Identification of a 90 000-dalton cell surface glycoprotein with elevated expression in human hepatoma cells

We describe a human-specific cell surface glycoprotein of molecular size 90 000-dalton (90K) and isoelectric point 5 defined by a monoclonal antibody prepared using human hepatoma-mouse hepatoma hybrid cells with a limited number of human chromosomes (6, 7, 14, 20, 21, and X) as immunogens in syngeneic mice. While detectable on cultured human cells of diverse origin, expression of the 90K protein is elevated in hepatoma cells. Moreover, a protein of identical molecular size and slightly more acidic isoelectric point is present in hepatoma culture supernatant. We sought to determine the identity of the 90K protein by comparing it to two hepatoma-expressed, major histocompatibility complex-linked proteins of similar molecular size, the α-chain of C4 and factor B; this comparison was also prompted by the presence of human chromosome 6 in the immunizing hybrids. We find no evidence, however, for these proteins being related. Melanoma-associated antigenic determinants carried by proteins of similar molecular size have been reported, and the possible relation of these proteins to the 90K protein is discussed.     

Radioimmunoassays for the 36,000-dalton glycoprotein of murine mammary tumor viruses demonstrate type, group, and interspecies determinants.

Mouse mammary tumor viruses (MMTVs) contain distinct membrane glycoproteins of 52,000 daltons (gp52) and 36,000 daltons (gp36). We report here the development of new radioimmunoassays for gp36, using gp36 purified by hydrophobic chromatography and gel filtration. These assays demonstrate that gp36 has both type-specific and group-specific antigenic determinants. The virus-coded nature of these determinants was shown by utilizing different MMTVs grown in the same feline cell line. Interspecies determinants on gp36 were demonstrated by the observations that (i) MC-MTV (a virus isolate from the Asian rodent Mus cervicolor, and morphologically identical to MMTVs) competed, with an altered slope, in the gp36 radioimmunoassay, and (ii) antisera raised against MC-MTV immunopreciptitated 125I-labeled gp36. The detection of gp36 in spontaneous mammary tumors of several strains of mice also facilitates further studies on the replication of MMTVs and the host's immune response to MMTV-mediated oncogenesis.     

Lateral diffusion of an 80,000-dalton glycoprotein in the plasma membrane of murine fibroblasts: relationships to cell structure and function

The lateral diffusion of an 80,000-dalton major cell surface glycoprotein of murine fibroblasts has been measured. This antigen, identified through the use of monoclonal antibodies, is an integral glycoprotein distributed through the plasma membrane as judged by immunofluorescence and immunoelectron microscopy (see preceding paper). Measurements of fluorescence recovery after photobleaching were performed on the antigen-antibody complex within the plasma membrane of C3H/10T1/2 and NIH/3T3 cells after labeling the monoclonal antibody with fluorescein. Measurements were performed as a function of temperature, for interphase, mitotic, and G0 C3H/10T1/2 cells. The mean lateral diffusion coefficients (D) for the antibody-protein complex in interphase cells were in the range of 0.7-3.5 X 10(-10) cm2/s between 9 degrees and 37 degrees C, while that for the lipid analog probe, dihexadecylindocarbocyanine was about two orders of magnitude greater. This comparison indicates that peripheral interactions other than bilayer fluidity limit the lateral mobility of the antigen. The mobile fraction of mitotic, G0, and interphase cells showed a monotonic increase with temperature with most of the antibody-antigen complexes being free to move about 25 degrees C. Semi-quantitative interpretations of both the slow glycoprotein diffusion and the immobile fraction are offered. Comparison of diffusion coefficients for cells in different phases of the cell cycle does not reveal striking differences. Mobile fractions for G0 cells at 25 degrees C or less are substantially lower than in interphase cells. In all cases, there was a remarkably broad range of the fluorescence recovery data between different cells, resulting in up to a 10-fold variation in diffusion coefficients, which is far greater than the precision limits of the experiment. Diffusion values and mobile fractions were generally well within a factor of two when measured at several arbitrary points on a single cell. The origins of this cellular heterogenity remain to be elucidated. Lateral mobility in cell fragments and specific regions of single cells was also examined. The glycoprotein was mobile in ventral surface cell fragments. Its mobility was not altered in regions of cell- cell underlapping. However, the diffusion coefficient was threefold higher near the leading edge of motile cells compared to the trailing region. This difference may reflect weaker coupling of the glycoprotein to the underlying cytoskeleton in the dynamic leading edge region.     

