Common antigenic determinants of the tubulin binding domains of the microtubule-associated proteins MAP-2 and tau.

The structural-functional aspects of the tubulin binding domain on the microtubule-associated protein MAP-2, and its relationship with the tubulin binding domain on tau, were studied using anti-idiotypic antibodies that react specifically with the epitope(s) on MAPs involved in their interaction with tubulin in addition to other tau and MAP-2 specific antibodies. Previous studies showed that MAP-2 and tau share common binding sites on tubulin defined by the peptide sequences alpha (430-441) and beta (422-434) of tubulin subunits. Furthermore, binding experiments revealed the existence of multiple sites for the interaction of the alpha- and beta-tubulin peptides with MAP-2 and tau. Most recent studies showed that the synthetic tau peptide Val187-Gly204 (VRSKIGSTENLKHQPGGG) from the repetitive sequence on tau defines a tubulin binding site on tau. Our present immunological studies using anti-idiotypic antibodies which interact with the synthetic tau peptide and antibodies against the Val187-Gly204 tau peptide indicate that MAP-2 and tau share common antigenic determinants at the level of their respective tubulin binding domains. These antigenic determinants appear to be present in the 35 kDa tubulin binding fragment of MAP-2 and in 18-20 kDa chymotryptic fragments containing the tubulin binding site(s) on MAP-2. These findings, along with structural information on these proteins, provide strong evidence in favor of the hypothesis that tubulin binding domains on MAP-2 and tau share similar structural features.     

Structural homology of microtubule-associated proteins 1 and 2 demonstrated by peptide mapping and immunoreactivity

The major high molecular weight microtubule-associated polypeptides from hog brain (MAP-1 and MAP-2) were compared by one- and two-dimensional peptide mapping under varied conditions and by immunological techniques. Partial digestion of MAP-1 and MAP-2 with Staphylococcus aureus V8 protease and analysis in one dimension gave rise to very similar peptide maps independent of whether 125I-, 3H-, or 32P-labeled proteins were used. One-dimensional cleavage patterns of significant similarity were also obtained by partial digestion of MAP-1 and MAP-2 using trypsin or chymotrypsin. Furthermore, a pronounced similarity, although clear nonidentity, of MAP-1 and MAP-2 was also revealed after exhaustive digestion of 125I-labeled proteins with S. aureus V8 protease or trypsin followed by analysis of peptides in two dimensions. For immunological comparison, antisera were used that had been raised in rabbits using electrophoretically purified MAP-1 and MAP-2 components as immunogens. As determined by immunoprecipitation, the antiserum raised to MAP-1 was equally reactive with MAP-1 and MAP-2 components, whereas the antiserum to MAP-2 reacted primarily with MAP-2. Indicating the presence of common as well as unique antigenic determinants on MAP-1 and MAP-2, these results, therefore, were in agreement with the peptide mapping data. Implications of these results for biosynthetic mechanisms as well as differential distribution and functions of MAPs in cells are discussed.     

Purification and characterization of the high molecular weight microtubule associated proteins from neonatal rat brain

The changes in the levels of microtubule-associated proteins (MAPs) during advanced embryonic stages, neonatal and adult organisms reflect the importance of these cytoskeletal proteins in relation to the morphogenesis of the central nervous system. MAP-1B is found in prenatal brains and it appears to have the highests levels in neonatal rat brains, being a developmentally-regulated protein. In this research, a fast procedure to isolate MAP-1B, as well as MAP-2 and MAP-3 from neonatal rat brains was designed, based on the differential capacity of poly L-aspartic acid to release MAPs during temperature-dependent cycles of microtubule assembly in the absence of taxol. The high molecular weight MAP-1B was recovered in the warm supernatants after microtubular protein polymerization in the presence of low concentrations of polyaspartic acid. Instead, MAP-2 and a 180 kDa protein with characteristics of MAP-3 remained associated to the polymer after the assembly. Further purification of MAP-1B was attained after phosphocellulose chromatography. Isolation of MAP-2 isoforms together with MAP-3 was achieved on the basis of their selective interactions with calmodulin-agarose affinity columns. In addition, MAP-2 and MAP-3 were also purified on the basis of their capacities to interact with the tubulin peptide -II (422–434) derivatized on an Affigel matrix. However, MAP-1B did not interact with the -II tubulin fragment, but it showed interaction with the Affigel-conjugated -I (431–444) tubulin peptide. The different MAPs componentes were characterized by western blots using specific monoclonal antibodies. A salient feature of neonatal rat brain MAP-3 was its interactions with site-directed antibodies that recognize binding epitopes on the repetitive sequences of tau and MAP-2. However, these site-specific antibodies did not interact with MAP-1B from the neonatal rat brain tissue.Abbreviations PAA poly (L-aspartic acid) - HMW-MAPs high molecular weight microtubule associated proteins     

