Production and Characterization of Monoclonal Antibodies against Aspartate Aminotransferase-P(2) from Lupin Root Nodules

Twenty-one monoclonal antibodies were raised against the aspartate aminotransferase-P₂ isoenzyme from root nodules of Lupinus angustifolius [L.] cv Uniharvest. Induction of this isoenzyme is positively correlated with the onset of N₂ fixation in effective root nodules and is associated with the assimilation of ammonia by the plant in the Rhizobium-legume symbiosis. The monoclonal antibodies produced were all of the IgG class, recognized five different epitopes on the protein, and represented greater than 90% of the available epitopes. These epitopes were not unique to lupin nodule aspartate aminotransferase-P₂ but were shown to be present on the enzyme from tobacco leaves and potato. Four of the epitopes were conformational with a fifth epitope recognized by the appropriate monoclonals in both its native and denatured forms. None of the monoclonal antibodies produced reacted with Rhizobium Iupini NZP2257 extracts. Antibodies against two epitopes showed some cross-reaction with the constitutive aspartate aminotransferase-P₁ isoenzyme also found in lupin root nodules. However, affinity of these monoclonals for AAT-P₁ was three orders of magnitude lower than for AAT-P₂. Monoclonals against the other epitopes appeared to be specific for aspartate aminotransferase-P₂.     

Production, characterization, and applications of monoclonal antibodies reactive with soybean nodule xanthine dehydrogenase

Seven monoclonal antibodies were produced against soybean nodule xanthine dehydrogenase, an enzyme involved in ureide synthesis. Specificity of the seven monoclonal antibodies for xanthine dehydrogenase was demonstrated by immunopurifying the enzyme to homogeneity from a crude nodule extract using antibodies immobilized to Sepharose 4B beads. Each monoclonal antibody was covalently bound to Sepharose 4B beads for the preparation of immunoaffinity columns for each antibody. All seven antibodies were found to be of the IgG1,K subclass. A competitive, indirect enzyme-linked immunosorbent assay demonstrated that two of the seven antibodies shared a common epitope while the remaining five antibodies defined unique determinants on the protein. Rapid, large scale purification of active xanthine dehydrogenase to homogeneity was performed by immunoaffinity chromatography. The presence of xanthine dehydrogenase activity and protein in every organ of the soybean plant was determined. Crude extracts of nodules, roots, stems, and leaves cross-reacted with all seven monoclonal antibodies in an indirect enzyme-linked immunosorbent assay. A positive correlation was observed between the degree of cross-reactivity of a given organ and the level of enzyme activity in that organ. These data demonstrate that xanthine dehydrogenase is not nodule specific. Antigenic variability of xanthine dehydrogenase present in crude extracts from nodules of soybean, wild soybean, cowpea, lima bean, pea, and lupin were detected in the indirect enzyme-linked immunosorbent assay which corresponded to six binding patterns for xanthine dehydrogenase from these plant species. These results correspond well with the epitope determination data which showed that the seven antibodies bind to six different binding determinants on the enzyme.     

Cloning and characterization of a cDNA encoding aspartate aminotransferase-P1 from Lupinus angustifolius root tips.

A root tip cDNA library, constructed in the lambda Zap II expression vector, was immunoscreened with a monoclonal antibody raised against aspartate aminotransferase-P1 from Lupinus angustifolius L. var Uniharvest. One 1452-base pair clone was isolated. The encoded protein sequence had high homology to both plant and animal aspartate aminotransferase sequences. The clone was converted to the phagemid form and expressed in Escherichia coli. The expressed protein was enzymically active and could be immunocomplexed with aspartate aminotransferase-P1-specific antibodies. The complete aspartate aminotransferase-P1 cDNA was cloned into the yeast expression vector pEMBL-yex4 and transformed into Saccharomyces cerevisiae strain BRSCS6, which possesses a mutated aspartate aminotransferase gene (the asp5 mutation). Complementation of the mutation was achieved using this construct.     

