The distribution and relative immunogenicity of calf alpha-crystallin antigenic determinants on different subunits.

In the native alpha-crystallin molecule, 45.9% of all reactive antigenic determinants were found to be located on SH-containing subunits. Of these, the majority (35.3%) were reaggregation dependent, and 10.6% were reactive on monomeric subunits. By contrast, only 10.9% of all antigenic determinants were located on SH-free subunits, and the ratio of aggregation-dependent determinants (4.4%) to those of monomeric subunits (6.5%) was reversed compared to SH-containing subunits. Among all antigenic determinants reactive in native alpha-crystallin, 44.1% were dependent on the presence of both types of subunits. These data indicate that the antigenic determinants requiring subunit interaction were formed from SH-containing and SH-free subunits in a ratio of 1:1. Direct analysis showed that in the alpha-crystallin molecule, the ratio of these subunits is 2:1. The experiments indicate that some conformations of subunits in the native molecule persist in separated subunits. The relative immunogenicity of each type of antigenic determinant expressed as the ratio of the percentage of the determinant reactive in the native calf lens alpha-crystallin to the percentage of corresponding antibodies induced by native alpha-crystallin was found to be close to 1.     

Epstein-Barr virus (EBV) antigenic determinants on subunits of the EBV determined nuclear antigen (EBNA)

In order to characterize the substructure of the Epstein-Barr virus determined nuclear antigen (EBNA) which is considered to have a molecular weight of 180 K in its native form, we have examined the antigenic specificity of the polypeptides obtained after denaturation of this molecule. Two procedures were employed; treatment by sodium dodecyl sulfate (SDS) and heat followed by gel electrophoresis, or denaturation by guanidine hydrochloride followed by gel filtration, which allowed us to detect a specific antigenic activity in the 50 K region, following dialysis. The denatured molecules could be reassociated into larger molecules (50 to 180 K) which retain the property of binding to fixed nuclei, as does native EBNA. These results indicate that EBNA has a polymeric structure and that 50 K subunits carry the antigenic determinants.     

Self-association and oxygen-binding characteristics of the isolated subunits of Limulus polyphemus hemocyanin

The hemocyanin of the horseshoe crab Limulus polyphemus is characteristic of arthropod hemocyanins in that it is a high-molecular-weight oligomer composed of functionally and structurally distinct subunits. The protein forms a 48-subunit complex, the largest form of arthropod hemocyanin, whose oxygen-binding characteristics are modulated by subunit interaction within the oligomer. It has previously been shown that a number of electrophoretic isozymes, which are identical immunochemically, are present in dissociated Limulus hemocyanin. In this study it is demonstrated that the electrophoretic differences in the antigenically identical subunits are not reflected in their oxygen-binding and self-assembly properties or in the roles they play in reassembly and function of the 48-subunit native molecule. The chloride-dependent modulation of the oxygen-binding properties of those Limulus subunits which do not self-assemble, as documented here, illustrates that this allosteric effect may be operable at the tertiary level. For each of the purified subunits the effects of pH and calcium ions on oxygen-binding characteristics and self-assembly reactions are reported, and the roles of specific subunits in reassembly of distinct aggregation states are further documented.     

Mouse antibodies to the insulin receptor developing spontaneously as anti-idiotypes. I. Characterization of the antibodies

Mice immunized to ungulate insulins were found to develop antibodies of two specificities: insulin antibodies that were mostly IgG1 and IgG2 antibodies that acted both as anti-idiotypes to specific mouse insulin antibodies and as antibodies to the insulin receptor. There was a negative association between the presence of anti-idiotypic receptor antibodies and insulin antibodies bearing the specific idiotype; the specific idiotypic antibodies were confined to the early phase of the primary response while the anti-idiotypic receptor antibodies were detected only after the idiotypic antibodies had disappeared. To map the insulin epitope that triggered the specific idiotypic response, we chemically altered the insulin molecule so as to inhibit its interaction with the insulin receptor. The altered insulins triggered high titers of antibodies binding to antigenic determinants on native insulin, but no anti-idiotypic receptor antibodies. Thus, the epitope responsible for the specific idiotypic-anti-idiotypic network was probably the part of the insulin molecule whose conformation is recognized by the insulin receptor.     

