Chinese Hamster Ovary Cell Mutants Research Articles

Tumour necrosis factor α-converting enzyme mediates ectodomain shedding of Vps10p-domain receptor family members

Several transmembrane molecules are cleaved at juxtamembrane extracellular sites leading to shedding of ectodomains. We analysed shedding of members of the Vps10p-D (Vps10p domain; where Vps is vacuolar protein sorting) family of neuronal type-I receptors with partially overlapping functions, and additional proteolytic events initiated by the shedding. When transfected into CHO (Chinese-hamster ovary) cells (CHO-K1), sorCS1a-sorCS1c isoforms were shed at high rates (approximately 0.61% x min(-1)) that were increased approx. 3-fold upon stimulation with phorbol ester. sorCS1c identified in the cultured neuroblastoma cell line SH-SY5Y was shed similarly. In CHO-K1 transfectants, constitutive and stimulated shedding of sorCS3 also occurred at high rates (0.29% and 1.03% x min(-1)). By comparison, constitutive and stimulated shedding of sorLA occurred at somewhat lower rates (0.07% and 0.48% x min(-1)), whereas sorCS2 and sortilin were shed at very low rates even when stimulated (approximately 0.01% x min(-1)). Except for sorCS2, shedding of the receptors was dramatically reduced in mutant CHO cells (CHO-M2) devoid of active TACE (tumour necrosis factor alpha-converting enzyme), demonstrating that this enzyme accounts for most sheddase activity. The release of sorCS1 and sorLA ectodomains initiated rapid cleavage of the membrane-tethered C-terminal stubs that accumulated only in the presence of gamma-secretase inhibitors. Purified shed sorLA bound several ligands similarly to the entire luminal domain of the receptor, including PDGF-BB (platelet-derived growth factor-BB) and amyloid-beta precursor protein. In addition, PDGF-BB also bound to the luminal domains of sorCS1 and sorCS3. The results suggest that ectodomains shed from a subset of Vps10p-D receptors can function as carrier proteins.

XBP1 is Critical to Protect Cells from Endoplasmic Reticulum Stress: Evidence from Site-2 Protease-deficient Chinese Hamster Ovary Cells

XBP1 is a transcription factor downstream of IRE1, a transmembrane protein in the endoplasmic reticulum (ER) which functions as a sensor and transducer of ER stress. XBP1 mRNA is constitutively expressed at a low level as an intron-containing precursor mRNA (unspliced mRNA), which is subject to IRE1-mediated splicing reaction upon ER stress to produce the active form of XBP1, pXBP1(S). Because the XBP1 promoter carries a perfect ER stress-response element, namely, the cis-acting element responsible for the induction of ER chaperones, and XBP1 mRNA is induced in response to ER stress with a time course similar to that of ER chaperone mRNAs, it is conjectured that transcription factor ATF6, activated immediately upon ER stress, induces the transcription of not only ER chaperone genes but also of XBP1 gene, such that pXBP1(S) produced by the splicing of an increased level of XBP1 mRNA escapes from proteasome-mediated degradation. Here, we examined this notion by determining the induction of XBP1 mRNA and pXBP1(S) in mutant Chinese hamster ovary (M19) cells deficient in Site-2 protease, which executes the last step of ER stress-induced activation of ATF6. We found that the induction of XBP1 mRNA and pXBP1(S) was greatly reduced in M19 cells as compared with wild-type cells, leading to a marked reduction in the extent of induction of XBP1-target gene. M19 cells were much more sensitive to ER stress than wild-type cells. Importantly, overexpression of XBP1 unspliced mRNA in M19 cells reversed all of these phenotypes. We concluded that ATF6-mediated induction of XBP1 mRNA is important to the production of pXBP1(S), activation of XBP1-target genes, and protection of cells from ER stress.

