Mapping of Distinct Structural Domains on Microtubule‐Associated Protein 2 by Monoclonal Antibodies

Abstract

Monoclonal antibodes against microtubule-associated protein 2 (MAP2) were prepared and their specificity was verified by visualization of the antigens using the antibody overlay technique and by radioimmunoassay. MAP2 was cleaved by a-chyrnotrypsin to generate a series of high-molecular-mass fragments ranging between 270 and 140 kDa. The precursor-product relationship of these fragments was suggested from the rate of their appearance and from the analysis of the tryptic peptide map of each fragment. A group of monoclonal antibodies was found to react predominantly with the intact 270-kDa MAP2 molecule and a fragment having a mass of 240 kDa and to some extent with a 215-kDa fragment. Another group of monoclonal antibodies reacted with an antigenic determinant which was located on the 270-kDa molecule as well as on fragments as small as 140 kDa. None of the two groups of monoclonal antibodies reacted with the microtubule-binding domain of MAP2. These results suggest that one group of antibodies reacts with sites located at or dependent upon a terminal 60-kDa domain(s) distal to the microtubule-binding site of MAP2. The second group of antibodies, which can still bind to smaller proteolytic products, appear to be associated with the central region of the MAP2 molecule. Indirect immunofluorescence experiments with the antibody preparations indicated that at least some of the antigenic determinants are exposed when MAP2 is associated with microtubules in the cell body and neurite outgrowths of differentiated rat brain neuroblastoma B104 cells.. Microtubules, isolated by successive cycles of assemblydisassembly, are composed of tubulin and several additional proteins designated microtubule-associated proteins or MAPs. Two major groups of MAPs have been identified in neutral tissues and in cultured cells. These include high-molecularmass components (350 and 270 kDa) termed MAP1 and MAP2, respectively [l -51 and a number of polypeptides in the molecular mass range of 55 -68 kDa, known collectively as z proteins [6 -8 1. Both MAP2 and z factors have been shown to promote nucleation and elongation of microtubules in vitro [I -7,9,10], but their role in the control of microtubule assembly and its interactions in the living cells is still uncertain.The most prominent MAP components in neuronal tissue are the high-molecular-mass proteins MAP1 and MAP2. The latter was found by electron microscopy to associate with microtubules at a fixed periodicity forming laterally projected arms [2,11,12]. The tissue and species distribution of MAP2 is still controversial at the present time. Immunocytochemical data suggest that this protein is associated with microtubules from a wide variety ofcells and tissues [I3 -171. Other groups, on the other hand, have used multispecific and monoclonal antibodies to show that MAP2 is present only in cells of neuronal origin [18], while other cells, such as HeLa, containAbbreviations. MAP1 and MAP2, microtubule-associated proteins 1 and 2; Mes, 4-morpholineethanesulfonic acid; SDS, sodium dodecyl su...

Common and distinct tubulin binding sites for microtubule-associated proteins.

“…Analysis of 1251-MAP2 and T fractions by gel electrophoresis indicated a complete separation ofthe two groups ofproteins. The MAP2 fraction displayed several additional high molecular mass bands, which are the products of limited endogenous proteolysis (9), while the major r band had an apparent molecular mass of 52 kDa. A specific binding assay of '251-MAPs to tubulin immobilized on nitrocellulose membrane was developed.…”

“…Thus, brain a-and 8-tubulins display extensive microheterogeneity when compared with tubulin from other tissues (6, 7). Brain microtubules are particularly rich in the variety of MAPs they contain and >20 distinct polypeptide bands have been identified by polyacrylamide gel electrophoresis (8,9). The best-studied brain MAPs are the high molecular mass MAPs termed MAP1 (350 kDa) and MAP2 (270 kDa) (10,11) and, more recently, MAP3 (180 kDa) and MAP4 (220 kDa) (12,13) as well as a number of proteins in the molecular mass range of 48-62 kDa, known collectively as 7 proteins (14).…”

“…Although MAP2 and 7 factors appear to be differentially compartmentalized in individual cells, they can compete in vitro for the same binding sites on microtubules (15,16). It also appears that in living cells the microtubule binding sites for MAP2 are conserved regardless of the presence or absence of this protein in the cells (17). Several reports have suggested that the highly charged carboxyl-terminal region of tubulin (3,4) may be involved in the binding of both MAP2 and r factors (18)(19)(20)(21)(22)(23) (10 international units/ml), pancreatic RNase (100 ,g/ml), and pancreatic DNase (100 ,ug/ml).…”

Anti-idiotypic antibodies that react with microtubule-associated proteins are present in the sera of rabbits immunized with synthetic peptides from tubulin's regulatory domain.

“…In general, previously reported antibodies against tubulin and MAPs do not appear to be effective in blocking microtubule formation in vitro and accordingly would not affect microtubules in cells (21)(22)(23)(24)). An anti-tubulin antibody has been reported to diminish the assembly of, and disrupt, cellular microtubules (25), but these observations are difficult to interpret because the tubulin epitope recognized by this particular anti-tubulin antibody is not known.…”

Characterization and Intracellular Distribution of Microtubule‐Associated Protein 2 in Differentiating Human Neuroblastoma Cells

“…Several types of high-molecular-mass MAPs were also reported in different cell lines of mouse neuroblastoma (Izant and McIntosh, 1980;Olmsted and Lyon, 1981;Scherson et al, 1982;Vallee, 1986) and the expression and organization of MAP2-like molecules was found to depend on the state of differentiation ofthese cells (Fischer et al, 1986). Very little, however, is known about the composition of MAPs in human neuroblastoma cells.…”