Hajime Fujio

The His-probe method: Effects of histidine residues introduced into the complementarity-determining regions of antibodies on antigen-antibody interactions at different pH values

We examined the effects or histidine residues that were artificially introduced into complementarity‐determining regions of antibodies on antigen‐antibody Interactions at different pH values. Using a monoclonal antibody specific ror hen egg‐white lysozyme and three mutant antibodies that contained a histidine residue. we measured binding constants for antibodies and lysozyme at different pH values (PH 5–8). No gross conformational changes were evident over this range of pH vulues, as determined by analysis of the spectra of circular dichroism. Since the charge on a histidine residue is the most likely factor that can vary over this range of pH values, differences on pH‐dependent antigen‐binding patterns observed between the wild‐type and mutant antibodies should be due mainly to the effects of the charges on the histidine residues. The three mutant antibodies showed different and characteristic patterns of pH‐dependent binding to lysozyme, which depended on the location of the artificially introduced histidine residues.

Regulation of delayed-type hypersensitivity (DTH) response to hen egg-white lysozyme (HEL).

Delayed-type hypersensitivity (DTH) can be demonstrated in A/J mice immunized with hen egg-white lysozyme (HEL) in complete Freunds adjuvant (CFA) by challenging primed animals in the ear with aqueous HEL. Normal A/J mice receiving soluble HEL derivative peptide, P-Ib, sequence 29-54 and 109-123 linked by a single S-S bond, 7 days prior to immunization with HEL showed much lower DTH response specific to the protein. The inhibition of DTH reactivity is due to active suppression and involves the generation of suppressor T-cells (Ts). Thus, the suppression induced with a single i.v. injection of P-Ib solution was able to be transferred into syngeneic recipients by the spleen cells from mice pretreated with P-Ib. These suppressor cells are T-cells, since their ability to suppress DTH is completely abrogated by treatment wit anti-Thy 1.2 and complement. Amongst HEL derivative peptides tested in the present study, only P-Ib could induce the tolerance.

Inhibition of tyrosine autophosphorylation of the solubilized insulin receptor by an insulin-stimulating peptide derived from bovine serum albumin

A polypeptide from a tryptic digest of bovine serum albumin potentiates glucose oxidation stimulated by insulin in isolated rat adipocytes. We studied whether this effect is related to a modification of the insulin receptor kinase. In a solubilized rat adipocytes receptor system, the peptide caused dose-dependent inhibition of the stimulation by insulin of phosphorylation of the 95,000 dalton subunit of insulin receptor. The peptide also inhibited stimulation by vanadate of tyrosine autophosphorylation of the beta subunit of the receptor, though it enhanced vanadate-stimulated glucose oxidation. During the phosphorylation reaction, no phosphorylated forms of the peptide could be detected. The peptide had no effect on dephosphorylation of the phosphorylated beta subunit of the insulin receptor. These results strongly suggest that the inhibition of phosphorylation by the peptide is due not to either simple substrate competition or activation of phosphoprotein phosphatase, but to specific inhibition of tyrosine-specific protein kinase.

Suppression of the delayed-type hypersensitivity response to hen egg white lysozyme (HEL) by HEL peptides in a genetically high-responder mouse strain: Evidence for requirement of the loop structure for induction of suppressor T cells

The delayed-type hypersensitivity (DTH) response of C3H/HeN mice to hen egg white lysozyme (HEL) can be blocked by a single iv injection of a solution of HEL in buffered saline 7 days before sensitization of animals with HEL in complete Freunds adjuvant (CFA). The minimal structure of HEL required for the suppression was examined by determining the abilities of various HEL-derivative peptides to inhibit HEL-DTH. Treatment of normal mice with Ploop I X II, sequence 57-107 (Cys64-Cys80, Cys76-Cys94), or P17 (sequences 1-27 and 123-129 linked by Cys6-Cys127) 7 days before immunization with HEL resulted in marked suppression of the DTH response. This inhibition of DTH involved generation of suppressor T cells (Ts). The results suggested that two suppressor pathways are involved. These data, together with another recent finding (1) that an entirely different portion of HEL is a suppressor determinant (SD) in A/J mice, indicate that different epitopes act as SDs in different strains of mice. Of the loop region peptides tested, Plc (intact loop I joined to a linear peptide, residues 84-97) was found to be the minimum structure capable of suppressing the HEL-DTH response; loop I or II alone did not cause suppression. Activation of Ts cells by the loop peptide depended on its conformational structure; completely reduced and carboxymethylated Ploop I X II did not cause suppression.

