Isolation and characterization of mouse FM3A cell mutants which are devoid of Newcastle disease virus receptors.

A method was developed to select host cell mutants which did not permit the replication of Newcastle disease virus (NDV), and 14 isolates of NDV-nonpermissive mutants of mouse FM3A cells were obtained. All these isolates were judged to be deficient in NDV receptors, since their ability to adsorb 3H-labeled NDV virions was markedly decreased. They were tested for genetic complementation in pairs by cell fusion and shown to fall into a single recessive complementation group, which was designated as Had-1. Vesicular stomatitis virus was able to replicate in this mutant to produce infectious progeny, but the glycoprotein of the released virion was abnormal in size, suggesting a defective processing of the asparagine-linked carbohydrate chains in the mutant cell. The Had-1 mutant was resistant to wheat germ agglutinin, but sensitive to a Griffonia simplicifolia lectin, GS-II, which recognizes terminal N-acetylglucosamine residues. The altered sensitivity to these plant lectins compared with that of the parental FM3A cells indicates that sialylated sugar chains on the cell surface are almost absent from the Had-1 cells, thereby rendering the cells NDV receptor deficient.     

Glycolipid composition of a mutant cell line of mouse FM3A cells, and the effect of exogenous glycolipids on cell growth

Had-1 isolated from mouse mammary tumour FM3A cells as a non-permissive cell line to Newcastle disease virus infection is deficient in NDV receptors, and galactosylation of the complex type sugar chains of the glycoproteins is extensively reduced compared to FM3A cells. It is also deficient in UDP-galactose transport into Golgi vesicles. The major neutral glycolipids in FM3A is Lac-Cer, whereas, in Had-1 cell, Glc-Cer is the major glycolipid and the concentration of neutral glycolipids is one-tenth as low as that in FM3A. GM3, GD3 and sialyl i- and I-type lactosaminylceramide are the gangliosides present in both FM3A and Had-1, although their presence in both cells is only in traces. Had-1 contains relatively high N-glycolyl-neuraminic acid. Among the several glycolipids tested, Lac-Cer, Gg-4-Cer and Glc-Cer showed inhibitory effect on proliferation of Had-1 cells, but did not show any appreciable effect on that of FM3A cells. Lac-Cer had the most potent inhibitory effect and this inhibitory effect was completely reversible. While mice injected with 5 x 10(6) cells of FM3A died in one month, those injected of Had-1 cells at the same dose survived for more than 6 months. Thus glycolipids on the cell surface play an essential role during cell growth both in vivo and in vitro.     

Complementation by a cloned human ubiquitin-activating enzyme E1 of the S-phase-arrested mouse FM3A cell mutant with thermolabile E1.

A temperature-sensitive growth mutant tsFS20 isolated from mouse FM3A cells was identified as a mutant with thermolabile ubiquitin-activating enzyme E1 by transfection with a full-length cDNA encoding the human E1 enzyme and cell-cell hybridization with an authentic E1 mutant ts85 previously isolated from FM3A cells. The resulting transformants produced thermoresistant E1 activity. Upon shift-up of temperature, asynchronously growing tsFS20 cells showed multiple points of cell-cycle arrest. At the nonpermissive temperature, tsFS20 cells that had been synchronized at the G1-S-phase progressed and accumulated in the mid-S-phase, as evidenced by the absence of G2-specific cdc2 kinase activity, while ts85 mutant cells, the widely used E1 mutant, reached the G2-phase and were arrested. Thus, the E1 mutation seemed to be involved in progression in the S-phase as well as in the G2-phase in the cell cycle. Degradation of short-lived abnormal proteins in tsFS20 cells was decreased to about 50% at the nonpermissive temperature, while the block was fully restored to the wild-type level in the transformant cells. Relevance of the unusually high incidence of the temperature-sensitive E1 mutation was discussed in terms of the E1 as a determinant of heat tolerance of cells.     

