Heterogeneity in the Phosphorylation of Micro tubule-Associated Protein MAP 1B During Rat Brair Development

Abstract: The patterns of isoforms and of immunoreactivity of the microtubule-associated protein MAP1 B toward a panel of antibodies to phosphorylation-sensitive epitopes are different in distinct rat brain regions and change during development. This suggests the occurrence of a considerable degree of heterogeneity in the phosphorylation state of rat brain MAP1 B. It appears that MAP1 B can be phosphorylated at multiple sites that may be conventionally classified into at least two modes of phosphorylation. Mode I of phosphorylation induces significant upward shifts in the electrophoretic mobility of the protein, giving rise to "high" MAP1B isoforms, whereas the mode II of MAP1B phosphorylation does not greatly affect the electrophoretic mobility of the protein. These MAP1B phosphorylation modes are differentially regulated throughout development and show some regional specificity. Cytosolic MAP1 B is highly phosphorylated both at mode I and mode II sites in the developing rat brain, as well as in the adult olfactory bulb, where axonal growth takes place. In most adult rat brain regions, cytosolic MAP1B is highly phosphorylated at mode II sites but largely dephosphorylated at certain mode I sites. However, MAP1 B present in the particulate fraction of most rat brain region homogenates may be partially dephosphorylated at certain mode II sites, although it contains some phosphorylated mode I sites. These data are compatible with the view that different protein kinases, possibly including casein kinase II and proline-directed protein kinases, might regulate the state of phosphorylation of MAP1B in distinct localizations along the development of different neuronal populations in the brain.     

Developmental Changes in τ Phosphorylation: Fetal τ Is Transiently Phosphorylated in a Manner Similar to Paired Helical Filament-τ Characteristic of Alzheimer's Disease

Rat and human fetal brain τ were probed with a panel of monoclonal antibodies (tau-1, AT8, 8D8, RT97, SMI31, SMI34) that distinguish between paired helical filament (PHF)-τ of Alzheimer's disease and normal adult brain τ. These antibodies discriminate between normal and PHF-τ because their epitopes are phosphorylated in PHF-τ. Although only one molecular isoform of τ was shown to be expressed in fetal brain, two fetal τ species could be distinguished on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the slower migrating species was recognized by all of the PHF-τ-specific antibodies. Moreover, this immunoreactivity was shown to be phosphorylation dependent. Our observations suggest that the abnormal phosphorylation of τ in Alzheimer's disease may be the result of reactivation of pathways governing the phosphorylation of τ in the developing brain.     

Immunochemical evidence that fragments of phosphorylated MAP5 (MAP1B) are bound to neurofibrillary tangles in Alzheimer's disease

To test the hypothesis that cortical neurons undergo massive sprouting in Alzheimer's disease brain, we investigated whether neurofibrillary tangles contain fetal antigens. Two monoclonal antibodies to tangles specifically labeled an approximately 300 kd protein in the neonatal brain homogenate, which was subsequently identified as MAP5 (MAP1B). Conversely, two monoclonal antibodies to MAP5 were found to stain tangles. All four reacted with only a phosphorylated species of MAP5. By careful immunochemical analysis, at least three independent phosphorylated epitopes that should have distinct conformations were shown to be shared by tangles and MAP5. However, several monoclonal antibodies to nonphosphorylated MAP5 did not stain tangles. From these observations, we conclude that fragments of phosphorylated MAP5 are bound to tangles. Since MAP5, in particular, a phosphorylated species, is known to be involved in neurite outgrowth, this result supports the sprouting hypothesis.     

Production and Characterization of Monoclonal Antibodies against Aspartate Aminotransferase-P1 from Lupin Root Nodules

