Antigens of alternaria. I. Isolation and partial characterization of a basic peptide allergen

A basic peptide allergen has been isolated from Alternaria extracts. Flat-bed gel preparative isoelectric focusing followed by dialysis and lyophilization allowed for concentration of substantial quantities of this heretofore unrecognized allergen. Rocket immunoelectrophoresis and autoradiography (using a 2 X concentrated, IgE serum fraction from Alternaria patient sera and 125I-anti human IgE), a RAST inhibition assay, and skin testing of Alternaria-sensitive patients were used to demonstrate the allergenicity of this fraction. The allergenicity of this protein was sufficient to give 50% RAST inhibition at a concentration of 100 micrograms/ml using standard RAST procedures. The immunoelectrophoresis autoradiography also indicated allergenicity. Skin tests indicated that over 90% of patients hypersensitive to Alternaria crude extracts were also hypersensitive to the basic allergen fraction.     

Biochemical comparison of the T6 antigen and HLA-A,B antigens

The human thymic differentiation antigen T6, which was found to be associated with beta 2-microglobulin, was compared to the HLA-A,B antigens. Using a heteroantiserum prepared against denatured heavy chains of HLA-A,B antigens, no cross-reactivity with denatured T6 could be detected. The molecular weight of the protein backbone of T6 was found to be 34,000 as compared to 40,000 for the HLA-A,B antigens. Also, not only was the percentage of carbohydrate of T6 (25-35%) different from the HLA-A,B antigens (10%), but lectin binding studies showed that their sugar composition may differ. The two forms of T6, which previously had been found on MOLT-4 cells, appeared to have different levels of glycosylation, but apparently had the same protein backbone. T6, like HLA, has a hydrophobic domain, since it could be labeled with [125I]iodonaphthylazide. We conclude from these studies that T6 may be a class I MHC antigen which is different from the classical HLA-A,B antigens.     

Biochemical characterization of the human T6 antigen: a comparison between T6 and murine TL

The human T6 antigen was studied by two monoclonal antibodies: OKT6 and Leu-6. A third monoclonal antibody, C56 (developed in our laboratory), was found to have similar properties to those of OKT6. On SDS-PAGE, all three antibodies precipitated a 48,000-12,000-dalton heterodimer. Two-dimensional gel electrophoresis and chymotryptic peptide map analysis revealed that these antibodies precipitated in identical 48,000-dalton heavy chain which was distinguishable from the HLA-A,B,C heavy chains. The single 12,000-dalton light chain precipitated with OKT6 antibody was shown to be distinct from beta 2-microglobulin by its pI. The two light chains precipitated with Leu-6 antibody were resolved by charge into beta 2-microglobulin and the more basic 12,000-dalton peptide identical to that precipitated with OKT6. In addition to beta 2-microglobulin, the latter component (presumably beta t) was also found in the light-chain fraction precipitated from the thymocytes with a monoclonal antibody recognizing the framework of HLA-A,B,C heavy chains. Using chymotryptic peptide mapping, no polymorphism was detected among the heavy chains of the T6 antigen isolated from thymocytes of four individuals. All three monoclonal antibodies failed to precipitate murine TL from ASL1 leukemia cell lysates. Similarly, none of the six monoclonal and two conventional anti-TL antibodies reacted with T6. Although a high degree of homology was found by peptide map analysis among the TL molecules encoded by the Tlaa, Tlad and Tlae alleles, a comparison between their peptide maps and that of T6 revealed no similarity. Despite previous suggestions that T6 is homologous to murine TL, the present biochemical studies do not support this hypothesis.     

