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M J Gething

Publications and source records attributed to M J Gething.

At least 73 records · Page 4Linked to original sources
PubMed1989

Identification of immunoglobulin heavy chain binding protein as glucose-regulated protein 78 on the basis of amino acid sequence, immunological cross-reactivity, and functional activity.

Immunoglobulin heavy chain binding protein (BiP) associates transiently with various proteins destined for the secretory pathway. To investigate the relationship between BiP and the 78K (K = 10(3) Mr) glucose-regulated protein (GRP78), we have determined a partial amino acid sequence of purified mouse BiP and isolated and sequenced a full-length cDNA clone encoding mouse GRP78. The 26 amino-terminal residues of the mature BiP protein are identical to a sequence of amino acids located near the start of the open reading frame encoding GRP78. A polyclonal antiserum raised against mouse GRP78 protein expressed in bacteria from the cloned GRP78 cDNA could immunoprecipitate complexes consisting of BiP and unfolded forms of immunoglobulin heavy chains. Furthermore, a monoclonal antibody raised against mouse BiP immunoprecipitated mouse GRP78 expressed in monkey CV-1 cells from an SV40-GRP78 recombinant vector. Finally, like the endogenous BiP of simian cells, mouse GRP78 associated with malfolded, non-glycosylated forms of influenza hemagglutinin (HA) when GRP78 and HA were co-expressed from SV40 vectors in CV-1 cells. These studies confirm that BiP is identical to GRP78. Comparison of the nucleic acid and deduced amino acid sequence of mouse GRP78 with those of other rodent and human GRP78s revealed an extremely high degree of sequence identity. BiP/GRP78 is closely related (approximately 60% identity) to the cytoplasmic 70K heat-shock proteins. Surprisingly, the carboxy-terminal 29 amino acids of BiP/GRP78, which are not conserved in HSP70 proteins, are almost identical in sequence to the steroidogenesis activator peptide found in the cytoplasm of rat Leydig tumor cells. Possible relationships between these polypeptides are discussed.

Amino Acid Sequence↗
PubMed1988

The presence of malfolded proteins in the endoplasmic reticulum signals the induction of glucose-regulated proteins.

Two glucose-regulated proteins, GRP78 and GRP94, are major constituents of the endoplasmic reticulum (ER) of mammalian cells. These proteins are synthesized constitutively in detectable amounts under normal growth conditions; they can also be induced under a variety of conditions of stress including glucose starvation and treatment with drugs that inhibit cellular glycosylation, with calcium ionophores or with amino-acid analogues. Unlike the closely-related heat shock protein (HSP) family, the GRPs are not induced significantly by high temperature. Recently, GRP78 has been identified as the immunoglobulin heavy chain binding protein (BiP) (ref. 5 and Y.K. et al., in preparation) which binds transiently to a variety of nascent, wild-type secretory and transmembrane proteins and permanently to malfolded proteins that accumulate within the ER. We have tested the hypothesis that the presence of malfolded proteins may be the primary signal for induction of GRPs by expressing wild-type and mutant forms of influenza virus haemagglutinin (HA) in simian cells. Only malfolded HAs, whose transport from the ER is blocked, induced the synthesis of GRPs 78 and 94. Additional evidence is presented that malfolding per se, rather than abnormal glycosylation, is the proximal inducer of this family of stress proteins.

Animals↗
PubMed1988

Variants of human tissue-type plasminogen activator that lack specific structural domains of the heavy chain.

The heavy chain of tissue plasminogen activator (t-PA) consists of four domains [finger, epidermal-growth-factor (EGF)-like, kringle 1 and kringle 2] that are homologous to similar domains present in other proteins. To assess the contribution of each of the domains to the biological properties of the enzyme, site-directed mutagenesis was used to generate a set of mutants lacking sequences corresponding to the axons encoding the individual structural domains. The mutant proteins were assayed for their ability to hydrolyze artificial and natural substrates in the presence and absence of fibrin, to bind to lysine-Sepharose and to be inhibited by plasminogen activator inhibitor-1. All the deletion mutants exhibit levels of basal enzymatic activity very similar to that of wild-type t-PA assayed in the absence of fibrin. A mutant protein lacking the finger domain has a 2-fold higher affinity for plasminogen than wild-type t-PA, while the mutant that lacks both finger and EGF-like domains is less active at low concentrations of plasminogen. Mutants lacking both kringles neither bind to lysine-Sepharose nor are stimulated by fibrin. However, mutants containing only one kringle (either kringle 1 or kringle 2) behave indistinguishably from one another and from the wild-type protein. We conclude that kringle 1 and kringle 2 are equivalent in their ability to mediate stimulation of catalytic activity by fibrin.

