Plasma membrane proteins and glycoproteins from chinese hamster cells sensitive and resistant to actinomycin D

Plasma membrane proteins and glycoproteins have been isolated from Chinese hamster cells of the spontaneously transformed DC-3F parental cell line and the DC-3F/AD X line with a high level of acquired resistance to actinomycin D. Plasma membrane preparations from both cell lines band at 1.16 g/ml after isopycnic centrifugation. We present evidence to indicate differences in the leucylpeptide backbones of the antibiotic-sensitive cells and the drug-resistant DC-3F/AD X cells. In addition, there are differences in the plasma membrane glycopeptides of the two cell lines as revealed by sodium dodecyl gel electrophoresis. Drug-resistant cells synthesize a surface glycopeptide which is much larger than the major one present on the drug-sensitive cells. Both of these cell lines are devoid of 5′-nucleotidase and alkaline phosphatase activities. The role of plasma membrane protein differences in drug-resistant cells is discussed.     

Analysis of the glycoproteins of murine tumour cell lines with 125I-concanavalin A in two-dimensional electrophoresis gels

The combination of two-dimensional gel electrophoresis and post-electrophoretic staining with 125I-labelled concanavalin A was used to compare the glycoproteins of murine tumour cell lines. Comparison between different cell lines showed that there were about eight common glycoproteins. The rest of the glycoproteins were generally unique to particular cells. Thus the P815 cell could be distinguished from 13 other murine cell lines by its glycoprotein pattern. The specific glycoproteins of each cell line were unaffected by culture in vivo, virus infection or hybridisation. Different clones from the same cell line gave identical patterns. Crude membrane preparations and glycoproteins purified from cell lysates by affinity chromatography on concanavalin/agarose gave the same patterns as whole cells. Thus the glycoproteins of murine tumour cells appear to be a stable characteristic which can provide specific markers for the identification of tumour cell lines.     

Temperature-sensitive transport of glycoproteins to the surface of a variant mouse lymphoma cell line.

The expression of mouse mammary tumor virus (MMTV) glycoproteins on the surface of stably infected mouse lymphoma cell line W7MG1 is dramatically increased by glucocorticoid hormones. A variant cell line, W7M.TS1, was selected from W7MG1 for its lack of expression of MMTV glycoproteins on the cell surface in response to treatment with glucocorticoid. Hormonal stimulation of MMTV RNA levels and hormone-induced cytolysis occurred normally in the variant cells. Furthermore, the rates of production of the precursor and mature forms of MMTV glycoproteins in the presence of glucocorticoid were similar in variant and wild-type cells. However, the accumulation of MMTV glycoproteins on the cell surface after hormone treatment was delayed by about 8 h in the variant relative to wild-type cells. The steady-state level of a constitutively expressed cellular protein, T200, on the variant cell surface was comparable to that on wild-type cells. However, in pulse-chase experiments, the appearance of newly synthesized T200 on the cell surface was delayed in the variant compared with wild-type cells. Another glucocorticoid hormone response, removal of H-2 class I antigens from the cell surface, was also delayed in the variant relative to wild-type cells, suggesting that turnover or internalization of cell surface glycoproteins may also be affected in the variant. The defects in the variant cell line were observed at 37 degrees C, but not at 31 degrees C; the variant cells grew normally at both temperatures. This variant phenotype defines a new genetic entity that is important for transport of glycoproteins between internal microsomal compartments and the cell surface.     

Mining of serum glycoproteins by an indirect approach using cell line secretome

Glycosylation is the most important and abundant post-translational modification in serum proteome. Several specific types of glycan epitopes have been shown to be associated with various types of disease. Direct analysis of serum glycoproteins is challenging due to its wide dynamic range. Alternatively, glycoproteins can be discovered in the secretome of model cell lines and then confirmed in blood. However, there has been little experi-mental evidence showing cell line secretome as a tractable target for the study of serum glycoproteins. We used a hydrazine-based glycocapture method to selectively enrich glycoproteins from the secretome of the breast cancer cell line Hs578T. A total of 132 glycoproteins were identified by nanoLC-MS/MS analysis. Among the identified proteins, we selected 13 proteins that had one or more N-glycosylation motifs in the matched peptides, which were included in the Secreted Protein Database but not yet in the Plasma Proteome Database (PPD), and whose antibodies were commercially available. Nine out of the 13 selected proteins were detected from human blood plasma by western analysis. Furthermore, eight proteins were also detected from the plasma by targeted LC-MS/MS, which had never been previously identified by data-dependent LC-MS/MS. Our results provide novel proteins that should be enrolled in PPD and suggest that analysis of cell line secretome with subfractionation is an efficient strategy for discovering disease-relevant serum proteins.     