Detection of unique peptides derived from the mu chains of cell surface but not secreted IgM

IgM is present on the surface of B lymphocytes in monomeric form and can also be secreted into the serum in pentameric form after differentiation of B cells into plasma cells.We have previously used a monoclonal population of murine tumor cells (BCL₁) which express surface IgM but can also secrete IgM after stimulation with lipopolysaccharide to study the primary structure of μ chains from cell surface and secreted IgM. Using cation exchange chromatography, we detected a peptide present only in μ chains from secreted IgM but not in cell surface IgM. The putative extra peptide(s) from the cell surface μ chain could not be identified perhaps due to its inability to bind to the exchange resin. In the present study we have used reverse phase high pressure liquid chromatography to analyse the peptides of μ chains from cell surface and secreted IgM from both BCL₁ tumor cells and from normal B lymphocytes. Our results demonstrate the presence of two peptides associated with the Fc portion of the cell surface but not with the secreted μ chain.     

Further chemical characterization of guinea pig Ia molecules derived from the three major classes of immunocompetent cells.

The guinea pig system is unique in that Ia molecules are readily demonstrable on the three major classes of immunocompetent cells. We were, therefore, able to characterize and compare the structures of Ia molecules from B and T lymphocytes and macrophages. T lymphocyte Ia molecules which bound to Lens culinaris (lentil) lectin co-electrophoresed on SDS-PAGE with lymph node cell Ia molecules (predominantly B lymphocyte Ia molecules) which also bound to lentil lectin. T lymphocyte and macrophage Ia molecules which did not bind to lentil lectin had a slower mobility on SDS-PAGE than lymph node cell Ia molecules which did bind to lentil lectin. One dimensional isoelectric focussing of Ia molecules derived from lymph nodes, T lymphocytes, or macrophages revealed discrete banding patterns for all Ia molecules examined. This result indicates that the Ia molecules which can be chemically isolated from B cells, T cells and macrophages do not have variable regions and probably do not possess antigen recognition capability analogous to immunoglobulin.     

Isolation and characterization of mouse C1q

C1q was isolated from mouse serum and ascites fluid by absorption onto human IgG-coated latex beads followed by seperation on 3–10% exponential gradient sodium dodecyl sulfate (SDS) polyacrylamide gels. Mouse C1q was also purified by low ionic strength precipitation of mouse serum. The purified C1q was heat-labile (56°C, 30 min) both structurally and functionally, contained 4.3% hydroxyproline, 1.38% hydroxylsine, and 18.5% glycine, had an apparent molecular weight of 380,000 daltons, and reconstituted the hemolytic complement activity of C1q-depleted mouse serum. The negatively stained ultrastructural appearance of this purified material consisted of 6 globular units connected by strands. These data demonstrate that mouse C1q structurally and functionally is similar to human and rabbit C1q. A portion of polyacrylamide gel containing mouse C1q was injected into rabbits resulting in the production of monospecific antisera against mouse C1q. Thus, this procedure is a new, rapid and simple method to obtain monospecific antisera against mouse C1q.     

A simple two-step procedure for the preparation of the first component of human complement (C1) in its native form

Native human C1 was purified from fresh human serum by affinity chromatography on protein A-bound Sepharose in the presence of 4-nitrophenyl-4-guanidinobenzoate hydrochloride (NPGB) taking advantage of the successive binding of IgG to protein A followed by C1 binding to IgG. After elution the C1 preparation contained IgG as a major contaminant as shown by SDS-PAGE. C1 was further purified by gel filtration. The yield of C1 was 12% and less than 4% of this C1 was activated during purification as assessed by a C4 consumption assay.     

Biochemical characterization of a p43,12 complex: comparison with human and murine class I molecules