Localization of 350K molecular weight and related proteins in both the cytoskeleton and nuclear flecks that increase during G1 phase

Monoclonal and polyclonal antibodies were raised against the highest molecular weight microtubule-associated protein (MAP-1) isolated from brain. Immunoblotting with the antibodies revealed the presence of cross-reactive protein of 350K or less on whole cells, isolated nuclei and cellular microtubules. Two-dimensional peptide maps showed substantial homology of immunoprecipitated cellular proteins of 350K, 80K and 51K with a 25K fragment of brain MAP-1. On antibody staining, immunofluorescence was seen on a cytoplasmic network, the mitotic spindle, the centrosome, and intranuclear flecks. The antibody causing immunofluorescence in all these sites was absorbed most effectively with slices of blotted membrane which contained the 350K protein. These results suggest that the cross-reactive molecules in diverse sites belong to the family of the 350K protein. The number of nuclear flecks and the amount of bound radioactivity of 125I-antibody almost doubled during G1 phase.     

Monoclonal antibody against microtubule associated protein-1 produces immunofluorescent spots in the nucleus and centrosome of cultured mammalian cells

A monoclonal antibody was raised against the highest molecular weight protein associated with microtubules (MAP-1). Its specific binding to MAP-1 was determined by immunoblotting of the gel electrophoretogram of microtubule proteins prepared from porcine brain. The antibody reacted only with MAP-1, not with MAP-2, tau or tubulin. Indirect immunofluorescent staining by this antibody showed bright intranuclear spots, the centrosome and the faint meshwork of the cytoplasm in several types of cultured mammalian cells; HeLa, PtK2, human skin fibroblasts, mouse melanoma cells, Chinese hamster ovary cells. The nuclear spots in the interphase cells, were replaced by diffuse enhanced fluorescence throughout the cell except for chromosomes during mitosis. They reappeared in late telophase, first in the cytoplasm, late in the nucleus. The punctate pattern of nuclear immunofluorescence was not affected by microtubule-depolymerizing agents. The result that it persisted on residual cell structures after extraction with a high salt concentration buffer containing Triton X-100 followed by digestion with DNase I and RNase A suggests that the antigen is associated with the nuclear skeleton.     

The primary structure of two molecular species of porcine organ-common type acylphosphatase.

Electrophoretically homogeneous preparations of organ-common type acylphosphatase from porcine testis and brain were separated into two molecular species by reversed-phase liquid chromatography. From tryptic peptide map analysis, it was inferred that each of the two testis proteins is the same as the corresponding one of the two brain proteins. The complete primary structures of the two acylphosphatases from testis were then determined. The one molecular species consists of 100 amino acid residues: [sequence; see text] The other consists of 98 amino acid residues identical to the 3rd-100th residues of the above sequence and is also acetylated at the amino-terminal alanine. The 98-residue sequence has only 59% homology with porcine muscle acylphosphatase, but has 92% homology with human erythrocyte acylphosphatase. It was thus confirmed that the major acylphosphatases in testis, brain, and erythrocyte belong to the same organ-common type isoenzyme, distinct from the muscle type isoenzyme.     