Profiling and comparative analysis of glycoproteins in Hs578BST and Hs578T and investigation of prolyl 4-Hydroxylase alpha polypeptide II expression and influence in breast cancer cells

To identify potential cancer related glycoproteins in breast cancer cells, we enriched N-linked glycoproteins by lentil lectin from the human breast cancer cell line Hs578T and the normal breast cell line Hs578BST for proteomic comparison. Glycoproteins were separated and compared by two-dimensional electrophoresis. Twenty-four glycoproteins were identified that expressed remarkably differently, among which nine were involved in the progress of collagen synthesis. Prolyl 4-hydroxylase alpha polypeptide II (P4HA2) expression and influence in breast cancer was further investigated. Immunohistochemistry revealed that P4HA2 was upregulated in breast tumor cells compared with its adjacent normal tissues. Moreover, overexpression and RNA interference of P4HA2 showed that P4HA2 expression suppressed cell proliferation and migration in Hs578T in vitro.     

Glucocorticoid-regulated and constitutive trafficking of proteolytically processed cell surface-associated glycoproteins in wild type and variant rat hepatoma cells

Glucocorticoids regulate the trafficking of mouse mammary tumor virus (MMTV) glycoproteins to the cell surface in the rat hepatoma cell line M1.54, but not in the immunoselected sorting variant CR4. To compare the localization of MMTV glycoproteins to another proteolytically processed glycoprotein, both wild type M1.54 cells and variant CR4 cells were transfected with a human insulin receptor (hIR) expression vector, pRSVhIR. The production of cell surface hIR was monitored in dexamethasone-treated and -untreated wild type M1.54 and variant CR4 cells by indirect immunofluorescence, direct plasma membrane immunoprecipitation, and by [125I] insulin binding. In both wild type and variant rat hepatoma cells, hIR were localized at the cell surface in the presence or in the absence of 1 microM dexamethasone. In contrast, the glucocorticoid-regulated trafficking of cell surface MMTV glycoproteins occurred only in wild type M1.54 cells. We conclude that the hIR, which undergoes posttranslational processing reactions similar to MMTV glycoproteins, does not require glucocorticoids to be transported to the plasma membrane and is representative of a subset of cell surface glycoproteins whose trafficking is constitutive in rat hepatoma cells. Thus, MMTV glycoproteins and hIR provide specific cell surface markers to characterize the glucocorticoid-regulated and constitutive sorting pathways.     

Use of carbohydrate probes in conjunction with fluorescence-activated cell sorting to select mutant cell lines of Chlamydomonas with defects in cell surface glycoproteins

Two carbohydrate-binding probes (the lectin concanavalin A and the anti-carbohydrate monoclonal antibody FMG-1) have been utilized in conjunction with fluorescence-activated cell sorting to select cell lines of Chlamydomonas reinhardtii that contain defects in cell surface-exposed glycoproteins. Two very different selection strategies (sorting cells with the lowest binding for the FMG-1 monoclonal antibody or the highest binding of concanavalin A) yield a class of mutant cells that exhibit a total lack of binding of the monoclonal antibody to cell wall and plasma membrane glycoproteins along with an increased affinity for concanavalin A. Detailed characterization of one such mutant cell line, designated L-23, is provided. The subtle glycosylation defect exhibited by this cell line does not alter the ability of the affected glycoproteins to be targeted to the flagellar membrane and does not affect the expression of flagellar surface motility, a phenomenon that appears to involve the major concanavalin A-binding glycoprotein of the flagellar membrane. This approach has general applicability for dissecting the role of carbohydrate epitopes in the targeting and function of any cell surface glycoprotein for which suitable carbohydrate probes are available.     