A monoclonal antibody designated as C21 reacting with a p43,12 complex was developed against human thymocytes. It stained predominantly the early hematopoietic cells of the lymphoid lineage and also thymocytes, peripheral B-cells and activated T- and B-cells similarly to OKT10. The heavy chain of this antigen was a glycoprotein of Mr 43,000 (p43). Sequential immunoprecipitation with C21 and OKT10 antibodies indicated that they both reacted with an identical heavy-chain molecule. This observation was further documented by two-dimensional analysis. Monoclonal antibody C21 was used to probe a p43,12 complex further. Structural polymorphism of the p43 heavy chain isolated from T- and B-cells of different individuals was not detected by chymotryptic peptide mapping, although molecules from these cell types possessed a different charge on two-dimensional gels. An unusual observation was made regarding this complex on MOLT4 cells. The light chain co-precipitated from these cells was 12,000 daltons and had a pI distinct from that of beta 2-microglobulin but similar to the pI of the beta t molecule. Comparison between chymotryptic peptide maps of the p43 heavy chain and those of the human and murine class I molecules such as HLA, T6, H-2K, Qa-2 and TL revealed no apparent homology. We have shown, however, that the peptide backbone of p43, as studied by both tunicamycin treatment of cells and endoglycosidase F digestion of immunoprecipitates, was identical in size to that of murine Qa-1. These results suggest that the p43 antigen may be homologous to murine Qa-1 or another class I antigen encoded in the murine TL:Qa region.     

Allergens in Hymenoptera venom XIII: Isolation and purification of protein components from three species of vespid venoms

Pure venoms were collected from individual insects of the species Dolichovespula maculata, white-faced hornet, Vespula squamosa, southern yellow jacket, and Polistes exclamans, paper wasp (one species). The venoms were first fractionated by high-resolution gel filtration on a 1.6 m column of Sephadex G-75 superfine, and the components were then purified by high-performance, ion-exchange chromatography on a Mono-S cation exchange column followed by a further gel filtration step. The isolated components were evaluated for purity by sodium dodecyl sulfate polyacrylamide gel electrophoresis by use of two different types of silver stains, by assays for enzyme activities, and by immunodiffusion with the use of rabbit antisera. The protein components were isolated in highly purified states by these techniques. Only three significant proteins were found in V. squamous venom: phospholipase (PL) A and B, hyaluronidase (HYAL), and antigen 5 (Ag 5). D. maculata venom contained HYAL, Ag 5, two isozymes of PL A and B, a high-molecular-weight protein, and several trace proteins. No significant amounts of proteases were found in D. maculata venom. P. exclamans venom contained HYAL, PL A and B, Ag 5, a high-molecular-weight protein, and several minor proteins. In all three venoms the PL A and B activities were found to be in the same molecule and did not separate. Trace components with apparent PL A activity were observed in the venoms. The venoms were screened for a variety of esterases, proteases, peptidases, glucosidases, and phosphatases, and none were detected in more than trace amounts. Vespid venoms do not appear to contain significant amounts of acid phosphatases as bee venoms do.     

Purification and partial characterization of two major allergens from the house dust mite Dermatophagoides pteronyssinus

Two major allergens of the house dust mite, Dermatophagoides pteronyssinus (Dp), were purified, and their molecular weight and isoelectric points (pIs) were determined. Dp 42 was purified from an acetone-precipitated mite-excrement extract by a combination of hydrophobic interaction chromatography on phenyl Sepharose and copper-chelate chromatography. The molecular weight was determined to be 18,000 and 25,000 to 30,000 by gel filtration (G-75) and sodium dodecyl sulphate-polyacrylamide gel electrophoresis, respectively, and pI values of 4.6, 5.6, and 6.6 were obtained by sucrose gradient isoelectric focusing (IEF). These values correspond well with those described for the identical allergen, P1. The pI 6.6 variant was considerably enriched in the purified material. Dp 42 constituted 6.4% of the dry weight of a reference whole mite-culture extract. Dp X was obtained partially purified by gel filtration (G-75), ammonium sulphate precipitation, and hydrophobic interaction chromatography. The molecular weight was 18,000 to 20,000 by gel filtration and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Multiple pIs in the range 5 to 7 were found by sucrose gradient IEF and crossed IEF. The two purified allergens carried clearly distinct activities toward human IgE and appeared as potent allergens in crossed radioimmunoelectrophoresis, RAST, and RAST inhibition.     