Subcellular localization of glycoprotein epitopes during the development of lupin root nodules

Summary The monoclonal antibodies MAC236 and MAC265, raised against a soluble component of pea nodules, were used to elucidate the presence and subcellular localization of glycoprotein epitopes during the development of lupin (Lupinus albus L. cv. Multolupa) nodules, by means of immunocytochemistry and Western blot analysis. These antibodies recognize a single band of 95 kDa in pea, soybean and bean nodules, whilst two different bands of 240 and 135 kDa cross-react with MAC236 and MAC265 respectively in lupin nodules. This fact may indicate that the recognized epitopes can be present in different subcellular compartments and/or play different roles through the development of functional nodules. The results show that MAC265 is mainly associated with Bradyrhizobium infection and with the development of nodule primordium, in the first stages of nodulation. MAC265 is also detected when glycoprotein transport takes place across the cytoplasm and the cell wall, and also in the intercellular spaces of the middle cortex, attached to cell walls. The amount of MAC265 remains constant through nodule development. In contrast the amount of MAC236 increases with nodule age, parallel to the establishment of nitrogenase activity. This antibody is localized in cytoplasmic globules attached to the inner side of cell walls in the middle cortex, and mainly in the matrix filling the intercellular spaces of the middle and inner cortex. This main site of localization of MAC236 may indicate a role in the functioning of the oxygen diffusion barrier.     

Temporal Relationships between Nitrogenase and Intercellular Glycoprotein in Developing White Lupin Nodules

The development of the N₂-fixing symbiosis between white lupin (Lupinus albusL.) cv. Multolupa andBradyrhizobiumstrain ISLU16 was followed using the acetylene reduction assay (ARA), immunoblots of protein extracts, and microscopy/immunogold labelling at 0, 8, 12, 17 and 20 d after infection. There was no ARA at 0, 8 and 12 d, although macroscopically visible nodule primordia had formed on roots by 8 d. The lack of nitrogenase at these times was confirmed by a negative signal to immunogold labelling with nitrogenase-specific antibodies. At 17 d three out of six plants had ARA, and nodules from these gave a positive signal with the nitrogenase antibody. By contrast, ARA⁻(fix⁻) nodules at 17 d were smaller (mean radius of 0.49 mm compared to 1.01 mm with fix⁺nodules) and gave a negative signal with the nitrogenase antibody. Western blots of nodule protein extracts using the monoclonal antibodies MAC236 and MAC265 (which recognize two epitopes on a glycoprotein which is considered to be involved in both rhizobial infection and the regulation of nodule oxygen diffusion) gave a strong signal with nodules (fix⁺) from 20 d plants and with 17 d fix⁺plants. The signal with MAC236/MAC265 was substantially weaker with nodules from 17 d fix⁻plants, and there was no signal apparent from nodules/nodulated roots from the 0, 8 and 12 d harvests. However, further investigation using immunogold labelling revealed that not only were MAC236 and MAC265 expressed within cortical intercellular spaces in 20 d and 17 d fix⁺/fix⁻nodules, but they were also strongly expressed in the developing cortex surrounding the newly-infected tissue in 8 d nodules, as well as in intercellular spaces within the cortex and infected tissue of 12 d nodules. These data demonstrate that the glycoprotein recognized by MAC236 and MAC265 is present before the onset of nitrogenase expression and function, but expression of the epitopes appears to be enhanced from the onset of N₂fixation. Nodules at all harvests were investigated for the presence of infection threads, as the MAC236/MAC265-recognized glycoprotein is also a component of the infection thread matrix in nodules from other legumes. Infection threads were not seen in nodules from any of the harvests except for the 20 d nodules, and then only after serial sectioning. The latter revealed occasional short wide infection threads entering and releasing rhizobia into small pockets of uninfected cells, within the infected tissue, but not within the meristems. The matrix of these infection threads labelled weakly, or not at all, with MAC236 and MAC265, and it was concluded that the majority of the MAC236/MAC265 detected in lupin nodule extracts originated from glycoprotein within cortical intercellular spaces.     

