Biomedical subjects
M T Filbin
Publications and source records attributed to M T Filbin.
Dominant-negative effect on adhesion by myelin Po protein truncated in its cytoplasmic domain.
The myelin Po protein is believed to hold myelin together via interactions of both its extracellular and cytoplasmic domains. We have already shown that the extracellular domains of Po can interact in a homophilic manner (Filbin, M.T., F.S. Walsh, B.D. Trapp, J.A. Pizzey, and G.I. Tennekoon. 1990. Nature (Lond.). 344:871-872). In addition, we have shown that for this homophilic adhesion to take place, the cytoplasmic domain of Po must be intact and most likely interacting with the cytoskeleton; Po proteins truncated in their cytoplasmic domains are not adhesive (Wong, M.H., and M.T. Filbin, 1994. J. Cell Biol. 126:1089-1097). To determine if the presence of these truncated forms of Po could have an effect on the functioning of the full-length Po, we coexpressed both molecules in CHO cells. The adhesiveness of CHO cells expressing both full-length Po and truncated Po was then compared to cells expressing only full-length Po. In these coexpressors, both the full-length and the truncated Po proteins were glycosylated. They reached the surface of the cell in approximately equal amounts as shown by an ELISA and surface labeling, followed by immunoprecipitation. Furthermore, the amount of full-length Po at the cell surface was equivalent to other cell lines expressing only full-length Po that we had already shown to be adhesive. Therefore, there should be sufficient levels of full-length Po at the surface of these coexpressors to measure adhesion of Po. However, as assessed by an aggregation assay, the coexpressors were not adhesive. By 60 min they had not formed large aggregates and were indistinguishable from the control transfected cells not expressing Po. In contrast, in the same time, the cells expressing only the full-length Po had formed large aggregates. This indicates that the truncated forms of Po have a dominant-negative effect on the adhesiveness of the full-length Po. Furthermore, from cross-linking studies, full-length Po, when expressed alone but not when coexpressed with truncated Po, appears to cluster in the membrane. We suggest that truncated Po exerts its dominant-negative effect by preventing clustering of full-length Po. We also show that colchicine, which disrupts microtubules, prevents adhesion of cells expressing only the full-length Po. This strengthens our suggestion that an interaction of Po with the cytoskeleton, either directly or indirectly, is required for adhesion to take place.
Myelin-associated glycoprotein: a role in myelination and in the inhibition of axonal regeneration?
Inhibitory molecules in CNS myelin affect axonal regeneration after injury. In the past year, myelin-associated glycoprotein (MAG), a well-characterized myelin protein, has been identified as an inhibitor of axonal regeneration. This finding, together with its established ability to promote outgrowth, defines MAG as a bifunctional molecule. MAG has also been included in a family of sialic acid binding proteins, providing a clue to the identity of the MAG receptor. MAG knockout mice reveal that MAG is not essential for the initiation of myelination; however, it plays an important role in maintaining a stable interaction between axons and myelin.
Sialoadhesin, myelin-associated glycoprotein and CD22 define a new family of sialic acid-dependent adhesion molecules of the immunoglobulin superfamily.
BACKGROUND: Protein-carbohydrate interactions are believed to be important in many biological processes that involve cell-cell communication. Apart from the selectins, the only well-characterized vertebrate sialic acid-dependent adhesion molecules are CD22 and sialoadhesin; CD22 is a member of the immunoglobulin superfamily that is expressed by B lymphocytes and sialoadhesin is a macrophage receptor. The recent cloning of the gene encoding sialoadhesin has shown that it is also immunoglobulin-like. Both proteins share sequence similarity with the myelin-associated glycoprotein, an adhesion molecule of oligodendrocytes and Schwann cells that has been implicated in the process of myelination, raising the important question of whether myelin-associated glycoprotein is also a sialic acid-binding protein. RESULTS: We have investigated the binding properties of these three receptors when expressed either in monkey COS cells or as chimaeric proteins containing the Fc portion of human immunoglobulin G. We demonstrate that, like sialoadhesin and CD22, myelin-associated glycoprotein mediates cell adhesion by binding to cell-surface glycans that contain sialic acid. We have dissected the specificities of these three adhesins further: whereas sialoadhesin binds equally to the sugar moieties NeuAc alpha 2-->3Gal beta 1-->3(4)GlcNAc or NeuAc alpha 2-->3Gal beta 1-->3GalNAc, myelin-associated glycoprotein recognizes only NeuAc alpha 2-->3Gal beta 1-->3GalNAc and CD22 binds specifically to NeuAc alpha 2-->6Gal beta 1-->4GlcNAc. Furthermore, we show that the recognition of sialylated glycans on the surfaces of particular cell types leads to the selective binding of sialoadhesin to neutrophils, myelin-associated glycoprotein to neurons and CD22 to lymphocytes. CONCLUSIONS: Our findings demonstrate that a subgroup of the immunoglobulin superfamily can mediate diverse biological processes through recognition of specific sialylated glycans on cell surfaces. We propose that this subgroup of proteins be called the sialoadhesin family.