DPM1, the Catalytic Subunit of Dolichol-phosphate Mannose Synthase, Is Tethered to and Stabilized on the Endoplasmic Reticulum Membrane by DPM3

Dolichol-phosphate mannose (DPM) synthase is required for synthesis of the glycosylphosphatidylinositol (GPI) anchor, N-glycan precursor, protein O-mannose, and C-mannose. We previously identified DPM3, the third component of this enzyme, which was co-purified with DPM1 and DPM2. Here, we have established mutant Chinese hamster ovary (CHO) 2.38 cells that were defective in DPM3. CHO2.38 cells were negative for GPI-anchored proteins, and microsomes from these cells showed no detectable DPM synthase activity, indicating that DPM3 is an essential component of this enzyme. A coiled-coil domain near the C terminus of DPM3 was important for tethering DPM1, the catalytic subunit of the enzyme, to the endoplasmic reticulum membrane and, therefore, was critical for enzyme activity. On the other hand, two transmembrane regions in the N-terminal portion of DPM3 showed no specific functions. DPM1 was rapidly degraded by the proteasome in the absence of DPM3. Free DPM1 was strongly associated with the C terminus of Hsc70-interacting protein (CHIP), a chaperone-dependent E3 ubiquitin ligase, suggesting that DPM1 is ubiquitinated, at least in part, by CHIP.

Heparan sulfate proteoglycans mediate Staphylococcus aureus interactions with intestinal epithelium

Staphylococcus aureus can be internalized by non-professional phagocytes, and may colonize the intestine in normal and antibiotic-treated individuals. Intestinal colonization may depend on the interactions of S. aureus with the intestinal epithelium. The best described mechanism of S. aureus binding to eukaryotic cells involves S. aureus fibronectin binding proteins (FnBPs), using fibronectin as a bridging molecule to beta1 integrins on the eukaryotic cell surface. Because S. aureus can be internalized by enterocytes, and because S. aureus is known to bind heparan sulfate (HS), we hypothesized that heparan sulfate proteoglycans (HSPGs) widely expressed on epithelia may mediate S. aureus interactions with intestinal epithelial cells. Internalization of S. aureus RN6390 by cultured intestinal epithelial cells was inhibited in a dose-dependent fashion by the HS mimic heparin, and by HS itself. Internalization of S. aureus DU5883, which lacks expression of staphylococcal FnBPs, was also inhibited by heparin. S. aureus adherence to ARH-77 cells, transfected to express the HSPG syndecan-1, was greatly increased when compared to adherence to plasmid control ARH-77 cells which have little detergent extractable HS. In addition, compared to wild-type HS-expressing Chinese hamster ovary (CHO) cells, internalization of S. aureus was decreased using mutant CHO cells with decreased HS expression. These findings are consistent with a model wherein S. aureus internalization by intestinal epithelial cells (and perhaps other epithelia) is mediated by S. aureus binding to the HS moiety of cell-surface HSPGs, and this interaction appears independent of fibronectin binding.

Tropoelastin Interacts with Cell-surface Glycosaminoglycans via Its COOH-terminal Domain

Using a biochemical and cell biological approach, we have identified a cell interaction site at the carboxyl terminus of tropoelastin. Cell interactions with the COOH-terminal sequence are not through the elastin-binding protein (EBP67) because neither VGVAPG-like peptides nor galactoside sugars altered adhesion. Our results also show that cell adhesion to tropoelastin is not promoted by integrins. Through the use of mutant Chinese hamster ovary cell lines defective in glycosaminoglycan biosynthesis, as well as competition studies and enzymatic removal of specific cell-surface glycosaminoglycans, the tropoelastin-binding moieties on the cell surface were identified as heparan and chondroitin sulfate-containing glycosaminoglycans, with heparan sulfate being greatly preferred. Heparin affinity chromatography combined with cell adhesion assays identified the last 17 amino acids as the sequence element at the carboxyl terminus of tropoelastin responsible for the adhesive activity.

A critical role for eukaryotic elongation factor 1A-1 in lipotoxic cell death.

The deleterious consequences of fatty acid (FA) and neutral lipid accumulation in nonadipose tissues, such as the heart, contribute to the pathogenesis of type 2 diabetes. To elucidate mechanisms of FA-induced cell death, or lipotoxicity, we generated Chinese hamster ovary (CHO) cell mutants resistant to palmitate-induced death and isolated a clone with disruption of eukaryotic elongation factor (eEF) 1A-1. eEF1A-1 involvement in lipotoxicity was confirmed in H9c2 cardiomyoblasts, in which small interfering RNA-mediated knockdown also conferred palmitate resistance. In wild-type CHO and H9c2 cells, palmitate increased reactive oxygen species and induced endoplasmic reticulum (ER) stress, changes accompanied by increased eEF1A-1 expression. Disruption of eEF1A-1 expression rendered these cells resistant to hydrogen peroxide- and ER stress-induced death, indicating that eEF1A-1 plays a critical role in the cell death response to these stressors downstream of lipid overload. Disruption of eEF1A-1 also resulted in actin cytoskeleton defects under basal conditions and in response to palmitate, suggesting that eEF1A-1 mediates lipotoxic cell death, secondary to oxidative and ER stress, by regulating cytoskeletal changes critical for this process. Furthermore, our observations of oxidative stress, ER stress, and induction of eEF1A-1 expression in a mouse model of lipotoxic cardiomyopathy implicate this cellular response in the pathophysiology of metabolic disease.