Changes in Cell Wall Properties Coincide with Overexpression of Extensin Fusion Proteins in Suspension Cultured Tobacco Cells

Extensins are one subfamily of the cell wall hydroxyproline-rich glycoproteins, containing characteristic SerHyp₄ glycosylation motifs and intermolecular cross-linking motifs such as the TyrXaaTyr sequence. Extensins are believed to form a cross-linked network in the plant cell wall through the tyrosine-derivatives isodityrosine, pulcherosine, and di-isodityrosine. Overexpression of three synthetic genes encoding different elastin-arabinogalactan protein-extensin hybrids in tobacco suspension cultured cells yielded novel cross-linking glycoproteins that shared features of the extensins, arabinogalactan proteins and elastin. The cell wall properties of the three transgenic cell lines were all changed, but in different ways. One transgenic cell line showed decreased cellulose crystallinity and increased wall xyloglucan content; the second transgenic cell line contained dramatically increased hydration capacity and notably increased cell wall biomass, increased di-isodityrosine, and increased protein content; the third transgenic cell line displayed wall phenotypes similar to wild type cells, except changed xyloglucan epitope extractability. These data indicate that overexpression of modified extensins may be a route to engineer plants for bioenergy and biomaterial production.     

糖基因在髓性白血病耐药性中特征性改变

目的通过研究糖基因在髓性白血病中的差异表达,明确这些糖基因与白血病耐药的相关性,从而为预测和诊断髓性白血病耐药性,寻求逆转药物提供新策略和靶点。方法采用real-time PCR技术筛选髓性白血病细胞及其耐药细胞株中差异表达的糖基因,筛选出两组细胞差异表达3倍以上的糖基因,初步探索糖基因在髓性白血病耐药性中的特征性改变;采用流式细胞仪分析髓性白血病耐药细胞株与多种FITC标记植物凝集素的结合能力,表征比较细胞膜表面糖链的特征。结果 12个糖基因在NB4和NB4/ADR细胞株中表达具有显著的差异;高表达的糖基因与FITC标记植物凝集素的结合能力增强。结论髓性白血病细胞及其耐药细胞株中糖基因、细胞膜表面糖链特征均有显著差异,这些特征性改变与白血病多药耐药具有相关性。     

Isolation of hydroxyurea-resistant CHO cells with altered levels of ribonucleotide reductase.

A Chinese hamster ovary cell line selected for resistance to hydroxyurea was serially cultivated in the absence of a selective agent, and cells with decreased resistance to the cytotoxic effects of hydroxyurea gradually accumulated in the population. Three stable subclones with differing drug sensitivities were isolated from this mixed population and were found to contain intracellular levels of drug-sensitive ribonucleotide reductase which correlated with the degree of cellular resistance to hydroxyurea. This new class of hydroxyurea resistance was expressed in a codominant fashion in cell-cell hybridization studies. Also, the enhanced enzyme activity in the drug-resistant cells was observed only during the cell cycle S phase of synchronized cells. The properties of these drug-resistant lines indicate that they will be useful for genetic and biochemical studies.     

Bovine natural killer-like cell responses against cell lines expressing recombinant bovine herpesvirus type 1 glycoproteins

The murine retrovirus shuttle vector pZipNeo SV(X)1 was used to construct plasmids encoding the three major surface glycoproteins of bovine herpesvirus-1 (BHV-1). Each plasmid was transfected into D17, a canine osteosarcoma cell line sensitive to lysis by bovine NK-like cells when infected with BHV-1. After selection in G418 sulfate, cell lines expressing the recombinant gene products were sorted by flow microfluorimetry, radioimmunoprecipitated, and analyzed by SDS-PAGE for fidelity as compared to the native viral glycoproteins. Two of the three genetically engineered cell lines (gI and gIV) could successfully serve as targets to detect bovine NK-like cytolysis. These findings support and extend a previous study from our laboratory indicating a role for BHV-1 glycoproteins in the cytolytic response by bovine NK-like cells. Additionally, this study demonstrated that individual proteins are recognized by these effector cells, and that recombinant glycoproteins can direct cytolytic activity in the absence of host cell infection-associated proteins. This is the first known report of Ag directed cytotoxicity by bovine null (non-B, non-T) cells.     

