共查询到20条相似文献,搜索用时 15 毫秒
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Ishida N Yoshioka S Iida M Sudo K Miura N Aoki K Kawakita M 《Journal of biochemistry》1999,126(6):1107-1117
UDP-galactose transporter is a membrane protein localized in the Golgi apparatus. It translocates UDP-galactose from the cytosol into the Golgi lumen, thus providing galactosyltransferases with their substrate. We characterized murine UDP-galactose transporter through molecular cloning for the following purposes: (i) to elucidate the molecular bases underlying the genetic defects of murine Had-1 mutants, which are deficient in UDP-galactose transporting activity, and (ii) to obtain information that would help us in planning rational approaches to identify functionally essential regions, based on comparison of primary structures between human and murine UDP-galactose transporters. We identified five nonsense mutations, one missense Gly178Asp mutation, and two aberrant splicing mutations. Although glycine178 is highly conserved among nucleotide-sugar transporters, a Gly178Ala variant was functional. The species-differences between human and murine UDP-galactose transporters were largely confined to the N- and C-terminal regions of the transporters. Substantial deletions in the N- and C-terminal regions did not lead to loss of UDP-galactose transporting activity, indicating that these cytosolic regions are dispensable for the transporting activity. The transporter was fused with green-fluorescent protein at the C-terminal cytosolic tail without impairing the functions of either protein. Our results demonstrate the importance of the transmembrane core region of the UDP-galactose transporter protein. 相似文献
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Sonja Wilke Joern Krausze Manfred Gossen Lothar Groebe Volker Jäger Ermanno Gherardi Joop van den Heuvel Konrad Büssow 《Protein science : a publication of the Protein Society》2010,19(6):1264-1271
Stable mammalian cell lines are excellent tools for the expression of secreted and membrane glycoproteins. However, structural analysis of these molecules is generally hampered by the complexity of N‐linked carbohydrate side chains. Cell lines with mutations are available that result in shorter and more homogenous carbohydrate chains. Here, we use preparative fluorescence‐activated cell sorting (FACS) and site‐specific gene excision to establish high‐yield glycoprotein expression for structural studies with stable clones derived from the well‐established Lec3.2.8.1 glycosylation mutant of the Chinese hamster ovary (CHO) cell line. We exemplify the strategy by describing novel clones expressing single‐chain hepatocyte growth factor/scatter factor (HGF/SF, a secreted glycoprotein) and a domain of lysosome‐associated membrane protein 3 (LAMP3d). In both cases, stable GFP‐expressing cell lines were established by transfection with a genetic construct including a GFP marker and two rounds of cell sorting after 1 and 2 weeks. The GFP marker was subsequently removed by heterologous expression of Flp recombinase. Production of HGF/SF and LAMP3d was stable over several months. 1.2 mg HGF/SF and 0.9 mg LAMP3d were purified per litre of culture, respectively. Homogenous glycoprotein preparations were amenable to enzymatic deglycosylation under native conditions. Purified and deglycosylated LAMP3d protein was readily crystallized. The combination of FACS and gene excision described here constitutes a robust and fast procedure for maximizing the yield of glycoproteins for structural analysis from glycosylation mutant cell lines. 相似文献
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Disrupting the function of the BRCA1 gene by mechanisms other than germline mutations is suspected to occur in cases of sporadic breast/ovarian cancers. Using ribonuclease protection assay and multiplex RT-PCR, we examined the change of the total BRCA1 mRNA pool and the expression profile of four predominant BRCA1 splice variants in asynchronous and in G1/S synchronized tumor cell populations compared to normal breast cells. Experiments were carried out on MCF-7 and MDA-MB-231 breast cancer, OVCAR-5 ovarian cancer, and K562 leukemia cell lines. The ratio of the full length, the delta(11q), the delta(9,10), and the delta(9,10,11q) BRCA1 isoforms showed different expression patterns in the examined breast and ovarian tumor cell lines as compared to the leukemia cell line. This observation raises the possibility that the dysregulation of alternative splicing of the BRCA1 gene could be involved in tumor formation in the breast and the ovary, even in the absence of germline mutations. 相似文献
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Eliminating tyrosine sequence variants in CHO cell lines producing recombinant monoclonal antibodies
