Impediment of Gpibα-Mediated Platelet Clearance By an Anti-Gpibβ Antibody Derivative

Abstract Background: Glycoprotein (GP)Ib-IX complex plays a critical role in mediating platelet activation and platelet clearance. Recently, we identified the mechanosensory domain (MSD) in the GPIbα subunit, and demonstrated that unfolding of the MSD and subsequent exposure of the Trigger sequence (residues 473-483) therein activates GPIb-IX and induces rapid platelet clearance. This mechanism could explain acute thrombocytopenia induced by activated VWF, anti-GPIbα antibodies, neuraminidase, and ectodomain shedding of GPIbα. Consistently, platelets in IL4R-IbαTg mice, a transgenic strain in which the entire extracellular domain of human GPIbα except the Trigger sequence was replaced with that of the α-subunit of interleukin-4 receptor, exhibit constitutively more filopodia and are cleared much faster than the wild type. Previously, an anti-GPIbβ antibody RAM.1 was developed. RAM.1 significantly inhibits GPIb-IX-mediated filopodia formation and Ca 2+ signaling in platelets. In addition, it could inhibit GPIb-dependent thrombin generation. These results suggest that targeting GPIbβ could inhibit activation of GPIb-IX induced by MSD unfolding. Objectives: To investigate whether targeting GPIbβ with RAM.1 can impede rapid platelet clearance induced by exposed Trigger sequence and ameliorate related thrombocytopenia. Methods: Spontaneous filopodia in platelets and transfected Chinese hamster ovary (CHO) cells were visualized by fluorescence staining of actin and confocal microscopy. Images were quantified by ImageJ. Platelet signaling events, like P-selectin exposure, β-galactose exposure, and Ca 2+ influx, were measured by flow cytometry. Endogenous platelet life span was tracked by Alexa 488-labeled anti-mouse GPIX antibody. Results: CHO cells stably expressing the same mutant GPIb-IX complex in IL4R-IbαTg mouse platelets have been successfully obtained. Like IL4R-IbαTg platelets, these IL4R-IbαTg CHO cells exhibited spontaneous filopodia in the absence of any GPIbα ligands. RAM.1 could inhibit spontaneous filopodia formation in these CHO cells and IL4R-IbαTg platelets (Fig. 1, 2). Compared to wild-type mouse platelets, IL4R-IbαTg platelets constitutively exhibited increased P-selectin exposure, increased β-galactose exposure, and elevated intracellular Ca 2+, all of which could be inhibited by treatment of RAM.1 (Fig. 3). Recombinant RAM.1-GCN4 protein (rRAM.1-GCN4), in which the Fc region of RAM.1 heavy chain was replaced with the GCN4 coiled coil dimerizing sequence, has been generated and used as an alternative of the divalent RAM.1-Fab2. It retained the ability of RAM.1 antibody to inhibit GPIb-IX signaling. Injecting rRAM.1-GCN4 into IL4R-IbαTg mice dramatically improved the life span of endogenous IL4R-IbαTg platelets (Fig. 4). Conclusion: These results demonstrate that the exposed Trigger sequence is sufficient to activate GPIb-IX in transfected CHO cells, and that RAM.1 derivatives can impede GPIbα-mediated rapid platelet clearance. Targeting GPIbβ may be a novel approach to treat GPIb-related thrombocytopenia. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Amelioration of the macrothrombocytopenia associated with the murine Bernard-Soulier syndrome

Blood ◽

10.1182/blood-2002-03-0997 ◽

2002 ◽

Vol 100 (6) ◽

pp. 2102-2107 ◽

Cited By ~ 123

Author(s):

Taisuke Kanaji ◽

Susan Russell ◽

Jerry Ware

Keyword(s):

