Properties and application to immunoassay of monoclonal antibodies to neuron-specific γγ enolase

Two monoclonal antibodies to human and bovine neuron-specific γγ enolase have been produced in the isolated hybrid cell lines, which were obtained by fusion between γγ-immunized mouse spleen cells and mouse myeloma cells (P3-NS-1/1-Ag4-1), followed by a screening procedure with an enzyme immunoassay. The monoclonal antibody to human γγ enolase (E1-G3) and that to bovine γγ enolase (B1-D6) consisted of γ2a/κ and γl/κ immunoglobulin chains, respectively. Both antibodies could bind with the respective antigen with a molar ratio of about 1:1, and were found to be specific for the γ subunit of enolase, showing reactivities with human γγ and αγ, rat γγ and αγ, and bovine γγ enolases. However, the antibodies did not cross-react with the α or β subunit of human and rat enolase isozymes. Both antibodies could partially inhibit the activity of γγ and αγ enolases. E1-G3 antibody inhibited γγ and αγ enolase activity by 70 and 30%, respectively, and B1-D6 antibody, by 90 and 40%, respectively. Both antibodies had no effect on the activity of αα and ββ enolases of human and rat origins. The applicability of E1-G3 and B1-D6 antibodies to the sandwich-type enzyme immunoassay for neuron-specific enolase (enolase γ subunit) was examined, and it was found that the assay system using E1-G3 and B1-D6 as the labeled antibodies were sufficiently sensitive for the assay of serum neuron-specific enolase concentrations.     

Developmental studies with the 14-3-2 antigen and the neuron specific enolase (NSE) associated activities—I

We have compared the rodent developmental pattern of the 14-3-2 antigen estimated by a microcomplement fixation technique with that of the cerebral enolases. Chromatographic separation of enolase isozymes on microcolumns demonstrates that the embryonic neuron specific enolase is firstly and mostly represented by the αγ isozyme. The most important increase in 14-3-2 antigen and γγ enolase occurs between post-natal days 7th and 15th. By post-natal day 30, adult levels have been reached. An interesting observation is—during embryonic development—the decrease in the specific activity of the cerebral enolase isozyme αα. This could be explained by the replacement—in neuroblasts—of αα enolase by neuron specific enolase. A comparison between 14-3-2 antigen and neuron specific enolase (γγ) purified by completely different methods is presented. The 14-3-2 antigen exhibits an enolase specific activity comparable to that of purified enzyme and has the same electrophoretic mobility. Antibodies raised against either antigen have an identical specificity. Pre and post-natal developmental pattern in rodent brains are similar for both proteins. Thus neuron specific 14-3-2 antigen is identical to neuron specific enolase.Thus we have precisely described the ontogenic transition between the three cerebral enolase isozymes at the tissue level. This study is completed by the analysis of these transitions at the neuronal cell level, using homogenous cell lines (Part II of this paper).     

Purification of two enolases from human brain using a chromatofocusing column

When a purified preparation of rat αγ enolase (2-phospho-D-glycerate hydrolyase, EC 4.2.1.11) was applied to a chromatofocusing column, the enolase was almost completely dissociated and recombined to form αα and γγ enolases, which were eluted at different fractions from the column. Using these phenomena, two homo-dimer forms (αα and γγ) of human brain enolase were purified from a crude preparation of the hybrid αγ enolase by the chromatofocusing, and subsequent chromatography on a QAE-Sephadex column (αα) or a DEAE-Sephadex column (γγ). Each purified preparation showed a single band on SDS-gel electrophoresis with a relative mobility corresponding to a molecular weight of about 50 000. Amino acid analysis, peptide mapping analysis with a limited proteolysis and immunochemical studies of the purified αα and γγ enolases revealed that the two subunits, α and γ, are distinct proteins. The antisera to human αα or γγ enolase cross-reacted with the respective form of rat enolase.     

Determination and characterization of neuron specific protein (NSP) associated enolase activity.

Fractionation of the 40–80% (NH₄)₂SO₄ fraction of a soluble rat brain extract on DEAE cellulose resolves three species of enolase activity, two of which react with antiserum to neuron specific protein from rat (NSP-R) and one which does not react. Direct assay of pure neuron specific protein from rat, cat and human brain (NSP-R, NSP-C, NSP-H) as well as bovine brain 14-3-2, using 2 different assay systems demonstrate that all these preparations display enolase activity of between 40 and 70 units/mg. This activity is Mg⁺⁺ dependent and inhibited by fluorophosphate in all cases. Kinetic parameters such as Km for Mg⁺⁺, 2 PGA, and pH optimum were determined for the 2 different NSP preparations and also for bovine brain 14-3-2 protein.     

