Comparison of the amino acid sequences of the variable regions of light chains derived from two homogeneous rabbit anti-pneumococcal antibodies

The amino acid sequence of the N-terminal 139 residues of the L (light) chain derived from a homogeneous rabbit antibody to type III pneumococci was determined. This L chain, designated BS-5, exhibits a greater degree of homology with the basic sequence of human kappa chains of subgroup I (72%) than with subgroups II and III. L-chain BS-5 differs from another L chain (BS-1), also derived from an antibody to type III pneumococci (Jaton, 1974), by eight amino acid residues, even though the chains are identical within the N-terminal 30 residues. Six of these eight substitutions are located within the three hypervariable sections of the variable half: Asn/Ser in position 31, Glu/Ala in position 55, Asx/Thr, Thr/Gly, Thr/Gly and Val/Tyr in positions 92, 94, 96 and 97 respectively. The two anti-pneumococcal L chains BS-1 and BS-5 are much more similar to each other than to an anti-azobenzoate L chain (Appella et al., 1973), from which they differ by 30 and 29 residues respectively. Of these interchanges 13-15 are confined to the three hypervariable sections, and 11 occur within the N-terminal 27 positions. The three chains have an identical sequence from residue 98 to residue 139, except for a possible inversion of two residues in positions 130-131 of the anti-azobenzoate chain.     

Circular dichroism and fluorescence studies of homogeneous antibodies to type III pneumococcal polysaccharide.

The near-ultraviolet circular dichroism (CD) of three homogeneous anti-type III pneumococcal antibodies in the absence and the presence of the specific hexasaccharide ligand was studied. In addition recombinations and hybridizations of H and L chains derived from two of these antibodies were carried out and the CD spectra of bound and free reconstituted IgG molecules were measured. The results indicate that the CD spectra of the native antibodies in the 260-310-nm range are very similar in shape and sign and exhibit a positive band at 285 nm. The homologous reconstituted antibody molecules exhibited CD spectra very similar in shape and sign to those of the native antibody molecules although recombinant molecules are no longer stabilized by interchain disulfide bonds. Upon addition of the hexasaccharide ligand, a significant decrease in amplitude of the CD spectra (18-21%) occurred in all three native antibodies and their Fab fragments as well as in the homologous recombinant molecules. No CD spectral changes could be detected upon interaction of the hapten ligand with the heterologous recombinants. All homogeneous antibodies studied exhibited fluorescence quenching upon oligosaccharide binding and a blue shift of the emission maximum. This property allowed the determination of the binding constant of one selected antibody to be made. Taken together, CD and fluorescence spectroscopic data suggest that oligosaccharide ligands induced detectable conformational changes in the Fab fragment of the antibody.     

Completion of the analysis of the primary structure of the variable domain of a homogeneous rabbit antibody to type III pneumococcal polysaccharide

The amino acid sequence between residues 70 and 116 of the V (variable) region of the H (heavy) chain derived from rabbit antibody BS-5, specific for type III pneumococcal polysaccharide, was determined. The sequence of this section of the H chain which includes the hypervariable residues 94 to about 112 was unique, although minor variant sequences present in the H chain preparation would not have been detected by the techniques used in this work. Taken together with the known sequences of the N-terminal 69 residues of H chain BS-5 (Jaton & Braun, 1972) and of the V region of the light chain (Jaton, 1974b), the data establish the complete sequence of the V domain of a rabbit immunoglobulin G. The V region of H chain BS-5 is compared with the basic sequences of the three human V region subgroups known to date, with one mouse H chain, and with guinea-pig pooled H chains. Even though chains from guinea pig and mouse clearly belong to the subgroup III of variability (V(HIII)), rabbit H chain BS-5 (allotypic variant a(1)) appears more closely related to the subgroup V(HII) than to the subgroups V(HIII) or V(HI). The homology between V(L) and V(H) regions of antibody BS-5 (28%) is not greater than that observed between the V(H) region of antibody BS-5 and the V(L) regions of different rabbit antibodies.     

Light chain variable region sequence of rabbit antipneumococcal type III polysaccharide antibody 3368.