Mouse Model ◽

Molecular Mechanisms ◽

Critical Role ◽

Chinese Hamster ◽

Interleukin 4 ◽

Platelet Glycoprotein ◽

Severe Bleeding ◽

Chimeric Receptor ◽

Α Subunit ◽

Bernard Soulier Syndrome

Abstract An absent platelet glycoprotein (GP) Ib-IX receptor results in the Bernard-Soulier syndrome and is characterized by severe bleeding and the laboratory presentation of macrothrombocytopenia. Although the macrothrombocytopenic phenotype is directly linked to an absent GP Ib-IX complex, the disrupted molecular mechanisms that produce the macrothrombocytopenia are unknown. We have utilized a mouse model of the Bernard-Soulier syndrome to engineer platelets expressing an α-subunit of GP Ib (GP Ibα) in which most of the extracytoplasmic sequence has been replaced by an isolated domain of the α-subunit of the human interleukin-4 receptor (IL-4Rα). The IL-4Rα/GP Ibα fusion is membrane expressed in Chinese hamster ovary (CHO) cells, and its expression is facilitated by the presence of human GP IX and the β-subunit of GP Ib. Transgenic animals expressing a chimeric receptor were generated and bred into the murine Bernard-Soulier syndrome–producing animals devoid of mouse GP Ibα but expressing the IL-4Rα/GP Ibα fusion sequence. The characterization of these mice revealed a 2-fold increase in circulating platelet count and a 50% reduction in platelet size when compared with platelets from the mouse model of the Bernard-Soulier syndrome. Immunoprecipitation confirmed that the IL-4Rα/GP Ibα subunit interacts with filamin-1 and 14-3-3ζ, known binding proteins to the GP Ibα cytoplasmic tail. Mice expressing the chimeric receptor retain a severe bleeding phenotype, confirming a critical role for the GP Ibα extracytoplasmic domain in hemostasis. These results provide in vivo insights into the structural elements of the GP Ibα subunit that contribute to normal megakaryocyte maturation and thrombopoiesis.

Download Full-text

Identification of Interacting Hot Spots in the αIIb Extracellular Stalk By Computational Alanine Scanning

Blood ◽

10.1182/blood.v128.22.2531.2531 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2531-2531

Author(s):

Karen P Fong ◽

Joanna Slusky ◽

Kyungchul Yoon ◽

Joel S. Bennett

Keyword(s):

Ligand Binding ◽

Cho Cells ◽

Hot Spots ◽

Conflicts Of Interest ◽

Binding Free Energy ◽

Chinese Hamster ◽

Computational Method ◽

Wild Type ◽

Alanine Scanning ◽

Computational Alanine Scanning

Abstract Binding of macromolecular ligands like fibrinogen to the active conformation of the integrin αIIbβ3 mediates platelet aggregation. Because platelets circulate in a fibrinogen-rich milieu, the αIIbβ3 on circulating platelets is constrained in an inactive conformation by intramolecular interactions involving the cytoplasmic, transmembrane, and stalk domains of αIIb and β3 to prevent the spontaneous formation of platelet aggregates. Following platelet stimulation, αIIbβ3 undergoes a global rearrangement during which the αIIb and β3 cytoplasmic, transmembrane, and stalk domains separate and the αIIbβ3 headpiece opens to expose its ligand binding site. Protein-protein interfaces, such as those of the αIIbβ3 stalks, are usually large complementary surfaces in which specific side chains, representing energetic "hot spots", contribute disproportionately to the binding free energy. Previously, we reported the use of a computational method, comprehensive interface design, as a way to control the direction of protein interactions by introducing changes in protein structure that destabilize undesired interactions. This enabled us to identify energetic hot spots in the β3 stalk, mutation of which caused constitutive αIIbβ3 activation. Here, we have extended our design strategy to the αIIb stalk. We used the Rosetta alanine scanning algorithm and the available crystal structure of the αIIbβ3 heterodimer to identify alanine substitutions in the αIIb region extending from residues 602-959 that would be predicted to destabilize the resting αIIbβ3 stalk interface. The functional consequences of the predicted mutations were confirmed by introducing them into wild type αIIb, expressing the mutant αIIb together with wild type β3 in Chinese hamster ovary (CHO) cells, and measuring fibrinogen binding to αIIbβ3 in the absence and presence of dithiothreitol by flow cytometry. We identified 5 alanine substitutions predicted to destabilize the αIIbβ3 stalk interface by >1.0 kcal/mol, 5 alanine substitutions predicted to destabilize the interface by 0.3-1.0 kcal/mol, and 2 neutral alanine substitutions having no effect on the interface as negative controls. Four of the 5 substitutions predicted to be the most destabilizing were located in the αIIb calf-2, rather than the calf-1, domain, and involved residues R751, N753, F755, and E785. It is noteworthy in this regard that the 5 destabilizing β3 substitutions we previously reported were located in the distal β3 stalk. To confirm the predictions of the computational alanine scanning, we introduced the four most destabilizing calf-2 substitutions and the two neutral controls into αIIb and co-expressed them with wild type β3 in CHO cells. There was little to no expression of αIIbR751Aβ3, implying that the presence of R751 is important for correct αIIb folding and/or αIIbβ3 assembly. By contrast, each of the other predicted destabilizing substitutions expressed and caused constitutive αIIbβ3 ligand binding activity. Moreover, as predicted, αIIbβ3 containing each of the neutral αIIb substitutions was inactive until the CHO cells were incubated with dithiothreitol. Thus, these results confirm the utility of our computational approach for identifying functionally significant regions in αIIbβ3. Further, they demonstrate that the interface between the distal αIIb stalk and the distal β3 stalk, much like the interface between the αIIb and β3 transmembrane domains, plays an important role in regulating the equilibrium between the inactive and active states of αIIbβ3. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Expression of human Mn SOD in Chinese hamster ovary cells confers protection from oxidant injury