Establishment of mutant murine mammary carcinoma FM3A cell strains transformed with the herpes simplex virus type 2 thymidine kinase gene

To establish cell systems appropriate for investigating the mode of action of antiherpetic nucleoside analogues, mutant cell strains were constructed from murine mammary carcinoma FM3A cells, which were deficient in TK, but were transformed with a recombinant plasmid DNA containing the HSV-2 TK gene. The transformed cells incorporated the viral DNA, expressed viral TK activity and showed unusually high sensitivity to the cytostatic action of the antiherpetic nucleoside analogues ACV and IVDU, both of which were only weakly inhibitory to the growth of the parent cells. Curiously, the FM3A cell strains transformed with HSV-2 TK gene showed a higher sensitivity to ACV and IVDU than the previously established cell line transformed with HSV-1 TK gene. This contrasts with the inhibitory effects of ACV and IVDU on acute HSV infection, since HSV-2 infection is slightly or considerably less susceptible than HSV-1 infection to inhibition by ACV or IVDU, respectively.     

Novel lectin-like proteins on the surface of human monocytic leukemia cell line THP-1 cells that recognize oxidized cells

Presence of lectin-like receptors on the membranes of human monocytic leukemia cell line THP-1 cells for clustered sialylated poly-N-acetyllactosaminyl sugar chains on the membranes of oxidized erythrocytes and T-lympoid cells was investigated. Membranes of THP-1 cells differentiated into macrophages were solubilized, and the membrane proteins obtained by affinity chromatographies using lactoferrin-Sepharose and band 3-Sepharose were purified by successive DE column chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins of 50, 60, and 80 kDa with specificity to bind to sialylated poly-N-acetyllactosaminyl sugar chains were detected in the chromatographic fractions. A 50-kDa protein was isolated in a pure form. N-Terminal amino acid sequence of the protein was Lys-Gln-Lys-Val-Ala-Gly-Lys-Gln-Pro-Val-, which has not been found in the N-terminal regions of the hitherto known proteins. The antibody, raised against the chemially synthesized peptide composed of the N-terminal amino acid sequence, bound to 50-, 60-, and 80-kDa proteins as analyzed by immunoblotting and immunoprecipitation, indicating that these proteins had the same N-terminal amino acid sequence. The results demonstrate that THP-1 cells have novel 50-, 60-, and 80-kDa lectin-like proteins with the same N-terminal amino acid sequence on the cell surface which would bind to clustered sialylated poly-N-acetyllactosaminyl sugar chains generated on oxidized erythrocytes and T-lymphoid cells.     

Downregulation of JNK/SAPK activity is associated with the cross-resistance to P-glycoprotein-unrelated drugs in multidrug-resistant FM3A/M cells overexpressing P-glycoprotein

In the present study, cross-drug resistance in multidrug-resistant (MDR) cells, which overexpress P-glycoprotein (Pgp), a mdr1 gene product, against Pgp-unrelated drugs, and its relevance to c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK) activity were examined. The multidrug-resistant FM3A/M cells overexpressing Pgp were resistant to apoptotic cell death induced either by Pgp-related drugs including vincristine and vinblastine, which are pumped out by Pgp, or by the Pgp-unrelated drugs including 5'-fluorouracil (5-FU) and bleomycin, which are not targets for Pgp, compared with the parental FM3A cells. Verapamil reversed the resistance of FM3A/M cells to apoptosis induced by the Pgp-related drugs but not that induced by the Pgp-unrelated drugs. Interestingly, FM3A/M cells have shown significantly lower basal and drug-stimulated JNK/SAPK activities than FM3A cells. After transfection with pEBG-SEK or pEBG-SAPK constructs, FM3A/M cells recovered the basal and Pgp-unrelated drug-stimulated activities of JNK/SAPK and the susceptibility to Pgp-unrelated drug-induced apoptotic cell death comparable to those of FM3A cells. Furthermore, FM3A cells became resistant to apoptotic cell death induced by vincristine and 5-FU after transfection with pEBG-SEK(K --> R), a dominant negative inhibitory mutant of SEK. These results suggest that downregulation of JNK/SAPK activity appears to confer on Pgp-associated FM3A/M cells a cross-resistance to Pgp-unrelated drugs.     

