Biochemistry and Pharmacology - Research Publications
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ItemPresentation of newly synthesized glycoproteins to CD4+ T lymphocytes. An analysis using influenza hemagglutinin transport mutants.(Rockefeller University Press, 1993-04-01)Human lymphoblastoid cells transiently expressing the hemagglutinin (HA) glycoprotein of influenza virus are rapidly and efficiently recognized by CD4+ HA-specific T lymphocytes. This endogenous presentation pathway is sensitive to chloroquine and is therefore likely related to the classical class II major histocompatibility complex (MHC) exogenous pathway of antigen presentation. In this study we have examined a series of transport-defective HA mutants. We correlate the intracellular fate of the native antigen with its presentation characteristics. We have found that the native antigen must enter the secretory pathway since a cytosolic form is not presented. However, surface expression and normal trafficking through the Golgi apparatus are not required for efficient presentation. Instead, escape of native antigen from the endoplasmic reticulum appears to be both necessary and sufficient for gaining access to a compartment where antigen is processed and binds class II MHC molecules.
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ItemDisulfide bond formation during the folding of influenza virus hemagglutinin.(Rockefeller University Press, 1992-07)To study the importance of individual sulfhydryl residues during the folding and assembly in vivo of influenza virus hemagglutinin (HA), we have constructed and expressed a series of mutant HA proteins in which cysteines involved in three disulfide bonds have been substituted by serine residues. Investigations of the structure and intracellular transport of the mutant proteins indicate that (a) cysteine residues in the ectodomain are essential both for efficient folding of HA and for stabilization of the folded molecule; (b) cysteine residues in the globular portion of the ectodomain are likely to form native disulfide bonds rapidly and directly, without involvement of intermediate, nonnative linkages; and (c) cysteine residues in the stalk portion of the ectodomain also appear not to form intermediate disulfide bonds, even though they have the opportunity to do so, being separated from their correct partners by hundreds of amino acids including two or more other sulfhydryl residues. We propose a role for the cellular protein BiP in shielding the cysteine residues of the stalk domain during the folding process, thus preventing them from forming intermediate, nonnative disulfide bonds.
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ItemCisplatin induced emesis: preliminary results indicative of changes in plasma levels of 5-hydroxytryptamine.(Springer Science and Business Media LLC, 1990-11)
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ItemNo Preview AvailablePOSTTRANSLATIONAL MODIFICATIONS DISTINGUISH CELL-SURFACE FROM GOLGI-RETAINED BETA-1,4 GALACTOSYLTRANSFERASE MOLECULES - GOLGI LOCALIZATION INVOLVES ACTIVE RETENTION(OXFORD UNIV PRESS UNITED KINGDOM, 1994-12-01)beta 1,4 Galactosyltransferase (GalT) is a membrane-bound enzyme localized predominantly to the trans-Golgi cisternae. Our previous studies have shown that the transmembrane domain of bovine GalT plays a critical role in Golgi localization (Teasdale, R.D., D'Agostaro, G. and Gleeson, P.A., J. Biol. Chem., 267, 4084-4096, 1992). Here we have compared the localization and post-translational modifications of full-length bovine GalT with a GalT/hybrid molecule where the transmembrane domain of GalT was replaced with that of the transferrin receptor. GalT/hybrid molecules were expressed on the surface of transfected cells; however, differences were observed in the distribution of the hybrid molecules between transfected COS and murine L cells. In transfected COS cells, the GalT/hybrid protein was expressed efficiently at the cell surface, with little Golgi-localized material, whereas in stable murine L cells, which expressed lower levels of the construct, hybrid molecules were detected both at the cell surface and within the Golgi apparatus. Expression of the GalT constructs in either COS or L cells produced two glycoprotein products which differed in molecular mass by 7 kDa. The difference in size between the two products is due to post-translational modifications which are inhibited by brefeldin A and are therefore likely to occur in the trans-Golgi network (TGN). Very little of the high-molecular-weight species was detected for full-length GalT, whereas it was a major product for the GalT/hybrid protein. Only the higher molecular weight species was expressed at the cell surface. Thus, this additional 7 kDa post-translational modification distinguishes molecules retained within the Golgi apparatus (lower M(r) species) from those transported through the TGN to the cell surface. These studies indicate that (i) the level of expression influences the intracellular distribution of GalT/hybrid molecules and (ii) the localization of full-length GalT involves active retention within the Golgi stack, and not retrieval from later compartments. After treatment of membrane preparations from stable L cell clones with a heterobifunctional cross-linking agent, full-length bovine GalT molecules were found almost exclusively as high-molecular-weight aggregates, suggesting that GalT exists as an oligomer or aggregate. This ability to oligomerize may be a requirement for Golgi retention.