Amino Acid Sequence↗
PubMed1988

Addition of carbohydrate side chains at novel sites on influenza virus hemagglutinin can modulate the folding, transport, and activity of the molecule.

We have constructed and expressed a series of mutant influenza virus hemagglutinins, each containing a new consensus site for glycosylation in addition to the seven sites found on the wild-type protein. Oligosaccharide side chains were added with high efficiency at four of the five novel sites, located on areas of the protein's surface that are not normally shielded by carbohydrate. Investigations of the structure, intracellular transport, and biological activities of the mutant hemagglutinin molecules indicated that (a) supernumerary carbohydrate side chains can be used to shield or disrupt functional epitopes on the surface of hemagglutinin, and (b) the presence of an additional oligosaccharide may cause temperature-dependent defects in the transport of the glycoprotein. We discuss the addition of supernumerary oligosaccharides as a general tool for shielding chosen areas of the surface of proteins that enter or traverse the secretory pathway.

Amino Acid Sequence↗
PubMed1987

On the role of the transmembrane anchor sequence of influenza hemagglutinin in target cell recognition by class I MHC-restricted, hemagglutinin-specific cytolytic T lymphocytes.

We have examined the requirement for the transmembrane hydrophobic anchor sequence of the influenza hemagglutinin (HA) in the formation of the antigenic moiety on the surface of target cells recognized by class I MHC-restricted murine CTL. For this analysis we have used a line of CV-1 monkey epithelial cells that express the transfected murine H-2Kd gene product as target cells and have used recombinant SV40-based late replacement vectors to achieve expression of genes encoding wild-type and mutant forms of HA. We have found that the majority of Kd-restricted HA-specific CTL clones recognize target cells that express a secreted HA molecule that lacks the transmembrane and cytoplasmic domains of the parent glycoprotein. Several Kd-restricted CTL clones that recognize subtype-specific and crossreactive epitopes on HA fail to recognize the anchor-negative, secreted HA or chimeric HA molecules containing the transmembrane and cytoplasmic domains of unrelated glycoproteins. These CTL clones appear to be directed to antigenic epitopes located within the transmembrane domain of HA, as defined by their capacity to recognize target cells sensitized with a synthetic 23-amino-acid peptide corresponding to sequences within this domain. The implications of these results for class I MHC-restricted CTL recognition are discussed.

Amino Acid Sequence↗
PubMed1987

Translocation in yeast and mammalian cells: not all signal sequences are functionally equivalent.

In Saccharomyces cerevisiae, nascent carboxypeptidase Y (CPY) is directed into the endoplasmic reticulum by an NH2-terminal signal peptide that is removed before the glycosylated protein is transported to the vacuole. In this paper, we show that this signal peptide does not function in mammalian cells: CPY expressed in COS-1 cells is not glycosylated, does not associate with membranes, and retains its signal peptide. In a mammalian cell-free protein-synthesizing system, CPY is not translocated into microsomes. However, if the CPY signal is either mutated to increase its hydrophobicity or replaced with that of influenza virus hemagglutinin, the resulting precursors are efficiently translocated both in vivo and in vitro. The implications of these results for models of signal sequence function are discussed.

Animals↗
PubMed1987

Antigen presentation pathways to class I and class II MHC-restricted T lymphocytes.

