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Binding Partners for the Myelin-Associated Glycoprotein of N2A Neuroblastoma Cells

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Binding Partners for the Myelin-Associated Glycoprotein of N2A Neuroblastoma Cells View metadata,FEBS 21505 citation and similar papers at core.ac.uk FEBS Letters 444brought (1999) to you 59^64 by CORE provided by Elsevier - Publisher Connector Binding partners for the myelin-associated glycoprotein of N2A neuroblastoma cells Karen Strenge, Roland Schauer, SÖrge Kelm* Institute of Biochemistry, University of Kiel, Olshausenstrasse 40, 24098 Kiel, Germany Received 11 November 1998; received in revised form 4 January 1999 indicating that MAG plays a role in long-term maintenance of Abstract The myelin-associated glycoprotein (MAG) has been proposed to be important for the integrity of myelinated axons. the integrity of both myelin and axons [12,13]. For a better understanding of the interactions involved in the An interesting aspect of MAG is its in£uence on neuronal binding of MAG to neuronal axons, we performed this study to growth. In vitro, MAG promotes neurite outgrowth of em- identify the binding partners for MAG on neuronal cells. bryonal dorsal root ganglion neurones [14^16], whereas it in- Experiments with glycosylation inhibitors revealed that sialy- hibits the neurite outgrowth of cerebellar neurones, adult dor- lated N-glycans of glycoproteins represent the major binding sal root ganglion neurones [15] and neuroblastoma cells [17]. sites for MAG on the neuroblastoma cell line N2A. From The role of MAG as a neurone regeneration inhibiting 3 extracts of [ H]glucosamine-labelled N2A cells several glyco- molecule in vivo has remained controversial [6,7] since ¢rst proteins with molecular weights between 20 and 230 kDa were experiments with MAG3=3 mice gave no clear evidence for a affinity-precipitated using immobilised MAG. The interactions signi¢cant contribution of MAG [18,19]. However, improved of these proteins with MAG were sialic acid-dependent and 3=3 specific for MAG. axonal regrowth has been demonstrated in MAG mice s z 1999 Federation of European Biochemical Societies. cross-bred with animals of the strain C57BL/Wld that have delayed lesion-induced myelin degradation and axonal re- Key words: Myelin-associated glycoprotein; Siglec; growth in the peripheral nervous system [20], indicating a Sialic acid; Cell-cell interaction signi¢cant inhibitory e¡ect of MAG on neurone regeneration processes also in vivo. Although the role of MAG as a cell adhesion molecule has 1. Introduction been under investigation for many years, and binding activ- ities to various extracellular components have been reported The myelin-associated glycoprotein (MAG) is a 100-kDa [21,22], no neuronal binding partners for MAG have been integral membrane glycoprotein expressed by myelinating characterised so far. First evidence for Sia-containing glyco- glia cells in the central and peripheral nervous system [1]. It conjugates as binding partners for MAG came from the ob- consists of ¢ve extracellular immunoglobulin-like (Ig-like) do- servation that MAG binds to sialylated glycans on erythro- mains with 8^9 potential glycosylation sites, and a carboxy- cytes [23]. The speci¢city of MAG for such glycans has been terminal intracellular domain with several phosphorylation investigated in detail using gangliosides [24^26] or synthetic sites [2]. Structurally most closely related proteins are the sialylated mono- and oligosaccharides [27,28]. Whereas ex- Schwann cell myelin protein (SMP), sialoadhesin (Sn), periments with resialylated red blood cells suggested that CD22 and CD33, which all bind to sialic acid (Sia)-containing MAG binds preferentially to Neu5Aca2,3GalL1,3GalNAc, a cell surface glycoconjugates [3,4]. Based on this speci¢city and structure typically found on O-glycans of glycoproteins and some unique structural features, the name siglecs has been on gangliosides [23], a direct comparison of free monovalent proposed for this group of Ig-like proteins [5]. oligosaccharides in hapten inhibition assays revealed that For MAG, several functions in interactions between glia structures found on N-glycans, i.e. Neu5Aca2,3GalL1,3Glc- cells and neurones have been proposed [6^8]. Whereas in vitro NAc and Neu5Aca2,3GalL1,4GlcNAc, were at least as well experiments provided evidence that MAG is important for the recognised as Neu5Aca2,3GalL1,3GalNAc [28]. initiation of myelination during development [9], myelination The functions of MAG mentioned above are likely to in- of axons occurs also in mice in which the MAG gene had been volve binding partners (counter-receptors) on neuronal cells. disrupted (MAG3=3 mice) [10,11]. However, several abnor- Although it is known that MAG binding is mediated by sia- malities were observed in the morphology of myelinated axons lylated glycans, it has remained unclear by which structures, in these animals. Animals older than 8 months show increased e.g. glycoproteins