Isolation and characterization of liver glycogen synthase from diabetic rats

NAD-specific pig heart isocitrate dehydrogenase is composed of three distinct types of subunits: α, β, and γ, which have molecular weights of about 40,000 but differ in amino acid composition and in isoelectric points. When the native enzyme is subjected to polyacrylamide gel electrophoresis under nondenaturing conditions, two major protein bands with M_r values of about 360,000 (band 1) and 100,000 (band 2) and two minor bands (bands 3 and 4) with M_r values of about 40,000 are consistently present. Enzymatic activity, as detected from NADH fluorescence, is distributed throughout the protein-staining region. Analytical isoelectric focusing in urea reveals that band 1 is composed of all three subunits in roughly the normal ratio of 2α:1β:1γ, and is probably an octamer, band 2 of an equal amount of α and β and is probably dimer, while bands 3 and 4 each consist of only the monomeric α subunit. The highest enzymatic specific activity is associated with a region intermediate between octamer and dimer, which includes the 160,000 tetramer. The protein pattern resulting from isoelectric focusing under nondenaturing conditions consists of protein bands comparable in pattern to those in the presence of urea along with bands of intermediate pI values, many of which are associated with enzymatic activity. Analysis of the subunit composition of these bands supports the activity of the α species in isolation and establishes the activity of the separated β component. No activity of the isolated γ subunit species has thus far been demonstrated. However, the highest apparent specific activity is observed when at least two types of subunits are present. These studies indicate that a range of oligomeric species of the enzyme are enzymatically active and that at least three of the four subunit chains comprising the minimum complete enzyme molecule (2α:1β:1γ) possess an active site.     

Monoclonal antibodies identify multiple epitopes on maize leaf nitrate reductase

Nine hybridoma cell lines secreting antibodies against the maize leaf nitrate reductase have been distinguished by reciprocal competition for binding to the antigenic site. Inhibition of enzymatic activities, and western blots of native enzyme and denatured subunits revealed different behaviors of individual antibodies towards the antigen. Two classes of monoclonal antibodies are inhibitory of NADH and methyl viologen nitrate reductase activities, but only one affects also NADH cytochrome c reductase activity. The associated epitopes are sensitive to antigen conformation. Among the 4 other classes, one is specific for the native conformation of the molecule, another binds more strongly to the denatured antigen, and two recognize equally well the two forms.     

Membrane-bound ribosomes of myeloma cells. II. Kinetic studies on the entry of newly made ribosomal subunits into the free and the membrane- bound ribosomal particles

The kinetics of appearance of newly made 60S and 40S ribosomal subunits in the free and membrane-bound ribosomal particles of P3K cells were explored by determining the specific radioactivities of their 18S and 28S RNA after various lengths of [3H]uridine pulse. Both 40S and 60S subunits enter free and membrane-bound polyribosomes at comparable rates from the cytoplasmic pool of newly made, free native subunits, the 40S subunits entering the native subunit pool and the polyribosomes slightly earlier than the 60S subunits. At all times, the specific radioactivity of the membrane-bound native 60S subunits was slightly lower than that of the polyribosomal 60S subunits. This indicates that the membrane-bound native 60S subunits are not precursors destined to enter membrane-bound polyribosomes and suggests that they result from the dissociation of ribosomes after chain termination. The results observed also suggest that the membrane-bound native 60S subunits are not reutilized before their release from the membranes, which probably takes place shortly after dissociation from their 40S subunits. The monoribosomes, both free and membrane-bound, had the lowest specific radioactivities in their subunits. Finally, a small amount of newly made native 40S subunits, containing 18S RNA of high specific radioactivity, and apparently also newly made messenger RNA were detected on the membranes. The high turnover of these membrane-bound native 40S subunits suggests that they may represent initiation complexes formed with mRNA which has just reached the membranes and which has not yet given rise to polyribosomes.     