Our observations on the cellular immune response to type-A influenza suggest the existence of two distinct pathways of protein antigen presentation to T lymphocytes. One of these pathways is involved with presentation of antigens introduced into the presenting cell from without. This exogenous presentation pathway is the well-recognized route of presentation of soluble and particulate antigens to T lymphocytes. This pathway probably involves uptake of antigen into endocytic vesicles, alteration of antigen within an intracellular compartment, and subsequent display of antigen on the presenting cell surface (Unanue 1984). The second pathway is one which we have tentatively designated as an endogenous presentation pathway. The constraints on this pathway have yet to be fully defined. At a minimum, this pathway appears to involve the presentation of antigens which are synthesized de novo in the presenting cell utilizing the cell's biosynthetic machinery. This pathway may also handle preformed antigens located within the cytosolic compartment of the presenting cell. Perhaps the most striking feature of these two antigen presentation pathways is the close association between the MHC restriction of an antigen-specific T lymphocyte and the pathway of antigen presentation to that T lymphocyte. Our data suggest that this association holds both at the effector level and at the level of induction of T lymphocytes. Thus, presentation of a given antigen by the endogenous pathway preferentially triggers a response from class I MHC-restricted T lymphocytes directed to that antigen. The molecular basis for this link of class I MHC-restriction to the endogenous pathway and MHC class II restriction to the exogenous pathway is unknown. It seems likely that interactions between MHC molecules and antigen within the presenting cell may be critical for the demarcation of these pathways. Thus, for example, antigen presented by the endogenous route may only be able to associate intracellularly with newly synthesized or recycling class I MHC molecules. An understanding of the molecular basis of this phenomenon will require detailed information on the expression, intracellular trafficking, and transport of class I and class II MHC molecules in the antigen-presenting cell. An unresolved issue, at least in the case of viral antigens, is the nature and form of the antigenic moieties presented by the exogenous and endogenous pathways. In the case of viral antigen presentation to class II MHC-restricted T lymphocytes, there is strong, albeit indirect, evidence for processing of antigen and recognition of fragments of viral polypeptides (Lamb et al. 1982, Hackett et al. 1983).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1987

Posttranslational translocation of influenza virus hemagglutinin across microsomal membranes.

The biosynthesis of influenza virus hemagglutinin (HA) and its translocation across microsomal membranes were studied in a mammalian cell-free system. All forms of HA could be cotranslationally translocated with high efficiency. However, only truncated forms of HA were translocated after protein synthesis has been terminated. The efficiency of this posttranslational translocation was dependent on the extent of the truncation. Posttranslational translocation was ribosome dependent and occurred only in the presence of a functional N-terminal signal sequence. The molecular mechanism of protein targeting and translocation across the membrane of the endoplasmic reticulum is discussed.

Biological Transport↗
PubMed1986

Expression of wild-type and mutant forms of influenza hemagglutinin: the role of folding in intracellular transport.

The hemagglutinin of influenza virus is synthesized as a monomeric subunit that is cotranslationally translocated across the membrane of the rough endoplasmic reticulum. We show that folding and assembly of hemagglutinin monomers into trimeric structures takes approximately 7-10 min and is completed before the protein leaves the endoplasmic reticulum. Mutants of hemagglutinin that fail to be transported from the endoplasmic reticulum are blocked at different stages of the folding pathway. Unfolded molecules of hemagglutinin are associated with a cellular protein of 77 kd that has been shown previously to bind to IgG heavy chain in the endoplasmic reticulum of certain myelomas. We discuss why assembly of native structures is required for transport of proteins through the exocytotic pathway.

Biological Transport↗
PubMed1986

Studies on the mechanism of membrane fusion: site-specific mutagenesis of the hemagglutinin of influenza virus.

Oligonucleotide-directed mutagenesis of a cDNA encoding the hemagglutinin of influenza virus has been used to introduce single base changes into the sequence that codes for the conserved apolar "fusion peptide" at the amino-terminus of the HA2 subunit. The mutant sequences replaced the wild-type gene in SV40-HA recombinant virus vectors, and the altered HA proteins were expressed in simian cells. Three mutants have been constructed that introduce single, nonconservative amino acid changes in the fusion peptide, and three fusion phenotypes were observed: substitution of glutamic acid for the glycine residue at the amino-terminus of HA2 abolished all fusion activity; substitution of glutamic acid for the glycine residue at position 4 in HA2 raised the threshold pH and decreased the efficiency of fusion; and, finally, extension of the hydrophobic stretch by replacement of the glutamic acid at position 11 with glycine yielded a mutant protein that induced fusion of erythrocytes with cells with the same efficiency and pH profile as the wild-type protein. However, the ability of this mutant to induce polykaryon formation was greatly impaired. Nevertheless, all the mutant proteins underwent a pH-dependent conformational change and bound to liposomes. These results are discussed in terms of the mechanism of HA-induced membrane fusion.

Animals↗
PubMed1986

Heterologous transmembrane and cytoplasmic domains direct functional chimeric influenza virus hemagglutinins into the endocytic pathway.

