Prediction and conformation by synthesis of two antigenic sites in human haemoglobin by extrapolation from the known antigenic structure of sperm-whale myoglobin.

The complete antigenic structure of sperm-whale myoglobin was previously determined in our laboratory. By structural analogy with myoglobin, two regions in human haemoglobin were predicted to comprise antigenic sites. One region was on the alpha-chain [alpha-(15-23)] and the other on the beta-chain [beta-(16-23)]. These two regions were synthesized, purified and characterized, and their immunochemistry was studied. Each peptide was able specifically to bind considerable amounts of haemoglobin antibodies. In a set of homologous proteins, barring any drastic conformational or electrostatic inductive effects exerted by the substitutions, and allowing for obstruction due to subunit interaction, the determination of the antigenic structure of one protein may serve as a useful starting model for the others.     

The antibody response to myoglobin is independent of the immunized species. Analysis in terms of replacements in the antigenic sites and in environmental residues of the cross-reactions of fifteen myoglobins with sperm-whale myoglobin antisera raised in different species.

The recent determination of the entire antigenic structure of sperm-whale myoglobin with rabbit and goat antisera has permitted the examination of whether the antigenic structure recognized by antibodies depends on the species in which the antisera are raised. Also, by knowledge of the antigenic structure, the molecular factors that determine and influence antigenicity can be better understood in terms of the effects of amino acid substitutions occurring in the antigenic sites and in the environmental residues of the sites. In the present work, the myoglobins from finback whale, killer whale, horse, chimpanzee, sheep, goat, bovine, echidna, viscacha, rabbit, dog, cape fox, mouse and chicken were examined for their ability to cross-react with antisera to sperm-whale myoglobin. By immunoadsorbent titration studies with radioiodinated antibodies, each of these myoglobins was able to bind antibodies to sperm-whale myoglobin raised in goat, rabbit, chicken, cat, pig and outbred mouse. It was found that the extent of cross-reaction of a given myoglobin was not dependent on the species in which the antisera were raised. This indicated that the antibody response to sperm-whale myoglobin (i.e. its antigenic structure) is independent of the species in which the antisera are raised and is not directed to regions of sequence differences between the injected myoglobin and the myoglobin of the immunized host. Indeed, in each antiserum from a given species examined, that antiserum reacted with the myoglobin of that species. The extent of this auto-reactivity for a given myoglobin was comparable with the general extent of cross-reactivity shown by that myoglobin with antisera raised in other species. The cross-reactivities and auto-reactivities (both of which are of similar extents for a given myoglobin) can be reasonably rationalized in terms of the effects of amino acid substitutions within the antigenic sites and within the residues close to these sites. These findings confirm that the antigenicity of the sites is inherent in their three-dimensional locations.     

Antibodies to synthetic antibody-combining sites. Antibodies against a surface-simulation peptide with antibody-combining activity toward lysozyme antigenic site 3 react with lysozyme antibodies

Recently, we reported the synthesis and immunochemistry of two peptides designed, by complementarity and surface-simulation synthesis, to mimic antibody-combining sites against two antigenic sites of lysozyme. In the present work antibodies were raised against one of these peptides, which is complementary to antigenic site 3 of lysozyme, to determine whether these antibodies will react with anti-lysozyme antibodies. Radioiodinated antipeptide antibodies were bound by immunoadsorbents of the immune IgG from two goats anti-lysozyme antisera but not by adsorbents of myoglobin, non-immune goat IgG or immune IgG of antisera against cytochrome c. The binding of anti-peptide antibodies to adsorbents of anti-lysozyme antibodies was fully inhibited by free lysozyme but not by bovine serum albumin, human hemoglobin A, horse cytochrome c or bovine ribonuclease A. Thus, antisera against an antibody-combining site can be raised by immunizing with a peptide which probably does not exist in the antibody but is designed by surface-simulation synthesis to mimic an antibody-combining site.     