Limited expression of nuclear pore membrane glycoprotein 210 in cell lines and tissues suggests cell-type specific nuclear pores in metazoans

The nuclear pore complex (NPC) is the only known gateway for nucleocytoplasmic traffic. The nuclear pore membrane glycoprotein 210 (POM210/gp210) is considered to be important for the assembly and structure of pore complexes in metazoan cells. However, here we demonstrate cell-type specific expression of the gp210 protein during mouse organogenesis. As shown previously for its mRNA, distinct expression of the gp210 was seen in developing epithelia and some other cell types, whereas it was undetectable in nuclei of several other embryonic tissue compartments. In sharp contrast, monoclonal antibody 414 recognizing four non-membrane nucleoporins, stained the nuclear envelope of all cell types. In four cultured mouse cell lines, gp210 mRNA and protein were below detection levels, in contrast to some other nucleoporins tested. Distinct expression of gp210 mRNA and protein was seen in cultured mouse embryonic stem (ES) cells. These findings support the view of cell-type specific NPCs in metazoans and that the gp210 gene is regulated by cell-type specific control elements not shared by other nucleoporins. Although it cannot be excluded that very low expression levels of gp210 are sufficient to allow attachment of NPCs, a more likely alternative is that it has cell-type specific functions.     

Synapsin IIa: expression in insect cells, purification, and characterization.

Synapsin IIa belongs to a family of neuron-specific phosphoproteins called synapsins, which are associated with synaptic vesicles in presynaptic nerve terminals. In order to examine the biochemical properties of synapsin IIa, and ultimately its physiological function, purified protein is required. Since attempts to purify significant quantities of synapsin IIa, an isoform of the synapsins, from mammalian brain have proven difficult, we undertook the production of recombinant synapsin IIa by utilizing the baculovirus expression system. Rat synapsin IIa cDNA was introduced into the baculovirus genome via homologous recombination, and the recombinant baculovirus was purified. Spodoptera frugiperda (Sf9) cells infected with this virus expressed synapsin IIa as 5% of the total cellular protein. The recombinant protein was extracted from the particulate fraction of the infected Sf9 cells with salt and a nonionic detergent and purified by immunoaffinity chromatography. The purified synapsin IIa was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase to a stoichiometry of 0.8 mol of phosphate/mol of protein. Metabolic labeling with [32P]Pi demonstrated synapsin IIa phosphorylation in infected Sf9 cells. Using a homogenate of uninfected Sf9 cells, a cAMP-dependent protein kinase activity which can phosphorylate synapsin IIa was detected. Limited proteolysis of recombinant synapsin IIa phosphorylated in vitro and in vivo resulted in identical phosphopeptide maps. Further, synapsin IIa, like synapsin I, binds with high affinity in a saturable manner to synaptic vesicles purified from rat cortex.     

Surface expression of influenza virus neuraminidase, an amino-terminally anchored viral membrane glycoprotein, in polarized epithelial cells.

We have investigated the site of surface expression of the neuraminidase (NA) glycoprotein of influenza A virus, which, in contrast to the hemagglutinin, is bound to membranes by hydrophobic residues near the NH2-terminus. Madin-Darby canine kidney or primary African green monkey kidney cells infected with influenza A/WSN/33 virus and subsequently labeled with monoclonal antibody to the NA and then with a colloidal gold- or ferritin-conjugated second antibody exhibited specific labeling of apical surfaces. Using simian virus 40 late expression vectors, we also studied the surface expression of the complete NA gene (SNC) and a truncated NA gene (SN10) in either primary or a polarized continuous line (MA104) of African green monkey kidney cells. The polypeptides encoded by the cloned NA cDNAs were expressed on the surface of both cell types. Analysis of [3H]mannose-labeled polypeptides from recombinant virus-infected MA104 cells showed that the products of cloned NA cDNA comigrated with glycosylated NA from influenza virus-infected cells. Both the complete and the truncated glycoproteins were found to be preferentially expressed on apical plasma membranes, as detected by immunogold labeling. These results indicate that the NA polypeptide contains structural features capable of directing the transport of the protein to apical cell surfaces and the first 10 amino-terminal residues of the NA polypeptide are not involved in this process.     

Herpes simplex viruses lacking glycoprotein D are unable to inhibit virus penetration: quantitative evidence for virus-specific cell surface receptors.