A novel role for myelin-associated glycoprotein as an inhibitor of axonal regeneration.
Following nerve injury, axons in the CNS do not normally regenerate. It has been shown that CNS myelin inhibits neurite outgrowth, though the nature of the molecules responsible for this effect are not known. Here, we demonstrate that the myelin-associated glycoprotein (MAG), a transmembrane protein of both CNS and PNS myelin, strongly inhibits neurite outgrowth from both developing cerebellar and adult dorsal root ganglion (DRG) neurons in vitro. This inhibition is reversed by an anti-MAG antibody. In contrast, MAG promotes neurite outgrowth from newborn DRG neurons. These results suggest that MAG may be responsible, in part, for the lack of CNS nerve regeneration in vivo and may influence, both temporally and spatially, regeneration in the PNS.
Formation of a disulfide bond in the immunoglobulin domain of the myelin P0 protein is essential for its adhesion.
It is widely accepted, although never demonstrated, that the formation of a disulfide bond in the majority of immunoglobulin (Ig)-like domains stabilizes their final conformation and thus is essential to their functioning as adhesion/recognition molecules. The myelin P0 protein, which has been shown directly to behave as a homophilic adhesion molecule, contains a single Ig-like domain, stabilized by a putative Cys21-Cys98 disulfide bond. To test if this bond is indeed necessary to the adhesive function of P0, the nucleotides in the P0 cDNA coding for Cys21 were altered to code for an alanine. The mutated P0 cDNA was transfected into Chinese hamster ovary cells, expression of the mutated P0 protein was characterized, and the adhesiveness of Cys21-mutated P0-expressing cells and that of cells expressing equivalent surface amounts of the unmutated protein were compared. It was found, as we previously reported, that incubation of a single cell suspension of the unmutated P0-expressing cells resulted in the rapid formation of large aggregates. In contrast, after a similar incubation the cells expressing the Cys21-mutated P0 were still mostly single cells, a result indistinguishable from that observed with the control transfected cells. This suggests that the P0 protein, when mutated at Cys21, does not behave as a homophilic adhesion molecule, which in turn implies that the formation of an Ig domain disulfide bond is essential to the functioning of this molecule.
The cytoplasmic domain of the myelin P0 protein influences the adhesive interactions of its extracellular domain.
The extracellular domain of the myelin P0 protein is believed to engage in adhesive interactions and thus hold the myelin membrane compact. We have previously shown that P0 can behave as a homophilic adhesion molecule through interactions of its extracellular domains (Filbin, M. T., F. S. Walsh, B. D. Trapp, J. A. Pizzey, and G. I. Tennekoon. 1990. Nature (Lond.) 344:871-872). To determine if the cytoplasmic domain of P0 must be intact for the extracellular domains to adhere, we compared the adhesive capabilities of P0 proteins truncated at the COOH-terminal to the full-length P0 protein. P0 cDNAs lacking nucleotides coding for the last 52 or 59 amino acids were transfected into CHO cells, and surface expression of the truncated proteins was assessed by immunofluorescence, surface labeling followed by immunoprecipitation, and an ELISA. Cell lines were chosen that expressed at least equivalent amounts of the truncated P0 proteins at the surface as did a cell line expressing the full-length P0. The adhesive properties of these three cell lines were compared. It was found that when a suspension of single cells was allowed to aggregate for a period of 60 min, only the cells expressing the full-length P0 had formed large aggregates, while the cells expressing the truncated P0 molecules were still mostly single cells indistinguishable from the control cells. Furthermore, 25-30% of the full-length P0 was insoluble in NP40, indicative of an interaction with the cytoskeleton, whereas only 5-10% of P0 lacking 52 amino acids and none of P0 lacking 59 amino acids were insoluble. These results suggest that for the extracellular domain of P0 to behave as a homophilic adhesion molecule, its cytoplasmic domain must be intact, and most probably, it is interacting with the cytoskeleton.
Homophilic adhesion of the myelin P0 protein requires glycosylation of both molecules in the homophilic pair.