Thyroid function and thyrotropin levels in rabbits immunized to produce antibodies against thyroid hormones

Various parameters of thyroid function were studied in 27 rabbits, out of which 10 were immunized to produce antibodies against triiodothyronine (T₃), 9 against thyroxine (T₄) and 8 were normals. Estimations of T₃, T₄, Free T₄ (FT₄) and thyrotropin (TSH) in blood, qualitative and quantitative analysis of iodoamino acids in serum, protein bound iodine-131 (PB¹³¹I), butanol extractable iodine-125 (BE¹²⁵I) and measurement of the disappearance rates of ¹²⁵I-labelled T₃ and T₄ from plasma were done. In addition, glandular changes were also studied by measurement of ¹³¹I uptake, thyroid scanning and chromatographic analysis of hydrolysate of soluble iodoproteins. In T₃ immunized animals, levels of T₃ in serum increased by 38 to 125 times, levels of TSH also showed a significant rise (7.4 ± 1.2 vs 28 ± 9 ng/mL). Chromatographic analysis of iodoamino acids in serum as well as in the hydrolysate of the thyroid gland demonstrated a selective increase in synthesis of T₃. Rate of disappearance of T₃ from blood showed a significant decline. Thyroid glands in the immunized rabbits showed signs of hypertrophy and hyperplasia. Identical studies done in rabbits immunized to produce antibodies against T₄ showed a similar pattern though of variable degree. Our studies indicate that the thyroid glands of the immunized rabbits undergo marked alterations resulting in selective increase in the synthesis and secretion of the particular thyroid hormone against which they were immunized. They do so under the influence of increased levels of TSH.     

Binding with lysozyme of antibodies against surface-simulation peptides representing the lysozyme antigenic sites.

Previously it had been shown that native lysozyme has three discontinuous antigenic sites (comprising spatially adjacent residues that may be distant in sequence) that were mimicked by surface-simulation synthetic peptides that had the capacity to bind the bulk (97-99%) of the antibody response against native lysozyme. In the present work these three surface-simulation synthetic peptides were coupled to succinoylated bovine serum albumin, and the conjugates were injected into rabbits. Antibodies against each peptide reacted, as expected, only with that peptide, but it was also found that the antibodies could bind with lysozyme, and the complete specificity of this binding was rigorously established. The advantages of these findings in conformational and immunological investigations are outlined.     

Exposure of antigenic sites during immunization. Monoclonal antibodies to monomer of lactose repressor protein

Monoclonal antibodies specific for the lactose repressor protein have been purified from three mouse hybridoma cell lines, and ascitic fluids from five other cell lines producing repressor antibodies have been assayed for immunoglobulin subclass and antigenic specificity. The chymotryptic core region (amino acids 57-360) of the repressor reacted with all antibodies examined, while no reaction with the NH2-terminal domain (1-56) could be detected. All of the purified antibodies and ascitic fluids reacted with the carboxyl-terminal fragment (amino acids 281-360) produced by cyanylation and base-catalyzed cleavage at the cysteine residues. Although none of the purified antibodies associated with native, tetrameric lac repressor, reaction was observed with repressor which had been denatured or dissociated into monomers by treatment with low levels of sodium dodecyl sulfate. Additionally, a mutant repressor which exists as a monomer in solution reacted with the antibodies in the absence of any denaturing treatments. These data indicate the carboxyl-terminal region is inaccessible in the intact repressor tetramer and further suggest that denaturation/dissociation of a protein during the initial immunologic challenge may result in the production of monoclonal antibodies to antigenic areas of the protein which are not exposed in the native conformation.     

Monoclonal antibodies against the fusion protein are protective in necrotizing mumps meningoencephalitis.

A monoclonal antibody against the fusion (F) protein of mumps virus was found to confer marked protection in mumps virus-induced encephalitis. Almost total prevention of extensive brain necrosis was found. This study indicates that the virus F protein is directly involved in the pathogenesis of brain necrosis.     

