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Previous reports demonstrated that monocarboxylate transporter-1 (MCT1) interacts with Basigin. It was hypothesized that the two proteins interact via the transmembrane domain of Basigin, specifically through the glutamate residue within the domain. We therefore sought to test this hypothesis and determine which amino acids of the Basigin protein are necessary for the interaction with MCT1. Probes consisting of the full-length putative transmembrane domain, as well as small regions of the domain, were generated for use in ELISA binding assays using endogenous mouse MCT1. Site directed mutagenesis of candidate residues was performed and probes were generated for ELISA analyses to determine the specific residues involved. The data suggest that hydrophobic residues at the N- and C-termini of the putative transmembrane domain of Basigin interact with MCT1, but the glutamate plays no role. The previously proposed hypothesis is partially correct, in that the putative transmembrane domain of Basigin does interact with MCT1. 相似文献
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Heller M von der Ohe M Kleene R Mohajeri MH Schachner M 《Journal of neurochemistry》2003,84(3):557-565
Recognition molecules that carry carbohydrate structures regulate cell interactions during development and play important roles in synaptic plasticity and regeneration in the adult. Glycans appear to be involved in these interactions. We have searched for binding proteins for oligomannosidic structures using the L3 antibody directed against high mannose-type glycans in an anti-idiotypic approach. A selected monoclonal anti-idiotype antibody was used for affinity chromatography and identified basigin as a binding protein from mouse brain detergent lysates. Basigin was found to bind to high mannose-carrying cell recognition molecules, such as myelin-associated glycoprotein, L1, the beta2-subunit of Na+/K+-ATPase and an oligomannosidic neoglycolipid. Furthermore, basigin was involved in outgrowth of astrocytic processes in vitro. A striking homology between the first immunoglobulin (Ig)-like domain of basigin and the fourth Ig-like domain of NCAM, previously shown to bind to oligomannosidic glycans, and the lectin domain of the mannose receptor confirms that basigin is an oligomannose binding lectin. To our knowledge this is the first report that anti-idiotypic antibodies can be used to identify binding partners for carbohydrates. 相似文献
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P450cam has long served as a prototype for the cytochrome P450 (CYP) gene family. But, little is known about how substrate enters its active site pocket, and how access is achieved in a way that minimizes exposure of the reactive heme. We hypothesize that P450cam may first bind substrate transiently near the mobile F-G helix that covers the active site pocket. Such a two-step binding process is kinetically required if P450cam rarely populates an open conformation-as suggested by previous literature and the inability to obtain a crystal structure of P450cam in an open conformation. Such a mechanism would minimize exposure of the heme by allowing P450cam to stay in a closed conformation as long as possible, since only brief flexing into an open conformation would be required to allow substrate entry. To test this model, we have attempted to dock a second camphor molecule into the crystal structure of camphor-bound P450cam. The docking identified only one potential entry site pocket, a well-defined cavity on the F-helix side of the F-G flap, 16 A from the heme iron. Location of this entry site pocket is consistent with our NMR T1 relaxation-based measurements of distances for a camphor that binds in fast exchange (active site camphor is known to bind in slow exchange). Presence of a second camphor binding site is also confirmed with [(1)H-(13)C] HSQC titrations of (13)CH3-threonine labeled P450cam. To confirm that camphor can bind outside of the active site pocket, (13)CH3-S-pyridine was bound to the heme iron to physically block the active site, and to serve as an NMR chemical shift probe. Titration of this P450cam-pyridine complex confirms that camphor can bind to a site outside the active site pocket, with an estimated Kd of 43 microM. The two-site binding model that is proposed based on these data is analogous to that recently proposed for CYP3A4, and is consistent with recent crystal structures of P450cam bound to tethered-substrates, which force a partially opened conformation. 相似文献
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Staphylococcal enterotoxin B and toxic shock syndrome toxin-1 bind to distinct sites on HLA-DR and HLA-DQ molecules 总被引:12,自引:0,他引:12
