Report |
Address correspondence to Richard Quarles, Lab of Molecular and Cellular Neurobiology, NINDS, NIH, Bldg. 49, Rm. 2A28, 49 Convent Dr., MSC 4440, Bethesda, MD 20892-4440. Tel.: (301) 496-6647. Fax: (301) 496-8244. E-mail: quarlesr{at}ninds.nih.gov
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Abstract |
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Key Words: axons; cytoskeleton; microtubule-associated proteins; myelin-associated glycoprotein; neuroglia
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Introduction |
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MAG has been shown to interact with a variety of neuronal glycoproteins (DeBellard and Filbin, 1999; Strenge et al., 1999) and gangliosides (Collins et al., 1999) based on its lectin specificity for 2,3-linked sialic acid (Kelm et al., 1994). However, there is evidence indicating that sialic acidbinding may reflect a docking mechanism and that a separate peptide-binding site in MAG is crucial for its effects on neurons (Tang et al., 1997). In this report, we describe the binding of MAG to proteins of neuronal membrane preparations from the peripheral and central nervous systems, respectively, that would be expected to contain physiological MAG binding partners, i.e., membranes from dorsal root ganglion neurons (DRGNs) and axolemma-enriched fractions (AEFs) from myelinated axons of brain. In both cases, MAG bound primarily to a high molecular weight protein that was identified as microtubule-associated protein 1B (MAP1B). A previous report from our laboratory (Tanner et al., 2000) and new findings in this paper demonstrate that some MAP1B is expressed as a plasma membrane glycoprotein in neurons, supporting the concept that MAP1B is an axonal binding partner for MAG.
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Results and discussion |
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The presence of MAP1B in AEFs isolated from myelinated axons, which had been described previously (Sapirstein et al., 1992), is consistent with it being a physiological axonal binding partner for MAG. We confirmed the well-known decrease in the total amount of MAP1B in brain during maturation (Gordon-Weeks and Fischer, 2000), but a high concentration of MAP1B is retained in the AEF even from adult brains when it could not be detected in whole brain homogenate (Fig. 1 C). This enrichment of MAP1B in the periaxonal compartment of adult myelinated axons supports the possibility that it has a functional role in gliaaxon interactions at this site.
Colocalization of MAP1B and MAG-binding sites on the surface of neurons
When the binding of MAG to MAP1B was first observed, it seemed that MAP1B was unlikely to be a physiological binding partner for MAG on the axonal surface because it is generally thought to be an intracellular cytoskeletal component (Gordon-Weeks and Fischer, 2000). However, MAP1B had been proposed to be a membrane glycoprotein based on a hydrophobic amino acid sequence that could be a transmembrane domain, potential sites for N-linked glycosylation and its role as a substrate for ecto-kinases (Muramoto et al., 1994). Subsequent studies in our laboratory supported this hypothesis that some MAP1B is expressed as a membrane glycoprotein on the surface of neurons (Tanner et al., 2000). Our results pointed to the expression of MAP1B as a type II transmembrane glycoprotein in which the COOH terminus and glycosylation sites are extracellular and the microtubule binding domains are in the neuronal cytoplasm. Immunostaining of DRGNs showed that MAP1B is concentrated in varicosities distributed along the neurites (Tanner et al., 2000). New evidence for the expression of MAP1B as a surface protein on neurons is shown in Fig. 2 A, demonstrating a similar localization of MAP1B at neuronal varicosities whether or not the neurons were permeabilized before immunostaining. The surface immunostaining was observed both with mAb AA6 (A1) and with the polyclonal antiserum recognizing amino acid sequence 20562070 (unpublished data), which is in the extracellular domain of MAP1B, according to its model as a transmembrane glycoprotein (Tanner et al., 2000).
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Fig. 2 E shows double immunostaining of a section of myelinated peripheral nerve for MAP1B and MAG. MAP1B was enriched at the periphery of axons in the region of the axolemma, and MAG appeared as rings corresponding to the periaxonal SC membrane. Colocalization of MAP1B and MAG, revealed by the overlapping yellow signal, is consistent with an interaction of MAG and MAB1B at the SC-axon junction in myelinated nerve.