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1993.264.6.l598 ◽

1993 ◽

Vol 264 (6) ◽

pp. L598-L605

Author(s):

B. Warner ◽

R. Papes ◽

M. Heile ◽

D. Spitz ◽

J. Wispe

Keyword(s):

Chinese Hamster Ovary ◽

Cho Cells ◽

Chinese Hamster Ovary Cells ◽

Lethal Dose ◽

Chinese Hamster ◽

Eukaryotic Expression ◽

Wild Type ◽

Oxidant Injury ◽

Sod Activity ◽

Recombinant Cho Cells

Manganese superoxide dismutase (Mn SOD) is an important component of antioxidant defense in aerobic cells because of its location in the mitochondria, a significant source of oxygen radicals and an important target of oxidant injury. To test the hypothesis that increased mitochondrial Mn SOD protects from oxidant injury, Chinese hamster ovary (CHO) cells were transfected with a eukaryotic expression vector containing the human Mn SOD cDNA. In recombinant CHO cells, Mn SOD activity was increased threefold over wild-type controls. Acute survival during paraquat exposure (0–500 microM) was significantly improved in CHO cells expressing human Mn SOD, with 71% of recombinant CHO cells surviving at the 50% lethal dose (LD50) for wild-type CHO controls. Cell growth following exposure to paraquat (100 microM) was also significantly improved in recombinant CHO cells. CHO cells expressing human Mn SOD continued to grow and divide after paraquat exposure, whereas growth of wild-type CHO cells was negligible. Protection against oxidant-induced injury was directly related to increased Mn SOD, occurring in the absence of changes in other antioxidant enzymes including catalase, Cu,Zn SOD, and glutathione associated cellular antioxidant mechanisms. We conclude that increased expression of human Mn SOD in vitro directly confers protection against oxidant injury.

Download Full-text

The Carboxyl-Terminal Region Is a Determinant for the Intracellular Behavior of the Chorionic Gonadotropin β Subunit: Effects on the Processing of the Asn-Linked Oligosaccharides

Molecular Endocrinology ◽

10.1210/mend.12.5.0116 ◽

1998 ◽

Vol 12 (5) ◽

pp. 766-772

Author(s):

Mesut Muyan ◽

Irving Boime

Keyword(s):