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ItemNo Preview AvailableTargeting of proteins to the Golgi apparatus(SPRINGER, 1998-05-01)The proteins that reside in the Golgi carry out functions associated with post-translational modifications, including glycosylation and proteolytic processing, membrane transport, recycling of endoplasmic reticulum proteins and maintenance of the structural organisation of the organelle itself. The latter includes Golgi stacking, interconnections between stacks and the microtubule-dependent positioning of the organelle within the cell. There are a number of distinct groups of Golgi membrane proteins, including glycosyltransferases, recycling trans-Golgi network (TGN) proteins, peripheral membrane proteins and receptors. Considerable effort has been directed at understanding the basis of the localisation of Golgi glycosyltransferases and recycling TGN proteins; in both cases there is increasing evidence that multiple signals may be involved in their specific localisation. A number of models for the Golgi retention of glycosyltransferases have been proposed including oligomerisation, lipid-mediated sorting and intra-Golgi retrograde transport. More information is required to determine the contribution of each of these potential mechanisms in the targeting of different glycosyltransferases. Future work is also likely to focus on the relationship between the localisation of resident Golgi proteins and the maintenance of Golgi structure.
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ItemNo Preview AvailableTARGETING OF PROTEINS TO THE GOLGI-APPARATUS(SPRINGER, 1994-10-01)The Golgi apparatus maintains a highly organized structure in spite of the intense membrane traffic which flows into and out of this organelle. Resident Golgi proteins must have localization signals to ensure that they are targeted to the correct Golgi compartment and not swept further along the secretory pathway. There are a number of distinct groups of Golgi membrane proteins, including glycosyltransferases, recycling trans-Golgi network proteins, peripheral membrane proteins, receptors and viral glycoproteins. Recent studies indicate that there are a number of different Golgi localization signals and mechanisms for retaining proteins to the Golgi apparatus. This review focuses on the current knowledge in this field.
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ItemCathepsin S controls the trafficking and maturation of MHC class II molecules in dendritic cells(ROCKEFELLER UNIV PRESS, 1999-11-15)Before a class II molecule can be loaded with antigenic material and reach the surface to engage CD4+ T cells, its chaperone, the class II-associated invariant chain (Ii), is degraded in a stepwise fashion by proteases in endocytic compartments. We have dissected the role of cathepsin S (CatS) in the trafficking and maturation of class II molecules by combining the use of dendritic cells (DC) from CatS(-/-) mice with a new active site-directed probe for direct visualization of active CatS. Our data demonstrate that CatS is active along the entire endocytic route, and that cleavage of the lysosomal sorting signal of Ii by CatS can occur there in mature DC. Genetic disruption of CatS dramatically reduces the flow of class II molecules to the cell surface. In CatS(-/-) DC, the bulk of major histocompatibility complex (MHC) class II molecules is retained in late endocytic compartments, although paradoxically, surface expression of class II is largely unaffected. The greatly diminished but continuous flow of class II molecules to the cell surface, in conjunction with their long half-life, can account for the latter observation. We conclude that in DC, CatS is a major determinant in the regulation of intracellular trafficking of MHC class II molecules.
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ItemDegradation of mouse invariant chain: Roles of cathepsins S and D and the influence of major histocompatibility complex polymorphism(ROCKEFELLER UNIV PRESS, 1997-08-18)Antigen-presenting cells (APC) degrade endocytosed antigens into peptides that are bound and presented to T cells by major histocompatibility complex (MHC) class II molecules. Class II molecules are delivered to endocytic compartments by the class II accessory molecule invariant chain (Ii), which itself must be eliminated to allow peptide binding. The cellular location of Ii degradation, as well as the enzymology of this event, are important in determining the sets of antigenic peptides that will bind to class II molecules. Here, we show that the cysteine protease cathepsin S acts in a concerted fashion with other cysteine and noncysteine proteases to degrade mouse Ii in a stepwise fashion. Inactivation of cysteine proteases results in incomplete degradation of Ii, but the extent to which peptide loading is blocked by such treatment varies widely among MHC class II allelic products. These observations suggest that, first, class II molecules associated with larger Ii remnants can be converted efficiently to class II-peptide complexes and, second, that most class II-associated peptides can still be generated in cells treated with inhibitors of cysteine proteases. Surprisingly, maturation of MHC class II in mice deficient in cathepsin D is unaffected, showing that this major aspartyl protease is not involved in degradation of Ii or in generation of the bulk of antigenic peptides.