Herpes simplex virus (HSV) glycoprotein D (gD) plays an essential role in the entry of virus into cells. HSV mutants unable to express gD were constructed. The mutants can be propagated on VD60 cells, which supply the viruses with gD; however, virus particles lacking gD were produced in mutant-infected Vero cells. Virus particles with or without gD adsorbed to a large number (greater than 4 x 10(4] of sites on the cell surface; however, virions lacking gD did not enter cells. Cells pretreated with UV-inactivated virions containing gD (approximately 5 x 10(3) particles per cell) were resistant to infection with HSV type 1 (HSV-1) and HSV-2. In contrast, cells pretreated with UV-inactivated virions lacking gD could be infected with HSV-1 and HSV-2. If infectious HSV-1 was added prior to UV-inactivated virus particles containing gD, the infectious virus entered cells and replicated. Therefore, virus particles containing gD appear to block specific cell surface receptors which are very limited in number. Particles lacking gD are presumably unable to interact with these receptors, suggesting that gD is an essential receptor-binding polypeptide.     

The lysine-rich arabinogalactan-protein subfamily in Arabidopsis: gene expression, glycoprotein purification and biochemical characterization

AtAGP17, AtAGP18 and AtAGP19 are homologous genes encoding three putative glycosylphosphatidylinositol (GPI)-anchored classical arabinogalactan-proteins (AGPs) in Arabidopsis. They are distinguished from other AGPs by a short, C-terminal lysine-rich region. Organ-specific expression of these genes was revealed by Northern blot analysis. AtAGP17 was strongly expressed in leaves and stems, and weakly expressed in flowers and roots; AtAGP18 was strongly expressed in flowers, and moderately expressed in roots, stems and young leaves; and AtAGP19 was strongly expressed in stems, moderately expressed in flowers and roots, and weakly expressed in young leaves. One of these genes, AtAGP17, was expressed and purified as a green fluorescent protein (GFP) fusion protein in transgenic tobacco cells using hydrophobic interaction chromatography, size exclusion chromatography and reverse phase high-performance liquid chromatography. The fusion (glyco)protein produced a characteristic AGP 'smear' with a molecular mass of 80-150 kDa when detected by Western blot analysis. Glycosyl composition and linkage analyses of purified GFP-AtAGP17 showed that carbohydrate accounted for approximately 86% of the molecule, with arabinose and galactose as major, and rhamnose and glucuronic acid as minor glycosyl residues and with 1,3,6-galactose, 1,4-glucuronic acid, 1,3-galactose and terminal arabinose as major linkages. GFP-AtAGP17 was also precipitated by beta-Yariv reagent, further confirming that AtAGP17 is a bona fide AGP. Confocal fluorescence microscopy of plasmolysed, transformed cells indicated that AtAGP17 is localized on the plasma membrane and in Hechtian strands. Hydroxyproline (Hyp) glycoside profiles of GFP-AtAGP17 in conjunction with the deduced protein sequence also served to corroborate the Hyp contiguity hypothesis, which predicts contiguous Hyp residues as attachment sites for arabinosides and clustered, non-contiguous Hyp residues as attachment sites for arabinogalactan polysaccharides.     

Isolation and characterization of a variant of mouse plasmacytoma J558 synthesizing a 110,000-dalton immunoglobulin heavy chain and of secondary variants synthesizing either a 55,000-dalton or an 80,000-dalton immunoglobulin heavy chain: possible implications.

A mutant has been isolated from the J558 (immunoglobulin A, lambda, anti-alpha 1 leads to 3 dextran) cell line which synthesizes a heavy-chain immunoglobulin twice the size of normal heavy chain. Secondary variants that synthesized heavy chains either 1.5 times as large as wild type or the same size as wild type were identified. All mutants were serologically immunoglobulin continued to bind antigen, and retained the individual idiotype of the parent. Northern blot analysis and in vitro synthesis studies showed that the large heavy chains were primary synthetic products and not the consequence of abnormal covalent bonds. Cleavage of genomic DNA with restriction endonucleases and molecular hybridization studies showed new fragments in the 2 X and 1.5 X mutants which disappeared in the 1 X revertant. These data cannot easily be reconciled with the mutants arising either by unequal recombination or gene conversion. Further molecular characterization of these mutants should give additional insight into immunoglobulin gene evolution.     

Extraction and purification of the chief glycoprotein of the erythrocyte membrane

The major glycoprotein of the human erythrocyte membrane has been released from ghosts, by the non-ionic detergent Tween 20 at pH 8,5 and at low ionic strength and further purified by successive passages through columns of DEAE Sephadex at pH 6,8 and CM Sephadex at pH 5 or by hydroxyapatite chromatography at pH 6,8. The purified glycoprotein thus obtained represents about 1 % of the membrane proteins, and shows two major bands upon polyacrylamide gel electrophoresis. These bands designed as PAS 1 and PAS 2 are the dimer and the monomer of the glycoprotein. Several other minor bands can also appear on SDS gels, according to experimental conditions of solubilization and purification and are probably oligomers of PAS 1 and PAS 2. This glycoprotein possess inhibitory activity against various phytohemagglutinins.     