Intramolecular Interaction ◽

Chinese Hamster ◽

Glycoprotein Hormones ◽

Wild Type ◽

Cho Cell ◽

Α Subunit ◽

Glycosylation Sites ◽

Carboxyl Terminal ◽

Complex Forms ◽

Placental Hormone

Abstract The placental hormone human CG (hCG) consists of two noncovalently linked α- and β-subunits similar to the other glycoprotein hormones LH, FSH, and TSH. These heterodimers share a common α subunit but differ in their structurally distinct β subunits. The CGβ subunit is distinguished among the β subunits by the presence of a C-terminal extension with four serine-linked oligosaccharides (carboxyl terminal peptide or CTP). In previous studies we observed that deleting this sequence decreased assembly of the truncated CGβ subunit (CGβ114) with the α-subunit and increased the heterogeneity of the secreted forms of the uncombined subunit synthesized in transfected Chinese hamster ovary (CHO) cells. The latter result was attributed to alterations in the processing of the two N-linked oligosaccharides. To examine at what step this heterogeneity occurs, the CGβ and CGβ114 genes were transfected into wild-type and mutant CHO cell lines that are defective in the late steps of the N-linked carbohydrate-processing pathway. We show here that removal of the CTP alters the processing of the core mannosyl unit of the subunit to complex forms at both glycosylation sites and that the oligosaccharides contain polylactosamine. Although it has been presumed that there is little intramolecular interaction between the CTP and the proximal domains of the subunit, our data suggest that the CTP sequence participates in the folding of the newly synthesized subunit, which is manifest by the posttranslational changes observed here.

Download Full-text

Identification of a protein altered in mutants resistant to microtubule inhibitors as a member of the major heat shock protein (hsp70) family

Molecular and Cellular Biology ◽

10.1128/mcb.10.10.5160-5165.1990 ◽

1990 ◽

Vol 10 (10) ◽

pp. 5160-5165

Author(s):

S Ahmad ◽

R Ahuja ◽

T J Venner ◽

R S Gupta

Keyword(s):

Heat Shock ◽

Heat Shock Protein ◽

Cho Cells ◽

Chinese Hamster ◽

Immunofluorescent Staining ◽

Mutant Form ◽

Two Dimensional ◽

Wild Type ◽

Cho Cell ◽

Microtubule Inhibitors

A major cellular protein (P2; approximately 70 kilodaltons) which is altered in Chinese hamster ovary (CHO) cell mutants resistant to the microtubule inhibitors colchicine and podophyllotoxin has been shown to correspond to the constitutive form of the 70-kilodalton heat shock protein (hsc70). The inference that P2 and hsc70 are the same protein is based on the following observations: (i) migration of P2 in two-dimensional polyacrylamide gels in the same position as that reported for hsc70; (ii) cross-reactivity of a monoclonal antibody which reacts with both the constitutive and induced forms of hsp70 with the P2 spot from wild-type CHO cells and with both P2 and a mutant form of P2 in a CHO cell mutant; (iii) specific reactivity of a polyclonal antibody to P2 with both the constitutive and heat-induced forms of hsp70 in human cells; (iv) identical immunofluorescent staining of dot/patchlike structures with both P2 and hsp70 antibodies in human and CHO cells; and (v) a cDNA clone for hsc70 has been isolated and sequenced from wild-type CHO cells. The in vitro transcription and translation product of this cDNA has been shown to comigrate with the P2 protein spot in two-dimensional gels, indicating their identity. The fact that there is an alteration in hsc70 in mutants resistant to antimitotic drugs suggests a role for this protein in the in vivo assembly and function of microtubules.

Download Full-text

Kinetics of endosome acidification in mutant and wild-type Chinese hamster ovary cells.

The Journal of Cell Biology ◽

10.1083/jcb.105.6.2713 ◽

1987 ◽

Vol 105 (6) ◽

pp. 2713-2721 ◽

Cited By ~ 64

Author(s):

D J Yamashiro ◽

F R Maxfield

Keyword(s):