Lipopolysaccharide epitope expression of Rhizobium bacteroids as revealed by in situ immunolabelling of pea root nodule sections.

To investigate the in situ expression of lipopolysaccharide (LPS) epitopes on nodule bacteria of Rhizobium leguminosarum, monoclonal antibodies recognizing LPS macromolecules were used for immunocytochemical staining of pea nodule tissue. Many LPS epitopes were constitutively expressed, and the corresponding antibodies reacted in nodule sections with bacteria at all stages of tissue infection and cell invasion. Some antibodies, however, recognized epitopes that were only expressed in particular regions of the nodule. Two general patterns of regulated LPS epitope expression could be distinguished on longitudinal sections of nodules. A radial pattern probably reflected the local physiological conditions experienced by endosymbiotic bacteria as a result of oxygen diffusion into the nodule tissue. The other pattern of expression, which followed a linear axis of symmetry along a longitudinal section of the pea nodule, was apparently associated with the differentiation of nodule bacteria and the development of the nitrogen-fixing capacity in bacteroids. Basically similar patterns of LPS epitope expression were observed for pea nodules harboring either of two immunologically distinct strains of R. leguminosarum bv. viciae, although these epitopes were recognized by different sets of strain-specific monoclonal antibodies. Furthermore, LPS epitope expression of rhizobia in pea nodules was compared with that of equivalent strains in nodules of French bean (Phaseolus vulgaris). From these observations, it is suggested that structural modifications of Rhizobium LPS may play an important role in the adaptation of endosymbiotic rhizobia to the surrounding microenvironment.     

Molecular cloning of a cDNA encoding aspartate aminotransferase-P2 from lupin root nodules

Two isoenzymic forms of aspartate aminotransferase are present in the plant fraction of developing lupin root nodules. One of these forms, aspartate aminotransferase-P₂ (AAT-P₂), increases dramatically with the onset of biological nitrogen fixation and is associated with the assimilation of ammonia by the plant in the Rhizobium-legume symbiosis. A day 18 lupin nodule cDNA library in the ZapII vector was immunoscreened with a monoclonal antibody specific for AAT-P₂ and yielded two near-full-length 1700 bp clones. These clones were sequenced. Amino acid sequences from three peptides derived from immunopurified AAT-P₂ were aligned, and showed 100% homology with the amino acid sequence deduced from the cDNA clones. The DNA sequence showed 50% homology with AAT sequences from a range of animal sources. Conversion of the clones to the phagemid form allowed their expression in Escherichia coli where both exhibited enzyme activity that could be immunoprecipitated with AAT-P₂-specific monoclonal antibodies. Western blot analysis revealed protein moieties with molecular masses of 39, 43, 45 and 55 kDa. The 5 end of the clones coded for a hydrophobic leader sequence of about 50 amino acids indicative of a targeting sequence and consistent with the plastid localisation of nodule AAT-P₂.     

Characterization and application of monoclonal antibodies directed to separate epitopes of glutathione-insulin transhydrogenase