Platelet derived growth factor recruits lactosylceramide to induce cell proliferation in UDP Gal:GlcCer: β1 → 4Galactosyltransferase (GalT-V) mutant Chinese hamster ovary cells

Recent molecular cloning studies have suggested the presence of at least two beta4Gal transferase genes (beta4GalT-V and beta4GalT-VI) that may encode lactosylceramide synthase but whether they are functional in vivo and whether they mediate growth factor induced phenotypic change such as cell proliferation is not known. Our previous studies lead to the suggestion that various risk factors in atherosclerosis such as oxidized LDL, shear stress, nicotine, tumor necrosis factor-alpha converge upon LacCer synthase to induce critical phenotypic changes such as cell proliferation and cell adhesion. However, whether platelet-derived growth factor also recruits LacCer synthase in mediating cell proliferation is not known. Here we have employed a Chinese hamster ovary mutant cell line Pro(-)5Lec20 to determine whether this enzyme physiologically functions to mediate cell proliferation. We show that PDGF stimulates the activity of UDP galactose:glucosylceramide, beta1,4galactosyltransferase. The activity of LacCer synthase increased about 2.5 fold within 2.5-5 min of incubation with PDGF in both wild type and Pro(-)5Lec20 cells. Concomitantly, there was an increase in the generation of superoxide radicals, p44MAPK phosphorylation and cell proliferation in CHO cells. D-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), a potent inhibitor of GlcCer synthase/LacCer synthase impaired PDGF mediated induction of LacCer synthase activity, superoxide generation, p44 MAPK activation and cell proliferation in Pro(-)5Lec20 cells. PDGF-induced superoxide generation was also mitigated by the use of diphenylene iodonium; an inhibitor of NADPH oxidase activity that is required for superoxide generation. This inhibition was bypassed by the addition of lactosylceramide. Thus, beta4GalT-V gene produces a bona fide LacCer synthase that can function in vivo to generate LacCer. Moreover, this enzyme alone can mediate PDGF induced activation of a signal transduction cascade involving superoxide generation, p44MAPK activation, phosphorylation of Akt and cell proliferation.

Characterization of ERCC3 mutations in the Chinese hamster ovary 27-1, UV24 and MMC-2 cell lines

Mutation of the XPB gene in humans gives rise to the distinct, autosomal recessive disorder, with a striking clinical heterogeneity: xeroderma pigmentosum associated with Cockayne's syndrome and trichothiodystrophy. XPB is a subunit of a multifunctional RNA polymerase II general initiation factor TFIIH and codes for 3′ → 5′ DNA helicase essential for both nucleotide excision repair (NER) and transcription. Since XPB defective human disease is extremely rare, Chinese hamster ovary (CHO) mutant cell lines belonging to the 3rd rodent complementation group (the hamster ERCC3 gene is the homologue of the human XPB gene) are a unique resource for analyzing structure-function relationships in the ERCC3/XPB protein. We have amplified, cloned and sequenced the ERCC3 genes from wild type and 27-1, UV24 and MMC-2 CHO mutant cell lines and identified the sites of the respective mutations. 27-1 mutant has an A1075G transition (K359E) located at the very beginning of the Ia helicase domain which causes deficiency in open complex formation and in 3′, 5′ and dual incisions during NER. UV24 cell line has two mutations. First, it is a T1144C transition (S382P) located behind the Ia helicase domain in a region responsible for ERCC3 binding to XPG, p62 and p44. Second mutation is identical with a mutation in MMC-2 mutant. It is a C2215T transition (Q739STOP) causing the truncation of the C-terminus of the protein, responsible for the 5′ incision, by 44 amino acids. All mutant cell lines are unable to recover RNA synthesis after 10 Jm −2 UV, suggesting a defect in transcription-coupled repair. Their limited global NER capacity measured by a single-cell gel electrophoresis assay (0.25 Jm −2) varies from 6% to 11%.