Six hybridoma clones were obtained that secreted monoclonal antibodies against the aspartate aminotransferase-P1 (AAT-P1) isoenzyme from root nodules of Lupinus angustifolius [L.] cv Uniharvest. This enzyme is found constitutively in the plant cytosol fraction. The monoclonal antibodies produced were all of the immunoglobulin G1 class, recognized two distinct epitopes on the protein, and represented the major paratopes found in the immunoglobulin fraction of sera taken from mice and rabbits immunized with the pure AAT-P1 protein. One of these epitopes was unique to lupin nodule AAT-P1. The other epitope was shown to be present on enzyme from lupin bean, white clover and tobacco leaves, lupin roots and nodules, and potato tubers. Both epitopes were recognized by the appropriate monoclonal antibodies in both their native and denatured forms. None of the monoclonal antibodies produced reacted with Rhizobium lupini NZP2257, Escherichia coli extracts, or with the inducible aspartate aminotransferase-P2 (AAT-P2) isoform also found in root nodules. A sandwich enzyme-linked immunosorbent assay utilizing two monoclonal antibodies recognizing the two distinct epitopes was developed and was capable of quantitating AAT-P1 in plant extracts. The limit of detection of AAT-P1 was less than 15 pg/mL and AAT-P1 protein could be quantified in the range 80 to 1000 pg/mL. Using this assay, AAT-P1 protein was shown to remain relatively constant during nodule development. Use of an AAT-P2-specific monoclonal antibody that inhibits the enzyme activity of this isoform enabled the direct determination of AAT-P1 enzyme activity in nodule extracts. Using these assays, specific activities of the individual isoforms were calculated; that of the AAT-P1 isoform was shown to be 7.5-fold higher than that of the AAT-P2 isoform.     

Expression and immunochemical analysis of rat and human fibroblast growth factor receptor (flg) isoforms.

Potentially 96 splice variants among four genes that code for the human heparin-binding fibroblast growth factor receptor family complicate study of structure, metabolism, and function of single isoforms in mammalian cells. As an alternative, we expressed structural subdomains and isoforms of the flg receptor gene in bacteria and baculoviral-infected insect cells. We developed and characterized a panel of 16 isoform and domain-specific polyclonal and monoclonal antibodies. The panel of antibodies was used to distinguish mature glycosylated ligand-binding and kinase-active and -inactive recombinant isoforms in baculoviral insect cells and transfected mammalian cells and natural isoforms in rat prostate and human liver cells. The results revealed a cell type-specific expression of the flg gene and isoforms that result from combinations of splice variations. Reactive epitopes of monoclonal antibodies against both the three (alpha) and two (beta) immunoglobulin-like disulfide loop extracellular domain isoforms were mapped by cross-reactivity with synthetic polypeptide sequences and deletion mutants expressed in bacteria. The native alpha and beta receptor isoforms differed in display of shared epitopes and suggested that the NH2-terminal Loop I and COOH-terminal Loops II and III of the alpha isoform are interactive. Although the common Loops II and III appear qualitatively sufficient for ligand binding, the results suggest that tertiary relationships among loops in the three and two loop isoforms are distinct and, therefore, the two isoforms may have distinct activities. Spatial models for arrangement of immunoglobulin-like loops in the extracellular domain of the two isoforms are presented.     

Localisation of Microtubule-Associated Protein 1B Phosphorylation Sites Recognised by Monoclonal Antibody SMI-31

Abstract: MAP 1B is a microtubule-associated phosphoprotein that is expressed early in neurons and plays a role in axon growth. MAP 1B has two types of phosphoisoforms, one of which is developmentally down-regulated after neuronal maturation and one of which persists into adulthood. Because phosphorylation regulates MAP 1B binding activity, characterisation of the phosphorylation sites and identification of the corresponding kinases/phosphatases are important goals. We have characterised the developmentally down-regulated phosphorylation sites recognised by monoclonal antibody (mAb) SMI-31. We purified MAP 1B from neonatal rat brain and mapped the mAb SMI-31 sites to specific MAP 1B fragments after chemical cleavage. We then developed an in vitro kinase assay by using a high-speed spin supernatant from neonatal rat brain in the presence of ATP and recombinant proteins encoding selective regions of the MAP 1B molecule. Phosphorylation of the recombinant protein was detected on western blots using mAb SMI-31. This analysis showed that mAb SMI-31 recognises two recombinant proteins corresponding to residues 1,109–1,360 and 1,836–2,076 of rat MAP 1B after in vitro phosphorylation. The former phosphorylation site was further defined in the in vitro kinase assay by inhibition with peptides and antibodies from candidate regions of the MAP 1B sequence. This approach identified a region of 20 amino acids, from 1,244 to 1,264, characterised by a high concentration of serines immediately upstream of prolines, indicating that the kinase responsible is a proline-directed serine kinase.     