Immuno-suppressive lymphocyte factors—III. Complete purification and partial characterization of human inhibitor of DNA synthesis

Inhibitor of DNA synthesis (IDS) is an immunoregulatory T lymphocyte factor first characterized in the rat. It appears to cause the non-specific immune suppression seen in certain tolerant disease states.We have produced IDS from human peripheral blood lymphocytes, purified it to homogeneity and partiallycharacterized it. Human IDS has an isoelectric point of 3.40 and a molecular weight of 20,000. However, in the supernatants containing IDS activity, it usually exists in an aggregated tetrameric form which is also biologically active. IDS is not cytotoxic and is glycoprotein in nature. The activity depends on an intact carbohydrate moiety. Human IDS is identical to rat IDS except for a slightly higher isoelectric point. The biologic role is undetermined at present, but might be similar to that of rat IDS.     

Identification of Schistosoma mansoni antigens by means of biologically active monoclonal antibodies

Two monoclonal antibodies of the IgE class (54.10) and of the IgG₁ class (27.21), that were shown previously to possess biological activity against Schistosoma mansoni larvae, were used for identification of surface antigens of the cercariae and schistosomula. This was performed by immunoprecipitation, immunoaffinity chromatography and immunoblotting. The epitope reactive with 27.21 mcIgG₁ is present on four polypeptides (60, 50, 27 and 19 kDa) derived from the parasite. The 60 kDa is specific to cercariae, whereas the 50 kDa is a glycoprotein shared both by cercariae and schistosomula. The antigen reactive with the 54.10 mcIgE was isolated by affinity chromatography on 54.10 column, and contained three major peptides of 125, 94 and 30 kDa. The 125 and 94 kDa band probably originate from the same protein, since they yield almost identical peptide maps. The isolated antigen retained its biological activity as demonstrated in the basophils degranulation assay.     

Biochemical characterization of I-Ak proteins from mutant antigen-presenting B-cell--B-lymphoma hybridomas

Chemically induced mutants of an I-Ak,d expressing antigen-presenting B-cell--B-lymphoma hybridoma have recently been generated by immunoselection in vitro and were found to possess alterations in some of their serologically and functionally defined I-Ak region dependent functions. In order to identify at the structural level the origin of the differences in serological and functional properties of these mutants, I-Ak molecules from several of these mutant hybridomas were compared biochemically to wild-type I-Ak polypeptides by two-dimensional gel electrophoresis and high-pressure liquid chromatographic tryptic peptide analyses. Two-dimensional gel electrophoresis indicated that no major structural alterations, resulting in changes in mol. wt or charge, had occurred in the Ak alpha or Ak beta polypeptides from the mutant cells. Likewise, Ak alpha peptide maps of the mutants were indistinguishable from the normal Ak alpha peptide maps. However, two of the three mutants studied did exhibit one additional peptide in their Ak beta peptide maps. These results suggest that the major deficiencies in T-cell-activating functions of these mutants are a result of a limited alteration in the Ak beta polypeptide primary structure.     

Identification of parasite proteins in a membrane preparation enriched for the surface membrane of erythrocytes infected with Plasmodium knowlesi

A subcellular fraction enriched in erythrocyte membranes has been isolated from rhesus monkey erythrocytes infected with Plasmodium knowlesi. Infected cells were lysed by centrifugation through a zone of hypotonic buffer and membranes isolated by equilibrium density gradient centrifugation in the same tube. The purified membrane fraction was shown to include the erythrocyte surface membrane by several methods: electron microscopy, identification of Coomassie Blue stained erythrocyte membrane proteins, identification of band 3 with a monoclonal antibody, and identification of radioiodinated cell surface proteins. The resulting ghosts were shown to be specifically reactive with monkey sera against the variant surface antigens of P. knowlesi by indirect immunofluorescence and membrane agglutination. No reactivity was seen with a monoclonal antibody (13C11) against the intracellular schizont surface. A number of metabolically labelled parasite proteins were enriched in this membrane function, including peptides of 277, 208, 173, 153, 134, 109, 80, 60 and 48 kDa and the variant surface antigens of variable molecular mass (180-207 kDa). These proteins were distinct from the major parasite proteins of total infected erythrocytes and isolated merozoites. The major glucosamine labelled glycoprotein of the internal schizont (230 kDa) was not found in this fraction. Moreover, no fragment of this parasite glycoprotein was found in this membrane fraction, indicating that no part of this molecule is transported to the erythrocyte surface. In contrast, the variant antigen of P. knowlesi, known to be on the erythrocyte surface, could be readily identified as peptides unique to specific cloned parasite lines. We propose that the other nine parasite proteins found within this membrane fraction represent a starting point for the identification of other parasite proteins transported to the surface membrane of the infected erythrocyte.     