Chimeric genes were created by fusing DNA sequences encoding the ectodomain of the influenza virus hemagglutinin (HA) to DNA coding for the transmembrane and cytoplasmic domains of either the G glycoprotein of vesicular stomatitis virus or the gC glycoprotein of Herpes simplex virus 1. CV-1 cells infected with SV40 vectors carrying the recombinant genes expressed large amounts of the chimeric proteins, HAG or HAgC on their surfaces. Although the ectodomains of HAG and HAgC differed in their immunological properties from that of HA, the chimeras displayed the biological functions characteristic of the wild-type protein. Both HAG and HAgC bound erythrocytes as efficiently as HA did and, after brief exposure to an acidic environment, induced the fusion of erythrocyte and CV-1 cell membranes. However, the behavior of HAG and HAgC at the cell surface differed from that of HA in several important respects. HAG and HAgC were observed to collect in coated pits whereas wild-type HA was excluded from those structures. In the presence of chloroquine, which inhibits the exit of receptors from endosomes, HAG and HAgC accumulated in intracellular vesicles. By contrast, chloroquine had no effect on the location of wild-type HA. HAG and HAgC labeled at the cell surface exhibited a temperature-dependent acquisition of resistance to extracellular protease at a rate similar to the rates of internalization observed for many cell surface receptors. HA acquired resistance to protease at a rate at least 20-fold slower. We conclude that HAG and HAgC are efficiently routed into the endocytic pathway and HA is not. However, like HA, HAG was degraded slowly, raising the possibility that HAG recycles to the plasma membrane.

Animals↗
PubMed1986

Analysis of progressive deletions of the transmembrane and cytoplasmic domains of influenza hemagglutinin.

Site-directed oligonucleotide mutagenesis has been used to introduce chain termination codons into the cloned DNA sequences encoding the carboxy-terminal transmembrane (27 amino acids) and cytoplasmic (10 amino acids) domains of influenza virus hemagglutinin (HA). Four mutant genes were constructed which express truncated forms of HA that lack the cytoplasmic domain and terminate at amino acids 9, 14, 17, or 27 of the wild-type hydrophobic domain. Analysis of the biosynthesis and intracellular transport of these mutants shows that the cytoplasmic tail is not needed for the efficient transport of HA to the cell surface; the stop-transfer sequences are located in the hydrophobic domain; 17 hydrophobic amino acids are sufficient to anchor HA stably in the membrane; and mutant proteins with truncated hydrophobic domains show drastic alterations in transport, membrane association, and stability.

Amino Acid Sequence↗
PubMed1986

Variant influenza virus hemagglutinin that induces fusion at elevated pH.

The hemagglutinin (HA) glycoprotein of influenza virus performs two critical roles during infection: it binds virus to cell surface sialic acids, and under mildly acidic conditions it induces fusion of the virion with intracellular membranes, liberating the genome into the cytoplasm. The pH dependence of fusion varies for different influenza virus strains. Here we report the isolation and characterization of a naturally occurring variant of the X31 strain that fuses at a pH 0.2 units higher than the parent strain does and that is less sensitive to the effects of ammonium chloride, a compound known to elevate endosomal pH. The bromelain-solubilized ectodomain of the variant HA displayed a corresponding shift in the pH at which it changed conformation and bound to liposomes. Cloning and sequencing of the variant HA gene revealed amino acid substitutions at three positions in the polypeptide. Two substitutions were in antigenic determinants in the globular region of HA1, and the third occurred in HA2 near the base of the molecule. By using chimeric HA molecules expressed in CV-1 cells from simian virus 40-based vectors, we demonstrated that the change in HA2 was solely responsible for the altered fusion phenotype. This substitution, asparagine for aspartic acid at position 132, disrupted a highly conserved interchain salt bridge between adjacent HA2 subunits. The apparent role of this residue in stabilizing the HA trimer is consistent with the idea that the trimer dissociates at low pH. Furthermore, the results demonstrate that influenza virus populations contain fusion variants, raising the possibility that such variants may play a role in the evolution of the virus.

Amino Acid Sequence↗
PubMed1986

Expression of human tissue-type plasminogen activator from lytic viral vectors and in established cell lines.

We have used two kinds of vectors to express a cDNA of human tissue plasminogen activator (t-PA) in mammalian cells. In one case, cDNAs inserted into vectors based on bovine papilloma virus were introduced into cultured murine cells and cell lines were established that efficiently and continuously secrete enzymatically active t-PA into the medium. Second, the t-PA gene was used to replace the sequences of the simian virus (SV40) genome that code for the viral coat proteins. Virus stocks were generated and used to infect a stable line of cultured simian cells. During the resulting lytic infection, expression of the t-PA gene is governed by the potent SV40 late promoter and enzymatically active t-PA accumulates rapidly in the medium. We have used these two vector systems to analyze the biosynthesis and transport of recombinant t-PA and to compare its properties with those of "natural" t-PA secreted by the Bowes line of human melanoma cells. t-PA secreted from all three sources is identical in specific activity (approximately 20,000 units/mg) despite differences in patterns of terminal glycosylation. Furthermore, non-glycosylated t-PA synthesized in the presence of tunicamycin was secreted efficiently and was indistinguishable in specific activity from glycosylated t-PAs.