Claustrin,an antiadhesive neural keratan sulfate proteoglycan,is structurally related to MAP1B

Our laboratory has recently identified a keratan sulfate proteoglycan (KSPG), named claustrin, that inhibits neural cell adhesion and neurite outgrowth in the chick nervous system. Antisera prepared against claustrin were used to screen a cDNA expression library from embryonic day 9 chick brain. Initial characterization of positive cDNAs revealed a high degree of homology to the mouse MAP1B gene, although these cDNAs represent a 5′ truncated fragment of MAP1B. Protein sequencing of three peptides derived from a tryptic digest of purified, keratanase-treated claustrin also revealed strong homology to MAP1B, and confirmed the authenticity of the 3.4 kb claustrin cDNA. To further determine the relationship between these two proteins, we used antibodies against MAP1B and KSPGs in immunoblotting and immunohistochemical studies. These studies demonstrated cross-reactivity between MAP1B and claustrin antibodies, and that monoclonal antibodies to cartilage keratan sulfate react with MAP1B in rat nervous tissue, and with claustrin in the chick nervous system. In addition, keratanase treatment of a taxol microtubule fraction from chick or rat brain eliminated MAP1B, as detected by immunoblotting with the MAP5 monoclonal antibody. These results suggest that MAP1B and claustrin are highly related, if not identical, proteins. 1994 John Wiley & Sons, Inc.     

Two Monoclonal Antibodies Recognize Alzheimer''s Neurofibrillary Tangles, Neurofilament, and Microtubule-Associated Proteins

Two monoclonal antibodies that recognize Alzheimer's neurofibrillary tangles (ANTs), AD10 and AB18, have been characterized by immunoblotting against human and calf spinal cord neurofilament (NF) and calf brain microtubule preparations. Both antibodies bind to the 200-kilodalton (kd) (NF-H) and 160-kd (NF-M) but not to the 68-kd (NF-L) NF triplet proteins. They also bind to high-molecular-weight microtubule-associated proteins (MAPs) and tau. AD10 immunostains MAP2 and MAP1 families, whereas AB18 stains mainly MAP1 bands. Preincubation of intact filament preparation or nitrocellulose strips containing electroblotted NF proteins with Escherichia coli alkaline phosphatase completely blocks AD10 binding and partially blocks binding of AB18. These results suggest that the determinants recognized by these antibodies are phosphorylated. Immunoblotting of peptide fragments generated by limited proteolysis of NF proteins with alpha-chymotrypsin and Staphylococcus aureus V8 protease shows that the localization of the antigenic determinants to AD10 and AB18 in NF-H is approximately 100 and 60 kd, respectively, away from the carboxy terminal, a region previously shown to form the NF projection side arm. In NF-M, the antigenic determinants to both antibodies are located also in the projection side arm, in a 60-kd polypeptide adjacent to the alpha-helical filament core. The results show that ANTs contain at least two phosphorylated antigenic sites that are present in NF and MAPs, a finding suggesting that ANTs may be composed of proteins or their fragments with epitopes shared by cytoskeletal proteins.     

Expression of actin isoforms in developing rat intestinal epithelium

A minimum of six very similar but distinct actin isoforms are encoded by the mammalian genome. Developmental regulation of these genes results in a tissue-specific distribution of the isoforms in the adult. Using a panel of actin specific monoclonal antibodies (MAb), we recently reported the expression of two unique actin isoforms in adult rat intestinal brush border. In this report, we examine the developmental expression of these and other actin isoforms in rat intestinal epithelial cells. Isoforms containing the HUC 1-1 and/or C4 epitopes are present by day 15 of gestation and are continuously expressed throughout adult life. Unexpectedly, the gamma-enteric smooth muscle isoactin, defined by the B4 epitope, is transiently expressed in these non-muscle cells late in gestation. The alpha-vascular smooth muscle isoform, however, is not expressed in intestinal epithelial cells during development and, as previously reported, both smooth muscle isoforms are absent in epithelial cells of adult intestine. In addition, we demonstrate that although multiple isoforms are expressed simultaneously in these cells, they are not uniformly distributed at the subcellular level, suggesting that the cell recognizes the actin isoforms as functionally distinct entities.     