Studies on structure-function relationships of human choriogonadotropins with C-terminally shortened alpha-subunits. I. Receptor binding and immunologic properties

Recombination products composed of the native beta-subunit and an alpha-subunit with an enzymatically shortened C-terminal region showed a diminished (less than 5 amino acids removed) or - in the case of des-(88-92)-alpha/native beta - a completely abolished ability to bind to testicular LH/hCG receptors of the rat. An antigenic determinant which is present in native hCG but not in the isolated subunits was not or incompletely expressed in the modified hormone species. Antigenic determinants which are characteristic for the isolated alpha-subunit, however, were not affected by removal of the C-terminal residues 88-92. The immunologic experiments indicate that hCG containing an alpha-subunit with a shortened C-terminal region differs from native hCG in its conformation. These conformational changes are probably responsible for the loss in receptor-binding ability.     

Radioimmunological study of the surface protein of the human serum low-density lipoprotein: comparison of the native particle and the products obtained by tryptic treatment.

The present report describes radioimmunological studies of the native low-density lipoprotein from human serum, and of the products obtained by limited tryptic treatment, i.e.a protein-depleted particle lacking some 20% of the original protein moiety and a peptide fraction of low molecular weight (<5000). The liberated peptides were highly immunogenic and elicited antibodies which reacted with both the native and protein-deficient lipoprotein particles. Moreover these peptides exhibited competitive reactivity with [¹²⁵I]-labelled low-density lipoprotein in binding with homologous antisera, and with antisera to the native and trypsin-treated lipoproteins. These findings suggest that the peptides liberated from low-density lipoprotein by tryptic digestion contain the major antigenic site(s) of the molecule. Consideration of the nature of the competitive displacement of radiolabelled low-density lipoprotein from antisera to low-density lipoprotein, to the trypsinised lipoprotein and to the peptide fraction indicate that a marked repetition of the antigenic site(s) occurs in the structure of the protein moiety, a possibility consistent with the recurrence of similar subunits in the apoprotein of low-density lipoprotein.     

Immunochemical analysis of Micrococcus lysodeikticus (luteus) F1-ATPase and its subunits

The F₁-ATPase from Micrococcus lysodeikticus has been purified to 95% protein homogeneity in this laboratory and as all other bacterial F₁s, possesses five distinct subunits with molecular weights ranging from 60 000 to 10 000 (Huberman, M. and Salton, M.R.J. (1979) Biochim. Biophys. Acta 547, 230–240). In this communication, we demonstrate the immunochemical reactivities of antibodies to native and SDS-dissociated subunits with the native and dissociated F₁-ATPase and show that: (1) the antibodies generated to the native or SDS-dissociated subunits react with the native molecule; (2) all of the subunits comprising the F₁ are antigenically unique as determined by crossed immunoelectrophoresis and the Ouchterlony double-diffusion techniques; (3) antibodies to the SDS-denatured individual δ- and ?-subunits can be used to destabilize the interaction of these specific subunits with the rest of the native F₁; and (4) all subunit antibodies as well as anti-native F₁ were found to inhibit ATPase activity to varying degrees, the strongest inhibition being seen with antibodies to the total F₁ and anti-α- and anti-β-subunit antibodies. The interaction of specific subunit antibodies may provide a new and novel way to study further and characterize the catalytic portions of F₁-ATPases and in general may offer an additional method for the examination of multimeric proteins.     