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ItemCHANGES IN THE REPERTOIRE OF PEPTIDES BOUND TO HLA-B27 SUBTYPES AND TO SITE-SPECIFIC MUTANTS INSIDE AND OUTSIDE POCKET-B(ROCKEFELLER UNIV PRESS, 1993-03-01)HLA-B27 subtypes share many structural features, including their pocket B, which interacts with a conserved Arg residue at the second position of B*2705-bound peptides. Subtypes differ among each other at other locations in the peptide binding site. In this study, metabolic labeling and radiochemical pool sequencing were used to address the following issues: (a) presence of the Arg 2 (R2) motif among peptides bound to the various HLA-B27 subtypes; (b) influence of mutations inside and outside pocket B on this motif; and (c) the degree of similarity among the peptide pools bound to the various B27 subtypes. Sequencing of Arg-labeled peptide pools extracted from B*2701 to B*2706, and from two site-directed mutants of B*2705 with changes outside pocket B, indicated that all of these molecules bind peptides with Arg at position 2. Peptides from several mutants with changes altering the structure of pocket B, and from one mutant at the pocket B rim, also retained the R2 motif. However, this was absent in the peptide pool extracted from the M45 mutant, in which the negative charge of pocket B, conferred to HLA-B27 by Glu45, was canceled. These results indicate that alterations outside pocket B, and even disruption of the network of hydrogen bonds that stabilizes Arg binding in pocket B, do not impair binding of peptides bearing the R2 motif, but a nonconservative substitution at position 45 does. As a substantial fraction of anti-B*2705 cytotoxic T lymphocyte (CTL) clones crossreact with the M45 mutant (Villadangos, J., B. Galocha, D. López, V. Calvo, and J. A. López de Castro. 1992. J. Immunol. 149:505) this result suggest that determinant mimicry by nonidentical peptides may frequently account for unexpected CTL crossreactions. Metabolic labeling with various other amino acids and radiochemical sequencing revealed similarities, but also substantial differences, among the peptide pools from the various HLA-B27 subtypes. This strongly suggests that many peptides bind to multiple subtypes, but significant subsets of peptides bound to a given HLA-B27 subtype do not bind to other subtypes or do so with greatly altered efficiency. These results indicate the importance of polymorphism outside pocket B in modulating peptide binding to HLA-B27.
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ItemAN AUTOIMMUNE-DISEASE WITH MULTIPLE MOLECULAR TARGETS ABROGATED BY THE TRANSGENIC EXPRESSION OF A SINGLE AUTOANTIGEN IN THE THYMUS(ROCKEFELLER UNIV PRESS, 1993-08-01)Many autoimmune diseases are characterized by autoantibody reactivities to multiple cellular antigens. Autoantigens are commonly defined as targets of the autoimmune B cell response, but the role, if any, of these autoantigens in T cell-mediated autoimmune diseases is generally unknown. Murine experimental autoimmune gastritis is a CD4+ T cell-mediated organ-specific autoimmune disease induced by neonatal thymectomy of BALB/c mice. The murine disease is similar to human autoimmune gastritis and pernicious anemia, and is characterized by parietal and chief cell loss, submucosal mononuclear cell infiltrates, and autoantibodies to the alpha and beta subunits of the gastric H/K ATPase. However, the specificity of T cells that cause the disease is not known. To examine the role of the H/K ATPase in this T cell-mediated disease, transgenic mice were generated that express the beta subunit of the H/K ATPase under the control of the major histocompatibility complex class II I-Ek alpha promoter. We show that transgenic expression of the gastric H/K ATPase beta subunit specifically prevents the onset of autoimmune gastritis after neonatal thymectomy. In addition, thymocyte transfer experiments suggest that tolerance of pathogenic autoreactive T cells is induced within the thymus of the transgenic mice. We conclude that the beta subunit of the gastric H/K ATPase is a major T cell target in autoimmune gastritis and that thymic expression of a single autoantigen can abrogate an autoimmune response to multiple autoantigens.