Demonstration of laminin,a basement membrane glycoprotein,in routinely processed formalin-fixed human tissues

Summary Laminin was demonstrated by immunoperoxidase and immunofluorescence staining in sections of normal human tissues fixed in formalin and routinely processed in paraffin. Exposure of the sections to a solution of pepsin (Burns et al. (1980) Histochemistry 6773–78) revealed the antigenicity of this basement membrane glycoprotein. Sections from paraffin blocks stored for years at room temperature could be stained with this procedure. Normal human tissues, developing fetal tissues and tumors could be stained with this method. The staining patterns were similar to those seen in unfixed frozen sections. It thus appears that basement membrane components can be detected by immunohistological means from routinely processed histological samples, once the sections are pretreated with proteases. Staining for laminin could be used in embryonic studies and in histopathology to study the relation of cells to basement membranes and for the visualization of normal and abnormal vascularization.To whom offprint requests should be sent     

Demonstration of laminin, a basement membrane glycoprotein, in routinely processed formalin-fixed human tissues

Laminin was demonstrated by immunoperoxidase and immunofluorescence staining in sections of normal human tissues fixed in formalin and routinely processed in paraffin. Exposure of the sections to a solution of pepsin (Burns et al. (1980) Histochemistry 67:73-78) revealed the antigenicity of this basement membrane glycoprotein. Sections from paraffin blocks stored for years at room temperature could be stained with this procedure. Normal human tissues, developing fetal tissues and tumors could be stained with this method. The staining patterns were similar to those seen in unfixed frozen sections. It thus appears that basement membrane components can be detected by immunohistological means from routinely processed histological samples, once the sections are pretreated with proteases. Staining for laminin could be used in embryonic studies and in histopathology to study the relation of cells to basement membranes and for the visualization of normal and abnormal vascularization.     

Crystals of the Chlamydomonas reinhardtii cell wall: polymerization, depolymerization, and purification of glycoprotein monomers

Two of the three major outer layers of the Chlamydomonas reinhardtii cell wall (W6 and W4) can be solubilized from living cells with sodium perchlorate or other chaotropes and will repolymerize in vitro to form milligram amounts of wall crystals. Conditions for optimal crystalization are presented, and conditions that fail to induce polymerization are exploited to maintain monomers in aqueous solution for ion-exchange chromatography. The four major glycoproteins of the complex (GP1, 1.5, 2, and 3) have in this way been purified to apparent homogeneity and have been characterized morphologically by transmission electron microscopy using the quick-freeze, deep-etch technique and by amino acid composition. Three of the four are hydroxyproline-rich species that copolymerize to form the W6 layer. The fourth (GP1.5) is a glycine-rich species that binds to the interior of the in vitro crystal; it is apparently equivalent to the granules within the W4 layer in situ.     

A 55,000-60,000 Mr receptor protein for urokinase-type plasminogen activator. Identification in human tumor cell lines and partial purification

The iodinated Mr approximately equal to 15,000 amino-terminal fragment (ATF) of the urokinase-type plasminogen activator (u-PA) molecule bound specifically to the cell surface of all of seven cultured human tumor cell lines studied. Cross-linking of iodinated ATF to the cell surface using a bifunctional amino-reactive reagent followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed with the four cell lines studied the occurrence of a single band migrating with an Mr of 70,000-75,000, indicating complex formation with an Mr of 55,000-60,000 u-PA receptor protein (u-PA-R). In the human monocyte cell line U937 cultivated in the presence of phorbol ester, the amount of complex was strongly increased, and a fraction of the complex had a slower electrophoretic mobility. Comparison between autoradiograms of reduced and unreduced samples suggests that u-PA-R consists of one polypeptide chain. Two forms of u-PA-R, which differed with respect to affinity to concanavalin A, were identified. u-PA-R retained its ability to bind to ATF after cell lysis, and it was purified approximately 2,200-fold from biosynthetically labeled U937 cells by affinity chromatography with proenzyme u-PA coupled to Sepharose. The purified Mr 55,000-60,000 protein was specifically bound and cross-linked to u-PA, proenzyme u-PA, and ATF, but not to tissue-type plasminogen activator or other unrelated proteins.     

Cytokine- and Ig-producing T cells in mucosal effector tissues: analysis of IL-5- and IFN-gamma-producing T cells, T cell receptor expression, and IgA plasma cells from mouse salivary gland-associated tissues.