and/or glycolipids, these glycans are pre- signs of a disturbed maintenance of their axon-myelin units sented. Speci¢c binding to gangliosides has been clearly dem- onstrated [23,24,26]. However, MAG binding to neuronal *Corresponding author. Fax: (49) (431) 880-2238. cells may be mainly to glycoproteins, since it is trypsin-sensi- E-mail: [email protected]. URL: http://www.uni-kiel.de/ tive [16]. The aim of this study has been the isolation of bind- biochemie/kelm/index.html ing partners for MAG from the neuroblastoma cell line N2A. Abbreviations: a2,3SL, a2,3-sialyllactose; benzyl-GalNAc, benzyl-N- acetyl-a-D-galactosamine; Con A, concanavalin A haemagglutinin; FBS, foetal bovine serum; Ig-like, immunoglobulin-like; MAG, 2. Materials and methods myelin-associated glycoprotein; MAG3=3 mice, mice in which the MAG gene has been disrupted; PBS, phosphate-buffered saline; PNA, 2.1. Reagents peanut haemagglutinin; Sia, sialic acid; SMP, Schwann cell myelin A¤nity-puri¢ed anti-human IgG antibodies were from BiÖdesign, protein; Sn, sialoadhesin; VCS, sialidase from Vibrio cholerae Kennebunk, ME, USA; carrier-free Na125I was from Amersham, 0014-5793/99/$19.00 ß 1999 Federation of European Biochemical Societies. All rights reserved. PII: S0014-5793(99)00029-0 FEBS 21505 6-2-99 60 K. Strenge et al./FEBS Letters 444 (1999) 59^64 Braunschweig, Germany; protein A beads were from Pharmacia Bio- 4³C, centrifuged and the supernatant was mixed with Fc-MAG- tech, Uppsala, Sweden; sialidase from Vibrio cholerae was purchased loaded protein A beads. After overnight incubation at 4³C under from Behringwerke, Marburg, Germany. gentle shaking, the beads were centrifuged and washed four times with lysis bu¡er containing 0.1% NP-40. Bound proteins were eluted 2.2. Fc chimeras from the beads by boiling for 5 min in the presence of 20 Wl of SDS- Plasmids encoding the Fc chimeras containing the N-terminal three PAGE sample bu¡er. domains of MAG (Fc-MAGd13) or the N-terminal ¢ve domains of MAG (Fc-MAGd15 [23], the N-terminal domain of sialoadhesin (Sn) 2.7. SDS-PAGE and Western blotting (Fc-Snd1) [29] or the N-terminal three domains of murine CD22 (Fc- SDS-PAGE was performed according to Laemmli using 10% or 8% CD22d13) [23] have been described. Fc chimeras were produced by polyacrylamide gels. For Western blot analysis proteins were trans- transient expression of the plasmid in COS cells followed by puri¢ca- ferred to nitrocellulose and detected by autoradiography or by phos- tion from the tissue culture supernatants by immunoa¤nity chro- phor imaging. matography on protein A agarose [30]. Fc chimeras containing the N-terminal part of NCAM [31] were a kind gift from Dr Paul R. Crocker. 3. Results 2.3. Binding assays For the isolation of neuronal binding partners of MAG a Puri¢ed Fc-MAG was labelled with 125I and complexed with equi- suitable source had to be found. Since neurones from primary molar concentration of anti-human IgG to be used in binding assays as described [23,30,32]. tissue culture are only available in limited amounts, several cell lines were compared for binding of MAG (Fig. 1). For all 2.3.1. Binding to adherent cells cell types, binding was Sia-dependent, since sialidase treat- Cells were grown on 10-mm coverslips, washed with phosphate- ment of the cells reduced the amount of bound Fc-MAG to bu¡ered saline (PBS) and ¢xed with 0.25% glutardialdehyde in PBS background levels. MAG showed high binding to the murine for 10 min at room temperature [33]. 200 Wl radio-iodinated Fc-MAG/ neuroblastoma cell line N A, whereas binding to PC12, C6, anti-human IgG complex was added to the cells, which were incu- 2 bated overnight at 4³C. Unbound radioactivity was removed by dip- L929 and S-16 cells was signi¢cantly lower. Therefore, N2A ping the coverslips three times in PBS containing 0.25% bovine serum cells were chosen for the isolation of counter-receptors for albumin. Bound radioactivity was quanti¢ed by gamma counting. As MAG. a control for Sia speci¢city, binding to sialidase-treated cells was Up to now it has not been clear whether natural counter- estimated. receptors for MAG are glycoproteins and/or glycolipids. In 2.3.2. Binding to cells in suspension order to investigate whether N- and/or O-glycans on glyco- Adherent cells were detached from the cell culture plates with PBS proteins are responsible for MAG binding to N2A cells, these containing 10 mM EDTA and 0.02% azide and ¢xed as described cells were cultivated in the presence of swainsonine, which above. 25 Wl radio-iodinated Fc-MAG/anti-human IgG complex was prevents processing of N-glycans by inhibiting the Golgi man- mixed with 25 Wl cell suspension. After overnight incubation at 4³C nosidase II [34], or the O-glycosylation inhibitor benzyl-Gal- bound radioactivity was quanti¢ed as described [30]. As a control for Sia speci¢city, binding to sialidase-treated cells was estimated and NAc, which inhibits competitively the extension of GalNAca- subtracted as background.
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