Biomedical subjects

L Puddington

Publications and source records attributed to L Puddington.

24 records · Page 2Linked to original sources

PubMed1987

Replacement of the cytoplasmic domain alters sorting of a viral glycoprotein in polarized cells.

The envelope glycoprotein (G protein) of vesicular stomatitis virus (VSV) is transported to the basolateral plasma membrane of polarized epithelial cells, whereas the hemagglutinin glycoprotein (HA protein) of influenza virus is transported to the apical plasma membrane. To determine if the cytoplasmic domain of VSV G protein might be important in directing G protein to the basolateral membrane, we derived polarized Madin-Darby canine kidney cell lines expressing G protein or G protein with its normal cytoplasmic domain replaced with the cytoplasmic domain from an influenza HA protein (GHA protein). Indirect immunofluorescence microscopy showed that G protein was present primarily on basolateral surfaces, whereas the GHA protein was present on the apical and basolateral membranes. These results suggest that the cytoplasmic domain can be an important determinant directing polarized expression of an integral membrane protein.

Amino Acid Sequence↗

PubMed1987

Effects of mutations in three domains of the vesicular stomatitis viral glycoprotein on its lateral diffusion in the plasma membrane.

The lateral mobility of the vesicular stomatitis virus spike glycoprotein (G protein) and various mutant G proteins produced by site-directed mutagenesis of the G cDNA has been measured. Fluorescence recovery after photobleaching results for the wild type G protein in transfected COS-1 cells yielded a mean diffusion coefficient (D) of 8.5 (+/- 1.3) X 10(-11) cm2/s and a mean mobile fraction of 75% (+/- 3%). Eight mutant proteins were also examined: dTM14, lacking six amino acids from the transmembrane domain; TA2, lacking an oligosaccharide in the extracellular domain; QN2, possessing an extra N-linked oligosaccharide in the extracellular domain; CS2, possessing a serine instead of a cysteine at residue 489 in the cytoplasmic domain, preventing palmitate addition to the glycoprotein; TMR-stop, lacking the entire cytoplasmic domain except an arginine at residue 483; and three chimeric proteins, G mu, G23, and GHA, containing in place of the 29 amino acid wild type cytoplasmic domain the cytoplasmic domains from the surface IgM from the spike protein of the infectious bronchitis virus or from the hemagglutinin protein of the influenza virus, respectively. The mean D for the mutant proteins varied over a relatively small range, with the slowest mutant, G23, exhibiting a value of 11.3 (+/- 1.4) X 10(-11) cm2/s and the fastest mutant, GHA, having a D of 28.6 (+/- 4.5) X 10(-11) cm2/s. The mean mobile fraction similarly varied over a small range, extending from 55 to 68%. None of the mutations resulted in the more rapid diffusion characteristic of membrane proteins embedded in artificial bilayers. Therefore, it appears that the cytoplasmic and transmembrane domains themselves contribute little to restraining the lateral mobility of this integral membrane protein when expressed in transfected cells.

Amino Acid Sequence↗

PubMed1986

Cytoplasmic domains of cellular and viral integral membrane proteins substitute for the cytoplasmic domain of the vesicular stomatitis virus glycoprotein in transport to the plasma membrane.

Oligonucleotide-directed mutagenesis was used to construct chimeric cDNAs that encode the extracellular and transmembrane domains of the vesicular stomatitis virus glycoprotein (G) linked to the cytoplasmic domain of either the immunoglobulin mu membrane heavy chain, the hemagglutinin glycoprotein of influenza virus, or the small glycoprotein (p23) of infectious bronchitis virus. Biochemical analyses and immunofluorescence microscopy demonstrated that these hybrid genes were correctly expressed in eukaryotic cells and that the hybrid proteins were transported to the plasma membrane. The rate of transport to the Golgi complex of G protein with an immunoglobulin mu membrane cytoplasmic domain was approximately sixfold slower than G protein with its normal cytoplasmic domain. However, this rate was virtually identical to the rate of transport of micron heavy chain molecules measured in the B cell line WEHI 231. The rate of transport of G protein with a hemagglutinin cytoplasmic domain was threefold slower than wild type G protein and G protein with a p23 cytoplasmic domain, which were transported at similar rates. The combined results underscore the importance of the amino acid sequence in the cytoplasmic domain for efficient transport of G protein to the cell surface. Also, normal cytoplasmic domains from other transmembrane glycoproteins can substitute for the G protein cytoplasmic domain in transport of G protein to the plasma membrane. The method of constructing precise hybrid proteins described here will be useful in defining functions of specific domains of viral and cellular integral membrane proteins.