The present study has focused on the analysis of cytokine- and Ig-producing mononuclear cells (MC) that reside in the salivary glands and their associated tissues (SGAT) in the oral region. The SGAT are located under the mandibular area and consist of submandibular glands, periglandular lymph nodes, and cervical lymph nodes. MC were isolated from individual SGAT and examined for T cell subsets and TCR expression, in comparison with T cells obtained from other mucosa-associated and systemic tissues. Forty to fifty percent of MC in submandibular glands were CD3+ T cells, equally divided into CD4+ CD8- and CD4- CD8+ T cell subsets. On the other hand, the intestinal lamina propria and Peyer's patches possessed a approximately 2 to 3:1 ratio of CD4+ CD8- to CD4- T cells. A high frequency of CD4- CD8- (double negative) (DN) T cells (approximately 6 to 10%) was also isolated from submandibular glands. In contrast, approximately 70 to 90% of MC in periglandular lymph nodes and cervical lymph nodes were CD3+ T cells and like the peripheral lymph nodes consisted of fivefold higher numbers of CD4+ CD8- than CD4- CD8+ T cells, with low numbers of DN cells (less than 5%). When expression of gamma/delta and alpha/beta TCR was examined in individual T cell subsets of submandibular glands, the CD4- CD8+ and DN T cell fractions contained 25% and 100% gamma/delta TCR+ cells, respectively. On the other hand, essentially all CD4+ CD8- T cells in SGAT as well as CD4- CD8+ cells in periglandular lymph nodes and cervical lymph nodes were alpha/beta TCR+ T cells. When cytokine production was examined by using IFN-gamma- and IL-5-specific enzyme-linked immunospot assays, the CD3+ CD4+ CD8- T cells in submandibular glands contained T cells spontaneously producing IFN-gamma and IL-5. Further, IL-5 spot-forming cells (SFC) were two- to threefold greater in number, compared with IFN-gamma SFC. The periglandular lymph node T cells contained cytokine producing cells with a ratio of 2:1 for IL-5 and IFN-gamma SFC cells, whereas cervical lymph node T cells did not produce cytokines unless stimulated with T cell mitogens. When the isotype distribution of Ig-producing cells was examined among SGAT, submandibular glands contained large numbers of IgA-producing cells, with few IgM- and IgG-producing cells, a pattern similar to that of the lamina propria. Further, elevated numbers of IgA-secreting cells were also seen in periglandular lymph nodes but not in cervical lymph nodes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification and partial purification of ABGP205, an integral membrane glycoprotein from brain that binds ankyrin.

1. A procedure was devised that allows the membrane-skeletal proteins brain spectrin and ankyrin to be extracted selectively from a membrane-skeletal preparation, together with some actin, an Mr-103,000 protein and a population of glycoproteins. 2. Ankyrin-binding activities of the glycoproteins were investigated by affinity chromatography. We detected only one, Mr 205,000, that binds ankyrin and is prevented from binding by the cytoplasmic domain of Band 3, the established erythrocyte-membrane-binding site for ankyrin. The Mr-205,000 glycoprotein, designated ABGP205, may be a candidate for a membrane-binding site for ankyrin.     

Role of a membrane glycoprotein in Friend virus erythroleukemia: nucleotide sequences of nonleukemogenic mutant and spontaneous revertant viruses.

We previously isolated spontaneous env gene mutants of Friend spleen focus-forming virus that are nonleukemogenic in adult mice but form leukemogenic revertants in newborns; we found that the revertants contain secondary env mutations. To identify sites in the encoded membrane glycoprotein that are important for its pathogenic function, we molecularly cloned and partially sequenced the env genes of two mutant viruses (clone 63 and clone 4) and one revertant (clone 4REV). Clone 63 contained three noncontiguous point mutations that caused nonconservative amino acid substitutions of Gly-119----Arg-119, Cys-180----Tyr-180, and Gly-203----Arg-203 in the xenotropic-related domain of the env glycoprotein. These substitutions were presumably responsible for the altered electrophoretic and pathogenic properties of the mutant glycoprotein. The presence of these and several other G-A nucleotide substitutions at different sites in one spontaneous mutant provided striking evidence that error-rich proviruses can form during retroviral replication. Clone 4 contained a point mutation that generated a premature termination condon at amino acid residue 304 (Gln-304----Ochre-304). This termination codon was located immediately after the proposed xenotropic-ecotropic recombination site and eliminated the ecotropic-related domain, including the putative membrane anchor of the glycoprotein. Clone 4REV was a true revertant derived from clone 4 in which the premature termination codon had back-mutated to re-form the wild-type sequence. These results confirm an essential role for the env gene in Friend spleen focus-forming virus pathogenesis and suggest that the encoded membrane glycoprotein contains different domains that contribute to its pathogenic function.