Chinese Hamster Ovary ◽

Cho Cells ◽

Ph Value ◽

Ph Regulation ◽

Chinese Hamster Ovary Cells ◽

Mutant Cell ◽

Chinese Hamster ◽

Wild Type ◽

Cell Biol ◽

Endocytic Compartments

Acidification of endocytic compartments is necessary for the proper sorting and processing of many ligands and their receptors. Robbins and co-workers have obtained Chinese hamster ovary (CHO) cell mutants that are pleiotropically defective in endocytosis and deficient in ATP-dependent acidification of endosomes isolated by density centrifugation (Robbins, A. R., S. S. Peng, and J. L. Marshall. 1983. J. Cell Biol. 96:1064-1071; Robbins, A. R., C. Oliver, J. L. Bateman, S. S. Krag, C. J. Galloway, and I. Mellman. 1984. J. Cell Biol. 99:1296-1308). In this and the following paper (Yamashiro, D. J., and F. R. Maxfield. 1987. J. Cell Biol. 105:2723-2733) we describe detailed studies of endosome acidification in the mutant and wild-type CHO cells. Here we describe a new microspectrofluorometry method based on changes in fluorescein fluorescence when all cellular compartments are equilibrated to the same pH value. Using this method we measured the pH of endocytic compartments during the first minutes of endocytosis. We found in wild-type CHO cells that after 3 min, fluorescein-labeled dextran (F-Dex) was in endosomes having an average pH of 6.3. By 10 min, both F-Dex and fluorescein-labeled alpha 2-macroglobulin (F-alpha 2M) had reached acidic endosomes having an average pH of 6.0 or below. In contrast, endosome acidification in the CHO mutants DTG 1-5-4 and DTF 1-5-1 was markedly slowed. The average endosomal pH after 5 min was 6.7 in both mutant cell lines. At least 15 min was required for F-Dex and F-alpha 2M to reach an average pH of 6.0 in DTG 1-5-4. Acidification of early endocytic compartments is defective in the CHO mutants DTG 1-5-4 and DTF 1-5-1, but pH regulation of later compartments on both the recycling pathway and lysosomal pathway is nearly normal. The properties of the mutant cells suggest that proper functioning of pH regulatory mechanisms in early endocytic compartments is critical for many pH-mediated processes of endocytosis.

Download Full-text

ICAM-1-CD18 interaction mediates neutrophil cytotoxicity through protease release

AJP Cell Physiology ◽

10.1152/ajpcell.1998.274.6.c1634 ◽

1998 ◽

Vol 274 (6) ◽

pp. C1634-C1644 ◽

Cited By ~ 36

Author(s):

Carlton C. Barnett ◽

Ernest E. Moore ◽

Gary W. Mierau ◽

David A. Partrick ◽

Walter L. Biffl ◽

...

Keyword(s):

Cho Cells ◽

Chinese Hamster ◽

Cell Lysis ◽

Polymorphonuclear Neutrophil ◽

Intercellular Adhesion Molecule ◽

Wild Type ◽

Myristate Acetate ◽

Intercellular Adhesion Molecule 1 ◽

Eglin C

Interaction of the β2-integrin complex on the polymorphonuclear neutrophil (PMN) with intercellular adhesion molecule-1 (ICAM-1) has been implicated in PMN-mediated cytotoxicity. This study examined interaction of the CD11a, CD11b, and CD18 subunits of the β2-integrin with ICAM-1, transfected into Chinese hamster ovarian (CHO) cells to avoid effects of other adhesion molecules. Incubation of quiescent PMNs with wild-type and ICAM-1-transfected CHO cells produced nominal cell lysis. Similarly, when phorbol myristate acetate (PMA)-activated PMNs were incubated with wild-type CHO cells, minimal cytotoxicity was produced. However, when ICAM-1-transfected CHO cells were incubated with PMA-activated PMNs, 40% cell lysis occurred. Blockade with a monoclonal antibody (MAb) to ICAM-1 or MAbs to CD11a, CD11b, or CD18 reduced PMN-mediated cytotoxicity to baseline. To examine the role of adhesion in cytotoxicity, we studied β2-integrin-mediated PMN adhesion to ICAM-1-transfected CHO cells and found that MAbs for CD11a, CD11b, and CD18 all abrogated PMN cytotoxicity despite disparate effects on adhesion. To assess the role of CD18, β2-integrin subunits were cross-linked, and CD18 alone mediated protease release. Moreover, ICAM-1 was immunoprecipitated from transfected CHO cells and incubated with PMNs. This soluble ICAM-1 provoked elastase release, similar to PMA, which could be inhibited by MAbs to CD18 but not MAbs to other β2-integrin subunits. In addition, coincubation with protease inhibitors eglin C and AAPVCK reduced PMN-mediated cytotoxicity to control levels. Finally, ICAM-1-transfected CHO cells were exposed to activated PMNs from a patient with chronic granulomatous disease that caused significant cell lysis, equivalent to that of PMNs from normal donors. Collectively, these data suggest that ICAM-1 provokes PMN-mediated cytotoxicity via CD18-mediated protease release.

Download Full-text

A single point mutation in Drosophila dihydrofolate reductase confers methotrexate resistance to a transgenic CHO cell line

Genome ◽

10.1139/g03-046 ◽

2003 ◽

Vol 46 (4) ◽

pp. 707-715 ◽

Cited By ~ 4

Author(s):

K Neumann ◽

K M Al-Batayneh ◽

M J Kuiper ◽

J Parsons-Sheldrake ◽

M G Tyshenko ◽

...