Lauren Feeney Veronica Carvalhal X. Christopher Yu Betty Chan David A. Michels Yajun Jennifer Wang Amy Shen Jan Ressl Brendon Dusel Michael W. Laird 《Biotechnology and bioengineering》2013,110(4):1087-1097
Amino acid sequence variants are defined as unintended amino acid sequence changes that contribute to product variation with potential impact to product safety, immunogenicity, and efficacy. Therefore, it is important to understand the propensity for sequence variant (SV) formation during the production of recombinant proteins for therapeutic use. During the development of clinical therapeutic products, several monoclonal antibodies (mAbs) produced from Chinese Hamster Ovary (CHO) cells exhibited SVs at low levels (≤3%) in multiple locations throughout the mAbs. In these examples, the cell culture process depleted tyrosine, and the tyrosine residues in the recombinant mAbs were replaced with phenylalanine or histidine. In this work, it is demonstrated that tyrosine supplementation eliminated the tyrosine SVs, while early tyrosine starvation significantly increased the SV level in all mAbs tested. Additionally, it was determined that phenylalanine is the amino acid preferentially misincorporated in the absence of tyrosine over histidine, with no other amino acid misincorporated in the absence of tyrosine, phenylalanine, and histidine. The data support that the tyrosine SVs are due to mistranslation and not DNA mutation, most likely due to tRNATyr mischarging due to the structural similarities between tyrosine and phenylalanine. Biotechnol. Bioeng. 2013; 110: 1087–1097. © 2012 Wiley Periodicals, Inc. 相似文献
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Variant forms of human alpha-1-proteinase inhibitor (alpha-1-PI), obtained by the treatment of human Hep G2 cells with specific inhibitors of glycosylation were tested for both inhibitory activity and heat stability. All were found to have the same second-order association rate with human neutrophil elastase, indicating a lack of importance of the carbohydrate moiety. In contrast, incompletely glycosylated forms of alpha-1-PI were found to be heat sensitive relative to the mature protein, suggesting a role for carbohydrate in protein stabilization. 相似文献
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Nucleosides are accumulated by cells via a family of equilibrative transport proteins (ENTs). An alternative splice variant of the most common subtype of mouse ENT (ENT1) has been identified which is missing a protein kinase CK2 (casein kinase 2) consensus site (Ser254) in the central intracellular loop of the protein. We hypothesized that this variant (mENT1a) would be less susceptible to modulation by CK2-mediated phosphorylation compared to the variant containing the serine at position 254 (mENT1b). Each splice variant was transfected into nucleoside transporter deficient PK15 cells, and stable transfectants assessed for their ability to bind the ENT1-selective probe [3H]nitrobenzylthioinosine (NBMPR) and to mediate the cellular uptake of [3H]2-chloroadenosine, with or without treatment with the CK2 selective inhibitor, 4,5,6,7-tetrabromobenzotriazole (TBB). mENT1a had a higher affinity for NBMPR relative to mENT1b – measured both directly by the binding of [3H]NBMPR, and indirectly via inhibition of [3H]2-chloroadenosine influx by NBMPR. Furthermore, incubation of mENT1b-expressing cells with 10 µM TBB for 48 h decreased both the KD and Bmax of [3H]NBMPR binding, as well as the Vmax of 2-chloroadenosine uptake, whereas similar treatment of mENT1a-expressing cells with TBB had no effect. PK15 cells transfected with hENT1, which has Ser254, was similar to mENT1b in its response to TBB. In conclusion, inhibition of CK2 activity, or deletion of Ser254 from mENT1, enhances transporter affinity for the inhibitor, NBMPR, and reduces the number of ENT1 proteins functioning at the level of the plasma membrane. 相似文献
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Differential regulation of mouse equilibrative nucleoside transporter 1 (mENT1) splice variants by protein kinase CK2 总被引:1,自引:0,他引:1