Interaction of phosphorylated tyrosine hydroxylase with 14-3-3 proteins: evidence for a phosphoserine 40-dependent association

Tyrosine hydroxylase (TH) has been reported to require binding of 14-3-3 proteins for optimal activation by phosphorylation. We examined the effects of phosphorylation at Ser19, Ser31 and Ser40 of bovine TH and human TH isoforms on their binding to the 14-3-3 proteins BMH1/BMH2, as well as 14-3-3 zeta and a mixture of sheep brain 14-3-3 proteins. Phosphorylation of Ser31 did not result in 14-3-3 binding, however, phosphorylation of TH on Ser40 increased its affinity towards the yeast 14-3-3 isoforms BMH1/BMH2 and sheep brain 14-3-3, but not for 14-3-3 zeta. On phosphorylation of both Ser19 and Ser40, binding to the 14-3-3 zeta isoform also occurred, and the binding affinity to BMH1 and sheep brain 14-3-3 increased. Both phosphoserine-specific antibodies directed against the 10 amino acids surrounding Ser19 or Ser40 of TH, and the phosphorylated peptides themselves, inhibited the association between phosphorylated TH and 14-3-3 proteins. This was also found when heparin was added, or after proteolytic removal of the N-terminal 37 amino acids of Ser40-phosphorylated TH. Binding of BMH1 to phosphorylated TH decreased the rate of dephosphorylation by protein phosphatase 2A, but no significant change in enzymatic activity was observed in the presence of BMH1. These findings further support a role for 14-3-3 proteins in the regulation of catecholamine biosynthesis and demonstrate isoform specificity for both TH and 14-3-3 proteins.     

Calpain-mediated proteolysis of microtubule associated proteins MAP1B and MAP2 in developing brain

Microtubule associated proteins MAP1B and MAP2 are important components of the neuronal cytoskeleton. During early development of the brain, MAP1B (340 kDa) is present as two isoforms that differ in their level of phosphorylation, while MAP2 is expressed as a single high molecular weight isoform (MAP2B, 280 kDa) and a low molecular weight form (MAP2C, 70 kDa). In this study we examined and compared the sensitivities of MAP1B and MAP2, obtained from MT preparations and brain homogenates of young rats, to degradation by calcium-activated neutral protease, calpain II. We found that in MAPs prepared from microtubules the two isoforms of MAP1B had comparable sensitivity to calpain-mediated proteolysis. Similarly, the high and low molecular weight forms of MAP2 were equally sensitive to digestion by calpain. However, although both MAPs were very susceptible to calpain-mediated proteolysis, MAP1B was more resistant to degradation by calpain than MAP2. Furthermore, the endogenous degradation of MAPs in neonate brain homogenates was calcium-dependent and inhibited by leupeptin, and the pattern of degradation products for MAP1B and MAP2 was similar to that of calpain-mediated proteolysis. These data suggest that calpain can play a role in the regulation of MAPs levels during brain development, in relation to normal neuronal differentiation and disorders associated with neurodegeneration.     

Hexokinase A from mammalian brain: comparative peptide mapping and immunological studies with monoclonal antibodies

Immunological reactivity of partially purified hexokinase A (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) from brain of several vertebrate species has been compared by using enzyme-linked immunosorbent assay and seven monoclonal antibodies raised against the rat brain enzyme. The epitopes recognized by three of these antibodies have been rather widely conserved among the species examined (rat, mouse, guinea pig, rabbit, cat, dog, sheep, cow, pig, chicken), while this was not the case for the epitopes recognized by the other antibodies, which differed markedly in their distribution among these species. The domain structure of these enzymes has been examined by peptide mapping (after limited tryptic digestion) in conjunction with immunoblotting techniques employing monoclonal antibodies. The results indicate that the overall domain structure of these enzymes is similar to that previously described for rat brain hexokinase A, but that there are significant differences in the size of these domains in enzymes from different species.     