Potent and specific inhibition of mammalian histone deacetylase both in vivo and in vitro by trichostatin A

(R)-Trichostatin A (TSA) is a Streptomyces product which causes the induction of Friend cell differentiation and specific inhibition of the cell cycle of normal rat fibroblasts in the G1 and G2 phases at the very low concentrations. We found that TSA caused an accumulation of acetylated histone species in a variety of mammalian cell lines. Pulse-labeling experiments indicated that TSA markedly prolonged the in vivo half-life of the labile acetyl groups on histones in mouse mammary gland tumor cells, FM3A. The partially purified histone deacetylase from wild-type FM3A cells was effectively inhibited by TSA in a noncompetitive manner with Ki = 3.4 nM. A newly isolated mutant cell line of FM3A resistant to TSA did not show the accumulation of the acetylated histones in the presence of a higher concentration of TSA. The histone deacetylase preparation from the mutant showed decreased sensitivity to TSA (Ki = 31 nM, noncompetitive). These results clearly indicate that TSA is a potent and specific inhibitor of the histone deacetylase and that the in vivo effect of TSA on cell proliferation and differentiation can be attributed to the inhibition of the enzyme.     

Isolation and partial characterization of the major sialoglycoprotein of human T-lymphoblastoid cells of a MOLT-4B cell line.

A sialoglycoprotein with an approx. mol.wt. of 95000 was isolated from human lymphoblastoid cells of a MOLT-4B cell line, which was of human T-lymphocyte origin, by ion-exchange chromatography, affinity chromatography on a column of wheat-germ agglutinin-Sepharose and preparative slab-gel electrophoresis. The localization of this glycoprotein on the cell surface was indicated by surface labelling by the periodate/NaB3H4 and lactoperoxidase-catalysed iodination methods. Carbohydrate analyses of this glycoprotein revealed that its total carbohydrate content is 28% (w/w), and it contains fucose, galactose, mannose, N-acetylglucosamine, N-acetylgalactosamine and sialic acid in molar proportions 1.0:4.0:3.7:3.5:1.2:2.5, suggesting that it has two types of sugar chain, i.e. sugar chains like those of serum glycoproteins and sugar chains of the type found in mucins. Actually, alkaline borohydride treatment of this glycoprotein yielded tri- and tetra-saccharide, the latter containing 1 molecule of fucose in addition to each molecule of galactose, N-acetylgalactosamine and sialic acid. This glycoprotein bound to Ricinus communis agglutinin and concanavalin A as well as to wheat-germ agglutinin.     

A mammalian cell mutant with temperature-sensitive thymidine kinase.

We have isolated a mutant clone from mouse FM3A cells with temperature-sensitive defects both in cytokinesis and in thymidine kinase enzyme activity. The clone, designated tsCl.B59, was isolated after mutagenesis at 33 degrees C followed by exposure to cytosine arabinoside at 39 degrees C. It was derived from a thymidine kinase deficient, 5-bromodeoxyuridine-resistant clone (S-BUCl.42) which was originally derived from wild-type clone H-5 of FM3A cells. The temperature-sensitive mutant clone grows normally at 33 degrees C, but not at 39 degrees C, where it exhibits an increased frequency of multinucleate cells due to defective cytokinesis. Unlike the parental S-BUCl.42 cells, which have negligible thymidine kinase activity and are unable to incorporate 3H-thymidine, the mutant in corporates substantial amounts of 3H-thymidine at 33 degrees C, although its thymidine kinase activity remains lower than that of wild-type H-5 cells. When cultures of tsCl.B59 cells are transferred to 39 degrees C, incorporation of 3H-thymidine decreases markedly. The decrease has been shown to be due to thermolability of the thymidine kinase in tsCl.B59 cells.     

Thymidylate synthetase-deficient mouse FM3A mammary carcinoma cell line as a tool for studying the thymidine salvage pathway and the incorporation of thymidine analogues into host cell DNA.

