Biomedical subjects
Publications and source records attributed to J C Jaton.
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The gel filtration behavior, in the presence of detergents, of membrane-bound IgM from normal mouse spleen B lymphocytes was compared to that of secretory IgM from mouse plasma cells. The proteins were labeled either by surface radioiodination or biosynthetically with radioactive amino acids. Cell lysates were fractionated on calibrated Sepharose 6B columns in the presence of the detergents Nonidet P-40 or deoxycholate. Eluted fractions were immunoprecipitated and the reduced or unreduced precipitates were analyzed by sodium dodecyl sulfate gel electrophoresis followed by radioautography. Surface (125)I-labeled 8S IgM exhibited a gel filtration pattern in Nonidet P-40 corresponding to much higher apparent molecular weight than that of secretory 8S IgM, a difference that almost disappeared when gel filtration was performed in the presence of deoxycholate, which forms much smaller micelles than does Nonidet P-40. Biosynthetically labeled lymphocytes contain two types of IgM molecules differing in their gel filtration behavior and fate: one identical to secretory 8S IgM of plasma cells and secreted in the medium during chase periods, and the other identical to surface (125)I-labeled IgM and remaining cell-associated. Because the surface-bound 8S IgM was not found to be associated with other labeled molecules, it is likely that the detergent-binding behavior of surface IgM is due to a hydrophobic segment carried by these Ig molecules. That lymphocytes synthesize two types of mu chains was also shown by the use of tunicamycin, an inhibitor of glycosylation. In its presence, two unglycosylated mu chains were observed: one identical in size to that made by tunicamycin-treated plasma cells, and the second slightly larger. Gel filtration in Nonidet P-40 of the cell lysates of tunicamycin-treated lymphocytes showed that the nonsecretory 8S IgM contains this second type of mu chains, whereas the IgM molecules of the secretory type contain plasma cell-like mu chains. It is suggested that membrane IgM mu chains contain a hydrophobic segment which is responsible for its association to the membrane.
Ribosomes from the reticulocyte lysate bind strongly and mainly to a region located in the 5' end of the Rous sarcoma virus RNA molecule between residues 9 and 53. This binding involves the participation of initiator tRNA and is sensitive to inhibitors of initiation of protein synthesis such as 7-methyl-GMP and aurintricarboxylic acid. The nucleotide sequence of this ribosome binding site has been determined: it conatains a GUG codon centered at position 26 that is not in phase with any termination codon within the 5' end nucleotide sequence of the RNA that we have analyzed (101 residues). However, the predicted N-terminal amino acid sequence starting from this GUG codon (or even from any AUG or GUG codon in the 5' end of the RNA) does not coincide with that of the in vitro-synthesized product of the 5' end proximal gag gene. Nevertheless, inhibition of ribosome binding to this site is accompanied by an inhibition of the in vitro translation of the gag gene.
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Temperature-jump experiments were performed with di-, tetra-, and hexasaccharides derived from type III pneumococcal polysaccharide using a homogeneous corresponding antibody IgG 45-394. A decrease in stability of the oligosaccharide-antibody complexes with decreasing chain length was observed and entirely reflected in the decrease of the association rate constants which were 1.7 X 10(4) M-1 s-1 for the di-, 3.7 X 10(5) M-1 s-1 for the tetra-, and 1.1 X 10(6) M-1 s-1 for the hexasaccharide at 23 degrees C. The dissociation rate constants for all oligomers were about 12 s-1. This marked chain-length dependence of the association rate constants as well as their low values are unexpected for a single binding step. A mechanism is proposed which consists of a fast formation of a labile oligosaccharide-antibody precomplex followed by a slow isomerization step which is induced by the oligosaccharide ligands but which is chain-length independent.
The binding of gadolinium [Gd(III)] to a homogeneous rabbit anti-(type-III pneumococcal polysaccharide) IgG (immunoglobulin G) and its Fab (N-terminal half of heavy and light chain) and Fc (C-terminal half of heavy-chain dimer) fragments was demonstrated by measurements of solvent-water proton relaxation rates in the appropriate Gd(III) solutions. At pH 5.5 the binding of Gd(III) to the Fc fragment is much tighter (KD approx. 5 micronM) than binding to the Fab fragment (KD approx. 250 micronM). The binding of Gd(III) to the whole IgG molecule (KD approx. 4 micronM) is very similar to that for the Fc fragment alone. This specificity of binding to the Fc region allows the use of Gd(III) as a probe of the Fc conformation. The environment of the Gd(III) in the Fc region of whole IgG is not affected by the presence of octasaccharide derived by hydrolysis of type-III pneumococcal polysaccharide, but the corresponding 28-unit saccharide does cause detectable changes. The addition of 16-unit saccharide to anti-(SIII polysaccharide) IgG in the presence of Gd(III) does not change the solvent water proton relaxation rate, although aggregation does occur. The effects of the 28-unit saccharide may be explained therefore by a change in the tumbling time of the IgG. From a study of the effect of various antigen/antibody ratios, it is concluded that the 28-unit-saccharide-induced changes in the Gd(III) environment in the Fc region are caused by the specific geometrical structure of the antigen-antibody complexes formed, and not simply by occupancy of the combining sites on the antibody.