Nucleosides are accumulated by cells via a family of equilibrative transport proteins (ENTs). An alternative splice variant of the most common subtype of mouse ENT (ENT1) has been identified which is missing a protein kinase CK2 (casein kinase 2) consensus site (Ser(254)) in the central intracellular loop of the protein. We hypothesized that this variant (mENT1a) would be less susceptible to modulation by CK2-mediated phosphorylation compared to the variant containing the serine at position 254 (mENT1b). Each splice variant was transfected into nucleoside transporter deficient PK15 cells, and stable transfectants assessed for their ability to bind the ENT1-selective probe [(3)H]nitrobenzylthioinosine (NBMPR) and to mediate the cellular uptake of [(3)H]2-chloroadenosine, with or without treatment with the CK2 selective inhibitor, 4,5,6,7-tetrabromobenzotriazole (TBB). mENT1a had a higher affinity for NBMPR relative to mENT1b - measured both directly by the binding of [(3)H]NBMPR, and indirectly via inhibition of [(3)H]2-chloroadenosine influx by NBMPR. Furthermore, incubation of mENT1b-expressing cells with 10 microM TBB for 48 h decreased both the K(D) and B(max) of [(3)H]NBMPR binding, as well as the V(max) of 2-chloroadenosine uptake, whereas similar treatment of mENT1a-expressing cells with TBB had no effect. PK15 cells transfected with hENT1, which has Ser(254), was similar to mENT1b in its response to TBB. In conclusion, inhibition of CK2 activity, or deletion of Ser(254) from mENT1, enhances transporter affinity for the inhibitor, NBMPR, and reduces the number of ENT1 proteins functioning at the level of the plasma membrane. 相似文献
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Michel Bergeron Georges Thiéry Frédéric Lenoir Marie-Cécile Giocondi Christian Le Grimellec 《Cell and tissue research》1994,277(2):297-307
The spatial organization of the endoplasmic reticulum has been studied in two renal cell lines, MDCK and LLC-PK1, which originate from the distal and proximal portions of the mammalian nephron, respectively, and which form a polarized epithelium when they reach confluence in tissue culture. The two renal cell lines, grown to confluence on either solid or permeable supports, were investigated by fluorescence microscopy, confocal microscopy, and transmission electron microscopy. Fluorescence labeling of the endoplasmic reticulum was achieved using the cationic fluorescent dye DIOC6 (3). In order to differentiate fluorescent labeling of the endoplasmic reticulum from that of the mitochondria, cells were also labeled with rhodamine 123. For electron microscopy, the spatial organization of the endoplasmic reticulum was examined in thick sections using the long-duration osmium impregnation technique or the ferrocyanide/osmium technique. In both cell lines, the endoplasmic reticulum formed an abundant tubular network of canaliculi that frequently abutted the basolateral domain of the plasma membrane and occasionally the apical membrane. Elements of the endoplasmic reticulum were also found in close proximity to mitochondria that, as in the nephron, formed branched structures. Canaliculi appeared circular or flattened and had an inner diameter of 10–70 nm for MDCK cells and 20–90 nm for LLC-PK1 cells. Such a three-dimensional organization might facilitate the translocation of defined lipid species between the endoplasmic reticulum and the plasma membrane, and between the endoplasmic reticulum and mitochondria. 相似文献
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Pascoe DE Arnott D Papoutsakis ET Miller WM Andersen DC 《Biotechnology and bioengineering》2007,98(2):391-410
Two-dimensional gel electrophoresis and tandem mass spectrometry were used to identify proteins associated with a metabolic shift during fed-batch cultures of two recombinant antibody-producing CHO cell lines. The first cell line underwent a marked change in lactate metabolism during culture, initially producing lactate and then consuming it, while the second cell line produced lactate for a similar duration but did not later consume it. The first cell line displayed a declining specific antibody productivity during culture, correlating to the 2-D gel results and the intracellular antibody concentration determined by HPLC. Several statistical analysis methods were compared during this work, including a fixed fold-change criterion and t-tests using standard deviations determined in several ways from the raw data and mathematically transformed data. Application of a variance-stabilizing transformation enabled the use of a global empirical standard deviation in the t-tests. Most of the protein spots changing in each cell line did not change significantly in the other cell line. A substantial fraction of the changing proteins were glycolytic enzymes; others included proteins related to antibody production, protein processing, and cell structure. Enolase, pyruvate kinase, BiP/GRP78, and protein disulfide isomerase were found in spots that changed over time in both cell lines, and some protein changes differed from previous reports. These data provide a foundation for future investigation of metabolism in industrially relevant mammalian cell culture processes, and suggest that along with differences between cell types, the proteins expressed in cultures with low lactate concentrations may depend on how those conditions were generated. 相似文献