Tightly bound nonprotamine proteins from ram sperm nuclei studied by one- and two-dimensional peptide mapping

The tightly bound proteins of ram sperm nuclei (TBSP) have been recovered as a fraction co-sedimenting with DNA after high salt-urea deprotamination of the nuclei. TBSP were studied by two-dimensional (2D)tryptic peptide mapping and by one-dimensional (1D)-partial proteolysis mapping. The 2D maps revealed a strong homology among the proteins, irrespective of substantial differences in their molecular masses. This homology was supported also by the 1D-mapping data.The 2D-tryptic maps of TBSP were compared to those of lamb liver lamins but no apparent similarity was detected. TBSP were found to react positively to a test for the presence of carbohydrate residues, suggesting that these proteins are glycoproteins as established earlier for the lamins.The 2D maps of several proteins of seminal plasma origin, used as a control, displayed completely different peptide profiles.     

Differential interaction of synthetic peptides from the carboxyl-terminal regulatory domain of tubulin with microtubule-associated proteins.

In previous studies we have demonstrated that a 4-kd tubulin fragment, including amino acid residues from Phe418 to Glu450 in alpha-subunit and Phe408-Ala445 of the beta-sequence, plays a major role in controlling tubulin interactions leading to microtubule assembly. The 4-kd carboxyl-terminal domain also constitutes an essential domain for the interaction of microtubule-associated proteins (MAPs). Removal of the 4-kd fragment facilitates tubulin self-association and renders the assembly MAP-independent. In order to define the substructure of the tubulin domain for MAP interaction, we have examined the binding of 3H-acetylated C-terminal peptides to MAP-2 and tau. Two synthetic peptides from the low-homology region within the 4-kd domain alpha (430-441) and beta (422-434) and the peptide, alpha (401-410) of the high-homology region adjacent to the 4-kd domain, were analyzed with respect to MAP interaction. The binding data showed a relatively strong interaction of MAP-2 with the beta (422-434) peptide and a weaker interaction of both MAPs components with alpha (430-441) tubulin peptide as analyzed by Airfuge ultracentrifugation and zone filtration chromatography. The homologous alpha (401-410) peptide did not bind to either MAP-2 or tau. Equilibrium dialysis experiments showed a co-operative binding of beta (422-434) peptide to multiple sites in tau. The alpha (430-441) peptide exhibited a stronger interaction for tau as compared with MAP-2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genetic variability in charge microheterogeneity and polypeptide composition of phaseolin, the major storage protein of Phaseolus vulgaris; and peptide maps of its three major subunits

Molecular polymorphism of phaseolin, the major storage protein of Phaseolus vulgaris L., and structural homology of its subunits were investigated. A high degree of charge heterogeneity was evidenced by isoelectric focusing. Comparison among phaseolins isolated from seven different cultivars showed genetic variability both in isoelectric focusing pattern and subunit composition. However tryptic peptide maps of these phaseolins were very similar. The three phaseolin subunits isolated from cultivar Greensleeves were compared by peptide mapping after limited enzymatic digestion and had very similar primary structures.     

Estramustine-phosphate binds to a tubulin binding domain on microtubule-associated proteins MAP-2 and tau.

Estramustine-phosphate (EMP), a phosphorylated conjugate of estradiol and nor-nitrogen mustard binds to microtubule-associated proteins MAP-2 and tau. It was shown that this estramustine derivative inhibits the binding of the C-terminal tubulin peptide beta-(422-434) to both MAP-2 and tau. This tubulin segment constitutes a main binding domain for these microtubule-associated proteins. Interestingly, estramustine-phosphate interacted with the synthetic tau peptides V187-G204 and V218-G235, representing two major repeats within the conserved microtubule-binding domain on tau and also on MAP-2. This observation was corroborated by the inhibitory effects of estramustine-phosphate on the tau peptide-induced tubulin assembly into microtubules. On the other hand, the nonphosphorylated drug estramustine failed to block the MAP peptide-induced assembly, indicating that the negatively charged phosphate moiety of estramustine-phosphate is of importance for its inhibitory effect. These findings suggest that the molecular sites for the action of estramustine-phosphate are located within the microtubule binding domains on tau and MAP-2.     