A thymidylate (dTMP) synthetase-deficient murine mammary carcinoma cell line (FM3A/TS-), auxotrophic for thymidine (dThd), proved extremely useful for studying the dependence of cell growth on the exogenous supply of dThd, the relation between cell growth and DNA synthesis, and the ability of a series of 25 5-substituted 2'-deoxyuridines (dUrd) to substitute for dThd in sustaining cell growth. FM3A/TS-cells did not proliferate unless dThd was supplied to the cell culture medium. The 5-halogenated dUrd derivatives 5-chloro-dUrd, 5-bromo-dUrd and 5-iodo-d Urd also sustained FM3A/TS- cell growth. The extents of incorporation of [methyl-3H]dThd and 5-iodo-[6-3H]dUrd into DNA were closely correlated with their stimulatory effects on FM3A/TS- cell growth. This suggests that the stimulatory effects of the dUrd analogues on the growth rate of FM3A/TS- cells may be considered as evidence for their incorporation into host cell DNA. Based on this premise it is postulated that, in addition to 5-chloro-dUrd, 5-bromo-dUrd, 5-iodo-dUrd and dThd itself, the following dThd analogues are also incorporated into FM3A/TS- cell DNA (in order of the extent to which they are incorporated): 5-hydroxy-dUrd greater than 5-propynyloxy-dUrd greater than 5-ethyl-dUrd greater than 5-ethynyl-dUrd approximately 5-vinyl-dUrd. Thus, the dTMP synthetase-deficient FM3A/TS- cell line represents a unique system to dissociate the de novo and salvage pathways of dTMP biosynthesis and to distinguish those dUrd analogues that are incorporated into DNA from those that are not.     

Monitoring of the tissue distribution of fibroblast growth factor containing a high mannose-type sugar chain produced in mutant yeast

Most therapeutic glycoproteins have been produced in mammalian cell lines. However, the mammalian cell culture system has various disadvantages, i.e., a high culture cost, difficulty in performing a large scale-up because of complicated handling requirements, and the risk of contamination by prion or other unknown pathogenic components through cultivation in the presence of bovine serum. There is thus a growing need for other host cells in which the recombinant glycoproteins can be produced. Recently, we successfully developed a mutant yeast strain engineered in a glycosylation system. The sugar chain produced in the mutant yeast is not immunogenic to the human immuno-surveillance system. In the present study, we selected fibroblast growth factor (FGF) as a model glycoprotein and assessed the bioactivity of FGF produced in yeast in terms of its proliferating activity and tissue distribution in mammalian cells and in the whole body. Structural changes in the sugar chains of FGFs derived from mutant yeast, as compared with those from mammalian cells, did not affect the proliferating activity remarkably. However, the tissue distribution in the mouse differed significantly; a high-mannose type sugar chain was the major determinant of the specific distribution of FGF to the kidney. The mechanism of this phenomenon is still unclear, but our observations suggest that recombinant glycoproteins derived from mutant yeasts producing high-mannose type sugar chains would be applicable for tissue-targeting therapy.     

Different effects on cell proliferation and migration abilities of endothelial cells by LPA1 and LPA3 in mammary tumor FM3A cells

Lysophosphatidic acid (LPA) receptors belong to G protein-coupled transmembrane receptors and mediate a variety of cellular responses through the binding of LPA. So far, six types of LPA receptors (LPA receptor-1 (LPA₁) to LPA₆) have been identified. Recently, it has been demonstrated that each LPA receptor has opposite effects on malignant property of cancer cells. In this study, to evaluate an involvement of LPA receptors on angiogenic process in mammary tumor cells, we generated Lpar1- and Lpar3-expressing (FM3A-a1 and FM3A-a3A9, respectively) cells from FM3A cells, and investigated the effects on cell proliferation and migration abilities of endothelial F-2 cells by those cells. In Vegf-A and Vegf-C genes, FM3A-a1 cells indicated high expression and FM3A-a3A9 cells showed low expression, compared with control cells. When F-2 cells were cultured with a supernatant from FM3A-a1 cells, the cell growth rate and migration ability of F-2 cells was significantly higher than control cells. By contrast, a supernatant from FM3A-a3A9 cells significantly inhibited those abilities of F-2 cells. These results suggest that LPA₁ and LPA₃ may play opposite roles on the regulation of endothelial cells in mouse mammary tumor FM3A cells.     