Characterization of a membrane protein from cholinergic synaptic vesicles isolated from the electric organ of Torpedo marmorata

Rabbits were immunized with cholinergic synaptic vesicles isolated from the electric organ of Torpedo marmorata. The resultant antiserum had one major antibody activity against an antigen called the Torpedo vesicle antigen. This antigen could not be demonstrated in muscle, liver or blood and is therefore, suggested to be nervous-tissue specific. The vesicle antigen was quantified in various parts of the nervous system and in subcellular fractions of the electric organ of Torpedo marmorata and was found to be highly enriched in synaptic vesicle membranes. The antigen bound to concanavalin A, thereby demonstrating the presence of a carbohydrate moiety. By means of charge-shift electrophoresis, amphiphilicity was demonstrated, indicating that the Torpedo vesicle antigen is an intrinsic membrane protein. The antigen was immunochemically unrelated to other brain specific proteins such as 14-3-2, S-100, the glial fibrillary acidic protein and synaptin. Furthermore, it was unrelated to two other membrane proteins, the nicotinic acetylcholine receptor and acetylcholinesterase, present in Torpedo electric organ. The antiserum against Torpedo synaptic vesicles did not react with preparations of rat brain synaptic vesicles or ox adrenal medullary chromaffin granules.     

Purification, Properties, and Immunohistochemical Localisation of Human Brain 14-3-3 Protein

Abstract: A protein has been purified from human brain that appears to be the human equivalent of bovine 14-3-3 protein. On polyacrylamide gel electrophoresis the protein migrates as a faster major component, termed 14-3-3-2 protein, and a slower minor component, termed 14-3-3-1 protein, which consists of approximately 12% of the total protein. Both 14-3-3-1 and 14-3-3-2 have a native molecular weight of approximately 67,000. 14-3-3-2 appears to have the subunit composition (αβ; 14-3-3-1 has the composition ββ. Peptide mapping with Stuphvlococcus aureus V8 proteinase shows that α and β subunits are unrelated but the β and β' subunits show some common peptides. Immunoperoxidase labelling shows that 14-3-3 is localised in neurones in the human cerebral cortex. 14-3-3 shows no enolase, creatine kinase, triose phosphate isomerase, ATPase, cyclic nucleotide-dependent protein kinase, or purine nucleoside phosphorylase activity. 14-3-3 does not bind calcium and does not appear to be related to calmodulin, calcineurin, tubulin, neurofilament proteins, clathrin-associated proteins, or tropomyosin. The functional significance of this neuronal protein remains obscure.     

Enolase isoenzymes as markers of differentiation in teratocarcinoma cells and normal tissues of mouse

The changing profile of enolase (EC 4.2.1.11) isoenzymes in differentiating mouse cells has been traced by the use of specific antisera to the three subunits α, β, and γ. The amounts of the isoenzymes were measured in a variety of tissues during normal mouse development and during the differentiation of mouse teratocarcinoma cells in culture and as tumors. One isoenzyme is predominant in the early cells of the developing mouse embryo, namely, the homodimer made up of α subunits. The same isoenzyme is also the sole form detected in undifferentiated teratocarcinoma (embryonal carcinoma) cells. The isoenzyme form remains unchanged in developing primitive and definitive endoderm of the embryo. Similarly, endoderm cells formed by differentiation of embryonal carcinoma cells contained only αα enolase. In contrast, during the development of striated muscle and of brain, increasing proportions of β and γ subunits, respectively, were detected. Thus enolase was found to be a marker of the differentiation of these tissues. This conclusion was substantiated by finding significant amounts of the β subunit in teratocarcinoma cell cultures which had formed beating striated muscle in vitro.     