Animals↗
PubMed1985

Lines of BPV-transformed murine cells that constitutively express influenza virus hemagglutinin.

We have developed and characterized several murine cell lines that constitutively express either the full-length, membrane-bound form of influenza virus hemagglutinin (HA) or a truncated version of the protein (HAsec) that lacks the carboxyterminal anchoring sequences and is secreted from cells. cDNAs encoding HA or HAsec were linked to the murine metallothionein-I promoter or the SV40 early promoter, and inserted into plasmids containing the transforming DNA fragment of bovine papilloma virus (BPV). The resulting vectors were introduced into three cultured lines of murine cells--C127, NIH3T3 and MME--either alone or in the presence of a plasmid that carries the aminoglycoside transferase gene of Tn5. The resulting lines of MME cells contained 1-5 copies of the vector in an integrated state and expressed low levels of HA (approximately 10(4) molecules/cell). In contrast, lines of C127 and NIH3T3 cells were obtained that express up to 5 X 10(6) molecules of HA per cell or secrete approximately 10(7) molecules of HAsec per cell per 24 h. Some of these cell lines carry multiple (30-200) copies of the vector in an integrated state; in others, the vector is propagated as unit-length episomes or as oligomers. Both the membrane-bound and secreted forms of HA expressed in these cell lines display a more extensive pattern of glycosylation than HA or HAsec synthesized in simian cells and they are transported to the cell surface more slowly. Pulse-chase experiments suggest that the step which limits the rate at which HA and HAsec travel down the secretory pathway occurs in the rough endoplasmic reticulum before the molecules are transferred to the Golgi apparatus. Using indirect immunofluorescence in combination with a cell sorter, we have shown that the level of expression of HA within cloned populations of producing cells can be variable. However, greater than 90% of the cells in certain cell lines display considerable quantities of HA on their surface, as judged by their ability to bind red blood cells in large numbers. We have taken advantage of the membrane fusion activity of HA to effect the fusion of erythrocytes to these cells and to deliver the contents of red cell ghosts into the cells' cytoplasm.

Animals↗
PubMed1985

SV40 T antigen and the exocytotic pathway.

A chimeric gene consisting of DNA coding for the 15-amino acid signal peptide of influenza virus hemagglutinin and the C-terminal 694 amino acids of SV40 large T antigen was inserted into a bovine papilloma virus (BPV) expression vector and introduced into NIH-3T3 cells. Cell lines were obtained that express high levels (approximately 5 X 10(6) molecules/cell) of the chimeric protein (HA-T antigen). The biochemical properties and intracellular localization of HA-T antigens were compared with those of wild-type T antigen. Wild-type T antigen. Wild-type T antigen is located chiefly in the cell nucleus, although a small fraction is detected on the cell surface. By contrast, HA-T antigen is found exclusively in the endoplasmic reticulum (ER). During biosynthesis, HA-T antigen is co-translationally translocated across the membrane of the ER, the signal peptide is cleaved and a mannose-rich oligosaccharide is attached to the polypeptide (T antigen contains one potential N-linked glycosylation site at Asn154). HA-T antigen does not become terminally glycosylated or acylated and little or none reaches the cell surface. These results suggest that T antigen is incapable of being transported along the exocytotic pathway. To explain the presence of wild-type T antigen on the surface of SV40-transformed cells, an alternative route is proposed involving transport of T antigen from the nucleus to the cell surface.

Amino Acid Sequence↗
PubMed1985

Host antigens associated with haemagglutinin.

A delayed-type hypersensitivity (DTH) test has been used to detect the presence of host-specific antigen(s) is highly purified egg-grown influenza virus, virus components and isolated haemagglutinin. Cyclophosphamide-treated mice, primed with an extract from allantoic fluid, showed DTH reactions when challenged with preparations containing envelope lipid or polymer-type subunits, whereas lipid-free derivatives such as internal virus protein complexes and monomer haemagglutinin preparations lacked the activity. This approach has been extended to demonstrate the presence of associated host antigens in SV40-vector expressed haemagglutinin produced in simian cells.

Allantois↗