Keyword(s):

Cell Line ◽

Point Mutation ◽

Cell Lines ◽

Dihydrofolate Reductase ◽

Cho Cells ◽

Chinese Hamster ◽

Single Point Mutation ◽

Parental Line ◽

Wild Type ◽

Drosophila Cell

Sequence analysis of a cDNA encoding dihydrofolate reductase (DHFR) from a selected methotrexate-resistant Drosophila melanogaster cell line (S3MTX) revealed a substitution of Gln for Leu at position 30. Although the S3MTX cells were ~1000 fold more resistant to methotrexate (MTX), the karyotype was similar to the parental line and did not show elongated chromosomes. Furthermore, kinetic analysis of the recombinant enzyme showed a decreased affinity for MTX by the mutant DHFR. To determine if the resistance phenotype could be attributed to the mutant allele, Drosophila Dhfr cDNAs isolated from wild type and S3MTX cells were expressed in Chinese hamster ovary (CHO) cells lacking endogenous DHFR. The heterologous insect DHFRs were functional in transgenic clonal cell lines, showing ~400-fold greater MTX resistance in the cell line transfected with the mutant Dhfr than the wild type Dhfr. Resistance to other antifolates in the CHO cells was consistent with the drug sensitivities seen in the respective Drosophila cell lines. ELevated Levels of Dhfr transcript and DHFR in transgenic CHO cells bearing the mutant cDNA were not seen. Taken together, these results demonstrate that a single substitution in Drosophila DHFR alone can confer Levels of MTX resistance comparable with that observed after considerable gene amplification in mammalian cells.Key words: dihydrofolate reductase, methotrexate, drug resistance, point mutation.

Download Full-text

Loss of transcriptional activation of three sterol-regulated genes in mutant hamster cells.

Molecular and Cellular Biology ◽

10.1128/mcb.13.9.5175 ◽

1993 ◽

Vol 13 (9) ◽

pp. 5175-5185 ◽

Cited By ~ 24

Author(s):

M J Evans ◽

J E Metherall

Keyword(s):

Transcriptional Regulation ◽

Cell Lines ◽

Transcriptional Activation ◽

Cho Cells ◽

Mutant Cell ◽

Chinese Hamster ◽

Wild Type ◽

Hmg Coa Reductase ◽

Low Levels ◽

Coa Reductase

Cholesterol biosynthesis and uptake are controlled by a classic end product-feedback mechanism whereby elevated cellular sterol levels suppress transcription of the genes encoding 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, HMG-CoA reductase, and the low-density lipoprotein receptor. The 5'-flanking region of each gene contains a common cis-acting element, designated the sterol regulatory element (SRE), that is required for transcriptional regulation. In this report, we describe mutant Chinese hamster ovary (CHO) cell lines that lack SRE-dependent transcription. Mutant cell lines were isolated on the basis of their ability to survive treatment with amphotericin B, a polyene antibiotic that kills cells by interacting with cholesterol in the plasma membrane. Four mutant lines (SRD-6A, -B, -C, and -D) were found to be cholesterol auxotrophs and demonstrated constitutively low levels of mRNA for all three sterol-regulated genes even under conditions of sterol deprivation. The mutant cell lines were found to be genetically recessive, and all four lines belonged to the same complementation group. When transfected with a plasmid containing a sterol-regulated promoter fused to a bacterial reporter gene, SRD-6B cells demonstrated constitutively low levels of transcription, in contrast to wild-type CHO cells, which increased transcription under conditions of sterol deprivation. Mutation of the SREs in this plasmid prior to transfection reduced the level of expression in wild-type CHO cells deprived of sterols to the level of expression found in SRD-6B cells. The defect in SRD-6 cells is limited to transcriptional regulation, since posttranscriptional mechanisms of sterol-mediated regulation were intact: the cells retained the ability to posttranscriptionally suppress HMG-CoA reductase activity and to stimulate acyl-CoA:cholesterol acyltransferase activity. These results suggest that SRD-6 cells lack a factor required for SRE-dependent transcriptional activation. We contrast these cells with a previously isolated oxysterol-resistant cell line (SRD-2) that lacks a factor required for SRE-dependent transcriptional suppression and propose a model for the role of these genetically defined factors in sterol-mediated transcriptional regulation.