The recently established mutant strain of rabbits, named BASILEA, lacks the k polypeptide L-chain which is replaced by a lambda-like type (bas). This is evident from the amino acid composition and peptide maps of the bas L-chain when compared to the L-chains of the rabbits suppressed for the expression of the b locus allotypes.
The amino acid sequence of the V (variable) region of the heavy (H) chain of rabbit antibody BS-1, raised against type III pneumococcal vaccine, is reported. Together with the sequence data of the V region of the light (L) chain previously determined [Jaton (1974a) Biochem. J. 141, 1-13], the present work completes the analysis of the V domain of the homogeneous antibody BS-1. The V domains (VL + VH regions) of this antibody are compared with those of two other anti-(type III) pneumococcal antibodies BS-5 and K-25 [Jaton (1975) Biochem. J. 147, 235-247]. Except for the second hypervariable section of the L chains, these antibodies have very different sequences in the hypervariable segments of the V domains. Within the third hypervariable region of the H chain, each antibody has a different length: BS-1 is three amino acids shorter than K-25 and two amino acids shorter than BS-5. When the sequences in that section are aligned for maximal homology, only two residues, glycine-97 and leucine-101, are common to the three antibodies. On the basis of the amino acid sequences of these three anti-pneumococcal antibodies, the results do not support the concept of a simple correlation between primary structure in the hypervariable sections (known to determine the shape of the combining site) and antigen-binding specificity.
The binding characteristics of an IgM Waldenström(FR) for the ligand phosphorylcholine has been studied by fluorescence spectroscopy. Upon phosphorylcholine addition, IgM FR exhibited 83% enhancement of the tryptophanyl fluorescence, which was associated with a red shift of the emission maximun (5nm). The same properties were observed with the 7S IgM subunits. The association constant KA for phosphorylcholine was 6X10(4) M-1 FOR IgM FR and the 7S subunit, as determined by fluorescence titration, a value in agreement with the obtained by equilibrium dialysis. No significant decrease in the KA value was found in the presence of 3 M urea; in 6 M urea, the increase in fluorescence intensity was 36% of the value obtained in the absence of denaturing agent. In contrast, only 4% of fluorescence enhancement was noted upon binding in 3 M GuHC1 and no enhancement could be seen when the concentration of GuHC1 was increased to 5 M, thus suggesting complete unfolding of the protein and subsequent loss of binding activity. The pH dependence study of the phosphorylcholine binding to IgM FR indicated no significant differences in the fluorescence enhancement between pH 5 and 8, whereas at more acidic or alkaline pH values, the enhancement became smaller. At pH 3.0 and 10.0, no enhancement was seen suggesting no binding of the ligand, a fact confirmed independently by equilibrium dialysis. When the spectroscopic properties of the IgM FR were compared with those of murine myeloma proteins that bind the same ligand large differences were recorded in the amplitude of the phosphorylcholine induced enhancement of the fluorescnece and in the shift of the emission maximum wavelength. This suggests that the human and murine proteins interact differently with the small ligand phosphorylcholine thus implying that the variable domains of these molecules are not identical
Protein synthesis elongation factor Tu from E. coli shares several physical, chemical, and functional properties with actin-like proteins. Limited tryptic degradation indicates that the two polypeptides have a similar molecular architecture. These observations suggest that they could have evolved from a common ancestor, although more information will be necessary to prove or disprove this hypothesis. A partial sequence, comprising 22 aminoacid residues from the aminoterminal end of the large tryptic fragment of elongation factor Tu is presented.