Biochemical characterization of the microsporidian Nosema bombycis spore proteins

Spore proteins of the microsporidian Nosema bombycis, from the silkworm Bombyx mori, were analysed by SDS–polyacrylamide gel electrophoresis. The protein profile of partially solubilized spores showed three major peptide bands of molecular weight 68, 94 and 100 kDa. On complete solubilization, it showed peptide bands ranging from 17 to 68 kDa. Attempts to purify the 17 kDa infection-specific protein showed aggregation of this protein to higher molecular size proteins. Partial peptide analysis of the different peptides exhibited similar patterns suggesting the probablity of processing during the infective cycle. Reconstitution assay showed the reversible nature of this processing. N-terminal sequencing showed homology to heat shock proteins. The low molecular weight 17 kDa protein also showed very high protease activity.     

Trypanosoma congolense: Surface glycoproteins of two early bloodstream variants: II. Purification and partial chemical characterization

Two sequential variant-specific glycoproteins have been purified from two variants of Trypanosoma congolense expressed during a relapsing infection. Isolation of the two glycoproteins, termed VSG-1 and VSG-2, respectively, employed glycerol lysis followed by purification on concanavalin A, Sephadex G-25, and gradient-eluted DE-52 columns. Partially purified VSG proteins were immunologically cross-reactive, but highly purified VSGs showed no cross-reactivity under the conditions employed. Both VSG-1 and VSG-2 consisted of a triplet of polypeptides. Although each member of a triplet subset could be distinguished by isoelectric focusing, all three gave identical N-terminal amino acid sequences and nearly identical tryptic peptide maps. The members of the VSG-1 polypeptide subset differed from those of the VSG-2 subset both with regard to N-terminal amino acid sequence and in tryptic peptide map patterns. Comparison of N-terminal sequences of VSG-1 and VSG-2 did, however, show that the sequences could be aligned to give a modest degree of amino acid homology (27%). This alignment also produced a minimum in the number of two-base changes, suggesting that the observed homology is not a coincidence and that these two proteins may well have arisen by gene duplication followed by retention of multiple point mutations.     

18 kDa microtubule-associated protein: identification as a new light chain (LC-3) of microtubule-associated protein 1 (MAP-1)

SDS gel electrophoresis of microtubule proteins obtained from bovine brain by polymerization cycles revealed a new protein of 18 kDa. This protein was copolymerized with tubulin and its stoichiometry to tubulin remained constant for at least 5 cycles of assembly. Moreover, this protein remained bound to microtubules stabilized with 10 μM taxol and pelleted through a 4 M glycerol cushion. The same 18 kDa protein was found in a purified preparation of the high molecular mass microtubule-associated protein 1 (MAP-1). The 18 kDa protein copurified with the MAP-1 heavy chains during column chromatography on phosphocellulose, DEAE-cellulose, hydroxyapatite and Bio-Gel A-15m. Incubation of the MAP-1 preparation with a mouse monoclonal antibody to the light chain 1 (LC-1) of MAP-1 and with a second precipitating antibody (a rabbit antibody to mouse IgG) immunoprecipitated from the solution all the known components of MAP-1 (heavy chains, LC-1, LC-2), as well as the 18 kDa protein. Immunoblotting showed, however, that this antibody does not interact directly with the 18 kDa protein. These results indicate that the 18 kDa protein forms a complex with all other components of MAP-1. This polypeptide, therefore, is a new light chain (LC-3) of M AP-1.     

Quantitation of microtubule-associated protein MAP-1B in brain and other tissues

1. The presence of microtubule-associated protein MAP-1B in all mammalian tissues tested, as well as in brain, has been demonstrated by immunoblotting using a monospecific polyclonal antibody. 2. The expression of brain MAP-1B is developmentally controlled, as it is less abundant in adult than in newborn rat brain, where it is a major microtubule assembly promoting factor. 3. The level of MAP-1B in tissues other than brain is lower than it is in brain; but the relative ratios of MAP-1B to tubulin are very similar in all tissues, thus differing from the observed for MAP-2 or tau. 4. The amount of MAP-1B in non-nervous tissues seems not to be under developmental control. 5. These results are consistent with a role for MAP-1B in the assembly of microtubules in most cells.     