FUNCTIONAL PROPERTIES OF NEURONAL AND GLIAL ISOENZYMES OF BRAIN ENOLASE

Two of the major brain enolase (EC 4.2.1.11) isoenzymes exist as cell specific forms. The neuron specific enolase (NSE) is localized in neurons and the non-neuronal enolase (NNE) in glial cells. A third enolase containing one subunit from each of the above species is also present in brain and has been designated hybrid enolase. The stabilities of the brain enolases towards incubation with chloride and bromide salts is markedly different. NNE is rapidly inactivated upon incubation in 0.5 M-KCI or KBr while NSE is minimally effected and the hybrid has an intermediate stability. The inactivation is temperature dependent and reversible by salt removal. Magnesium exerts a stabilizing effect on each enzyme form. The mechanism of the reversible salt inactivation involves dissociation of the enolase subunits with reassociation occurring during reactivation. The brain enolases also display marked stability differences during incubation in 3 M-urea. with the neuronal form again being more stable. The urea inactivation was highly reversible for NNE but only marginally so for NSE. The neuronal enolase is also by far the most stable of the brain enolases at 50°C.     

Application of proteomics for comparison of proteome of Neospora caninum and Toxoplasma gondii tachyzoites

Protein profiles of two isolates of Neospora caninum (KBA-2 and JPA1) and Toxoplasma gondii RH strain were investigated by proteomic approach. Approximately, 78% of protein spots on two-dimensional gel electrophoresis (2-DE) profiles and 80% of antigen spots on 2-DE immunoblotting profiles were exhibited to share the same pI and M(r) between KBA-2 and JPA1 of N. caninum. On the other hand, a total of 30 antigen spots of T. gondii were recognized on 2-DE immunoblotting profile using rabbit antiserum against N. caninum KBA-2. A number of homologue proteins, such as heat shock protein 70, tubulin alpha- and beta-chain, putative protein disulfide isomerase, actin, enolase and 14-3-3 protein homologue are believed as the conserved proteins in both N. caninum and T. gondii. On the contrary, NcSUB1, NcGRA2 and NCDG1 (NcGRA7) might be the species-specific proteins for N. caninum tachyzoites. The present study showed that the high degree of similarity between N. caninum isolates (KBA-2 and JPA1), whereas large differences between N. caninum and T. gondii were noticed by proteome comparisons.     

Isolation and some physico-chemical properties of a new neurospecific protein 10-40-4 from human brain

A method for isolation of a neurospecific protein 10-40-4 from human brain has been elaborated. This procedure includes immunoaffinity chromatography of a Sepharose 4B-IgG fraction of rabbit antisera against the protein fraction containing the antigen. The isolated protein cannot be detected in protein extracts of various organs and human blood serum by immunochemical methods. This indicates that the protein is specific for nervous tissue. The values of molecular weight (74 000) and pI (4.7) of the isolated protein suggest that the protein does not contain the carbohydrate component and reveals limited tissue specificity. The properties of protein 10-40-4 differ from those of the well-known neurospecific proteins, such as S-100, enolase 14-3-2 and glial fibrillar acid protein GFA.     

Neurospecific and neuron-specific enolase levels in human nerve tissue at early stages of embryogenesis

Neurospecific and neuro-nonspecific isozymes of glycolytic enzyme enolase known to be molecular markers of neuronal and glial cells respectively were isolated from human brain. Using immunoenzyme assay, the content of these proteins was measured in the brain of 7-13-week human embryos and adult subjects. The content of both isoenzymes gradually increases attaining constant level in 10-13-week embryos. Relative content of neuro-nonspecific enolase in the brain of 10-13-week embryos is 1 1/2 times higher than that in adults, whereas the content of neurospecific enolase amounts only to 2% of its level in the adult brain.     

Developmental profile of three enolase isozymes in rat brain determination from one-cell embryo to adult brain

Levels of three enolase isozymes (αα, αγ and γγ) were determined in rat tissues from one-cell embryo to adult brain with a sensitive enzyme immunoassay system. Each embryo of the early stage (gestational age, 0–3 days) contained about 5 × 10^?17 mol of αα enolase. The nervous system-specific αγ and γγ enolases would be detected in the embryos of 6–8 days, which contain no histologically recognizable neurones. The 8-day embryos contained 4.3 × 10^?17 and 3.4 × 10^?16 mol of αγ and γγ enolases. Amounts of all the three enolases were increased with growth of the embryo. The nervous system-specific enolases (αγ and γγ) in the brain kept increasing until 1–2 months of postnatal age, whereas the αα enolase level in the brain was relatively constant after the 15-day embryo through the adult rat.     