Preparation and biologic characterization of fragments containing dimeric and monomeric C gamma 2 domain of rabbit IgG

A number of fragments derived from acid-treated rabbit IgG by digestion with plasmin have been separated by high-resolution gel filtration. Fragments isolated included a dimer and monomer Facb, named F(acb)2 and Facb, respectively and a heterodimer composed of Facb and Fab subunits, named F(acb)(ab). A C gamma 2 fragment was obtained by papain digestion of Facb. A heterodimer composed of Facb and Fab', named F(acb)(ab'), was also prepared by oxidizing a reduced mixture of these fragments. Fragments thus obtained are classified into two groups--those carrying paired C gamma 2 domains, i.e. F(acb)2, and the disulfide-linked dimeric C gamma 2 fragment; and those having a single C gamma 2 domain, i.e. reduced, alkylated Facb and C gamma 2 fragment, F(acb)(ab) and F(acb)(ab'). These fragments exhibited marked differences in their capacity to activate complement in assay systems of hemolysis and complement consumption by immune complexes or aggregates on polystyrene latex. Fragments of the former group could activate complement but with a definitely reduced efficiency (50%) compared to intact IgG, whereas fragments of the latter group were practically inactive. Although it was not determined whether the C1-binding capacity itself is changed by monomerization of the C gamma 2 domain, the results suggested that the intact paired C gamma 2 module is required at least for the activation process of complement.     

Evidence that the 32, 38 and 20 kilodalton surface antigens of schistosomula and schistosomes are a family of conserved proteins

The structures of the 38, 32 and 20 kDa surface antigens isolated from schistosomula and adult worms of Schistosoma mansoni were compared by two-dimensional peptide mapping, by immunological analysis and by one- and two-dimensional electrophoresis. Peptide mapping showed a high degree of similarity between the isolated antigens from both parasite stages. The NIMP/M47 monoclonal antibody showed cross-reactivity between the 32 and the 20 kDa antigens under denaturating and non-denaturating conditions, as demonstrated by immunoprecipitation and Western blotting. It is concluded that these antigens constitute a homologous family of surface antigens.     

A Mr 90 000 surface polypeptide of Trypanosoma cruzi as a candidate for a Chagas' disease diagnostic antigen

Trypanosoma cruzi (Peru strain) trypomastigotes and epimastigotes were biosynthetically labeled with [35S]methionine, and the proteins were analyzed by two dimensional polyacrylamide gel electrophoresis (2D-PAGE). 2D-PAGE analysis of the trypomastigotes showed a complex array of polypeptides with distinct clusters at Mr 88 000-92 000, isoelectric point (pI) 5.6-6.0, and Mr 72 000-76 000, pI 5.6-5.8. 2D-PAGE analysis of the epimastigotes did not show the cluster of polypeptides at Mr 90 000. When the trypomastigote lysate was reacted with sera from either mice or humans chronically infected with T. cruzi, 10-50 polypeptides were immunoprecipitated. Five of these polypeptides were recognized by all sera tested. However, of these polypeptides, only three, two of Mr 90 000 and one of Mr 150 000, can be identified by immunoreaction of [35S]methionine-labeled live parasites as surface proteins of T. cruzi trypomastigotes. 125I-iminobiotinylated surface proteins isolated from T. cruzi trypomastigotes were immunoprecipitated with the same series of sera as described above. Chagasic sera immunoprecipitated an antigen of Mr 90 000. The [35S]methionine and 125I-labeled Mr 90 000 polypeptides were not immunoprecipitated with sera from individuals infected with Leishmania donovani, Leishmania braziliensis, Leishmania tropica or Leishmania mexicana. These data indicate that a surface polypeptide of Mr 90000, pI 5.8-5.9 is a viable candidate for a Chagas' disease diagnostic antigen.     