Localization of antigenic sites for monoclonal antibodies against alpha-subunit of Go-protein from the bovine brain]

The properties of monoclonal antibodies (MA) specifically raised against the alpha-subunit of the GTP-binding protein from bovine brain, G0, were studied. The hybridoma clones were found to secrete MA capable to interact with different antigenic sites of G0 alpha. Clone 1D2 MA interacted with the N-terminal domain of G0 alpha. The antigenic sites for clones 3DE. 1H6 and 2E3 MA were localized in the C-terminal domain of the protein molecule. Using clone 1H6 MA, the site of G0 alpha involved in the interaction with the beta gamma complex located in the C-terminal domain of the alpha-subunit, was revealed. It was found that the interaction of the alpha-subunit with the beta gamma complex changed the conformation of the C-terminal fragment of G0 alpha (Mr5000) together with an increase in the alpha-subunit affinity for clone 2E3 MA. It was concluded that the observed conformational changes may be the reason for the increased affinity of the alpha-subunit for the receptor.     

The topology of the brown adipose tissue mitochondrial uncoupling protein determined with antibodies against its antigenic sites revealed by a library of fusion proteins.

The uncoupling protein (UCP) of brown adipose tissue mitochondria is a specialized member of the family of evolutionarily related mitochondrial membrane transporters, which also includes the ADP/ATP translocator and the phosphate carrier. We have generated a library of bacterial clones randomly expressing short subsequences of the UCP fused to the MalE periplasmic protein of Escherichia coli. Anti-UCP sera were used to select clones expressing antigenic sequences of the UCP. Ten different fusion proteins representing eight non-overlapping subsequences of the UCP were obtained. The ability of fusion proteins to select antibodies directed against a short segment of the UCP was used to study the topological organization of the UCP in the inner mitochondrial membrane. Four different fusion proteins were used to determine the orientation of the N-terminal extremities of the first, second, third and fourth predicted alpha-helices of the UCP. This topological study together with previous data on the UCP provides an experimental basis for the predicted structure of the UCP and for other homologous carrier proteins.     

Evidence that the protein components of bovine erythrocyte green heme binding protein and flavin reductase are identical

Bovine erythrocyte green heme binding protein and bovine erythrocyte flavin reductase have been isolated in highly purified forms and subjected to amino acid analysis and N-terminal amino acid sequence analysis. The two proteins possess similar amino acid compositions and identical N-terminal amino acid sequences. Moreover, the two proteins are immunochemically cross-reactive and are indistinguishable when compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by double diffusion technique. This study provides evidence that the protein components of bovine erythrocyte green heme binding protein and flavin reductase are identical.     

Identification of the sites in myelin basic protein that are phosphorylated by meiosis-activated protein kinase p44mpk.

Myelin basic protein serves as a convenient substrate for detection of a 44 kDa protein-serine/threonine kinase (p44mpk) that is activated near the time of germinal vesicle breakdown in maturing echinoderm and amphibian oocytes. In vitro phosphorylation by purified p44mpk from sea star oocytes was primarily on threonine residues on a single tryptic peptide of bovine brain myelin basic protein. Amino acid composition analysis of the isolated posphopeptide revealed that it was rich in proline residues. Automated solid-phase sequencing by Edman degradation identified the major site as Thr-97 in the sequence NIVTPRTPPPSQGK, which corresponds to residues 91-104 in bovine brain myelin basic protein. Thr-94 was also phosphorylated by p44mpk to a very minor extent.     

Inhibition of lobster-muscle arginine kinase by homologous antibodies and study of an antibody population directed against a tyrosine-containing antigenic structure.

The effect of highly inhibitory rabbit antibodies on the enzymic activity of lobster-muscle arginine kinase has been studied. 100% inhibition was obtained at a 30-fold molar excess of antibody. The guanidine substrate L-arginine did not protect the enzyme from subsequent inhibition by its antibodies. On the other hand, the metal-nucleotide substrate Mg-ATP, or the substrate analog Mg-AMP, protected about 50% the enzyme activity, the extent of hte protection being irrespective of hte presence or of the absence of L-arginine and of the value of the molar ration Ab/Ag/ An antibody population directed against the antigenic determinant containing the essential tyrosine residue of the enzyme has been isolated by the affinity chromatography. No inhibitory acitivity was found in that fraction. Most of the inhibitory activity was found in the remaining antibody populations...