Five monoclonal antibodies specific for glutathione-insulin transhydrogenase were characterized. None of the monoclonal antibodies cross-reacted with another insulin-degrading enzyme, neutral thiopeptidase. The isotype of four antibodies was IgG1 and of the fifth IgG2b. Affinity studies, competitive binding studies and immunoblot analysis of CNBr and trypsin cleavage products of glutathione-insulin transhydrogenase demonstrated that the four IgG1 antibodies were directed to an epitope of the enzyme which was distinct from the epitope recognized by the IgG2b antibody. Inhibition studies indicated that each monoclonal antibody, when added singly to glutathione-insulin transhydrogenase, was unable to inhibit the insulin-degrading activity of the enzyme. However, when monoclonal antibodies directed against separate epitopes of glutathione-insulin transhydrogenase were presented together (i.e., the IgG2b with any one of the four IgG1 antibodies), a loss in enzymatic activity was noted. Immunoblot analysis of rat organ extracts with the IgG1 antibodies demonstrated one immunoreactive protein band of Mr 56,000 in all tissues examined (liver, fat, pancreas and kidney) except the spleen, which demonstrated two immunoreactive protein bands of Mr 56,000 and 51,000. The same immunoblots, when probed with the IgG2b antibody, demonstrated the same immunoreactive protein banding pattern as above plus an additional immunoreactive protein band of Mr 67,000 in all tissues. Studies with spleen extracts from steptozotocin-induced diabetic rats demonstrated that there was a loss of the 51,000 immunoreactive band in diabetes. This 51,000 protein was restored upon insulin treatment of the diabetic rats and nullified upon concomitant administration of cycloheximide or actinomycin D with insulin. Immunoblots of human liver, adipose and skeletal muscle extracts indicated that each monoclonal antibody cross-reacted with the human form of the enzyme which had a molecular weight of Mr 63,000; a second minor immunoreactive band of 67,000 was detected with the IgG2b antibody. The physiological significance of additional molecular forms of the enzyme (i.e., 67,000 and 51,000) remains to be determined.     

The aberrant cell walls of boron-deficient bean root nodules have no covalently bound hydroxyproline-/proline-rich proteins.

B-deficient bean (Phaseolus vulgaris L.) nodules examined by light microscopy showed dramatic anatomical changes, mainly in the parenchyma region. Western analysis of total nodule extracts examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that one 116-kD polypeptide was recognized by antibodies raised against hydroxyproline-rich glycoproteins (HRGPs) from the soybean (Glycine max) seed coat. A protein with a comparable molecular mass of 116 kD was purified from the cell walls of soybean root nodules. The amino acid composition of this protein is similar to the early nodulin (ENOD2) gene. Immunoprecipitation of the soybean ENOD2 in vitro translation product showed that the soybean seed coat anti-HRGP antibodies recognized this early nodulin. Furthermore, we used these antibodies to localize the ENOD2 homolog in bean nodules. Immunocytochemistry revealed that in B-deficient nodules ENOD2 was absent in the walls of the nodule parenchyma. The absence of ENOD2 in B-deficient nodules was corroborated by performing hydroxyproline assays. Northern analysis showed that ENOD2 mRNA is present in B-deficient nodules; therefore, the accumulation of ENOD2 is not affected by B deficiency, but its assembly into the cell wall is. B-deficient nodules fix much less N2 than control nodules, probably because the nodule parenchyma is no longer an effective O2 barrier.     

Partial purification and characterization of NADH-glutamate synthase from faba bean (Vicia faba) root nodules

The NADH-dependent glutamate synthase (EC 1.4.1.14) from the plant fraction of N₂-fixing faba bean (Vicia faba) nodules has been purified 74-fold to a specific activity of about 3 μmol min⁻¹ mg protein⁻¹ with a final yield of 32%. The NADH-GOGAT activity was associated with a single form of the enzyme that behaved as a monomeric protein with a subunit molecular weight of 195 kDa and a native molecular weight from 222 to 236 kDa estimated by gel filtration or PAGE, respectively. The NADH-GOGAT band on SDS-PAGE was cut out and used to produce antibodies. Western blots of SDS-PAGE of crude nodule proteins revealed a 195 kDa polypeptide in root extracts but not in those of leaves or bacteroids. The antiserum also cross-reacted with a polypeptide of camparable molecular weight (195 kDa) from both amide and ureide transporting species legume nodules, indicating that some antigenic epitopes have been conserved between nodule NADH-GOGAT of different species.     