Troponin T switching in the developing rat heart

A monoclonal antibody specific for cardiac troponin T has been used to investigate troponin changes during development in the rat heart. Specificity of the antibody was determined by immunoblot analysis with purified bovine cardiac troponin. In the rat heart, immunoblot analysis shows that anticardiac troponin T reacts with a 42.5-kDa band in fetal ventricles and with a 41-kDa band in adult ventricles. The faster migrating troponin T is present in traces in the fetal heart and increases markedly during the first 2 weeks after birth, concomitantly with the progressive decrease of the slower migrating form that is no longer detectable in the adult. The pattern of reactivity of the monoclonal antibody is not modified by alkaline phosphatase pretreatment, suggesting that the antibody is not specific for a phosphorylated epitope. Conditions known to affect cardiac myosin composition, such as hypothyroidism and hypertrophy secondary to systemic hypertension, do not change the troponin T isoform profile of adult rat ventricles. The expression and accumulation of the adult isoforms of troponin T are not suppressed by propylthiouracil treatment of pregnant and nursing rats.     

Monoclonal antibodies against salt-resistant rat liver lipase. Cross-reactivity with lipases from rat adrenals and ovaries

To obtain monoclonal antibodies against rat salt-resistant liver lipase, mice were immunized with enzyme purified from heparin-containing rat liver perfusates. Hybridomas were screened for antibody production by means of an enzyme-linked immunosorbent assay (ELISA) and an immunoprecipitation assay. Five hybridoma cell lines secreting antibodies against rat liver lipase indicated as A, B, C, D and E, have been obtained. All antibodies possess gamma one (gamma 1) heavy chains and kappa (kappa) light chains. The antibodies precipitate salt-resistant lipase from rat post-heparin plasma, are positive in ELISA, inhibit liver lipase activity and bind monospecifically with the enzyme as shown by immunoblotting. The monoclonal antibodies showed no significant reactivity with human liver lipase. The salt-resistant lipases of rat adrenals and ovaries are also precipitated by the monoclonal antibodies directed against the liver enzyme. Therefore, the heparin-releasable lipases of the liver, adrenals and ovaries possess identical epitopes.     

Studies on species cross-reactivity of hemopexin by use of monoclonal and polyclonal antibodies

The extent of immunological cross-reactivity between hemopexins of four species (rat, human, rabbit and chicken) was assessed with four affinity purified polyclonal antibodies and three monoclonal antibodies using RIA, Western blotting and rocket immunoelectrophoresis. Neither the two monoclonal antibodies to rabbit hemopexin (Rb3D11 and Rb3H9), the monoclonal antibody (R4B3) to rat hemopexin nor any of the polyclonal antibodies showed shared antigenic determinants between avian and mammalian hemopexins as judged by RIA or rocket immunoelectrophoresis. Western blotting with polyclonal antibodies revealed some reactivity raising the possibility of a few shared, though distantly related, epitopes. Polyclonal antibodies, raised to the mammalian hemopexins cross-reacted to variable extents with the respective antigens by RIA, results paralleled by data obtained by Western blotting. Anti-rat monoclonal antibodies reacted only with rat hemopexin in Western blots and minimally with rabbit hemopexin in RIA. The anti-rabbit monoclonal antibodies recognized two distinct epitopes one of which is shared with human hemopexin and presumably highly conserved.     

Rapid Dephosphorylation of τ in Heat-Shocked Fetal Rat Cerebral Explants: Prevention and Hyperphosphorylation by Inhibitors of Protein Phosphatases PP1 and PP2A