The myelin P0 protein is glycosylated at a single site, asparagine 93, within its only immunoglobulin (Ig)-like domain. We have previously shown that P0 behaves like a homophilic adhesion molecule (Filbin, M. T., F. S. Walsh, B. D. Trapp, J. A. Pizzey, and G. I. Tennekoon. 1990. Nature (Lond.). 344:871-872). To determine if the sugar residues of this molecule contribute to its adhesiveness, the glycosylation site was eliminated by replacing asparagine 93 with an alanine, through site-directed mutagenesis of the P0 cDNA. The mutated P0 cDNA was transfected into CHO cells and surface expression of the mutated P0 was assessed by immunofluorescence, limited trypsinization and an ELISA. A cell line was chosen which expressed approximately equivalent amounts of the unglcosylated P0 (UNGP0) at the cell surface as did a cell line expressing the fully glycosylated P0 (GPo); the adhesive properties of these two cell lines were compared. It was found that when a single cell suspension of the UNGPo cells were incubated, by 60 min, unlike the GP0 cells, they had not formed large aggregates; they were indistinguishable from the control transfected cells. This suggests that the UNGP0 protein does not behave like an adhesion molecule. To establish if only one molecule in the P0:P0 homophilic pair must be glycosylated for adhesion to occur, the ability of UNGP0 cells to adhere to GP0 cells was assessed both qualitatively and quantitatively. The results of both types of assay imply that, indeed, both P0 molecules in the homophilic pair must be glycosylated for adhesion to take place.
Myelin P0-protein, more than just a structural protein?
The protein P0 has long been proposed to be responsible for the compact nature of peripheral myelin through interactions of both its extracellular and cytoplasmic domains. Recent studies support such a role for P0's extracellular region while more precise mapping of its adhesive domains are ongoing. As P0 is a member of the immunoglobulin gene superfamily and perhaps bears the closest similarity to the ancestral molecule of this whole family, these studies may also have more general implications for adhesive interactions. In addition, although long believed to be purely an inert, structural molecule, P0 has been reported to promote neurite outgrowth, which suggests a more dynamic role for this interesting molecule.
The role of complex carbohydrates in adhesion of the myelin protein, P0.
The most abundant protein of peripheral nerve myelin, a glycoprotein termed P0, is believed to be involved in the compaction of the myelin sheath and is postulated to be the closest relative to the ancestral gene for the immunoglobulin superfamily. Recently, P0 has indeed been shown to behave like a homophilic adhesion molecule via interactions of its extracellular domains. Here we demonstrate the importance of the oligosaccharide moieties of P0 in its functioning as a homophilic adhesion molecule. Expression of the complex form of P0 glycoprotein in transfected Chinese hamster ovary cells greatly increased the adhesiveness of those cells, whereas expression of the high-mannose form of P0 glycoprotein did not. This is the first step in the dissection of P0-P0 interaction at the molecular level.
Role of myelin P0 protein as a homophilic adhesion molecule.
Peripheral nervous system myelin is an extension of the Schwann cell's plasma membrane that tightly enwraps axons in many layers and permits nerve impulses to be rapidly conducted. It is not known how these multiple membrane layers are held together in this compact form. Here we present evidence supporting the hypothesis that the extracellular leaflets of myelin are held together by the most abundant protein of myelin of the peripheral nervous system, P0, by homophilic interaction of its extracellular domains. Transfected Chinese hamster ovary cells expressing P0 protein adhere to each other in suspension, to form large aggregates, whereas cells that are identical but which do not express P0 do not. We also show that this aggregation is mediated by homophilic binding between P0-expressing cells and that the apposing plasma membranes of these cells specifically form desmosomes, whereas control transfected cells do not. As the only difference between the two cell populations is the expression of P0, this protein is apparently responsible for the changes in morphology and adhesion in the cells that express it. The idea that P0 is a homophilic adhesion molecule is supported by its inclusion in the immunoglobulin supergene family, all members of which are involved in recognition and/or adhesion.
High level of expression of the myelin protein P0 in Chinese hamster ovary cells.
The major PNS myelin protein, P0, has been expressed in Chinese hamster ovary cells by transfection with a plasmid containing the P0-cDNA. The expression of P0 at both the RNA and the protein level was greatly increased, without detriment to the cell, by the dihydrofolate reductase-methotrexate strategy of gene amplification. The P0 expressed by these cells was glycosylated (containing approximately equal amounts of the complex and high-mannose type glycoproteins) and reached the plasma membrane. This system is suitable not only for addressing the function of P0 directly, but it also applicable to any protein of which an abundance is needed.
Purification and characterization of plasma membranes obtained from rat neurons prepared by bulk-isolation.