Staphylococcal enterotoxins (SE) activate human T cells in vitro. This requires the presence of Ia+ accessory cells but does not require processing of the toxin by the accessory cell. We and others have recently demonstrated direct binding of SE to human MHC class II molecules. In this study, we have compared in detail the ability of class II molecules to bind two SE, toxic shock syndrome toxin-1 (TSST-1) and SEB. Scatchard analysis of equilibrium binding data indicate that SEB binds to Ia+ human cell lines with a 10-fold lower affinity than TSST-1. Likewise, SEB precipitates HLA-DR alpha- and beta-chains from detergent lysates of Ia+ cells less efficiently than TSST-1. The binding of TSST-1 and SEB to transfected L cells expressing HLA-DR and HLA-DQ gene products was differentially inhibited by anti-HLA-DR mAb. There was virtually no cross-inhibition of TSST-1 and SEB in competitive binding assays. We conclude that TSST-1 and SEB bind to two MHC class II sites which can be distinguished by their relative accessibility to blocking by anti-class II mAb and heterologous toxin. 相似文献
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Lactate release by astrocytes is postulated to be of importance for neuroenergetics but its regulation is poorly understood. Basigin, a chaperone protein for specific monocarboxylate transporters (MCTs), represents a putatively important regulatory element for lactate fluxes. Indeed, basigin knockdown by RNA interference in primary cultures of astrocytes partially reduced both proton-driven lactate influx and efflux. But more strikingly, enhancement of lactate efflux induced by glutamate was prevented while the effect of sodium azide was significantly reduced by treatment of cultured astrocytes with anti-basigin small interfering RNA. Enhancement of glucose utilization was unaffected under the same conditions. Basal lactate uptake and release were significantly reduced by MCT1 knockdown, even more so than with basigin knockdown, whereas glutamate-driven or sodium azide-induced enhancement of lactate release was not inhibited by either MCT1, 2, or 4 small interfering RNAs. In conclusion, MCT1 plays a pivotal role in the control of basal proton-driven lactate flux in astrocytes while basigin is only partly involved, most likely via its interaction with MCT1. In contrast, basigin appears to critically regulate the enhancement of lactate release caused by glutamate (or sodium azide) but via an effect on another unidentified transporter at least present in astrocytes in vitro. 相似文献
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L1 mediated homophilic binding and neurite outgrowth are modulated by alternative splicing of exon 2
The neural cell adhesion molecule (CAM) L1 is a member of the immunoglobulin superfamily that has been implicated in neuronal adhesion, neurite outgrowth, and axon guidance. The clinical importance of L1 is illustrated by pathological mutations that lead to hydrocephalus, mental retardation, motor defects, and early mortality. The L1 gene is composed of 28 exons, including exons 2 and 27 that are spliced alternatively, and mutations in exon 2 are associated with severe neurological abnormalities in humans. To elucidate the role of L1 exon 2, a recombinant Fc fusion protein called Delta2L1 was constructed lacking the second exon in the extracellular domain of L1. When bound to fluorescent beads, L1 exhibited homophilic binding while Delta2L1 did not. However, L1 beads coaggregated with the Delta2L1 beads. Similarly, in cell binding studies, L1 bound to L1 and Delta2L1 did not bind to Delta2L1 but it bound moderately to L1. Given the reduced binding of Delta2L1, we tested its effect on neurons. By comparison to L1, a lower percentage of dissociated neurons extended neurites on Delta2L1, and there was a modest decrease in the length of the neurites that grew. Neurite outgrowth from reaggregated neurons was much less robust on Delta2L1 than on L1. The combined results indicate that Delta2L1 does not bind homophilically but it can interact with L1 containing exon 2. The reduced binding and neurite promoting activity of Delta2L1 provides an explanation for certain pathological mutations in L1 that lead to clinically apparent disease in the absence of the normal form of L1 in the nervous system. 相似文献
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Halestrap AP 《IUBMB life》2012,64(1):1-9
Monocarboxylate transporters (MCTs) catalyze the proton-linked transport of monocarboxylates such as L-lactate, pyruvate, and the ketone bodies across the plasma membrane. There are four isoforms, MCTs 1-4, which are known to perform this function in mammals, each with distinct substrate and inhibitor affinities. They are part of the larger SLC16 family of solute carriers, also known as the MCT family, which has 14 members in total, all sharing conserved sequence motifs. The family includes a high-affinity thyroid hormone transporter (MCT8), an aromatic amino acid transporter (T-type amino acid transporter 1/MCT10), and eight orphan members yet to be characterized. MCTs were predicted to have 12 transmembrane helices (TMs) with intracellular C- and N-termini and a large intracellular loop between TMs 6 and 7, and this was confirmed by labeling studies and proteolytic digestion. Site-directed mutagenesis has identified key residues required for catalysis and inhibitor binding and enabled the development of a molecular model of MCT1 in both inward and outward facing conformations. This suggests a likely mechanism for the translocation cycle. Although MCT family members are not themselves glycosylated, MCTs1-4 require association with a glycosylated ancillary protein, either basigin or embigin, for their correct translocation to the plasma membrane. These ancillary proteins have a single transmembrane domain and two to three extracellular immunoglobulin domains. They must remain closely associated with MCTs1-4 to maintain transporter activity. MCT1, MCT3, and MCT4 bind preferentially to basigin and MCT2 to embigin. The choice of binding partner does not affect substrate specificity or kinetics but can influence inhibitor specificity. 相似文献