Molecular evolution and subunit structure of cattle lens alpha crystallin

Summary The present studies were based on the premise that any common determinants in homologous proteins must have originated with the common ancestor of all of the taxonomic groups in which that determinant occurs. Cross-reacting antigenic determinants of lens alpha crystallin in various classes of modern vertebrates were used to trace their evolutionary relationships.For quantitation of evolutionarily distinct determinants, equimolar amounts of alpha crystallin or its subunits, in either monomeric or reaggregated form, were bound to a matrix, then saturated with^{1 2 5}I-labeled Fab fragments of anti-cattle alpha crystallin antibodies having phylogenetically restricted specificities. This quantitative procedure has the important advantage of independence from variation in antibody responses to different determinants of the same antigenic molecule. The procedure is not impaired by steric hindrance.Both the SH-containing and SH-free subunits of cattle lens alpha crystallin were found to contain common antigenic determinants with the cyclostomata alpha crystallin. Such determinants originated in evolution with the first vertebrates, the primitive agnatha. Antigenic determinants transferred from ancestral aquatic and land vertebrates to the mammals were found to constitute 93% of all determinants reactive in the monomeric SH-free subunits of cattle alpha crystallin. These determinants constitute only 76.5% of all determinants which are reactive in the SH-containing subunits. The antigenic determinants on both types of subunits were all found to be different. These findings indicate that evolutionary changes must have occurred more slowly in SH-free subunits than in SH-containing subunits.Significant decreases or increases were found in the content of various evolutionarily distinct determinants reactive in the reaggregated subunits as compared to the ones reactive in monomeric subunits. These differences can result from the formation of new conformational antigenic determinants during aggregation as well as from the burial or exposure of other determinants after aggregation.Different amounts of evolutionarily distinct antigenic determinants were found to be reactive in the molecules dissociated into subunits than in the intact molecules one of the reasons being that the intact molecules contain phylogenetically distinct determinants which depend on the quaternary structure of the protein molecule. The data obtained indicate that the quaternary structure of cattle alpha crystallin has, to a large degree, remained unchanged since the origin of vertebrates.     

Specificity of the precipitation reaction of one-zone diphtheria antitoxin for toxicity determination ofCorynebacterium diphtheriae strains

In immunodiffusion analysis of crude diphtheria toxin, one-zone diphtheria antitoxin may give one or two subsidiary lines in addition to the main precipitation line. The subsidiary lines belong to antigenic fragments of the toxin molecule. These fragments are formed from the complete molecule, probably by proteolytic degradation by bacterial enzymes. Other forms of fragment production were not demonstrated. When testing the toxicity of strains ofCorynebacterium diphtheriae by means of one-zone antitoxin, any precipitation reaction observed can thus be regarded as specific evidence of the toxicity of the test strain.     

Immunochemical characterization of human plasma fibronectin.

Human plasma fibronectin has been purified by a non-denaturing affinity chromatography procedure [Vuento & Vaheri, (1979) Biochem.J. 183, 331--337], and antisera have been raised by immunizing rabbits with the native protein. The antisera reacted strongly with native fibronectin, but only weakly with reduced and alkylated fibronectin or with heat-denaturated fibronectin. Denaturation also affected the haemagglutinating and gelatin-binding activities of fibronectin and increased its susceptibility to proteolytic degradation. The antisera reacted with fragments of fibronectin obtained by proteolysis with plasmin. Large fragments (mol.wt. 180000--200000), lacking the region harbouring the interchain disulphide bridges but containing the sites responsible for gelatin-binding and haemagglutinating activity, showed as intense a reaction with the antisera as intact fibronectin. Smaller peptides showed a weaker reaction. All fragments tested showed sensitivity to denaturation in their reaction with the antisera. The results were interpreted as showing that: (1) native fibronectin has an ordered conformation that is easily perturbed by denaturation; (2) most of the antigenic determinants of the protein are dependent on conformation; (3) the region of the fibronectin molecule containing the interchain disulphide bridges has only few antigenic determinants; and (4) covalent interaction of the two subunits does not contribute to the antigenic structure recognized by rabbit antisera. The observed correlation between the antigenic activity and a structural and functional intactness of fibronectin suggests that the antibodies to native fibronectin could be used as a conformational probe in studies on this protein.     