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Chinese hamster ovary (CHO) cells are widely used for the production of biopharmaceuticals; however, our understanding of several physiological elements that contribute to productivity is limited. One of these is amino acid transport and how its limitation and/or regulation might affect productivity. To further our understanding, we have examined the expression of 40 mammalian amino acid transporter genes during batch cultures of three CHO cell lines: a non-producer and two antibody-producing cell lines with different levels of productivity. In parallel, extracellular and intracellular levels of amino acids were quantified. The aim was to identify differences in gene regulation between cell lines and within culture. Our results show that three transporters associated with transport of taurine and β-alanine, acidic amino acids and branched chain amino acids, are highly upregulated in both antibody-producing cell lines but not in the non-producer. Additionally, genes associated with the transport of amino acids related to the glutathione pathway (alanine, cysteine, cystine, glycine, glutamate) were found to be highly upregulated during the stationary phase of cell culture, correlating well with literature data on the importance of the pathway. Our analysis highlights potential markers for cell line selection and targets for process optimization. 相似文献
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HIV-1 Tat protein is a crucial element for viral replication; therefore, its inhibition might be exploited against the AIDS infection. To gain insights on the natural variability of this protein, we present a comparative investigation on the relationship between the primary sequences and the experimentally available three-dimensional structures from the HIV-1 Tat variants Z2, BRU, and MAL. Our computational tools include sequence conservation algorithms, structural analysis, electrostatic modeling, and molecular dynamics (MD) simulations. We find that two regions located between residues 10-18 and 41-52 display the highest primary sequence conservation, while the most conserved region among the available structures corresponds approximately to the segment between positions approximately 44 and 50. Furthermore, in spite of their large structural divergence, Tat variants share a common mode for long-range intramolecular interactions. Finally, the flexibility of the Z2, BRU, and MAL variants, as emerging from multinanosecond MD simulations, is rather similar. Based on this work, we conclude that the turnlike region between amino acids 44 and 50 is structurally most conserved, emerging as an important motif for pharmaceutical targeting aimed toward inhibiting Tat action. 相似文献
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The development and production of recombinant monoclonal antibodies is well established. Although most of these are IgGs, there is also great interest in producing recombinant IgAs since this isotype plays a critical role in providing immunologic protection at mucosal surfaces. the choice of expression system for production of recombinant antibodies is crucial because they are glycoproteins containing at least one N-linked carbohydrate. these glycans have been shown to contribute to the stability, pharmacokinetics and biologic function of antibodies. We have produced recombinant human IgA1 and all three allotypes of IgA2 in murine myeloma and CHo cell lines to systematically characterize and compare the N-linked glycans. Recombinant IgAs produced in murine myelomas differ significantly from IgA found in humans in that they contain the highly immunogenic Galα(1,3)Gal epitope and N-glycolylneuraminic acid residues, indicating that murine myeloma is not the optimal expression system for the production of human IgA. In contrast, IgAs produced in CHo cells contained glycans that were more similar to those found on human IgA. expression of IgA1 and IgA2 in Lec2 and Lec8 cell lines that are defective in glycan processing resulted in a less complex pool of N-glycans. In addition, the level of sialylation of rIgAs produced in murine and CHo cells was significantly lower than that previously reported for serum IgA1. these data underscore the importance of choosing the appropriate cell line for the production of glycoproteins with therapeutic potential.Key words: recombinant antibody, IgA, glycosylation, expression system, mass spectrometry 相似文献
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P. A Poland C. L Kinlough M. D Rokaw J Magarian-Blander O. J Finn R. P Hughey 《Glycoconjugate journal》1997,14(1):89-96
