Myelin-associated glycoprotein (MAG) binds to the nerve cell surface and inhibits nerve regeneration. The nerve cell surface ligand(s) for MAG are not established, although sialic acid-bearing glycans have been implicated. We identify the nerve cell surface gangliosides GD1a and GT1b as specific functional ligands for MAG-mediated inhibition of neurite outgrowth from primary rat cerebellar granule neurons. MAG-mediated neurite outgrowth inhibition is attenuated by (i) neuraminidase treatment of the neurons; (ii) blocking neuronal ganglioside biosynthesis; (iii) genetically modifying the terminal structures of nerve cell surface gangliosides; and (iv) adding highly specific IgG-class antiganglioside mAbs. Furthermore, neurite outgrowth inhibition is mimicked by highly multivalent clustering of GD1a or GT1b by using precomplexed antiganglioside Abs. These data implicate the nerve cell surface gangliosides GD1a and GT1b as functional MAG ligands and suggest that the first step in MAG inhibition is multivalent ganglioside clustering.
Lateral assemblies of sphingolipids, glycosphingolipids and cholesterol, termed rafts, are postulated to be present in biological membranes and to function in important cellular phenomena. We probed whether rafts are heterogeneous by determining the relative distribution of two gangliosides, GM1 and GD3, in artificial supported monolayers, in intact rat primary cerebellar granule neurones, and in membrane rafts isolated from rat cerebellum. Fluorescence resonance energy transfer (FRET) using fluorophore-labelled cholera toxin B subunit (which binds GM1) and mAb R24 (which binds GD3) revealed that GM1 spontaneously self-associates but does not co-cluster with GD3 in supported monolayers and on intact neurones. Cholera toxin and immunocytochemical labelling of isolated membrane rafts from rat cerebellum further demonstrated that GM1 does not co-localise with GD3. Furthermore, whereas the membrane raft resident proteins Lyn and caveolin both co-localise with GD3 in isolated membrane rafts, GM1 appears in separate and distinct aggregates. These data support prior reports that membrane rafts are heterogeneous, although the mechanisms for establishing and maintaining such heterogeneity remain to be determined.
Enzyme I (EI) is the first protein in the phosphotransfer sequence of the bacterial phosphoenolpyruvate:glycose phosphotransferase system. This system catalyzes sugar phosphorylation/transport and is stringently regulated. Since EI homodimer accepts the phosphoryl group from phosphoenolpyruvate (PEP), whereas the monomer does not, EI may be a major factor in controlling sugar uptake. Previous work from this and other laboratories (e.g. Dimitrova, M. N., Szczepanowski, R. H., Ruvinov, S. B., Peterkofsky, A., and Ginsburg A. (2002) Biochem. 41, 906 -913), indicate that K a is sensitive to several parameters. We report here a systematic study of K a determined by sedimentation equilibrium, which showed that it varied by 1000-fold, responding to virtually every parameter tested, including temperature, phosphorylation, pH (6.5 versus 7.5), ionic strength, and especially the ligands Mg 2؉ and PEP. This variability may be required for a regulatory protein. The phosphoenolpyruvate:glycose phosphotransferase system (PTS), 3 is widely distributed in bacteria and has several important roles in these cells, the most general being PTS sugar uptake where these substrates are translocated across the cytoplasmic membrane concomitant with their phosphorylation. Indeed, the PTS was first discovered as a sugar "kinase" (2) and only somewhat later recognized to be a translocase (3).The system has been extensively studied and reviewed (4, 5). Although variations of the basic motif are known, the most general phosphotransfer sequence is as follows. PEP3 Enzyme I 3 HPr 3 Sugar-specific Enzymes II SugarsEach step is physiologically reversible except for the last, phosphotransfer to the sugar. The phosphotransfer potential of PEP is 14.7 kcal/ mol, about twice that of ATP and greater than any other naturally occurring phosphate derivative. Since the phosphotransfer potentials of the PTS proteins are close to that of PEP, the energetics of the system strongly favor sugar uptake (6). From these considerations alone, it is apparent that the PTS must be stringently regulated, and indeed it is. Even the earliest results on the glucose permease by Kepes (7), before the PTS was discovered (2), showed that when a noncatabolizable Glc analogue, methyl ␣-D-glucopyranoside, is taken up by intact cells, the rate of uptake declined virtually immediately. Thus, the progress curves for uptake of PTS sugars resemble hyperbolas. These results are observed not only with intact cells but also with membrane vesicles supplied with unlimited quantities of PEP (8). We originally suggested Enzyme I as a potential candidate for governing the system (6). This idea is based on the facts that EI monomer forms a homodimer (9, 10), that the dimer but not the monomer is phosphorylated by PEP in the presence of Mg 2ϩ , and that the rate of association/dissociation is surprisingly slow, much slower than sugar uptake (11)(12)(13). This difference in rates suggests that regulation of sugar transport could be affected by factors or ligands (e.g. metabolites or other prote...