Genetic Analysis of the Subunit Organization and Function of the Conserved Oligomeric Golgi (COG) Complex: STUDIES OF COG5- AND COG7-DEFICIENT MAMMALIAN CELLS

The conserved oligomeric Golgi (COG) complex is an eight-subunit (Cog1-8) peripheral Golgi protein involved in Golgi-associated membrane trafficking and glycoconjugate synthesis. We have analyzed the structure and function of COG using Cog1 or Cog2 null Chinese hamster ovary cell mutants, fibroblasts from a patient with Cog7-deficient congenital disorders of glycosylation, and stable Cog5-deficient HeLa cells generated by RNA interference. Although the dilation of some Golgi cisternae in Cog5-deficient cells resembled that observed in Cog1- or Cog2-deficient cells, their global glycosylation defects (less severe) and intracellular processing and function of low density lipoprotein receptors (essentially normal) differed from Cog1- and Cog2-deficient cells. Immunoblotting, gel filtration, and immunofluorescence microscopy analyses of the COG-deficient cells and cell extracts indicated that 1) Cog2-4 and Cog5-7 form stable subcomplexes, 2) Cog1 mediates Golgi association of a Cog2-4 plus Cog8 subcomplex, 3) Cog8 associates stably with both Cog5-7 and Cog1-4 subcomplexes, and thus 4) Cog8 helps assemble the Cog1-4 and Cog5-7 subcomplexes into the complete COG complex. This model of the subunit organization of COG is in excellent agreement with in vitro data presented in an accompanying paper (Ungar, D., Oka, T., Vasile, E., Krieger, M., and Hughson, F. M. (2005) J. Biol. Chem. 280, 32729-32735). Only one or two of the seven Cog1- or Cog2-dependent Golgi membrane proteins called GEARs are also sensitive to Cog5 or Cog7 deficiency, indicating that the COG subunits play distinctive roles in controlling Golgi structure and function.

Cell Surface Heparan Sulfate Promotes Replication of Toxoplasma gondii

Previous work suggests that cell surface heparan sulfate acts as a receptor for the Apicomplexan parasite Toxoplasma gondii. Using Chinese hamster ovary cell mutants defective in heparan sulfate biosynthesis, we show that heparan sulfate is necessary and sufficient for infectivity. Further, we demonstrate that the parasite requires N sulfation of heparan sulfate initiated by N-deacetylase/N-sulfotransferase-1, but 2-O sulfation and 6-O sulfation appear to be dispensable. In order to study the role of heparan sulfate in other cell types, we created a conditional allele for N-deacetylase/N-sulfotransferase-1 by using Cre-loxP technology. Mammary tumor cells lacking N-deacetylase/N-sulfotransferase-1 exhibited reduced toxoplasma infectivity like Chinese hamster ovary cell mutants. Surprisingly, heparin, chemically modified heparinoids, and monoclonal antibodies to heparan sulfate had no effect on toxoplasma infection. T. gondii attachment and invasion were unchanged in N-deacetylase/N-sulfotransferase-1-inactivated cells as well, but replication was reduced. Thus, heparan sulfate does not appear to function as a receptor for T. gondii but instead facilitates parasite replication postinvasion.

New mutant Chinese hamster ovary cell representing an unknown gene for attachment of glycosylphosphatidylinositol to proteins

Aerolysin, a secreted bacterial toxin from Aeromonas hydrophila, binds to glycosylphosphatidylinositol (GPI)-anchored protein and kills the cells by forming pores. Both GPI and N-glycan moieties of GPI-anchored proteins are involved in efficient binding of aerolysin. We isolated various Chinese hamster ovary (CHO) mutant cells resistant to aerolysin. Among them, CHOPA41.3 mutant cells showed several-fold decreased expression of GPI-anchored proteins. After transfection of N-acetylglucosamine transferase I ( GnT1) cDNA, aerolysin was efficiently bound to the cells, indicating that the resistance against aerolysin in this cells was mainly ascribed to the defect of N-glycan maturation. CHOPA41.3 cells also accumulated GPI intermediates lacking ethanolamine phosphate modification on the first mannose. After stable transfection of PIG- N cDNA encoding GPI-ethanolamine phosphate transferase1, a profile of accumulated GPI intermediates became similar to that of GPI transamidase mutant cells. It indicated, therefore, that CHOPA41.3 cells are defective in GnT1, ethanolamine phosphate modification of the first mannose, and attachment of GPI to proteins. The GPI accumulation in CHOPA41.3 cells carrying PIG- N cDNA was not normalized after transfection with cDNAs of all known components in GPI transamidase complex. Microsomes from CHOPA41.3 cells had normal GPI transamidase activity. Taken together, there is an unknown gene required for efficient attachment of GPI to proteins.