Isolation and analysis of a mammalian temperature-sensitive mutant defective in G2 functions

A temperature-sensitive (ts) mutant, designated tsFT210, was isolated from a mouse mammary carcinoma cell line, FM3A. The tsFT210 cells grew normally at 33 degrees C (permissive temperature), but more than 80% of the cells were arrested at the G2 phase at 39 degrees C (non-permissive temperature) as revealed by flow-microfluorimetric analysis. DNA replication and synthesis of other macromolecules by this mutant seemed to be normal at 39 degrees C for at least 10 h. However, in this mutant, hyperphosphorylation of H1 histone from the G2 to M phase, which occurs in the normal cell cycle, could not be detected at the non-permissive temperature. This suggests that a gene product which is temperature-sensitive in tsFT210 cells is necessary for hyperphosphorylation of H1 histone and that this gene product may be related to chromosome condensation.     

Structural study of the sugar chains of human leukocyte cell adhesion molecules CD11/CD18.

Leu-CAMs (CD11/CD18) consisting of LFA-1, Mac-1, and p150/95 are leukocyte cell surface glycoproteins that are involved in various leukocyte functions. The asparagine-linked sugar chains were released as oligosaccharides from Leu-CAMs by hydrazinolysis. About 12 mol of sugar chains was released from 1 mol of Leu-CAMs. These sugar chains were converted to radioactive oligosaccharides by reduction with sodium borotritide and separated into neutral and acidic fractions by paper electrophoresis. All of the acidic oligosaccharides were converted to neutral ones by digestion with sialidase, indicating that they are sialyl derivatives. The neutral and sialdase-treated acidic oligosaccharides were fractionated by chromatography on lectin columns followed by Bio-Gel P-4 column chromatography. Structural studies of each oligosaccharide by sequential exo- and endoglycosidase digestion and by methylation analysis revealed that Leu-CAMs contain mainly high mannose type and high molecular weight complex type sugar chains. The latter sugar chains were of bi-, tri-, and tetraantennary complex types with the Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----and/or the Gal beta 1----3GlcNAc beta 1----groups together with the Gal beta 1----4GlcNAc group in their outer-chain moieties. In addition to these sugar chains, a small amount of monoantennary complex type and hybrid type sugar chains was found in Leu-CAMs. Furthermore, analysis of the asparagine-linked sugar chains released from the beta-subunit of Leu-CAMs by a series of lectin chromatography showed that subunit-specific glycosylation is not observed between the alpha- and beta-subunits of Leu-CAMs.     

Monitoring of the tissue distribution of fibroblast growth factor containing a high mannose-type sugar chain produced in mutant yeast

Most therapeutic glycoproteins have been produced in mammalian cell lines. However, the mammalian cell culture system has various disadvantages, i.e., a high culture cost, difficulty in performing a large scale-up because of complicated handling requirements, and the risk of contamination by prion or other unknown pathogenic components through cultivation in the presence of bovine serum. There is thus a growing need for other host cells in which the recombinant glycoproteins can be produced. Recently, we successfully developed a mutant yeast strain engineered in a glycosylation system. The sugar chain produced in the mutant yeast is not immunogenic to the human immuno-surveillance system. In the present study, we selected fibroblast growth factor (FGF) as a model glycoprotein and assessed the bioactivity of FGF produced in yeast in terms of its proliferating activity and tissue distribution in mammalian cells and in the whole body. Structural changes in the sugar chains of FGFs derived from mutant yeast, as compared with those from mammalian cells, did not affect the proliferating activity remarkably. However, the tissue distribution in the mouse differed significantly; a high-mannose type sugar chain was the major determinant of the specific distribution of FGF to the kidney. The mechanism of this phenomenon is still unclear, but our observations suggest that recombinant glycoproteins derived from mutant yeasts producing high-mannose type sugar chains would be applicable for tissue-targeting therapy. Published in 2004. This revised version was published online in July 2006 with corrections to the Cover Date.     