Heparin and heparan sulfate have recently been shown to bind to snake cardiotoxin (CTX) and to potentiate its penetration into phospholipid monolayer under physiological ionic conditions. Herein we analyze the heparinbinding domain of CTX using 10 CTXs from Taiwan and African cobra venom. We also performed computer modeling to obtain more information of the binding at molecular level. The results provide a molecular model for interaction of CTX-heparin complex where the cationic belt of the conserved residues on the concave surface of three finger -sheet polypeptides initiates ionic interaction with heparin-like molecules followed by specific binding of Lys residues near the tip of loop 2 of CTX. The dissociation constants of CTXs differ by as much as 4 orders of magnitude, ranging from ϳ140 M for toxin ␥ to ϳ20 nM for CTX M3, depending on the presence of Lys residues near the tip of loop 2. High affinity heparin binding becomes possible due to the presence of Arg-28, Lys-33, or the socalled consensus heparin binding sequence of XKKXXXKRX near the tip of the loop. The well defined three-finger loop structure of CTX provides an interesting template for the design of high affinity heparin-binding polypeptides with -sheet structure. The finding that several cobra CTXs and phospholipase A2 bind to heparin with different affinity may provide information on the synergistic action of the two venom proteins.
The bacterial phosphoenolpyruvate (PEP):glycose phosphotransferase system (PTS) mediates uptake/phosphorylation of sugars. The transport of all PTS sugars requires Enzyme I (EI) and a phosphocarrier histidine protein of the PTS (HPr). The PTS is stringently regulated, and a potential mechanism is the monomer/dimer transition of EI, because only the dimer accepts the phosphoryl group from PEP. EI monomer consists of two major domains, at the N and C termini (EI-N and EI-C, respectively). EI-N accepts the phosphoryl group from phospho-HPr but not PEP. However, it is phosphorylated by PEP(Mg 2؉ ) when complemented with EI-C. Here we report that the phosphotransfer rate increases ϳ25-fold when HPr is added to a mixture of EI-N, EI-C, and PEP(Mg 2؉ ). A model to explain this effect is offered. Sedimentation equilibrium results show that the association constant for dimerization of EI-C monomers is 260-fold greater than the K a for native EI. The ligands have no detectable effect on the secondary structure of the dimer (far UV CD) but have profound effects on the tertiary structure as determined by near UV CD spectroscopy, thermal denaturation, sedimentation equilibrium and velocity, and intrinsic fluorescence of the 2 Trp residues. The binding of PEP requires Mg 2؉ . For example, there is no effect of PEP on the T m , an increase of 7°C in the presence of Mg 2؉ , and ϳ14°C when both are present. Interestingly, the dissociation constants for each of the ligands from EI-C are approximately the same as the kinetic (K m ) constants for the ligands in the complete PTS sugar phosphorylation assays.The best characterized function of the phosphoenolpyruvate:glycose phosphotransferase system (PTS) 4 is in the uptake of sugars by bacterial cells, which are phosphorylated during their translocation across the cytoplasmic membrane (for reviews see Refs. 1-3). There are many variants of the PTS, but all of them require the first two proteins in the phosphotransfer process, Enzyme I (EI) and the low molecular weight phosphocarrier protein, HPr. The first two steps in the reaction sequence are depicted in Reactions 1 and 2.The phosphoryl group is then transferred from P-HPr to the sugarspecific proteins. The properties of EI have been reviewed previously (4, 5).As discussed in an earlier report (6), the phosphotransfer potential of PEP is so high that the PTS must be stringently regulated or cells would burst with accumulated sugar phosphate. One potential mechanism for this regulation is dependent on the properties of the monomer/dimer transition of EI. The dimer accepts the phosphoryl group from PEP, whereas the monomer does not, and association/dissociation is a very slow process relative to the phosphotransfer reactions. A report on the kinetics of EI phosphorylation and dimerization has recently been published (7), and the hydrodynamic properties of the monomer/dimer transition are described in the companion paper (36).Early work on thermal unfolding of EI, using high sensitivity scanning calorimetry and partial proteolysis,...
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