Abstract: We heat shocked 21- and 35-day-old fetal rat cerebral explants at 45°C for 18 min and performed immunocytochemistry and immunoblot analysis of sodium dodecyl sulfate extracts using the monoclonal anti-τ antibodies Tau-1, Tau-5, Tau-46, and PHF-1 and the peroxidase-antiperoxidase technique or ¹²⁵I-labeled protein A. Tau-1 and PHF-1 recognize nonphosphorylated and phosphorylated epitopes, respectively, and both Tau-5 and Tau-46 recognize phosphate-independent epitopes. τ immunoreactivity was confined to neurons and increased in heat-shocked perikarya but not axons. At 0 h after heat shocking, there was dephosphorylation of τ exemplified by (1) faster migration of τ isoforms with resultant loss or attenuation of the 60- and 52-kDa τ isoforms recognized by all four anti-τ antibodies and concomitant accentuation of the fastest moving 50-kDa τ isoform recognized by Tau-1, Tau-5, and Tau-46; and (2) significant increase in the nonphosphorylated Tau-1 epitope with resultant decreases in the ratio of total (phosphorylated plus nonphosphorylated) τ to nonphosphorylated τ and the difference of total τ minus nonphosphorylated τ. τ was phosphorylated back to the control level by 12 h and remained so at 24 and 48 h after heat shocking. Treatment of explants with cycloheximide, a protein synthesis inhibitor, did not prevent the heat shocking-induced dephosphorylation of τ. Treatment of explants with the inhibitors of protein phosphatases PP1 and PP2A, okadaic acid or calyculin A, produced hyperphosphorylated τ polypeptides, prevented the heat shocking-induced dephosphorylation of τ, and intensified the immunoreactivity of the neurofilament subunit H with the only antiphosphoneurofilament antibody that reacts with intraneuronal neurofibrillary tangles. In 35-day-old explants, in addition to the three 50-, 52-, and 60-kDa τ isoforms seen in 21-day-old explants, a 66-kDa τ polypeptide was also present.     

Identification of two distinct microtubule binding domains on recombinant rat MAP 1B.

A set of partially overlapping cDNA clones covering 9 kb of continuous sequence encoding the high molecular weight microtubule-associated protein (MAP) 1B, was isolated from a rat brain library in lambda gt11. The protein encoded was immunoreactive with monoclonal antibodies raised against calf MAP 1B, rat MAP 1X, and rat MAP 5, as shown by immunoblotting. Using Northern blot analysis, it was shown that the level of MAP 1B mRNA increased dramatically upon nerve growth factor-induced PC12 cell differentiation. The expression of polypeptides encoded by cDNA constructs, in conjunction with microtubule binding assays, revealed two separate microtubule binding domains, corresponding to sequences at the 5' and 3' end of the mRNA. As shown by DNA sequencing, the binding domain encoded by 5' terminal sequences consisted of the basic repeat motif KKEE(I/V), previously identified in mouse MAP 1B (Noble, M., S. A. Lewis, N. J. Cowan, J. Cell Biol. 109, 3367-3376 (1989)). The second binding domain, too, was found to be basic, but without any apparent repeat structure. It is concluded that single proteolytically unprocessed MAP 1B molecules would have the potential to function as microtubule cross-linkers.     

Distribution of anti-Leu-7, anti-Leu-11a and anti-Leu-M1 immunoreactivity in the brain of the adult rat

Summary This study reports a specific cross-reactivity of the three anti-human-hematopoetic-cell monoclonal antibodies, anti-Leu-7 (HNK-1), anti-Leu-11a (NKP-15), and anti-Leu-M1 (MMA), with different epitopes in the brain of the adult rat. The distribution of these epitopes in rat brain is determined by means of immunohistochemistry in paraffin-embedded frontal serial sections.The reaction pattern of anti-Leu-11a monoclonal antibody is very similar to that of polyclonal antibodies against the myelin basic protein. Both antisera give a specific reaction with myelinated fibers. Immunoreaction products with the anti-Leu-7 monoclonal antibody are found as diffuse, mostly punctiform material in the neuropil and even more evident as small granules coating the cell surface of many neurons. In the white matter anti-Leu-7 reveals a moderate reactivity, which occurs predominantly as spots and fine-stranded material within the myelinated fiber tracts.Anti-Leu-M1 immunoreactivity is present between myelinated fiber bundles of the white matter, where it has a reticulate appearance, and as fine-granulated material within the grey matter of the cortex and the nuclei. The characteristic feature in the grey matter is that of irregularly shaped immunopositive plaques, which are often located around small blood vessels. The cytoplasm of glial and neuronal cells appeared negative with this MAB.The exact topographical distribution of the Leu-7 and Leu-M1 epitopes throughout the rat brain is described. The present hypotheses concerning the nature of this shared antigenicity between hematopoetic cells and nervous tissue are discussed.Supported by the Deutsche Forschungsgemeinschaft, SFB 200