Immunogold localization of nodule-enhanced phosphoenolpyruvate carboxylase in alfalfa

Phosphoenolpyruvate carboxylase (PEPC; EC 4-1-1-31) plays a paramount role in providing carbon for synthesis of malate and aspartate in alfalfa (Medicago sativa L.) root nodules. PEPC protein and activity levels are highly enhanced in N₂-fixing alfalfa nodules. To ascertain the relationship between the cellular location of PEPC and root nodule metabolism, enzyme localization was evaluated by immunogold cytochemistry using alfalfa nodule PEPC antibodies. Gold labelling patterns in effective nodules showed that PEPC is a cytosolic enzyme and is distributed relatively equally in infected and uninfected cells of the nodule symbiotic zone. A high amount of labelling was also observed in pericycle cells of the nodule vascular system. Labelling was also detected within inner cortical cells, but the density was reduced by 60%. When Lotus corniculatus was transformed with a chimeric gene consisting of the 5′-upstream region of the PEPC gene fused to β-glucuronidase (GUS), GUS staining in nodules was consistent with immunogold localization patterns. The occurrence of PEPC in both infected and uninfected cells of the symbiotic zone of effective nodules coupled to the reduced amounts in ineffective nodules suggests a direct role for this enzyme in supporting N₂-fixation. PEPC localization in the uninfected, interstitial cells of the symbiotic zone indicates that these cells may also have a role in nodule carbon metabolism. Moreover, the association of PEPC with the nodule vascular system implies a role for the enzyme in the transport of assimilates to and from the shoot.     

Lectin-like glycoprotein PsNLEC-1 is not correctly glycosylated and targeted in boron-deficient pea nodules

Symbiosome development was studied in pea root nodules from plants growing in the absence of boron (B). Rhizobia released into the host cells of nodules from B-deficient plants developed to abnormal endophytic forms with an altered electrophoretic lipopolysaccharide pattern. Immunostaining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotting of nodule homogenates with antibodies that recognize glycoprotein components showed that two previously described lectin-like glycoproteins (PsNLEC-1A and PsNLEC-1B) did not harbor the carbohydrate epitope normally recognized by specific monoclonal antibodies. Material derived from B-deficient nodules, however, still contained three antigenic isoforms with similar electrophoretic mobilities to PsNLEC-1 isoforms A, B, and C. These could be detected following immunoblotting and immunostaining with a specific antiserum originating from the purified PsNLEC protein that had been heterologously expressed in Escherichia coli. Immunogold localization of PsNLEC-1 sugar epitopes in B-deficient nodules showed that they were associated mostly with cytoplasmic vesicles rather than normal localization in the symbiosome compartment of mature infected cells. These results suggest that a modification of the glycosyl-moieties of PsNLEC-1 and an alteration of vesicle targeting occur during the development of pea nodules in the absence of B, and that these changes are associated with the development of aberrant nonfunctional symbiosomes.     

Preparation and characterization of monoclonal antibodies against native membrane-bound penicillin-binding protein 1B of Escherichia coli.

We prepared monoclonal antibodies against penicillin-binding protein 1B (PBP 1B) of Escherichia coli to study the membrane topology, spatial organization, and enzyme activities of this protein. The majority of the antibodies derived with PBP 1B as the immunogen reacted against the carboxy terminus. To obtain monoclonal antibodies recognizing other epitopes, we used PBP 1B lacking the immunodominant carboxy-terminal 65 amino acids as the immunogen. Eighteen monoclonal antibodies directed against membrane-bound PBP 1B were isolated and characterized. The epitopes recognized by those monoclonal antibodies were located with various truncated forms of PBP 1B. We could distinguish four different epitope areas located on different parts of the molecule. Interestingly, we could not isolate monoclonal antibodies against the amino terminus, although they were specifically selected for. This is attributed to its predicted extreme hydrophilicity and flexibility, which could make the amino terminus very sensitive to proteolytic degradation. All antibodies reacted against native PBP 1B in a dot-blot immunobinding assay. One monoclonal antibody also recognized PBP 1B in a completely sodium dodecyl sulfate-denatured form. This suggests that all the other monoclonal antibodies recognize conformational epitopes. These properties make the monoclonal antibodies suitable tools for further studies.     

Protection against Streptococcus equi infection by monoclonal antibodies against an M-like protein.