Three Antigenic Regions in p17 of Human Immunodeficiency Virus Type 1 (HIV-1) Revealed by Mouse Monoclonal Antibodies and Human Antibodies in HIV-1 Carrier Sera

We investigated the murine antibody response to recombinant p17 (rp17) of human immunodeficiency virus type 1 (HIV‐1) and the human antibody response directed to p17 in HIV‐1 infection. Three large peptides covering residues 12‐29, 53‐87 and 87‐115 of p17 were synthesized. The cysteine residues 57 and 87 of peptide 53‐87 were reoxidized to form a disulfide bridge. Eighteen out of 19 murine monoclonal anti‐rp17 antibodies had relatively high affinities (KA = 1.9 × 105−1.4 × 108 M−1) with one of the 3 p17 peptides in the liquid phase. Each monoclonal antibody reacted only with one particular peptide and had no reactivity with the other 2 p17 peptides. All the monoclonal antibodies reacted with rp17 in the liquid phase with a reasonable degree of affinity (KA = 2.0 × 105−1.8 × 107 M−1). Four HIV‐1 carrier sera, which were positive in ELISA using rp17 as the antigen, reacted positively in an ELISA using 3 p17 peptides which were used to titrate murine monoclonal antibodies. Murine monoclonal antibodies having specificity for the 3 p17 peptides stained live HIV‐1‐infected cells by means of indirect membrane immunofluorescence, irrespective of their specificity. This suggests that the various portions of p17 (at least 3 regions of p17) were exposed on the surface of live infected cells, probably as short polypeptide chains.

Idiotypic Analysis of Antibodies to Hen Egg-White Lysozyme (HEL): III. Further Studies on the Idiotypic Cross-Reactivity among Anti-HEL Antibodies in Mice

An isolated antibody preparation directed to the native hen egg‐white lysozyme (HEL) from a single A/J mouse (#a‐11), termed IdHELa‐11, was inoculated into rabbits to produce anti‐idiotypic sera (anti‐Id). The antisera were extensively absorbed with normal A/J Ig to render them idiotype specific. Radioimmunoassay utilizing 125IdHELa‐11 and the anti‐Id sera (R103 and R104) was performed to examine the idiotypic cross‐reactivity of the humoral immune response to HEL in various mouse strains and other animal species. Idiotypes shared by IdHELa‐11 were detected in the sera of five mouse strains tested, but not in any of the examined sera of other animal species such as rats, goats, guinea pigs, or sheep, indicating the occurrence of species‐specific cross‐reactive idiotypes (CRI) of antibodies to HEL. Our experiments also suggested the presence of intrastrain CRI. The present experiments, along with our earlier results (15), suggest that idiotypic cross‐reactivity among murine antibodies to HEL appears to be weak. Thus when R103 and R104 were the anti‐Id sera used, the frequency of occurrence of CRI shared by IdHELa‐11 in 5 μg of anti‐HEL antibody in strain A mice was 74 ng and 111 ng; in other strains it was 25–44 ng and 60–98 ng, respectively.

Association of specific tyrosine phosphorylation with stages of B-cell differentiation in human lymphoid leukemias

In vitro protein phosphorylation in various types of human fresh lymphoid leukemic cells (C-ALL, B-CLL, HCL and PCL: B-cell lineage and T-ALL, ATL and T-CLL: T-cell lineage) were studied. In cases of B-CLL and HCL, tyrosine protein kinase (TPK) activity was at least 5-fold higher than that in other cases of B- and T-cell lineages. B-cell leukemic cells at various differentiation stages had different endogenous substrates in tyrosine phosphorylation as well as distinct TPK activity. The P-tyr-containing proteins of 68K, 59K and 56K were detected commonly in all the cases of B-cell lineage. The phosphorylated protein of 32K was present only in cases of PCL. On the other hand, in T-ALL and ATL, the major substrate in tyrosine phosphorylation was 58K. These results suggest that the characterization of in vitro tyrosine phosphorylation provides a new means not only to distinguish T- and B-lymphoid leukemia, but also to differentiate stages of lymphoid development.