Identification and characterization of a hapten-modifiable TEPC 15 cross-reactive idiotype in swine

Rabbits and swine immunized with TEPC 15 IgA, goats immunized with T15-positive IgM and swine immunized with affinity-pure swine anti-phosphorylcholine (PC) all produce antibodies which recognize a hapten-inhibitable idiotypic determinant on swine anti-PC. The similarity in reactivity and order of inhibitability with various PC analogs of the heterologous (swine anti-TEPC 15) and isologous (swine anti-swine anti-PC) reagents indicates that they recognize a related idiotype and suggest it may be the predominant idiotype expressed on swine anti-PC antibodies. The heterologous and isologous anti-idiotypic reagents generated in this study recognize swine and mouse anti-PC but not normal swine IgM, IgG or MOPC 460. Only reactions with swine anti-PC and mouse T15-positive anti-PC proteins are hapten-inhibitable. The greater inhibitory capacity of trimethylammonium and acetylcholine than PC suggests that the idiotope(s) recognized on swine anti-PC by the anti-idiotypic reagents is integral rather than peripheral to the anti-PC binding site. The nearly exclusive IgM anti-PC response of swine to Streptococcus pneumoniae R36A and PC-Brucella have so far hindered attempts to study the isotypic distribution of the idiotype.     

Differences in the surface radioiodinated proteins of skin and uterine microfilariae of Onchocerca gibsoni

Surface labeling studies using two populations of Onchocerca gibsoni microfilariae revealed important differences in major radioiodinated proteins. Small numbers of microfilariae harvested from the skin of cattle or the uteri of adult worms from skin nodules were purified, radioiodinated, solubilized and the proteins analysed by two dimensional gel electrophoresis and autoradiography. As reported previously, uterine microfilariae showed a complex profile of radioiodinated proteins, none of which appeared to be bovine albumin or immunoglobulin. In contrast, application of the same techniques to skin microfilariae demonstrated only one major labeled protein complex of approximate Mr 67 000. This protein complex was immunoprecipitated with an antiserum to bovine serum albumin. Surprisingly, fluorescence techniques failed to show bovine serum albumin on the surface of living microfilariae. Although the evidence is circumstantial at present, acquisition of host albumin (perhaps oriented in a particular way) may be a means whereby skin microfilariae evade immune effector mechanisms and, when living, generally fail to elicit inflammatory reactions in the skin of the host.     

In vitro biosynthesis and core glycosylation of the histidine-rich protein of Plasmodium lophurae

We have characterized the early biosynthetic forms of the histidine-rich protein (HisRP), a major, granule-bound protein (Mr 58 000) of the avian malarial parasite Plasmodium lophurae. We have translated poly(A)-containing, size-selected parasite mRNA in the wheat germ cell-free system in the presence of [3H]histidine. HisRP was synthesized as a larger precursor (Mr 63 000). When dog pancreas microsomal membranes were present in the cell-free system during translation, a still larger form of HisRP (Mr 66 000) was detected. This larger form was segregated into the dog pancreas microsomal vesicles and was core glycosylated. Presumably, it corresponds to an intermediate form located in the parasite rough endoplasmic reticulum (RER). The difference in the Mr of approx. 8 000 between this RER associated 'pro' form and the granule-bound, mature form of HisRP suggests that proteolytic processing occurs upon transport from the RER to the granule. Segregation and core glycosylation were strictly coupled to translation and were not observed upon posttranslational addition of microsomal membranes. Thus, the early events in the biosynthesis of HisRP are similar to those established for secretory and lysosomal proteins.