Lack of binding of MAG to glial MAP1B
Because MAP1B is also expressed by SCs (Ma et al., 1999) and oligodendrocytes (Vouyiouklis and Brophy, 1993), a possibility was that the MAG binding to MAP1B represents a cis interaction within myelinating glia. Fig. 3 A shows that primary SCs and immortalized S16 SCs express MAP1B, but it is of lower molecular weight than the primary neuronal isoform and does not react with the SMI-31 antibody to phosphorylated epitopes in neuronal MAP1B. Ma et al. (1999) also reported that phosphorylation of MAP1B in SCs is different from that in neurons. Furthermore, blot overlay and coimmunoprecipitation experiments with SCs and oligodendrocytes gave no evidence for an interaction of MAG with glial MAP1B. For example, Fig. 3 B shows an overlay experiment in which binding of MAG to the higher molecular weight form of MAP1B in DRGNs was clearly demonstrated, but there was no binding to MAP1B of S16 SCs. This appears to be due to a difference in the isoforms expressed by neurons and SCs, respectively, because the MAG binding corresponds to the higher molecular weight phosphorylated MAP1B in neurons, which is not expressed in SCs (Fig. 3 A, second panel, and B, third panel). Coimmunoprecipitation experiments also demonstrated the specificity of the interaction of MAG with neuronal MAP1B (Fig. 3 C). MAG was in the immunoprecipitate when MAP1B was immunoprecipitated from the AEF with anti-MAP1B antiserum. This is the inverse of the experiment shown in Fig. 1 B in which coimmunoprecipitation was with anti-MAG antiserum and further documents the interaction between MAG and neuronal MAP1B. Although the immortalized S16 SC line is differentiated toward myelination and expresses a substantial amount of MAG (Toda et al., 1994), there was no MAG coimmunoprecipitated with MAP1B from S16 SC extracts by the same antiserum.
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MAP1B appears to play roles in neuronal differentiation (Gordon-Weeks and Fischer, 2000) and axonal formation (Gonzalez-Billault et al., 2001) by regulating cytoskeletal organization, but its exact functions remain unclear. Disruption of MAP1B expression in vivo by gene targeting has generally supported such roles (Edelmann et al., 1996; Takei et al., 1997; Meixner et al., 2000). Of particular interest, with regard to the potential role of MAP1B as a neuronal binding partner for MAG, is the impaired myelination reported for two of these mutants (Takei et al., 1997; Meixner et al., 2000).
It is well established that the cytoskeletal structure of axons is modulated by surrounding myelin sheaths, based on studies of dysmyelinating mutants and comparisons of myelinated and nonmyelinated regions of the same axon (Kirkpatrick and Brady, 1999). For example, the neurofilaments in peripheral nerves of Trembler mutants exhibit decreased phosphorylation and increased density in comparison to normally myelinated nerves. The similar changes observed in MAG-null mice (Yin et al., 1998) suggest that MAG may be involved in the molecular mechanism by which myelin affects the axonal cytoskeleton. Furthermore, the stability of microtubules and the phosphorylation of several MAPs, including MAP1B, are decreased in the absence of normal myelin in Trembler mice (Kirkpatrick and Brady, 1994). Here, we demonstrate that the expression and phosphorylation of MAP1B are increased in DRGNs cocultured with MAG-expressing cells. These findings are all consistent with an effect of MAG on the phosphorylation and structure of cytoskeletal elements in the axon, including MAP1B. Based on the findings reported here, we hypothesize that a MAGMAP1B interaction could provide a structural link between the periaxonal membrane of the myelin-forming cell and the axonal cytoskeleton, thereby contributing to the known capacity of myelin to affect the structure and stability of myelinated axons.
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Materials and methods |
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Antibodies.
Mouse mAbs to MAP1B were AA6 that reacts with all isoforms (Sigma-Aldrich) and SMI-31 that is specific for mode I phosphorylated epitopes (Sternberger). Rabbit polyclonal antisera to synthetic peptides corresponding to MAP1B sequences included one to the NH2 terminus from Dr. P. Brophy (University of Edinburgh, Edinburgh, Scotland) and another to amino acids 20562070 raised by Zymed Laboratories. Antibodies to MAG included an mAb 513 to native MAG (Poltorak et al., 1987) and an mAb (B11F7) and rabbit polyclonal antisera raised in our laboratory.
