other structural methods.