Properties of the human erythrocyte glucose transport protein are determined by cellular context

Human erythrocyte hexose transfer is mediated by the glucose transport protein GLUT1 and is characterized by a complexity that is unexplained by available hypotheses for carrier-mediated sugar transport [Cloherty, E. K., Heard, K. S., and Carruthers, A. (1996) Biochemistry 35, 10411-10421]. The study presented here examines the possibility that the operational properties of GLUT1 are determined by host cell environment. A glucose transport-null strain of Saccharomyces cerevisiae (RE700A) was transfected with the p426 GPD yeast expression vector containing DNA encoding the wild-type human glucose transport protein (GLUT1), mutant GLUT1 (GLUT1(338)(-)(A3)), or carboxy-terminal hemagglutinin-polyHis-tagged GLUT1 (GLUT1-HA-H6). GLUT1 and GLUT1-HA-H6 are expressed at the yeast cell membrane and restore 2-deoxy-d-glucose, 3-O-methylglucose, and d-glucose transport capacity to RE700A. GLUT1-HA-H6 confers GLUT1-specific sugar transport characteristics to transfected RE700A, including inhibition by cytochalasin B and high-affinity transport of the nonmetabolized sugar 3-O-methylglucose. GLUT1(338)(-)(A3), a catalytically inactive GLUT1 mutant, is expressed but fails to restore RE700A sugar uptake capacity or growth on glucose. In contrast to transport in human red cells, K(m(app)) for 2-deoxy-d-glucose uptake equals K(i(app)) for 2-deoxy-d-glucose inhibition of 3-O-methylglucose uptake. Unlike transport in human red cells or transport in human embryonic kidney cells transfected with GLUT1-HA-H6, unidirectional sugar uptake in RE700A-GLUT1-HA-H6 is not inhibited by reductant and is not stimulated by intracellular sugar. Net uptake of subsaturating 3-O-methylglucose by RE700A-GLUT1-HA-H6 is a simple, first-order process. These findings support the hypothesis that red cell sugar transport complexity is host cell-specific.     

Flow cytometric assay for cytotoxic activity of crude Clostridium perfringens enterotoxin using non-adherent cell FM3A

Abstract The flow cytometric assay method was tested for the cytotoxic activity of Clostridium perfringens enterotoxin (CPE) in culture using mouse mammary carcinoma cell line FM3A stained with propidium iodide (PI). From the results obtained, FM3A cells proved to be susceptible to CPE. A reproducible dose-response curve with FM3A was obtained between crude CPE at 13.9–109 ng/ml and between purified CPE at 40–400 ng/ml, respectively. These findings indicate that non-adherent FM3A is preferable to determine the cytotoxic activity of CPE because it can be used without detachment procedures with trypsinin compared with adherent African monkey kidney cell line (Vero cells). Furthermore, the flow cytometry with non-adherent cell FM3A stained with PI only proved to be a useful method to determine the biological activity of CPE in culture isolates.     

Comparative study of the asparagine-linked sugar chains of natural human interferon-beta 1 and recombinant human interferon-beta 1 produced by three different mammalian cells