The properties of neuronal plasma membranes are highly specialized, since they include the receptors by which many hormones, neurotransmitters, drugs, and toxins initiate their actions, as well as the components necessary for receiving synaptic input from a variety of sources and for generating the appropriate pattern of action potentials. To be able to study plasma membranes of neurons, it is necessary to separate sufficient quantities of neurons from the other cell types, processes, and myelin found in brain. The methodology for obtaining neurons by bulk-isolation from 10-day-old rat brain has been established, and techniques for purifying the plasma membranes are presented here. The procedure involves swelling the cells in a buffered solution, homogenizing the cells vigorously, and separating the membrane fragments on discontinuous sucrose gradients. The plasma membrane fraction is enriched in plasma membrane marker enzymes and has low activity for contaminating enzymes for subcellular organelles. Electron micrographs show that the fraction consists of membrane vesicles and profiles of various sizes. The protein composition reveals over 60 proteins with molecular weights as high as 100,000 to a low of 14,000. This plasma membrane fraction now can be the focus for studying receptors, glycoproteins, and cell-specific markers of neurons in health and disease.
A comparison of the glycoproteins and the proteins from multiple sclerosis and normal brain tissue.
Two highly sensitive techniques were used to identify glycoproteins and proteins in several tissue fractions from two normal brains and five brains from multiple sclerosis (MS) patients. Comparison of glycoproteins and proteins in white and gray matter, myelin and a myelin-related fraction between normal and MS brains not only showed the presence in all fractions of many more minor components than has previously been reported, but also subtle differences in some of these components. However, no change was unequivocally MS-specific. This provides additional evidence that MS is a demyelinating rather than a dysmyelinating disorder and may lead to some insight into the etiology and/or progression of this disease. Moreover, this study has served to characterize further the proteins and glycoproteins of human brain tissue.
Biochemical identification of a putative glutamate receptor in housefly thoracic membranes.
Specific stereoselective binding of [3H]L-glutamate was detected to membranes prepared from housefly thorax to which were added several antiproteases. A single high affinity binding site was detected (KD 0.5 +/- 0.04 microM), but total binding varied from preparation to preparation (5-60 pmoles/mg protein). Specific binding was inhibited by preincubation of the membranes with trypsin, chymotrypsin or protease, or by exposure to 70 degrees C for 5 min. It was also inhibited by several compounds, the most potent being L-glutamate and L-aspartate, followed by L-glutamate diethylester, then D-glutamate, N-methyl-D-aspartate and ibotenate. Quisqualate had little effect, while kainate, proctolin and D-aspartate had none. d-Tubocurarine stimulated [3H]L-glutamate binding. The data suggest that [3H]L-glutamate is binding to an L-glutamate receptor in housefly thoracic muscle membranes.
Partial purification and characterisation of an acetylcholine receptor with nicotinic properties from the supraoesophageal ganglion of the locust (Schistocerca gregaria).
An alpha-bungarotoxin-binding component has been partially purified from the supraoesophageal ganglion of the locust, (Schistocerca gregaria). The component binds alpha-bungarotoxin with a Kd of about 1.7 nM and this value changes little throughout the purification procedure. The specific binding activity ranges from 1.18 pmol alpha-bungarotoxin bound/mg protein for the membrane-bound site up to a maximum of 230 pmol bound/mg protein for the partially purified component. The pharmacological properties of the membrane-bound site are predominantly nicotinic. Affinity labelling of the binding species with 4-(N-maleimido)-[3H]benzyltrimethylammonium suggests that the binding is associated with a peptide of Mr 58000. Polyacrylamide gel electrophoresis of the partially purified of binding component shows three major bands corresponding to Mr of 60000, 41000 and 25000. We suggest that the binding component can be tentatively identified as a nicotinic acetylcholine receptor.
Glutamate receptor binding in insects and mammals.
High affinity stereospecific binding sites for L-glutamate have been reported in several regions of mammalian brain. The binding sites in the hippocampus and cerebellum have been studied more extensively than binding in other brain regions. The hippocampal and cerebellar binding sites show similar properties with respect to their pharmacology and their independence of Na+. There is evidence, particularly good in the case of hippocampus, of mechanisms that may regulate the availability of the binding sites in both brain areas. Some progress has been made with the isolation of the hippocampal binding site but the protein has not been extensively characterised. In the case of insect muscle, high-affinity stereospecific binding of L-glutamate to whole membrane preparations, to detergent-solubilised membranes and to isolated proteolipids has been reported. Much greater variability in the binding characteristics is seen than is the case with the mammalian brain preparations. Preliminary experiments suggest that at least four distinct binding sites may be present on insect muscle. The complete characterisation of glutamate binding sites is at present precluded by a lack of potent agonists and antagonists. However, recent advances in the pharmacological classification of receptor sites for the excitatory amino acids in mammalian brain could provide sufficient information to permit the identification of the binding sites as synaptic receptors. Invertebrate toxins whose site of action is the insect neuromuscular junction may well prove to be useful tools with which to isolate and characterise the synaptic receptor proteins.