Proteolytic Cleavage of Subunits of the Nucleocapsid of the Paramyxovirus Simian Virus 5

The protein subunits of the nucleocapsid of the parainfluenza virus simian virus 5 isolated from infected cells after dispersion with trypsin, chymotrypsin, or ficin are cleaved proteolytically. The molecular weights of the subunits which result from cleavage depend on the enzyme used, but are around 43,000, compared to the native subunit of 61,000. In most instances cleavage of the subunit appears to be due to the protease used to disperse the cell, and follows cell disruption. Nucleocapsids composed of native, uncleaved subunits can frequently be obtained from infected cells dispersed without a proteolytic enzyme; however, cleavage occasionally occurs even under those conditions, indicating that cellular proteases can at times cleave this protein. Nucleocapsids containing uncleaved subunits can be isolated from cells persistently infected with simian virus 5, indicating that persistent infection is not invariably associated with intracellular cleavage of this protein. Nucleocapsids composed of native subunits are hydrophobic, whereas those composed of the cleaved subunit can be dispersed in aqueous solution. It is suggested that the portion of the molecule removed by cleavage may be responsible for a specific interaction during virus assembly between the nucleocapsid and those areas of plasma membrane which contain the non-glycosylated viral membrane protein, which is also hydrophobic. An amino acid analysis of native and cleaved subunits has been done. The portion of the subunit removed by cleavage does not have a high proportion of hydrophobic residues, suggesting that those present are arranged together to form a hydrophobic domain.The N termini of both the native and cleaved subunits are blocked. This suggests that the portion of the molecule which is externally disposed and removed by cleavage contains the C terminus, and the cleaved subunit which reacts with the viral RNA contains the N terminus.     

A major part of the polypeptide chain of tobacco mosaic virus protein is antigenic

Eighteen synthetic peptides representing virtually the entire length of the polypeptide chain of tobacco mosaic virus coat protein (TMVP) have been analyzed for their ability to bind in an enzyme immunoassay to 30 monoclonal antibodies raised against the dissociated viral subunits. Only five of the monoclonal antibodies were able to bind a number of peptides while the other 25 antibodies recognized only the complete molecule and seemed to be specific for conformational features that are absent in the peptide fragments. The 18 peptides were also tested for their ability to bind to several antisera to TMVP. Virtually the entire sequence of TMVP possessed antigenic activity. Four new epitopes were identified in the vicinity of residues 19–32, 90–95, 115–134 and 134–146. These results bring to 11 the number of continuous epitopes that have been identified in the TMVP molecule and show that the entire surface of the molecule is antigenic. When peptides of TMVP of a length of 6–8 residues were tested for antigenic activity previously a correlation was found between the location of short continuous epitopes and mobile segments of the protein. In the present study, in which longer peptides as well as monoclonal antibodies were used to probe the antigenicity of TMVP, additional conformation-dependent epitopes were shown to be present. Our results illustrate the operational nature of any definition of antigenicity and caution against the use of any single criterion for distinguishing between antigenic and non-antigenic regions of a protein.     

