The Rab6 GTPase regulates intracellular transport at the level of the Golgi apparatus, probably in a retrograde direction. Here, we report the identification and characterization of a novel human Rab6-interacting protein named human GAPCenA (for 'GAP and centrosome-associated'). Primary sequence analysis indicates that GAPCenA displays similarities, within a central 200 amino acids domain, to both the yeast Rab GTPase activating proteins (GAPs) and to the spindle checkpoint proteins Saccharomyces cerevisiae Bub2p and Schizosaccharomyces pombe Cdc16p. We demonstrate that GAPCenA is indeed a GAP, specifically active in vitro on Rab6 and, to a lesser extent, on Rab4 and Rab2 proteins. Immunofluorescence and cell fractionation experiments showed that GAPCenA is mainly cytosolic but that a minor pool is associated with the centrosome. Moreover, GAPCenA was found to form complexes with cytosolic γ-tubulin and to play a role in microtubule nucleation. Therefore, GAPCenA may be involved in the coordination of microtubule and Golgi dynamics during the cell cycle.

M ultiple sclerosis (MS) is a chronic inflammatory and demyelinating disease of the central nervous system (CNS) associated with autoaggressive T and B cell responses to various myelin proteins. Myelin oligodendrocyte glycoprotein (MOG) was identified as a candidate autoantigen in MS because it induces a demyelinating antibody response in laboratory animals with experimental autoimmune encephalomyelitis (EAE), an animal model of MS (1). MOG is a quantitatively minor type I transmembrane CNS protein of unknown function with a single extracellular Ig-like domain (2). In contrast to other CNS proteins, MOG is found only in mammals and is highly conserved across species. It is expressed exclusively in the CNS by myelin-forming oligodendrocytes and is preferentially localized at the outermost surface of the myelin sheath thus directly exposed to autoantibodies in the extracellular milieu. MOG is the only antigen that can induce both a pathogenic demyelinating autoantibody response and an encephalitogenic T cell response in experimental animals (3). In MOG-induced EAE this combination of immune effector mechanisms reproduces the demyelinating pathology seen in the majority of patients with MS. The clinical relevance of autoimmune responses to MOG in MS is supported by reports that MS is associated with enhanced MOG-specific T and B cell responses and by the identification of MOG-specific antibodies associated with myelin debris in actively demyelinating MS lesions (4). Experimental evidence indicates that the autoantibody response to MOG is heterogeneous; demyelinating MOG-specific autoantibodies recognize purely conformation-dependent epitopes whereas MOG peptide-specific antibodies fail to recognize the native protein (5-7). To examine the structural basis of the pathogenic autoantibody response to MOG we determined the crystal structures of the extracellular domain of rat MOG (residues 1-126, MOG Igd ) and a complex of MOG Igd with the chimeric antigen-binding fragment (Fab) of the demyelinating MOG-specific monoclonal antibody 8-18C5 (8). Materials and Methods Preparation of Recombinant MOG Igd and the MOG Igd -(8-18C5)-FabComplex. The cDNA of the extracellular domain of rat MOG (residues 1-125) was subcloned into the His-tag expression vector pQE-12. The protein was overexpressed in inclusion bodies in Escherichia coli, refolded, and further purified by nickel-nitrilotriacetic acid affinity chromatography and gel filtration. Selenomethionine (SeMet)-labeled protein was produced in the methionine-auxotrophic E. coli strain B834(DE3) and grown in minimal medium with SeMet (0.3 mM) substituted for methionine. The cDNA of the variable domains of the mouse monoclonal antibody 8-18C5 was subcloned into the expression vector pASK107, yielding the chimeric (8-18C5)-Fab composed of the 8-18C5 variable domains, the human IgG constant domains, and the Strep tag II fused to the C terminus of the heavy chain (9, 10). (8-18C5)-Fab was produced by periplasmic secretion in E. coli and purified by streptavidin affinity chromatogr...