We have developed an in vivo passive transfer assay using mice to identify monoclonal antibodies (mAbs) which offer protection against Streptococcus equi infection. The assay was developed using serum antibodies collected from horses convalescing from strangles. In this study, we show that a preparation of M-like protein, acid-extracted from S. equi, affords 80% protection to mice immunized with it. A number of mouse mAbs directed against a preparation of M-like protein were then assessed for their ability to passively protect mice against challenge with a lethal dose of the bacteria. Two mAbs, 1D10 and 2A6, were shown to be highly protective. It was also demonstrated, by means of a competitive enzyme immunoassay, that these mAbs recognized different epitopes in the preparation. Examination of a dose-response curve for mAbs 1D10 and 2A6 revealed that optimal levels of protection were achieved using 1 mg of either 1D10 or 2A6, or 0.5 mg 1D10 and 0.5 mg 2A6 given together. Immunological reactivity of these mAbs with a preparation of M-like protein showed that the antigens they recognized were comparable in size to some of the antigens recognized by convalescent horse serum antibodies. The role of immunoglobulin isotype in conferring protection is discussed.     

Immunological evidence for a conformational difference between recombinant bovine rhodanese and rhodanese purified from bovine liver

Rhodanese has been utilized as a model enzyme for the study of protein structure-function relationships. The enzyme has recently been cloned and the recombinant enzyme is now available for investigation. However, prior to use in structure-function studies, the recombinant enzyme must be shown to have the same structure and activity as the bovine liver enzyme used in the previous studies. An immunological study of the conformations of these enzyme conformers is described. Three antibodies (two monoclonal and one polyclonal, site-directed antibody) were shown to detect distinct and nonoverlapping epitopes. The epitopes of the monoclonal antirhodanese antibodies (R207 and MAB11) were mapped to the same CNBr digest fragment of the amino terminal domain of rhodanese, and the epitope of the site-directed antibody prepared against the interdomain tether sequence of rhodanese (PAT-T1) was mapped to that region of rhodanese (residues 142–156). The rhodanese conformers were studied by monitoring the accessibility of the epitopes recognized by each antibody in each conformer using an indirect ELISA. None of the antibodies could detect its epitope on the purified liver enzyme. Two of the antibodies (R207 and PAT-T1) could also not detect their epitopes on the recombinant enzyme. However, MAB11 did detect a conformational difference between the natural and recombinant rhodanese conformers, indicating the conformational difference is localized in the first 73 amino acids of rhodanese. This difference presumably reflects the difference in the histories of the two enzymes and may be due to differences in enzyme folding, differences in the purification procedures, and differences in storage conditions—all of which could influence the final conformation of the enzyme.     

Identification and synthesis in vitro of plant-specific proteins in yellow lupin root nodules

Fifteen to twenty specific polypeptides of Mr ranging from 15 000 to 90 000 were detected using immunochemical techniques in the lupin root nodules and cell-free translation products of the nodule polysomal RNA. These polypeptides were characteristic for symbiotic state of the host plant and represented 9-18% of total nodule proteins. They were absent in uninfected roots and in protein extracts of Rhizobium lupini.     

Neutralizing human monoclonal antibodies to conformational epitopes of human T-cell lymphotropic virus type 1 and 2 gp46.

Ten human monoclonal antibodies derived from peripheral B cells of a patient with human T-cell lymphotropic virus (HTLV)-associated myelopathy are described. One monoclonal antibody recognized a linear epitope within the carboxy-terminal 43 amino acids of HTLV gp21, and two monoclonal antibodies recognized linear epitopes within HTLV type 1 (HTLV-1) gp46. The remaining seven monoclonal antibodies recognized denaturation-sensitive epitopes within HTLV-1 gp46 that were expressed on the surfaces of infected cells. Two of these antibodies also bound to viable HTLV-2 infected cells and immunoprecipitated HTLV-2 gp46. Virus neutralization was determined by syncytium inhibition assays. Eight monoclonal antibodies, including all seven that recognized denaturation-sensitive epitopes within HTLV-1 gp46, possessed significant virus neutralization activity. By competitive inhibition analysis it was determined that these antibodies recognized at least four distinct conformational epitopes within HTLV-1 gp46. These findings indicate the importance of conformational epitopes within HTLV-1 gp46 in mediating a neutralizing antibody response to HTLV infection.     