Glycoproteomic analysis of glioblastoma stem cell differentiation

Cancer stem cells are responsible for tumor formation through self-renewal and differentiation into multiple cell types and thus represent a new therapeutic target for tumors. Glycoproteins play a critical role in determining the fates of stem cells such as self-renewal, proliferation, and differentiation. Here we applied a multilectin affinity chromatography and quantitative glycoproteomics approach to analyze alterations of glycoproteins relevant to the differentiation of a glioblastoma-derived stem cell line HSR-GBM1. Three lectins including concanavalin A (Con A), wheat germ agglutinin (WGA), and peanut agglutinin (PNA) were used to capture glycoproteins, followed by LC-MS/MS analysis. A total of 73 and 79 high-confidence (FDR < 0.01) glycoproteins were identified from the undifferentiated and differentiated cells, respectively. Label-free quantitation resulted in the discovery of 18 differentially expressed glycoproteins, wherein 9 proteins are localized in the lysosome. All of these lysosomal glycoproteins were up-regulated after differentiation, where their principal function was hydrolysis of glycosyl residues. Protein-protein interaction and functional analyses revealed the active involvement of lysosomes during the process of glioblastoma stem cell differentiation. This work provides glycoprotein markers to characterize differentiation status of glioblastoma stem cells that may be useful in stem-cell therapy of glioblastoma.     

Differential transport requirements of HLA and H-2 class I glycoproteins

Transport of human and mouse major histocompatibility complex class I glycoproteins has been examined in a transport deficient B-lymphoblastoid cell line × T-lymphoblastoid cell line (B-LCL × T-LCL) hybrid, 174 × CEM. T2 (T2). This cell line expresses no detectable endogenous HLA-B5 and reduced levels of HLA-A2 on its surface although these molecules are synthesized. In order to study this defect further, either HLA-Bw58 or HLA-B7 genomic clones were transfected into T2. Metabolic labeling and immune precipitation demonstrated biosynthesis of the Bw58 or 137 glycoprotein. However, like the endogenous HLA-B5 molecule, neither HLA-Bw58 nor HLA-B7 was expressed at the cell surface. The cloned genes were properly expressed on the surface of C1R, a control B-LCL. To determine if mouse class I alleles had the same transport requirements as the human class I glycoproteins, either mouse H-2D ^p or H-2K ^b class I genes were introduced into T2. Surprisingly, the H-2 class I glycoproteins were transported to the cell surface normally. These data suggest a fundamental difference between human and mouse histocompatibility antigens in their requirements for intracellular transport.     

两株毛筒腔菌属真菌逆转人乳腺癌细胞多药耐药性

高水平的多药耐药性（multidrug resistance,MDR）基因在肿瘤细胞中过量表达是肿瘤细胞耐药的内在原因,是导致肿瘤化疗失败的主要原素。寻求一种抑制MDR活性的抑制剂是提升抗肿瘤药物药效的重要途径。本研究采用低浓度持续诱导方法建立人乳腺癌细胞（MCF-7）耐药细胞系,结果显示,阿霉素（ADM）、紫杉醇和顺铂对MCF-7耐药细胞系有交叉耐药性,耐药指数（resistance index,RI）分别为5.11、3.55和1.79。菌株对肿瘤细胞的逆转活性筛选表明,红棕毛筒腔菌Tubeufia rubra PF02-2和河池毛筒腔菌T. hechiensis XSL05具有逆转肿瘤细胞多药耐药性为敏感性的活性,逆转倍数（reversion fold,RF）分别为3.79和1.07。结果表明,T. rubra和T. hechiensis具有开发为MDR逆转剂的潜能。     

Engineering lepidopteran insect cells for sialoglycoprotein production by genetic transformation with mammalian beta 1,4-galactosyltransferase and alpha 2,6-sialyltransferase genes

Recombinant mammalian glycoproteins produced by the baculovirus-insect cell expression system usually do not have structurally authentic glycans. One reason for this limitation is the virtual absence in insect cells of certain glycosyltransferases, which are required for the biosynthesis of complex, terminally sialylated glycoproteins by mammalian cells. In this study, we genetically transformed insect cells with mammalian beta 1,4-galactosyltransferase and alpha 2,6-sialyltransferase genes. This produced a new insect cell line that can express both genes, serve as hosts for baculovirus infection, and produce foreign glycoproteins with terminally sialylated N-glycans.     