B-Lymphocytes↗

PubMed1986

N protein is the predominant antigen recognized by vesicular stomatitis virus-specific cytotoxic T cells.

The specificity of anti-vesicular stomatitis virus (VSV)-specific cytotoxic T cells was explored with cell lines expressing VSV genes introduced by electroporation. Low levels of nucleocapsid (N) protein were detected on the surface of VSV-infected cells, but N protein could not be detected on the plasma membrane of transfected EL4 cells. Intracellular N protein was detectable by enzyme-linked immunosorbent assay or immunoprecipitation in some of the transfected cell lines but not in others, unless the transfected genes were induced by sodium butyrate. However, all of the stably transfected EL4 cell lines expressing the VSV-Indiana N protein were efficiently lysed by serotype-specific and cross-reactive anti-VSV cytotoxic T cells (CTLs). Primary cross-reactive anti-VSV CTLs appeared to be specific solely for N protein, based on cold-target competition assays using infected and transfected target cells. Cell lines expressing 100- to 1,000-fold less N protein than did VSV-infected cells were efficiently lysed by both primary and secondary anti-VSV CTLs. Cell lines expressing 100-fold less G protein than did VSV-infected cells were not lysed by either population of effectors. Significantly, cold-target competition studies with secondary CTLs demonstrated that N protein-expressing cell lines were more efficient competitors than were VSV-infected cells even though the latter expressed 100- to 1,000-fold more N protein. This was not an artifact of viral infection since infection of the transfected cell lines did not affect their ability to compete. The possibility that cell lines constitutively expressing internal virus proteins present antigen more effectively than infected cells do is discussed.

Antibodies, Monoclonal↗

PubMed1985

Acquisition of cytotoxic T lymphocyte-specific carbohydrate differentiation antigens.

Cytotoxic T cell (CTL)-specific activation antigens, termed CT determinants, have been detected by monoclonal antibodies (mAb) that inhibit CTL function. At the cell surface, the CT antigens are associated with the T200 glycoproteins and two other proteins of Mr 140,000 and 85,000 and are present on a secreted protein, gp155. Periodate treatment followed by binding analysis and immunoprecipitation experiments using tunicamycin-treated cells indicated that carbohydrate is necessary for CT antigen expression. Furthermore, gp155 is secreted in the presence of tunicamycin while retaining the CT antigens, and the CT determinants are added late in T200 biosynthesis, suggesting that the CT glycans are O-linked. Finally, interleukin 2 was shown to dramatically influence the expression of the CT mAb-reactive oligosaccharides present at the CTL cell surface.

Animals↗

PubMed1985

Role of the nuclear envelope in synthesis, processing, and transport of membrane glycoproteins.

The outer nuclear membrane is morphologically similar to rough endoplasmic reticulum. The presence of ribosomes bound to its cytoplasmic surface suggests that it could be a site of synthesis of membrane glycoproteins. We have examined the biogenesis of the vesicular stomatitis virus G protein in the nuclear envelope as a model for the biogenesis of membrane glycoproteins. G protein was present in nuclear membranes of infected Friend erythroleukemia cells immediately following synthesis and was transported out of nuclear membranes to cytoplasmic membranes with a time course similar to transport from rough endoplasmic reticulum (t 1/2 = 5-7 min). Temperature-sensitive mutations in viral membrane proteins which block transport of G protein from endoplasmic reticulum also blocked transport of G protein from the nuclear envelope. Friend erythroleukemia cells and NIH 3T3 cells differed in the fraction of newly synthesized G protein found in nuclear membranes, apparently reflecting the relative amount of nuclear membrane compared to endoplasmic reticulum available for glycoprotein synthesis. Nuclear membranes from erythroleukemia cells appeared to have the enzymatic activities necessary for cleavage of the signal sequence and core glycosylation of newly synthesized G protein. Signal peptidase activity was detected by the ability of detergent-solubilized membranes of isolated nuclei to correctly remove the signal sequence of human preplacental lactogen. RNA isolated from the nuclear envelope was highly enriched for G protein mRNA, suggesting that G protein was synthesized on the outer nuclear membrane rather than redistributing to nuclear membranes from endoplasmic reticulum before or during cell fractionation. These results suggest a mechanism for incorporation of membrane glycoproteins into the nuclear envelope and suggest that in some cell types the nuclear envelope is a major source of newly synthesized membrane glycoproteins.

Animals↗