Alfalfa root nodule phosphoenolpyruvate carboxylase: characterization of the cDNA and expression in effective and plant-controlled ineffective nodules

Phosphoenolpyruvate carboxylase (PEPC) plays a key role in N₂ fixation and ammonia assimilation in legume root nodules. The enzyme can comprise up to 2% of the soluble protein in root nodules. We report here the isolation and characterization of a cDNA encoding the nodule-enhanced form of PEPC. Initially, a 2945 bp partial-length cDNA was selected by screening an effective alfalfa nodule cDNA library with antibodies prepared against root nodule PEPC. The nucleotide sequence encoding the N-terminal region of the protein was obtained by primer-extension cDNA synthesis and PCR amplification. The complete amino acid sequence of alfalfa PEPC was deduced from these cDNA sequences and shown to bear striking similarity to other plant PEPCs. Southern blots of alfalfa genomic DNA indicate that nodule PEPC is a member of a small gene family. During the development of effective root nodules, nodule PEPC activity increases to a level that is 10- to 15-fold greater than that in root and leaf tissue. This increase appears to be the result of increases in amount of enzyme protein and PEPC mRNA. Ineffective nodules have substantially less PEPC mRNA, enzyme protein and activity than do effective nodules. Maximum expression of root nodule PEPC appears to be related to two signals. The first signal is associated with nodule initiation while the second signal is associated with nodule effectiveness. Regulation of root nodule PEPC activity may also involve post-translational processes affecting enzyme activity and/or degradation.     

Changes in Microtubule-Associated Protein MAP1B Phosphorylation During Rat Brain Development

Microtubule-associated protein MAP1B from neonatal rat brain was separated on sodium dodecyl sulfate-containing polyacrylamide gels into two isoforms (high and low MAP1B), both of which were recognized by a panel of monoclonal and polyclonal antibodies against MAP1B. In addition, SMI31, a monoclonal antibody directed against phosphorylated epitopes of the neurofilament proteins, showed phosphatase-sensitive reactivity against the high isoform of MAP1B. The antigenic relationship between the phosphorylated isoform of MAP1B and neurofilaments was confirmed by the reactivity of SMI31 with the immunoprecipitated MAP1B protein. After dephosphorylation of MAP1B with alkaline phosphatase, the higher-molecular-weight isoform of MAP1B was no longer detectable with phosphate-insensitive anti-MAP1B antibodies, whereas there was a significant increase in the immunoreactivity of the lower-molecular-weight MAP1B isoform. These data suggest that the structural microheterogeneity of MAP1B is due to differences in phosphorylation. The two isoforms were present in all brain regions of the young rat. During brain development, the general decrease in MAP1B levels was accompanied by changes in the relative amount of the two isoforms. In particular, the phosphorylated isoform of MAP1B decreased dramatically to almost undetectable levels in adult brain. This conclusion was further supported by immunoblotting analysis that showed the disappearance of phosphorylated epitopes of MAP1B early during brain development. In addition, dephosphorylation experiments demonstrated the phosphatase sensitivity of the phosphorylated isoform throughout development.     

Identification of two novel B-cell epitopes on the nucleocapsid protein of porcine deltacoronavirus

Highlights
1. Two monoclonal antibodies against newly emerged porcine deltacoronavirus nucleocapsid protein were prepared.
2. The epitopes that these two monoclonal antibodies recognized on nucleocapsid protein were identified.
3. The monoclonal antibody 6B7 recognized a linear epitope of N protein, while the 7F8 recognized a conformational epitope.
4. Conservation of the identified epitopes between different coronaviruses was analyzed.