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M Oleszewski S Beer S Katich C Geiger Y Zeller U Rauch P Altevogt 《The Journal of biological chemistry》1999,274(35):24602-24610
The cell adhesion molecule L1, a 200-220-kDa type I membrane glycoprotein of the Ig superfamily, mediates many neuronal processes. Originally studied in the nervous system, L1 is expressed by hematopoietic and many epithelial cells, suggesting a more expanded role. L1 supports homophilic L1-L1 and integrin-mediated cell binding and can also bind with high affinity to the neural proteoglycan neurocan; however, the binding site is unknown. We have dissected the L1 molecule and investigated the cell binding ability of Ig domains 1 and 6. We report that RGD sites in domain 6 support alpha5beta1- or alphavbeta3-mediated integrin binding and that both RGD sites are essential. Cooperation of RGD sites with neighboring domains are necessary for alpha(5)beta(1). A T cell hybridoma and activated T cells could bind to L1 in the absence of RGDs. This binding was supported by Ig domain 1 and mediated by cell surface-exposed neurocan. Lymphoid and brain-derived neurocan were structurally similar. We also present evidence that a fusion protein of the Ig 1-like domain of L1 can bind to recombinant neurocan. Our results support the notion that L1 provides distinct cell binding sites that may serve in cell-cell or cell-matrix interactions. 相似文献
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Isolation and characterization of mutants with lesions affecting pellicle formation and erythrocyte agglutination by type 1 piliated Escherichia coli. 总被引:9,自引:5,他引:4
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S L Harris D A Elliott M C Blake L M Must M Messenger P E Orndorff 《Journal of bacteriology》1990,172(11):6411-6418
The product of the pilE (also called fimH) gene is a minor component of type 1 pili in Escherichia coli. Mutants that have insertions in the pilE gene are fully piliated but unable to bind to and agglutinate guinea pig erythrocytes, a characteristic of wild-type type 1 piliated E. coli. In this paper we describe the isolation of 48 mutants with point lesions that map to the pilE gene. Such mutants were isolated by using mutT mutagenesis and an enrichment procedure devised to favor the growth of individuals that could form a pellicle in static broth containing alpha-methylmannoside, an inhibitor of erythrocyte binding and pellicle formation. Results indicated that the enrichment favored mutants expressing pilE gene products that were defective in mediating erythrocyte binding. Characterization of 12 of the mutants in greater detail revealed that certain lesions affected pilus number and length. In addition, a mutant that was temperature sensitive for erythrocyte binding was isolated and used to provide evidence that pellicle formation relies on the intercellular interaction of pilE gene products. Our results suggest a molecular explanation for the old and paradoxical observations connecting pellicle formation and erythrocyte agglutination by type 1 piliated E. coli. 相似文献
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Treatment of mice by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridene hydrochloride (MPTP) is a well established animal model for Parkinson's disease (PD), while overexpression of L1 cell adhesion molecule (L1cam) has been proposed to attenuate the degeneration of dopaminergic neurons induced by MPTP. To gain insight into the role of L1cam in the pathomechanism of PD, we investigated protein expression patterns after MPTP-treatment in both C57BL/6 (wild-type) and transgenic mice overexpressing L1cam in astrocytes. Our results showed that during the acute phase, proteins in functional complexes responsible for mitochondrial, glycolysis, and cytoskeletal function were down-regulated in MPTP-treated wild-type mice. After a recovery phase, proteins that were down-regulated in the acute phase reverted to normal levels. In L1cam transgenic mice, a much higher number of proteins was altered during the acute phase and this number even increased after the recovery phase. Many proteins involved in oxidative phosphorylation were still down-regulated and glycolysis related protein were still up-regulated. This pattern indicates a lasting severely impaired energy production in L1cam mice after MPTP treatment. 相似文献