Secretion of Hepatitis C Virus Envelope Glycoproteins Depends on Assembly of Apolipoprotein B Positive Lipoproteins

The density of circulating hepatitis C virus (HCV) particles in the blood of chronically infected patients is very heterogeneous. The very low density of some particles has been attributed to an association of the virus with apolipoprotein B (apoB) positive and triglyceride rich lipoproteins (TRL) likely resulting in hybrid lipoproteins known as lipo-viro-particles (LVP) containing the viral envelope glycoproteins E1 and E2, capsid and viral RNA. The specific infectivity of these particles has been shown to be higher than the infectivity of particles of higher density. The nature of the association of HCV particles with lipoproteins remains elusive and the role of apolipoproteins in the synthesis and assembly of the viral particles is unknown. The human intestinal Caco-2 cell line differentiates in vitro into polarized and apoB secreting cells during asymmetric culture on porous filters. By using this cell culture system, cells stably expressing E1 and E2 secreted the glycoproteins into the basal culture medium after one week of differentiation concomitantly with TRL secretion. Secreted glycoproteins were only detected in apoB containing density fractions. The E1–E2 and apoB containing particles were unique complexes bearing the envelope glycoproteins at their surface since apoB could be co-immunoprecipitated with E2-specific antibodies. Envelope protein secretion was reduced by inhibiting the lipidation of apoB with an inhibitor of the microsomal triglyceride transfer protein. HCV glycoproteins were similarly secreted in association with TRL from the human liver cell line HepG2 but not by Huh-7 and Huh-7.5 hepatoma cells that proved deficient for lipoprotein assembly. These data indicate that HCV envelope glycoproteins have the intrinsic capacity to utilize apoB synthesis and lipoprotein assembly machinery even in the absence of the other HCV proteins. A model for LVP assembly is proposed.     

Mucin-carbohydrate directed monoclonal antibody

To raise monoclonal antibodies recognizing cancer-associated alterations of the carbohydrate structure of glycoproteins, Balb/c mice were immunized with human colonic cancer cells (LS 180 from ATCC). One of the generated hybridomas produced a monoclonal antibody that bound to the carbohydrate moiety of mucin-type glycoproteins from LS 180. The antibody did not bind to glycoproteins from another colonic cancer cell line, SW 1116, or to glycolipids from any of the colonic cancer cell lines. The antibody bound to ovine and bovine submaxillary mucins (OSM and BSM). NeuAc alpha 2----6Ga1NAc seemed to be involved in the epitope.     

Surface distribution of wheat germ agglutinin binding sites of human melanoma cell lines with low and high tumorigenicity

Wheat germ agglutinin (WGA) binding patterns of human malignant melanoma cell lines with a high or a low ability to grow subcutaneously in nude mice were compared. SDS-PAGE analysis of WGA binding glycoproteins revealed similar qualitative but different quantitative profiles. Striking differences were observed in the density of WGA binding sites, twice as high in the low tumorigenic cell line as in the high tumorigenic cell line. Differences were also observed in the topographical organization of these WGA binding sites patched on the low tumorigenic cell line and diffuse on the high tumorigenic cell line. Fluorescence photobleaching measurements showed clear-cut differences in their motion patterns.     

Intracellular trafficking of cell surface sialoglycoconjugates

Recent reports have suggested that the majority of the molecular traffic through the Golgi apparatus is comprised of recycling, rather than newly synthesized, molecules. To evaluate the importance of this recycling pathway in greater detail, we examined the internalization and recycling of cell surface glycoproteins on EL-4 cells, a murine T-cell lymphoma, using sialic acids as covalent markers. Sialic acids were removed from the surface of living cells by exhaustive treatment with Vibrio cholerae sialidase at 4 degrees C and shown to be derived primarily from glycoproteins (93%), with only a small amount from glycolipids (7%). Cells were recultured at 37 degrees C over time and monitored for the resialylation of the cell surface using a sensitive high pressure liquid chromatography adaptation of the thiobarbituric acid assay for sialic acids. The return of sialic acid to the cell surface was found to be contingent upon de novo protein synthesis indicating that the bulk of plasma membrane sialoglycoconjugates do not recycle to an endogenous sialyltransferase-containing compartment for oligosaccharide reprocessing. Identical results were found for K562 cells, a human erythroleukemia cell line. The movement of specific glycoproteins was followed using the enzyme rat liver alpha 2-6Gal beta 1-4GlcNAc sialyltransferase together with CMP-[3H]NeuAc as an impermeant probe of the cell surface. Surface sialoglycoproteins were internalized slowly, a process unaffected by cycloheximide treatment. Only a few of these internalized glycoproteins were found to return to a trans-Golgi compartment followed by recycling to the cell surface. Taken together, these data indicate that the majority of replacement of sialic acids on the cell surface is due to de novo synthesis of glycoproteins and that only a small number of glycoproteins recycle through a trans-Golgi compartment.