The amino acid sequence of the amino-terminal 111 residues (variable region) for the light chain of the homogeneous rabbit antipneumococcal type III polysaccharide antibody 3368 was determined. This sequence was obtained principally through automated Edmann degradations of the intact light chain and of peptides generated by tryptic digestion of the citraconylated light chain. With these methods only 2 mumol of purified light chain was required to determine the reported sequence. When compared with the light chains of four other antipneumococcal type III polysaccharide antibodies, the 3368 light chain exhibits a unique sequence in those segments of the variable region that contribute to formation of the antigen binding site (complementarity-determining regions) (10 or 11 residue differences in 12 positions). The 3368 light chain also demonstrates an insertion of three residues relative to the other four light chains in the complementarity-determining region at positions 89 to 98. These five light chains have greater than 80% sequence homology for the portion of the variable region which is not involved in antigen binding (framework).     

Lectin-induced modulation of the antibody response to type III pneumococcal polysaccharide

Several lectins were tested for their capacity to alter the antibody response to type III pneumococcal polysaccharide (SSS-III). The antibody response was enhanced by concanavalin A (Con A), phytohemagglutinin (PHA), as well as lectins from Phytolacca americana (Pa-2), Pisum sativum (PSA), and Lens culinaris (LCH), when these lectins were given 2 days after immunization with SSS-III; however, suppression was obtained when Con A and Pa-2 were given at the time of immunization. By contrast the lectins from Vicia villosa (VVL) and Bauhinia purpurea (BPA) did not alter the antibody response. Since the lectins PSA and LCH bind to the same monosaccharide as Con A, whereas the other lectins bind to different monosaccharides, these findings indicate that there is no relationship between nominal monosaccharide specificity and the capacity to modulate the antibody response. Substantial increases in the magnitude of the IgG₁ antibody response was noted after the administration of Con A whereas profound enhancement of IgG_2a antibody response was noted after PHA was given.     

Genetic control of the immune response of mice to type III pneumococcal polysaccharide

BALBc mice immunized with Type III pneumococcal polysaccharide (SIII) had higher numbers of IgM plaque-forming cells (PFC) in the spleen than similarly immunized C57BL/Ks mice. The F₁ hybrids of these two strains had intermediate numbers of SIII-specific PFC. Analysis of the responses of F₂ and backcross strains indicated that the observed responses were compatible with results expected for control of the immune response to SIII at a single autosomal locus.     

Secondary IgG responses to type III pneumococcal polysaccharide. I. Kinetics and antigen requirements.

Mice primed with a thymus- (T)3 dependent form of Type III pneumococcal polysaccharide (S3), i.e., S3 coupled to sheep or horse erythrocytes (S3-RBC), produce S3-specific IgG antibody after secondary challenge with either the T-dependent (S3-RBC) or T-independent (S3) form of the antigen. The potential to produce IgG antibody after challenge with S3-RBC appears earlier after priming than the potential to produce IgG after challenge with S3, suggesting that different "memory" cells may be involved in the two responses. The "memory" cells were shown to be S3-specific since S3 had to be present on the carrier in order for priming to occur and carrier specificity was not required for elicitation of the secondary response by S3-RBC.     

The effect of Bordetella pertussis on the antibody response in mice to type III pneumococcal polysaccharide.

The effect of an i.p. injection of Bordetella pertussis on the primary humoral immune response in mice to the thymus-independent antigen SIII has been studied. Suppression of the antibody response occurred when pertussis cells were injected at the same time as an optimal immunizing dose of SIII. In contrast, the antibody response to high doses of SIII was enhanced by B. pertussis. When SIII alone was injected, only 19S antibody was detected. However, when B. pertussis was administered with either optimal or high doses of SIII, 7S as well as 19S antibody against SIII was produced.     

Sensitivity of amplifier T cells involved in the antibody response to type III pneumococcal polysaccharide to anti-lymphocyte serum.

Amplifier T cells responsible for enhancement of the antibody response to type III pneumococcal polysaccharide have been shown to be resistant to the effects of antilymphocyte serum (ALS) given at the time of immunization, a treatment that eliminates suppressor T cell activity. The resistance of amplifier T cells to ALS can be attributed to the fact that their activity develops after that of suppressor T cells. ALS given 1 or 2 days after immunization does abrogate amplifier T cell activity, independent of the mode by which that activity is elicited. The data emphasize the importance of kinetic considerations in understanding the effects produced by immunologically active agents such as ALS.     