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E1A induces phosphorylation of the retinoblastoma protein independently of direct physical association between the E1A and retinoblastoma products. 总被引:26,自引:7,他引:19
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We have studied the initial effects of adenovirus E1A expression on the retinoblastoma (RB) gene product in normal quiescent cells. Although binding of the E1A products to pRB could, in theory, make pRB phosphorylation unnecessary for cell cycle progression, we have found that the 12S wild-type E1A product is capable of inducing phosphorylation of pRB in normal quiescent cells. The induction of pRB phosphorylation correlates with E1A-mediated induction of p34cdc2 expression and kinase activity, consistent with the possibility that p34cdc2 is a pRB kinase. Expression of simian virus 40 T antigen induces similar effects. Induction of pRB phosphorylation is independent of the pRB binding activity of the E1A products; E1A domain 2 mutants do not bind detectable levels of pRB but remain competent to induce pRB phosphorylation and to activate cdc2 protein kinase expression and activity. Although the kinetics of induction are slower, domain 2 mutants induce wild-type levels of pRB phosphorylation and host cell DNA synthesis and yet fail to induce cell proliferation. These results imply that direct physical interaction between the RB and E1A products does not play a required role in the early stages of E1A-mediated cell cycle induction and that pRB phosphorylation is not, of itself, sufficient to allow quiescent cells to divide. These results suggest that the E1A products do not need to bind pRB in order to stimulate resting cells to enter the cell cycle. Indeed, a more important role of the RB binding activity of the E1A products may be to prevent dividing cells from returning to G0. 相似文献
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Interleukin-1 (IL-1) receptors can be solubilized from murine cell surfaces and immunoprecipitated with a xenogeneic rat antiserum raised in this laboratory. We demonstrated first that this antiserum contains antibodies directed against IL-1 receptors. We have now successfully used this antiserum as a reagent to immunopurify polysomes along with their messenger RNA from a murine leukemic cell line known to express relatively high levels of IL-1 receptors. The immunoselected mRNA was translated into proteins in vitro. The translation products contained an IL-1 binding protein which could specifically bind to immobilized IL-1 but not to other immobilized ligands such as interleukin-2 or tumor necrosis factor-alpha. The translation products which bound to IL-1 could be acid-eluted from the immobilized ligand, and the proteins released could still specifically bind to IL-1 in a receptor-ligand binding reaction. The eluted IL-1 binding proteins, as well as soluble receptor-ligand complexes derived from them, could also be immunoprecipitated with the xenogeneic rat antiserum. The xenogeneic rat antiserum could, furthermore, immunoprecipitate the IL-1 binding proteins from the translated products before ligand was added. The residual translated products no longer interacted with IL-1. We conclude that our antiserum contains antibodies that recognize determinants expressed on the following proteins: on nascent chains of IL-1 binding proteins; on soluble translated IL-1 binding proteins; on soluble complexes of IL-1 binding proteins that had been cross-linked with IL-1 ligand; and on cell surface-associated IL-1 receptors. The translated and unprocessed IL-1 binding proteins have a molecular mass of approximately 52,000-56,000 daltons. 相似文献
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Becker HM Hirnet D Fecher-Trost C Sültemeyer D Deitmer JW 《The Journal of biological chemistry》2005,280(48):39882-39889
Injection of carbonic anhydrase isoform II (CA) into Xenopus frog oocytes increased the rate of H+ flux via the rat monocarboxylate transporter isoform 1 (MCT1) expressed in the oocytes. MCT1 activity was assessed by changes of intracellular H+ concentration measured by pH-selective microelectrodes during application of lactate. CA-induced augmentation of the rate of H+ flux mediated by MCT1 was not inhibited by ethoxyzolamide (10 microM) and did not depend on the presence of added CO2/HCO3- but was suppressed by injection of an antibody against CA. Deleting the C terminus of the MCT1 greatly reduced its transport rate and removed transport facilitation by CA. Injected CA accelerated the CO2/HCO3(-)-induced acidification severalfold, which was blocked by ethoxyzolamide and was independent of MCT1 expression. Mass spectrometry confirmed activity of CA as injected into the frog oocytes. With pulldown assays we demonstrated a specific binding of CA to MCT1 that was not attributed to the C terminus of MCT1. Our results suggest that CA enhances MCT1 transport activity, independent of its enzymatic reaction center, presumably by binding to MCT1. 相似文献