The membrane-bound mucin-like protein MUC1 with a specified number of tandem repeats has been expressed by transfection of the cDNAs in both the epithelial cell lines MDCK and LLC-PK1, and human lymphoblastoid cell lines T2 and C1R. The structure and glycosylation states of the MUC1 in these four lines were compared with that of the endogenous MUC1 found in the human pancreatic (HPAF) and breast (BT-20) tumor cell lines using flow cytometry and Western blot analysis with anti-MUC1 antibodies, which are either sensitive or insensitive to the glycosylation state of the tandem repeat, and pretreatment of cells with phenyl--galactosaminide, an inhibitor of mucin sialylation. A similar analysis of MUC1 expression in transfected normal and O-glycosylation defective CHO cells reveals that the addition of galactose to the core oligosaccharide structure is apparently responsible for the anomalous difference in Mr between the mature and propeptide forms of the MUC1. Both the tumor cells and the transfected lymphoblastoid cells consistently express significant steady state levels of both the heavily glycosylated mature forms and the poorly glycosylated propeptide forms of the MUC1, whereas MUC1 is found predominantly as the mature extensively glycosylated species in the transfected epithelial cells. Immunofluoresence microscopy of cross sections of the polarized epithelial cells grown on culture filter inserts reveals that the MUC1 is clearly present at the apical surface of the cells, consistent with its expression in normal tissues. Thus, the successful expression of the MUC1 by transfection of either lymphoblastoid cells or epithelial cells yields model systems both for studying the natural structure/function relationships of the protein domains within the MUC1 molecule and for further elucidating the previously reported MHC-independent T-cell recognition of the MUC1. 相似文献
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Huang C Zhou Q Liang P Hollander MS Sheikh F Li X Greaser M Shelton GD Evans S Chen J 《The Journal of biological chemistry》2003,278(9):7360-7365
Previously, we reported two splice variants of Cypher, a striated muscle-specific PDZLIM domain protein, Cypher1 and Cypher2. We have now characterized four additional splice isoforms, two of which are novel. The six isoforms can be divided into skeletal or cardiac specific classes, based on the inclusion of skeletal or cardiac specific domains. Short and long isoforms share an N-terminal PDZ domain, but the three C-terminal LIM domains are unique to long isoforms. By RNA and protein analysis, we have demonstrated that Cypher isoforms are developmentally regulated in both skeletal and cardiac muscle. We have previously shown that knockout of Cypher is neonatal lethal. To investigate the function of splice variants in vivo, we have performed a rescue experiment of the Cypher null mutant by replacing the endogenous Cypher gene with cDNAs encoding either a short or long skeletal muscle isoform. In contrast to Cypher null mice, a percentage of mice that express only a short or a long skeletal muscle-specific isoform can survive to at least 1 year of age. Although surviving mice exhibit muscle pathology, these results suggest that either isoform is sufficient to rescue the lethality associated with the absence of Cypher. 相似文献
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《MABS-AUSTIN》2013,5(3):320-334
The development and production of recombinant monoclonal antibodies is well established. Although most of these are IgGs, there is also great interest in producing recombinant IgAs since this isotype plays a critical role in providing immunologic protection at mucosal surfaces. The choice of expression system for production of recombinant antibodies is crucial because they are glycoproteins containing at least one N-linked carbohydrate. These glycans have been shown to contribute to the stability, pharmacokinetics and biologic function of antibodies. We have produced recombinant human IgA1 and all three allotypes of IgA2 in murine myeloma and CHO cell lines to systematically characterize and compare the N-linked glycans. Recombinant IgAs produced in murine myelomas differ significantly from IgA found in humans in that they contain the highly immunogenic Galα(1,3)Gal epitope and N-glycolylneuraminic acid residues, indicating that murine myeloma is not the optimal expression system for the production of human IgA. In contrast, IgAs produced in CHO cells contained glycans that were more similar to those found on human IgA. Expression of IgA1 and IgA2 in Lec2 and Lec8 cell lines that are defective in glycan processing resulted in a less complex pool of N-glycans. In addition, the level of sialylation of rIgAs produced in murine and CHO cells was significantly lower than that previously reported for serum IgA1. These data underscore the importance of choosing the appropriate cell line for the production of glycoproteins with therapeutic potential. 相似文献