A 205 kDa protein from non-neuronal cells in culture contains tubulin binding epitopes

Microtubule-associated proteins (MAPs) interact with tubulinin vitro andin vivo. Despite that there is a large amount of information on the roles of these proteins in neurons, the data on non-neuronal MAPs or MAPs-related proteins is scarce. There is an increasing number of microtubule-interacting proteins that have been identified in different cultured cell lines, and some of them share common functional epitopes with the most well-known MAPs, MAP-2 and tau. In a search for tubulin-interacting proteins in non-neuronal cells we identified a 205 kDa protein in the monkey kidney Vero cells in culture, on the basis of immunological studies and affinity chromatography. This protein interacts with the C-terminal moiety of -tubulin and cosediments with taxol assembled microtubules, but it was not recovered after successive cycles of assembly and disassembly. The presence of neuronal MAPs such as MAP-1, MAP-2 and tau was not detected in these cells. Interestingly, the studies showed that the 205 kDa protein contained a tubulin binding motif which was recognized by site-directed antibodies that also tag tubulin binding epitopes on MAP-2 and tau. This characteristic led us to designate this protein as MBD-205, a component that shares binding domains with these MAPs, rather than as a marker of the MAPs family. On the other hand, immunofluorescence experiments using site-specific antibodies, i.e. MAP-reacting monoclonal anti-idiotypic reagent MTB6.22 and a polyclonal antibody to the second tau repeat, revealed a MBD-205 co-localization with membrane structures and microtubule-organizing centers in Vero cells. Microinjection studies along with studies on the cell distribution suggest that MBD-205 appears to play a structural role at the level of the microtubule interactions in these cells.     

The two components of spectrin, filamin, and the heavy chain of smooth muscle myosin show no detectable homologies to one another by two-dimensional mapping of iodinated tryptic peptides.

The possible structural relationships among four high molecular weight mechanochemical proteins has been examined using two-dimensional mapping of the tryptic peptide fragments prepared from ¹²⁵I-labeled proteins (Elder et al., J. Biol. Chem. $\text{252}$:6510–6515 (1977)). Erythrocyte spectrin bands 1 and 2 protein, the heavy chain of smooth muscle (uterine) myosin, a filamin from human and rabbit were studied. The maps of the four proteins within each species differed considerably from each other, with no apparent homologies evident among them, whereas maps of the same individual protein between the two species showed a high degree of homology.     

Characterization and sequencing of a 40-amino-acid peptide from human thyroglobulin inducing experimental autoimmune thyroiditis.

We previously demonstrated that: a) a cytotoxic T cell hybridoma (HTC2) was able to induce lysis of syngeneic macrophages pulsed with either porcine thyroglobulin (pTg) or the tryptic fragments (TF) from pTg less than 10 kDa (M(r)) and that b) these low M(r) pTg TF included pathogenic epitopes because their injection into CBA/J mice induces thyroid lymphocytic infiltration typical of experimental autoimmune thyroiditis. Therefore the biochemical analysis of the TF preparation from pTg less than 10 kDa M(r) was undertaken and the characterized peptides were tested for their ability to be recognized or not by HTC2 cells. The sequencing of the selected peptides showed a 70% sequence homology with a portion of human thyroglobulin (hTg). The lack of a published sequence of pTg led us to synthesize a 40-amino acid peptide (F40D) similar to that portion of hTg. This F40D peptide was able to generate lymphocytic infiltrations in CBA/J mice thyroid glands, as was the native pTg molecule. Although the lymphocytic infiltrations were similar in the pTg or F40D-immunized mice, auto-antibodies to pTg or to hTg were only detectable in mice immunized with pTg. In contrast, autoantibodies levels to F40D peptide were significantly increased in serum from mice in which EAT had been induced by the F40D peptide. This highly hydrophobic peptide shows a M(r) of 4,492 kDa; it is located at the end of the second-third of the thyroglobulin molecule and up to now represents a unique sequence from the hTg molecule inducing experimental autoimmune thyroiditis.     