The asparagine-linked sugar chains of natural interferon-beta 1 secreted from human foreskin fibroblasts by poly I:poly C induction and of three recombinant human interferon-beta 1 produced by Chinese hamster ovary cells, mouse epithelial cells (C127), and human lung adenocarcinoma cells (PC8) were released quantitatively as oligosaccharides by hydrazinolysis followed by N-acetylation. After being reduced with either NaB3H4 or NaB2H4, their structures were comparatively analyzed. More than 80% of the sugar chains of natural interferon-beta 1 occur as biantennary complex-type sugar chains, approximately 10% of which contain N-acetyllactosamine repeating structure in their outer chain moieties. The remainders are 2,4- and 2,6-branched triantennary complex-type sugar chains. The sugar chains of the recombinant interferon-beta 1 derived from Chinese hamster ovary cells were very similar to those of its natural counterpart. In contrast, two other recombinant proteins contain quite different sugar chains. The protein derived from C127 cells contains complex-type sugar chains with the Gal alpha 1----3Gal beta 1----4GlcNAc group in their outer chain moieties. Their sialic acid residues occur solely as the Sia alpha 2----6Gal group, where Sia is sialic acid. In contrast, the sialic acid residues of other interferon-beta 1 occur as the Sia alpha 2----3Gal group only. A part of the sugar chains of the protein derived from PC8 cells contains bisecting N-acetylglucosamine residue in addition to the Gal alpha 1----3Gal beta 1----4GlcNAc group.     

Structure and cell surface maturation of the attachment glycoprotein of human respiratory syncytial virus in a cell line deficient in O glycosylation.

The synthesis of the extensively O-glycosylated attachment protein, G, of human respiratory syncytial virus and its expression on the cell surface were examined in a mutant Chinese hamster ovary (CHO) cell line, ldlD, which has a defect in protein O glycosylation. These cells, used in conjunction with an inhibitor of N-linked oligosaccharide synthesis, can be used to establish conditions in which no carbohydrate addition occurs or in which either N-linked or O-linked carbohydrate addition occurs exclusively. A recombinant vaccinia virus expression vector for the G protein was constructed which, as well as containing the human respiratory syncytial virus G gene, contained a portion of the cowpox virus genome that circumvents the normal host range restriction of vaccinia virus in CHO cells. The recombinant vector expressed high levels of G protein in both mutant ldlD and wild-type CHO cells. Several immature forms of the G protein were identified that contained exclusively N-linked or O-linked oligosaccharide side chains. Metabolic pulse-chase studies indicated that the pathway of maturation for the G protein proceeds from synthesis of the 32-kilodalton (kDa) polypeptide accompanied by cotranslational attachment of high-mannose N-linked sugars to form an intermediate with an apparent mass of 45 kDa. This step is followed by the Golgi-associated conversion of the N-linked sugars to the complex type and the completion of the O-linked oligosaccharides to achieve the mature 90-kDa form of G. Maturation from the 45-kDa N-linked form to the mature 90-kDa form occurred only in the presence of O-linked sugar addition, confirming that O-linked oligosaccharides constitute a significant proportion of the mass of the mature G protein. In the absence of O glycosylation, forms of G bearing galactose-deficient truncated N-linked and fully mature N-linked oligosaccharides were observed. The effects of N- and O-linked sugar addition on the transport of G to the cell surface were measured. Indirect immunofluorescence and flow cytometry showed that G protein could be expressed on the cell surface in the absence of either O glycosylation or N glycosylation. However, cell surface expression of G lacking both N- and O-linked oligosaccharides was severely depressed.     

Structures of the sugar chains of interferon-gamma produced by human myelomonocyte cell line HBL-38

Interferon-gamma produced by the human myelomonocyte cell line HBL-38 contained galactose, mannose, fucose, N-acetylglucosamine, and N-acetylneuraminic acid as sugar components. Sugar chains were liberated from interferon-gamma by hydrazinolysis. Free amino groups of the sugar chains were acetylated and the reducing-end sugar residues were tagged with 2-aminopyridine under new reaction conditions in which no sialic acid residue was hydrolyzed. The pyridylamino (PA-) derivatives of the sugar chains thus obtained were purified by gel filtration and reversed-phase HPLC. Seven major PA-sugar chains were isolated and the structure of each purified PA-sugar chain was identified by stepwise exoglycosidase digestion and 500-mHz 1H-NMR spectroscopy. The results indicated that the structures of the major PA-sugar chains were of the biantennary type, to which 0 to 2 mol of fucose and 1 to 2 mol of N-acetylneuraminic acid were linked as shown below. (formula; see text)