Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.5.1.4 (deaminase)
 5,113 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To evaluate the functional role of the N-linked oligosaccharides of major histocompatibility complex (MHC) class II molecules, affinity-purified murine IAs class II molecules were deglycosylated in the presence of asparagine amidase enzyme. The deglycosylated IAs molecules were characterized by 12% SDS-polyacrylamide gel analysis under reduced and native conditions and the complete enzymatic removal of all three N-linked sugar components from the alpha/beta heterodimer was confirmed by lectin-link Western blot analysis. Like the native IAs molecules, the deglycosylated IAs molecules were fully capable of binding an antigenic peptide from myelin basic protein MBP(89-101). The kinetics of dissociation of preformed complexes of IAs.MBP(89-101) and deglycosylated IAs.MBP(89-101) were compared at 4 and at 37 degrees C. Both complexes were equally stable at 4 degrees C; however, at 37 degrees C the deglycosylated IAs.MBP(89-101) complexes showed an increased rate of dissociation as compared with the native IAs.MBP(89-101) complexes. When tested for their ability to recognize the T cell receptor on T cells, both complexes bound to cloned HS-1 T cells that recognize and respond to IAs.MBP(89-101). Finally, the complexes of deglycosylated IAs.MBP(89-101) were tested for the induction of in vitro nonresponsiveness and compared with native IAs.MBP(89-101) complexes. Both complexes were capable of inducing 95-100% nonresponsiveness in a proliferation assay. These results suggest that the N-linked oligosaccharide of MHC class II molecules may not be essential for either antigenic peptide binding or T cell recognition. In addition results obtained here provide evidence that the carbohydrate moities of MHC class II molecules may not be involved in induction of T cell clonal anergy.
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PMID:N-linked oligosaccharides of murine major histocompatibility complex class II molecule. Role in antigenic peptide binding, T cell recognition, and clonal nonresponsiveness. 138 2

A specific increase in T cell extracellular acidification rate has been demonstrated recently when complexes of purified MHC class II molecules and antigenic peptides interact with T cell receptors (TCRs) on cloned T cells. The present study shows that such measurements of an increase in extracellular acidification rate can be used to evaluate the functional role of various N-linked oligosaccharides of MHC class II antigens. Affinity-purified murine IAk and IAs were deglycosylated in the presence of aspargine-amidase enzyme and were characterized by SDS-polyacrylamide gel electrophoresis. The complete removal of all three N-linked oligosaccharides from the alpha/beta heterodimer was confirmed by four different lectin-linked Western blot analyses. Similar to the native heterodimer, both deglycosylated IAk and deglycosylated IAs were fully capable of binding synthetic antigenic peptides derived from myelin basic protein (MBP). When equivalent amount of glycosylated and deglycosylated class II-peptide complexes were exposed to restricted cloned T cells, identical increases in T cell extracellular acidification rates were observed. The specificity of such increases in extracellular acidification rate was demonstrated by exposing cloned T cells to irrelevant complexes of glycosylated and deglycosylated class II and antigenic peptides. These results show how measurement of extracellular acidification rate can be used to study structure-function correlations of ligand-receptor interactions, and support an earlier observation that N-linked oligosaccharides of murine MHC class II molecules are not involved in either antigenic peptide binding or T cell recognition.
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PMID:The role of N-linked oligosaccharides of MHC class II antigens in T cell stimulation. 751 35

Major histocompatibility (MHC) class II antigens are heterodimeric cell surface glycoproteins consisting of an alpha and a beta chain. Although one-dimensional SDS-polyacrylamide gel electrophoresis analysis of purified MHC class II antigens shows a single diffuse band for each chain, multiple spots of identical molecular size were observed for each chain when analyzed by two-dimensional electrophoresis. The basis of this heterogeneity has not been clearly defined and has been predicted partially to be due to glycosylation and/or phosphorylation of the mature protein. To investigate the role of the three N-linked oligosaccharides of the alpha and beta chains in determining the isoelectric point of each chain, affinity-purified MHC class II antigens from human and rat sources were deglycosylated using asparagine amidase. The complete enzymatic removal of all three N-linked oligosaccharides was confirmed by SDS-polyacrylamide gel electrophoresis as well as by four different lectin-linked Western blot analyses. Two-dimensional gel analysis of the deglycosylated molecules shows no significant difference from the fully glycosylated chains. We have expressed truncated forms of the HLA DR2 chains which lack the transmembrane and cytoplasmically exposed regions in Escherichia coli. Two-dimensional electrophoresis of these single chains also reveal multiple banding patterns. The two-dimensional banding patterns described are unaffected by exposure to acidic or basic conditions, increased gel running time in the first dimension, treatment of the proteins with alkaline phosphatase to remove any potential phosphorylation, or preincubation in the presence of iodoacetamide. Multiple forms of recombinant alpha and beta chains were also observed in Tris-glycine-urea gels which merged into a single band in the presence of SDS. In addition, partially fractionated bands from preparative isoelectric focusing gels, when refocused, showed an identical number of multiple spots spanning the same range of isoelectric points. These results together suggest that each polypeptide chain of MHC class II antigens may exist in multiconformational forms, and the observed charge heterogeneity is independent of glycosylation and phosphorylation of the proteins.
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PMID:Intramolecular charge heterogeneity in purified major histocompatibility class II alpha and beta polypeptide chains. 814 5

Arachidonylethanolamide (anandamide, AEA) has been identified as an endogenous ligand for cannabinoid receptors CB1 and CB2. Characterization of the direct cannabimimetic actions of anandamide has been hampered by its short duration of action and rapid degradation in in vivo and in vitro systems to arachidonic acid, a precursor in the biosynthesis of a broad range of biologically active molecules. In the present studies, we utilized 2-methylarachidonyl-(2'-fluoroethyl)amide (F-Me-AEA), an analog of anandamide resistant to enzymatic degradation, to determine whether F-Me-AEA modulated T cell function similar to that of plant-derived cannabinoids. Indeed, F-Me-AEA at low micromolar concentrations exhibited a marked inhibition of phorbol ester plus calcium ionophore (PMA/Io)-induced IL-2 protein secretion and steady state mRNA expression. Likewise, a modest suppression of the mixed lymphocyte response was observed in the presence of F-Me-AEA indicating an alteration in T cell responsiveness to allogeneic MHC class II antigens. F-Me-AEA was also found to modestly inhibit forskolin-stimulated adenylate cyclase activity in thymocytes and splenocytes, a hallmark of cannabinoid receptor agonists. Further characterization of the influence of F-Me-AEA on the cAMP signaling cascade revealed an inhibition of CREB-1/ATF-1 phosphorylation and subsequently, an inhibition of CRE DNA binding activity. Characterization of nuclear binding proteins further revealed that NF-AT and, to a lesser extent, NF-kappaB DNA binding activities were also suppressed. These studies demonstrate that F-Me-AEA modulates T cell function in a similar manner to plant-derived and endogenous cannabinoids and therefore can be utilized as an amidase- and hydrolysis-resistant endogenous cannabinoid.
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PMID:Inhibition of leukocyte function and interleukin-2 gene expression by 2-methylarachidonyl-(2'-fluoroethyl)amide, a stable congener of the endogenous cannabinoid receptor ligand anandamide. 1589 38