Immune complexes formed between a homogeneous rabbit antibody to type III pneumococcal polysaccharide and a series of oligosaccharides of varying size derived from it were prepared and tested for their ability to fix guinea pig hemolytic complement. Antibody and either tetra-, hexa-, or octasaccharide formed only monomeric antibody-hapten complexes and did not show any complement binding. A dodecasaccharide and a 16-sugar residues oligomer formed dimer and trimer immune complexes. These complexes were also unable to fix complement. However, as the size of the sugar oligomers was increased to about 21 sugar residues per oligosaccharide molecule or more, the resulting complexes exhibited substantial complement binding, concomitant with the formation of antigen-antibody aggregates higher than trimers. On the other hand, an independent study carried out with the same material suggested changes in the conformation of the Fc moiety in the antibody molecule upon addition of oligosaccharide ligands as small as a 16-residue unit. Since the resulting complexes hardly ehibited any complement binding, ligand-induced conformational changes in the Fc part of the antibody molecule appears to be an insufficient condition per se for triggering complement fixation.
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.
The circular polarization of luminescence (CPL) emitted by tryptophan residues was used as a sensitive probe for measuring ligand-induced structural changes in a homogeneous type III pneumococcal antibody. A series of oligosaccharide ligands of increasing size derived from type III polysaccharide by partial acid hydrolysis was assayed. Ligand-induced changes in the circular polarization of fluorescence of the antibody were observed for all antigens tested, including tetra-, hexa-, and octasaccharides and a 16-residue oligomer, the largest changes being recorded upon interaction with the intact soluble type III pneumococcal (SIII) polysaccharide. When Fab' or F(ab')2 fragments were used instead of the antibody IgG for binding of SIII polysaccharide, the extent of conformational changes was decreased. This suggests interactions between Fab and Fc portions in the IgG molecule and subsequent conformational changes in Fc part upon antigen binding. Reduction of interchain disulfide bonds abolished the additional spectral changes attributed to the Fc part but not the changes observed in the Fab part, thus suggesting that the presence of the interchain disulfide bond in the hinge region is required for maximal CPL changes to occur. Small monovalent ligands, i.e., the tetra-, hexa-, and octasaccharides, were capable of inducing CPL changes in the Fab part of the antibody molecule as well as CPL changes attributed to the Fc portion. A multivalent ligand containing about 16 sugar residues appears to be the minimal antigenic size required for triggering conformational changes attributed to the Fc part, similar to those seen in the interaction with the whole polysaccharide antigen.
Recombinations and hybridizations of H and L-chains derived from several homogeneous rabbit antibodies to type 3 pneumococcal polysaccharide (SIII) were carried out. All reconstitution experiments performed gave rise to genuine IgG molecules. Antigen-binding studies and affinity measurements for a hexasaccharide ligand derived from SIII were made. In addition, heterologous antiidiotypic serum raised against one rabbit anti-SIII antibody was used to measure the reconstitution of idiotypic determinants in hybrid immunoglobulin molecules. The results show that full recovery of the antigen-binding properties was obtained only when chains derived from the same antibody molecules were reassociated. Similarly, the complete regain of idiotypic determinants (studied in one antibody system) could only be demonstrated in the homologous recombinants. The pairing of an H-chain with several heterologous L-chains, which differed in 6-11 positions in the 3 hypervariable sections, led to the formation of hybrid IgG molecules which had an affinity at least 100-fold lower than that of the parent anti-body molecule and a number of hapten-binding sites which did not exceed 0.30.
The amino acid sequences of the V (variable) regions of the H (heavy) and L (light) chains derived from rabbit antibody K-25, specific for type III pneumococci, were determined; this is the second homogeneous rabbit antibody besides antibody BS-5 whose complete sequence of the V domain has been established (Jaton, 1974d). The V regions of L chains BS-5 and K-25 (both of allotype b4) differ from each other by 19 amino acid residues; 11 of these 19 substitutions are located within the three hypervariable sections of the V region. On the basis of seven amino acid differences within the N-terminal 28 positions, it is suggested that L chain K-25 belongs to a different subgroup of rabbit K chains and L chain BS-5. H chain K-25 (allotype a2) differs from another H chain of the same allotype by one amino acid substitution within the N-terminal 70 positions in addition to interchanges occurring in the first two hypervariable sections. H chain K-25 was compared with H chain BS-5 (allotype a1) and with the known V-region rabbit sequences. Allotype-related differences between a1, a2 and a3 chains appear to occur within the N-terminal 16 positions and possibly in scattered positions throughout the V-region. In the hypervariable positions, variability between the two antibodies is remarkably more pronounced within the third hypervariable section of both H and L chains than within the first two.