Suppression of IgM responses to type III pneumococcal polysaccharide by lipopolysaccharide (LPS).

Lipopolysaccharide (LPS) suppressed the primary IgM response of mice to Type III pneumococcal polysaccharide (S3). LPS and S3 had to be given together in order for maximum suppression to occur and suppression was not due to a change in the time of the peak response in mice which received LPS. Suppression was not due to an effect of LPS on T cells since S3 responses of nude mice were suppressed by LPS. LPS did not suppress S3 responses of C₃H/HeJ mice and base hyrolysis of LPS destroyed the suppressive effect, i.e., suppression was dependent of B cell mitogenic activity of the LPS. The suppressive effect of LPS was presumably directed against virgin B cells since LPS did not suppress the S3 response of primed mice and did not suppress the development of IgG memory responses to S3.     

New monoclonal antibodies to the Ebola virus glycoprotein: Identification and analysis of the amino acid sequence of the variable domains

We determined the nucleotide and amino acid sequences of variable domains of three new monoclonal antibodies to the glycoprotein of Ebola virus capsid. The framework and hypervariable regions of immunoglobulin heavy and light chains were identified. The primary structures were confirmed using massspectrometry analysis. Immunoglobulin database search showed the uniqueness of the sequences obtained.     

Amino terminal amino acid sequences and carbohydrate of the two major forms of rabbit plasminogen

Two major forms of plasminogen exist in the plasma of many animal species and are distinguished by their affinities for certain antifibrinolytic amino acids. Quantitative end group analysis demonstrated that each isolated form of rabbit plasminogen possessed a single amino terminal residue of glutamic acid. Amino acid sequence analysis indicated that at least the first twelve amino terminal amino acids were identical in the two forms. The unique amino terminal sequence obtained for each form was NH₂-glu-pro-leu-asp-asp-tyr-val-asn-thr-gln-gly-ala-. Analysis of the carbohydrate content of each major plasminogen form revealed some striking differences. The first major form of rabbit plasminogen isolated from affinity chromatography columns contained 1.5–1.7 percent neutral carbohydrate and 3.0–3.3 moles of sialic acid per mole of protein. The second major form of rabbit plasminogen isolated from affinity chromatography columns contained 0.6–0.8 percent neutral carbohydrate and 1.8–2.2 moles of sialic acid per mole of protein.     

Suppression of the antibody response to type III pneumococcal polysaccharide with antigen coupled to syngeneic lymphoid cells

Nine allophenic mice of the type C57BL/10Sn … A were analyzed quantitatively, at weekly intervals over a period of 6 weeks, for the relative parental contributions to their red blood cell and white blood cell populations. It was found that four of the mice showed a significant change (termed “chimeric drift”) in the parental composition of their peripheral white blood cells, as determined by cytotoxicity testing. Six of the mice analyzed showed chimeric drift in their red blood cell population, as determined by hemoglobin analysis on isoelectric focusing gels. The isoelectric points of the hemoglobins of six inbred strains of mice were determined as an outgrowth of this study. Chimeric drift was observed in the direction of either parental cell type, and was found to be independent of the coat color, age, or sex of the mice.     

Requirements for activation of contrasuppressor T cells by type III pneumococcal polysaccharide

Either S3-coupled spleen cells (S3-SC) or soluble S3 activates two populations of regulatory T cells, T suppressor cells (Ts) and contrasuppressor T cells (Tcs). The latter cells function to mask the activity of Ts in unfractionated T cell populations, so that Ts can be detected only after removal of Tcs. Activation of Tcs by S3 may be required for induction of an antibody response to S3. This is suggested by the findings that Tcs are activated only by immunogenic doses of S3, that Tcs are not detectable in the spleens of mice tolerant to S3, and that (CBA/N X BALB/c)F1 male (xid) mice, which are genetically unresponsive to S3, do not develop Tcs after immunization with S3. Moreover, the kinetics of activation of Tcs by S3 closely parallels the kinetics of the antibody response to S3. Tcs have no detectable activity in the absence of Ts, indicating that these cells do not function as amplifier or helper T cells.