Download Full-text

Characterization of β-galactosidase mutations Asp332→Asn and Arg148→Ser, and a polymorphism, Ser532→Gly, in a case of GM1 gangliosidosis

Biochemical Journal ◽

10.1042/bj3480621 ◽

2000 ◽

Vol 348 (3) ◽

pp. 621-632 ◽

Cited By ~ 19

Author(s):

Sunqu ZHANG ◽

Richard BAGSHAW ◽

William HILSON ◽

Yuko OHO ◽

Alina HINEK ◽

...

Keyword(s):

Catalytic Activity ◽

Cho Cells ◽

Chinese Hamster ◽

Nucleotide Position ◽

Missense Mutations ◽

Wild Type ◽

Gm1 Gangliosidosis ◽

Cultured Fibroblasts ◽

The Impact ◽

Perinuclear Area

We have identified and characterized three missense mutations in a patient with type 1 GM1 gangliosidosis, namely a substitution of G for A at nucleotide position 1044 (G1044 → A; in exon 10) on one allele, which converts Asp332 into asparagine, and both a mutation (C492 → A in exon 4, leading to the amino acid change of Arg148 → Ser) and a polymorphism (A1644 → G in exon 15, leading to a change of Ser532 → Gly) on the other allele. This patient had less than 1% residual β-galactosidase activity and minimally detectable levels of immunoreactive β-galactosidase protein in fibroblasts. To account for the above findings, a series of expression and immunolocalization studies were undertaken to assess the impact of each mutation. Transient overexpression in COS-1 cells of cDNAs encoding Asp332Asn, Arg148Ser and Ser532Gly mutant β-galactosidases produced abundant amounts of precursor β-galactosidase, with activities of 0, 84 and 81% compared with the cDNA clone for wild-type β-galactosidase (GP8). Since the level of vector-driven expression is much less in Chinese hamster ovary (CHO) cells than in COS-1 cells, and we knew that exogenous β-galactosidase undergoes lysosomal processing when expressed in these cells, transient expression studies were performed of Arg148Ser and Ser532Gly, which yielded active forms of the enzyme. In this case, the Arg148Ser and Ser532Gly products gave rise to 11% and 86% of the control activity respectively. These results were not unexpected, since the Arg148Ser mutation introduced a major conformational change into the protein, and we anticipated that it would be degraded in the endoplasmic reticulum (ER), whereas the polymorphism was expected to produce near-normal activity. To examine the effect of the Asp332Asn mutation on the catalytic activity, we isolated CHO clones permanently transfected with the Asp332Asn and Asp332Glu constructs, purified the enzymes by substrate-analogue-affinity chromatography, and determined their kinetic parameters. The Vmax values of both mutant recombinant enzymes were markedly reduced (less than 0.9% of the control), and the Km values were unchanged compared with the corresponding wild-type enzyme isolated at the same time. Both the Arg148Ser β-galactosidase in CHO cells and Asp332Asn β-galactosidases (in COS-1 and CHO cells) produced abundant immunoreaction in the perinuclear area, consistent with localization in the ER. A low amount was detected in lysosomes. Incubation of patient fibroblasts in the presence of leupeptin, which reduces the rate of degradation of lysosomal β-galactosidase by thiol proteases, had no effect on residual enzyme activity, and immunostaining was again detected largely in the perinuclear area (localized to the ER) with much lower amounts in the lysosomes. In summary, the Arg148Ser mutation has no effect on catalytic activity, whereas the Asp332Asn mutation seriously reduces catalytic activity, suggesting that Asp332 might play a role in the active site. Immunofluorescence studies indicate the expressed mutant proteins with Arg148Ser and Asp332Asn mutations are held up in the ER, where they are probably degraded, resulting in only minimum amounts of the enzyme becoming localized in the lysosomes. These results are completely consistent with findings in the cultured fibroblasts. Our results imply that most of the missense mutations described in GM1 gangliosidosis to date have little effect on catalytic activity, but do affect protein conformation such that the resulting protein cannot be transported out of the ER and fails to arrive in the lysosome. This accounts for the minimal amounts of enzyme protein and activity seen in most GM1 gangliosidosis patient fibroblasts.

Download Full-text