A local antigenic determinant distribution in a continuous antigenic region at residues 38-54 of hen egg white lysozyme.

The precise location of the antigenic determinants in a continuous antigenic region at residues 38–54 of hen egg white lysozyme (lysozyme) was investigated using the inhibition test of binding of Nα-[14C]acetyl fragment 38–54 with goat (three individuals) and sheep (four individuals) anti-lysozyme antisera by various synthetic and proteolytic fragments of lysozyme. From these inhibition studies, we found that in this region there were three independent antigenic determinants, consisting of residues 38–45, 40–48, and 44–54, respectively. The existence and the specificity of the antibodies directed to these determinants were further examined with isolating the specific antibodies by affinity chromatography on columns to which the fragment 38–45, 44–48, and 46–54 were bound. The results indicated that these determinants partially overlapped one another in amino acid sequence, but the antibodies directed to them could recognize only each corresponding determinant. These antibodies were also shown to be reactive with native lysozyme as well as a reduced and S-carboxymethylated derivative of lysozyme, and to be found in goat and sheep anti-lysozyme antibodies. The amounts of these antibodies calculated from the binding capacities were in the range from 0 to 48 μg/ml of antisera. These values corresponded to a small fraction of the total precipitable anti-lysozyme antibodies and were as high as 0.8% of the total. The ratios of the amounts of these antibodies differ in individuals or in different species of animals. The binding affinities of Nα-[14C]acetyl fragment 38–54 with these antibodies were in the range from 1.3 × 107 to 2.6 × 108m−1. The double-reciprocal plots of the antigen binding with these antibodies drew almost a straight line compared with those of a mixture of several antibody populations, that is, whole antisera.

Recognition of the Self Idiotype by T Cells: Induction of a Rapid Increase in Cytoplasmic Free Calcium in T Cells Recognizing a Variable L Chain Determinant

To investigate the initial stages of recognition of the self idiotype (Id) by T cells, we examined the early increase in cytoplasmic free calcium ([Ca2+]i) occurring in murine CD4+ T cells specific for a model Id, Id315, following their interaction with the Id. The changes in [Ca2+]i were monitored with stopped‐flow fluorometry bv loading T cells with fura 2, a Ca2+‐binding fluorescent dye. An increase of [Ca2+]i in the Id‐specific T cell line was dependent on the presence of both antigen‐presenting cells (APC) and Id315. When T cells were mixed with APC pulsed with M315 for 90 min at 37 C, a significant increase in T cell [Ca2+]i was observed within one second. A pronounced elevation in [Ca2+]i was also observed in T cells after their interaction with APC which had been pulsed for 90 min with VL‐315 Id‐containing proteins (such as VL‐315, L315, FV‐315 or Fab′‐315 fragments). In contrast, pulsing APC for 5 min with the VL fragment produced little or no change in the [Ca2+]i. These results suggest that VL must be further processed by APC before it can be recognized by T cells. Indeed, a synthetic VL region peptide (positions 91–108, designated as P18) produced an elevation in T cell [Ca2+]i when mixed with APC without pulsing.

Monoclonal Antibodies to O4 Antigen of Vibrio parahaemolyticus

Four monoclonal antibodies were prepared against O4 antigen of Vibrio parahaemolyticus. All the antibodies were shown to be specific for O4 antigen by agglutination with heat‐killed O‐cells of the organism and precipitation with LPS preparations. The inhibition experiments of the precipitations with various sugars and oligosaccharides suggested that the combining sites of these hybridoma antibodies were directed to an antigenic determinant structure containing →3 and →6 linked d‐glucose, d‐galactose, and N‐acetyl‐d‐galactosamine.