Heterodimeric Barnase-Barstar Vaccine Molecules: Influence of One versus Two Targeting Units Specific for Antigen Presenting Cells

It is known that targeting of antigen to antigen presenting cells (APC) increases immune responses. However, it is unclear if more than one APC-specific targeting unit in the antigenic molecule will increase responses. To address this issue, we have here made heterodimeric vaccine molecules that each express four different fusion subunits. The bacterial ribonuclease barnase and its inhibitor barstar interact with high affinity, and the barnase-barstar complex was therefore used as a dimerization unit. Barnase and barstar were fused N-terminally with single chain fragment variable (scFv)s targeting units specific for either MHC class II molecules on APC or the hapten 5-iodo-4-hydroxy-3-nitrophenylacetyl (NIP). C-terminal antigenic fusions were either the fluorescent protein mCherry or scFv³¹⁵ derived from myeloma protein M315. The heterodimeric vaccine molecules were formed both in vitro and in vivo. Moreover, the four different fused moieties appeared to fold correctly since they retained their specificity and function. DNA vaccination with MHC class II-targeted vaccine induced higher mCherry-specific IgG1 responses compared to non-targeted control. Since mCherry and MHC class II are in trans in this heterodimer, this suggests that heterodimeric proteins are formed in vivo without prior protein purification. Surprisingly, one targeting moiety was sufficient for the increased IgG1 response, and addition of a second targeting moiety did not increase responses. Similar results were found in in vitro T cell assays; vaccine molecules with one targeting unit were as potent as those with two. In combination with the easy cloning strategy, the heterodimeric barnase-barstar vaccine molecule could provide a flexible platform for development of novel DNA vaccines with increased potency.     

Monoclonal antibodies against tryptophanyl-tRNA-synthetase

Monoclonal antibodies designated as Am1 and Am2 were prepared against purified beef pancreas tryptophanyl-tRNA synthetase (EC 6.1.1.2). Both antibodies were able to bind the native enzyme in a solid-phase assay and to precipitate enzyme activity in immune complexes. Am2 inhibited the tryptophanyl-tRNA synthetase activity in ATP-[32P]pyrophosphate exchange and in tRNATrp aminoacylation reactions; Am1 had no influence on both the enzyme activity and the inhibiting action of Am2. Only Am2, not Am1, bound elastase-modified form of the enzyme which consists of two subunits shortened by 20 000 daltons from the N-end of the molecule. These results were interpreted as an evidence for non-overlapping of Am1 and Am2 antigenic determinants along the polypeptide chains of the enzyme.     

Determination of the native subunit pattern of gizzard tropomyosin using antibodies specific to each subunit

The gizzard tropomyosin molecule is composed of two subunits at 1:1 molar ratio. Possible composites of the tropomyosin molecule are two kinds of homodimer (one for each subunit), a heterodimer of two subunits, or a mixture of heterodimer and homodimer(s). We tried to evaluate the native subunit composition of gizzard tropomyosin by cross-linking experiments and immunological methods using specific antibodies to each subunit. For the cross-linking experiment we used dimethyl suberimidate, an amino group-specific cross-linker, in the presence of dithiothreitol to avoid artificial oxidative intersubunit cross-linking. When gizzard tropomyosin was cross-linked, it generated several products which might correspond to dimers formed by intersubunit cross-linkage. When the reaction was carried out for a long time, non-cross-linked subunits completely disappeared and two or three major cross-linked products arose. All of these cross-linked products were recognized by both of the specific antibodies to each subunit. These results indicated that the predominant part, if not all, of gizzard tropomyosin is present as heterodimer.     

Recombinant subunits as tools for the structural and functional characterization of Echinococcus granulosus antigen B

Antigen B (AgB) is a major protein component of the Echinococcus granulosus metacestode. It is oligomeric and this raises several questions regarding the subunit structure and composition of AgB. Several genes that encode different AgB subunits have been identified, and some of these have been cloned and expressed to produce recombinant subunits. The study of these recombinant subunits may provide new insights into the structure, physical-chemical properties, and functional aspects of AgB. Like native AgB, the AgB8/1, AgB8/2, and AgB8/3 recombinant subunits produced in our laboratory form 120-160 kDa oligomers that have stable secondary structures, are strongly antigenic and immunogenic, and selectively bind hydrophobic compounds. Here, we review these results and discuss their implications for the elucidation of the structure and function of AgB. This includes a possible role for AgB in host-parasite interactions.