STRUCTURAL AND IMMUNOLOGICAL PROPERTIES OF NEURON SPECIFIC PROTEIN (NSP) FROM RAT, CAT AND HUMAN BRAIN: COMPARISON TO BOVINE 14-3-2

Abstract— Neuron specific protein (NSP) has been isolated from cat (NSP-C) and human (NSP-H) brain utilizing the purification procedure described for rat brain 14-3-2 (M arangos et al. , 1975a,b,c), a protein which is now designated NSP-R. The protein as isolated from cat and human brain has a molecular weight of approx 80,000 as determined by sedimentation equilibrium. Sedimentation studies done in the presence of 6mg-HCl and 0.2%β mercaptoethanol yields a protomer M.W. of approx 40,000 for both preparations establishing the dimeric nature of each. The subunits appear identical in each case since one band is observed upon electrophoresis of either preparation in the presence of 8 M-urea. NSP-C and NSP-H have identical isoelectric points of 4.7 making them slightly more acidic than NSP-R (pi = 5.0).
Comparison of NSP-C and NSP-H with NSP-R and bovine 14-3-2 by electrophoretic and immunological criteria revealed that the cat, human and bovine proteins were very similar. NSP-R can be distinguished from the other three preparations electrophoretically and immunologically. The protomer unit of NSP-R differs in amino acid composition from that of the cat, human or bovine proteins since the former can be completely resolved from any of the latter three preparations on 8 M-urea polyacrylamide gels. The data indicate that NSP and bovine 14-3-2 are probably homologous proteins, and establish the general structural properties of NSP.     

Human 14-3-3 Protein: Radioimmunoassay, Tissue Distribution, and Cerebrospinal Fluid Levels in Patients with Neurological Disorders

Abstract: An antiserum to human 14-3-3 protein has been produced in rabbits. The protein was a poor antigen and attempts to improve immunogenicity were unsuccessful. A radioimmunoassay was developed using the antiserum, ¹²⁵I- 14-3-3-2, and unlabelled 14-3-3-2 as standards. The assay had a sensitivity limit of 2.5 ng.m1⁻¹. The minor component of human 14-3-3 protein (14-3-3-1 protein) cross-reacted to approximately 10% in the assay. Human tissues were surveyed for 14-3-3 protein by two-dimensional electrophoresis and by radioimmunoassay. Two-dimensional electrophoresis showed a 14-3-3 protein complex in brain, intestine, and testis, but not in other tissues. Radioimmunoassay showed that although brain had the highest concentration of 14-3-3 (13.3 μg. mg⁻¹ soluble protein), immunoreactivity was present in all tissues, with the concentration in intestine and testis approaching 50% of the brain level. Lower levels (less than 1.0 μg. mg⁻¹ soluble protein) were seen in liver, kidney, skeletal muscle, and erythrocytes. The immunoreactivity present in tissues other than brain showed the same molecular weight and charge characteristics as authentic 14-3-3 protein. The radioimmunoassay also detected 14-3-3 protein in serum (50 ng.m1⁻¹) and in CSF (5-130 ng.ml⁻¹). The immunoreactivity present in CSF appeared to be intact 14-3-3 protein. CSF 14-3-3 levels were measured in 82 patients with various neurological disorders. Measurements of this protein did not appear sufficiently discriminating to be o f diagnostic value.     

Immunological Reactivity of Antisera Prepared Against the Sodium Dodecyl Sulfate-Treated Structural Polypeptides of Adenovirus-Associated Virus

The preparation of antisera to the three purified sodium dodecyl sulfate (SDS)-treated polypeptide components (VP1, VP2, VP3) of adenovirus-associated virus (AAV) type 3H is described. In immunofluorescence tests (FA), these antisera stained heat-stable antigens with distinct morphologies in cells co-infected with either adenovirus or herpes simplex virus. Kinetic studies of antigen formation showed that VP1 antiserum first stained the cytoplasm (14 hr) and later (by 18 hr) stained both cytoplasmic and intranuclear areas. VP2 antiserum stained only discrete intranuclear areas, and VP3 antiserum stained nearly the entire nucleus. All three VP antigens appeared at about the 14th hr postinfection, about 2 hr prior to the appearance of whole virion antigen. The VP antisera cross-reacted in FA with AAV types 1 and 2 (all at one-eighth of the homologous titer), but did not react with other parvoviruses, i.e., rat virus, hemadsorbing enteric virus of calves, minute virus of mice, or H-1 virus. These non-neutralizing antisera reacted specifically with SDS-treated AAV virion antigens in complement fixation and immunodiffusion tests, and antiserum prepared against SDS-treated helper adenovirus structural polypeptides reacted with adenovirus polypeptide antigens. All antisera to SDS-treated polypeptides were specific for new antigens revealed on the dissociated peptides and did not react with whole virions, whereas whole-virion antisera did not cross-react with the polypeptide antigens. These findings suggest that antigens unique to the polypeptides of AAV are revealed by SDS treatment and that these antigens can be detected in cells prior to the folding of the polypeptides into the molecular configuration they possess as virion subunits. These results also indicate that at least one AAV polypeptide component is synthesized in the cell cytoplasm.     