Purification of native MAG and binding to proteins on Western blots
Nondenatured MAG was purified from adult rat brain myelin by immunoaffinity with the 513 mAb by a procedure similar to that described by Poltorak et al. (1987) using the Immunopure Protein G IgG Orientation Kit from Pierce Chemical Co. MAG binding to the antibody beads was at 4°C for 16 h, and it was eluted with a pH 11.5 solution of 50 mM diethanolamine and 5 mM ß-octylglucoside. Proteins in membrane fractions were separated by SDS-PAGE and transferred to nitrocellulose membranes. The blots were incubated overnight at 4°C in a blocking reagent (from the DIG Glycan Differentiation Kit; Roche) and washed twice in TBS (pH 7.5). After rinsing once in TBS containing 1 mM MgCl2, 1 mM MnCl2 and 1mM CaCl2 blots were incubated for 3 h at room temperature with 10 µg per ml of purified MAG in the same buffer. After washing with TBS, bound MAG was detected by immunostaining (overnight at 4°C) with the 513 mAb followed by peroxidase labeled antimouse IgG (ICN Biomedicals) and ECL (NEN Life Science Products).
Coimmunoprecipitation
MAG was immunoprecipitated from the proteins of AEFs (50 µg solubilized in PPi buffer) with polyclonal anti-MAG antiserum overnight at 4°C followed by protein A Sepharose (Amersham Pharmacia Biotech) for 1 h at 4°C. MAP1B was immunoprecipitated similarly from the AEF and S16 SC protein extracts with the polyclonal antibody to its NH2 terminus. Immunoprecipitates were washed three times in PPi buffer, solubilized, and fractionated by SDS-PAGE. After transfer to blots and blocking, coimmunoprecipitated proteins were detected by immunostaining with ECL.
Immunostaining and localization of MAG-binding sites
Cultured DRGNs fixed with 2% paraformaldehyde and 0.1% glutaraldehyde were immunostained for MAP1B either without permeabilization or with permeabilization by exposure to 1% Triton X-100 in PBS for 10 min. After blocking for 30 min in 10% normal goat serum (NGS) in PBS, cells were incubated for 1 h with primary antibodies to MAP1B, rinsed with PBS, and incubated with appropriate Texas redcoupled secondary antibodies (Jackson ImmunoResearch Laboratories). Sections of rat sciatic nerve were immunostained for MAG and MAP1B as described previously (Tanner et al., 2000).
For localization of MAG-binding sites on cultured DRGNs, fixation was with 1% paraformaldehyde in PBS. After blocking in 2% FBS and 2% NGS in PBS, the cells were incubated either with a MAG-Fc chimera protein (5 µg/ml; R&D Systems) or with human-Fc protein (5 µg/ml; ICN Biomedicals) for 3 h at 4°C. After three PBS washes, FITC-coupled antihuman Fc antibody was applied for 1 h. The cells were blocked again as above either with or without prior treatment with ice-cold methanol and then incubated with polyclonal antibody to MAP1B followed by rhodamine-coupled antirabbit IgG.
Protein analysis of DRGNs cocultured with MAG-expressing COS cells
COS-7 cells were stably transfected by lipofectamine reagent (GIBCO-BRL) with either 2 µg L-MAGpcDNA3.1 (provided by M. Filbin, Hunter College, New York, NY) or 2 µg pcDNA3.1 (Invitrogen) without the L-MAG insert (mock transfected). E17 DRGNs were plated on 6-well collagen-coated plates at a density of 5 x 105 cells/well and maintained in defined medium (DMEM + N2 + 2% FBS + 100 ng/ml NGF) for 5 d. DRGNs were then seeded onto 105 COS-7 cells for 4 d. Cell lysates were fractionated by SDS-PAGE, blotted, and immunostained for MAP1B with ECL. Densitometry was with NIH Image software (version 1.6), and statistical comparisons were by a two-tailed paired t test.
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Footnotes |
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Acknowledgments |
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Submitted: 27 August 2001
Revised: 18 October 2001
Accepted: 22 October 2001
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References |
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