Immunological characterization of microtubule-associated proteins specific for the immature brain

Immunoblotting analysis was used to detect the microtubule-associated proteins present at different stages of rat brain development. Polyclonal antibodies were raised against the two main adult brain microtubule-associated proteins: MAP-2 (300 kDa) and TAU (60-70 kDa). Whatever the stage of development, anti-MAP-2 serum detected high molecular mass proteins and at immature stages a protein of 62 kDa. This protein which has previously been referred to as 'young TAU slow' is, therefore, immunologically related to MAP-2. The anti-TAU serum (but not the anti-MAP-2 serum) detected at immature stages of development a 48 kDa protein which also disappears at adulthood. This 48 kDa entity which has been referred to as 'young TAU fast' is progressively replaced by the closely spaced bands (60-70 kDa) of adult TAU proteins. The 62 and 48 kDa proteins appear therefore to be immunologically distinct and represent two microtubule-associated proteins specific to the immature brain.     

Structural analysis of NADPH-cytochrome P-450 reductase from porcine hepatic microsomes. Sequences of proteolytic fragments, cysteine-containing peptides, and a NADPH-protected cysteine peptide

Detergent-solubilized NADPH-cytochrome P-450 reductase was purified from porcine hepatic microsomes and compared to the rabbit enzyme isolated under identical conditions. The porcine enzyme had an equivalent specific activity toward cytochrome c compared to the rabbit enzyme. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the porcine enzyme exhibited a major band at Mr = 80,000 and two additional bands at Mr = 20,000 and 60,000. The 20-kDa fragment was shown to be the COOH-terminal portion of the protein which contains a hydrophobic sequence of 28 residues homologous to the pyrophosphate-binding portion of the FAD-binding protein p-hydroxybenzoate hydroxylase. The 60-kDa fragment corresponded to the NH2-terminal portion of the protein since this peptide and the intact protein have blocked NH2 terminal. The trypsin-solubilized porcine enzyme has an NH2-terminal sequence which is homologous to the equivalent trypsin-solubilized enzymes from rat and rabbit (80% sequence homology). Eight cysteine-containing peptides were isolated from a tryptic digest of the S-carboxymethylated pig enzyme. Significant sequence homology was not found between these peptides and other flavoproteins, except for one peptide (Glu-Val-Gly-Glu-Thr-Leu-Leu-Tyr-Tyr-Gly-Cys-Arg) which exhibited partial homology with the known NADPH-binding site of glutathione reductase. When the NADPH-protected enzyme was first S-alkylated with unlabeled iodoacetate, NADPH depleted, and further alkylated with 14C-labeled iodoacetate, the above radiolabeled peptide was isolated from a tryptic digest. The equivalent peptide was also isolated by a similar procedure from rabbit liver cytochrome P-450 reductase.     

Fyn phosphorylates human MAP-2c on tyrosine 67

The Src homology 3 (SH3) domain of Fyn binds to a conserved PXXP motif on microtubule-associated protein-2. Co-transfections into COS7 cells and in vitro kinase assays performed with Fyn and wild-type, or mutant MAP-2c, determined that Fyn phosphorylated MAP-2c on tyrosine 67. The phosphorylation generated a consensus sequence for the binding of the SH2 domain of Grb2 (pYSN). Pull-down assays with SH2-Grb2 from human fetal brain homogenates, and co-immunoprecipitation of Grb2 and MAP-2 confirmed the interaction in vivo, and demonstrated that MAP-2c is tyrosine-phosphorylated in human fetal brain. Filter overlay assays confirmed that the SH2 domain of Grb2 binds to human MAP-2c following incubation with active Fyn. Enzyme-linked immunosorbent assays confirmed the interaction between the SH2 domain of Grb2 and a tyrosine-phosphorylated MAP-2 peptide spanning the pY(67)SN motif. Thus, MAP-2c can directly recruit multiple signaling proteins important for central nervous system development.