Transition between isozymic forms of enolase during in vitro differentiation of neuroblastoma cells—II

An analysis of enolase expression during differentiation of neuroblastoma clones in homogeneous culture is presented. The enolases expressed in these neuroblast-like cells are identical to those of mouse brain with respect to the examined properties.Our biochemical investigation has premitted us to demonstrate formally that neuroblastoma cells undergo a transition from the embryonic αα form to the neuronal γγ form and contain both enolases as well as the αγ hybrid form during maturation. These results suggest that the same phenomenon must exist in vivo for neuroblasts. In neuroblastoma cells, an increase in both αγ and γγ neuron specific enolases is related to cell maturation and expression of the αγ form precedes that of the γγ form during differentiation. Modulation of neuronal enolase activities is similar in the various conditions of differentiation studied and appears not to be necessarily related with morphological differentiation, although concomitant with an arrest of cell division. The evolution of specific neuronal enolases in neuroblastoma cells parallels that observed in vivo, in brain from embryonic day 15 to post-natal day 7. Moreover, at least one treatment (dimethylsulfoxide) causes an important decrease in the high specific αα activity of these cells as occurs in vivo. This enolase can therefore also be considered as a biochemical marker for neuroblastoma maturation.As observed with other markers and other cell types, neuroblastoma cells in culture express an immature biochemical differentiation of the enolase isozymes.     

Evidence for a new form of enolase in rat brain.

Rat brain enolase (2-phospho-D-glycerate hydrolyase, EC 4.2.1.11) is unaffected by an antiserum raised against rat muscle enolase (isoenzyme 3) and an antiserum raised against rat liver enolase (isoenzyme 1) affects only 70% of the total brain activity. By gradient elution of QAE-Sephadex at pH 8.5, brain enolase is separated into two major peaks. The first is chromatographically and immunochemically identical with isoenzyme 1 whilst the second more complex peak is apparently specific for brain. Gel filtration chromatography on G-150 Sephadex shows no significant difference in molecular weight between these two components.     

Pituitary autoantibodies in lymphocytic hypophysitis target both gamma- and alpha-Enolase - a link with pregnancy?

The first target autoantigen to have been identified in lymphocytic hypophysitis is a 49 kDa protein, identified as alpha-enolase. Pituitary autoimmunity is strongly associated with pregnancy and we have shown that pituitary autoantibodies from patients with peripartum lymphocytic hypophysitis also recognise enolase in the placenta. Enolase exists in different forms as a number of isoenzymes, which are homo- or heterodimers of three subunits, alpha, beta and gamma. alphaalpha-enolase is ubiquitous, betabeta-enolase is muscle-specific and gammagamma-enolase, which is restricted to neuronal tissue and neuroendocrine cells, is known as neuron-specific enolase (NSE). NSE is expressed in normal human pituitary and pituitary neoplasms. The current study investigated which isoforms of enolase in pituitary and placenta reacted with the sera of patients with lymphocytic hypophysitis. Immunoblotting of two-dimensional gels of human pituitary cytosolic proteins showed that autoantibodies in patient sera react with both an acidic form, and more neutral forms of enolase. Immunoblotting with a monoclonal antibody to NSE confirmed the identity of the acidic enolase isoform as the gammagamma-isoform in both pituitary and placental samples. Gamma-enolase, i.e. NSE, was detected by immunohistochemistry in term placenta in decidua, syncytiotrophoblasts, anchoring villi and terminal villi. Our study is the first to describe the cellular localisation of NSE in normal human placenta, thus establishing a direct link between pituitary and placental autoantigens. This link provides a theoretical basis for the strong prediliction of lymphocytic hypophysitis to occur during or after pregnancy.