Isolation of Sterol-resistant Chinese Hamster Ovary Cells with Genetic Deficiencies in Both Insig-1 and Insig-2

Insig-1 and Insig-2, a pair of endoplasmic reticulum (ER) membrane proteins, mediate feedback control of cholesterol synthesis through their sterol-dependent binding to the following two polytopic ER membrane proteins: sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP) and 3-hydroxy-3-methylglutaryl-coenzyme A reductase. Sterol-induced binding of Insigs to SCAP prevents the proteolytic processing of SREBPs, membrane-bound transcription factors that enhance the synthesis of cholesterol, by retaining complexes between SCAP and SREBP in the ER. Sterol-induced binding of Insigs to reductase leads to the ubiquitination and ER-associated degradation of the enzyme, thereby slowing a rate-controlling step in cholesterol synthesis. Here we report the isolation of a new line of mutant Chinese hamster ovary cells, designated SRD-15, deficient in both Insig-1 and Insig-2. The SRD-15 cells were produced by gamma-irradiation of Insig-1-deficient SRD-14 cells, followed by selection in high levels of the oxysterol, 25-hydroxycholesterol. Sterols neither inhibit SREBP processing nor promote reductase ubiquitination/degradation in SRD-15 cells. Sterol regulation of SREBP processing and reductase ubiquitination/degradation is fully restored in SRD-15 cells when they are transfected with expression plasmids encoding either Insig-1 or Insig-2. These results demonstrate an absolute requirement for Insig proteins in the regulatory system that mediates lipid homeostasis in animal cells.

StarD10, a START Domain Protein Overexpressed in Breast Cancer, Functions as a Phospholipid Transfer Protein

We originally identified StarD10 as a protein overexpressed in breast cancer that cooperates with the ErbB family of receptor tyrosine kinases in cellular transformation. StarD10 contains a steroidogenic acute regulatory protein (StAR/StarD1)-related lipid transfer (START) domain that is thought to mediate binding of lipids. We now provide evidence that StarD10 interacts with phosphatidylcholine (PC) and phosphatidylethanolamine (PE) by electron spin resonance measurement. Interaction with these phospholipids was verified in a fluorescence resonance energy transfer-based assay with 7-nitro-2,1,3-benzoxadiazol-4-yl-labeled lipids. Binding was not restricted to lipid analogs since StarD10 selectively extracted PC and PE from small unilamellar vesicles prepared with endogenous radiolabeled lipids from Vero monkey kidney cells. Mass spectrometry revealed that StarD10 preferentially selects lipid species containing a palmitoyl or stearoyl chain on the sn-1 and an unsaturated fatty acyl chain (18:1 or 18:2) on the sn-2 position. StarD10 was further shown to bind lipids in vivo by cross-linking of protein expressed in transfected HEK-293T cells with photoactivable phosphatidylcholine. In addition to a lipid binding function, StarD10 transferred PC and PE between membranes. Interestingly, these lipid binding and transfer specificities distinguish StarD10 from the related START domain proteins Pctp and CERT, suggesting a distinct biological function.

Adherence of Plasmodium falciparum infected erythrocytes to CHO-745 cells and inhibition of binding by protein A in the presence of human serum

Adhesion of erythrocytes infected with the malaria parasite Plasmodium falciparum to human host receptors is a process associated with severe malarial pathology. A number of in vitro cell lines are available as models for these adhesive processes, including Chinese hamster ovary (CHO) cells which express the placental adhesion receptor chondroitin-4-sulphate (CSA) on their surface. CHO-745 cells, a glycosaminoglycan-negative mutant CHO cell line lacking CSA and other reported P. falciparum adhesion receptors, are often used for recombinant expression of host receptors and for receptor binding studies. In this study we show that P. falciparum-infected erythrocytes can be easily selected for adhesion to an endogenous receptor on the surface of CHO-745 cells, bringing into question the validity of using these cells as a tool for P. falciparum adhesin expression studies. The adhesive interaction between CHO-745 cells and parasitized erythrocytes described here is not mediated by the known P. falciparum adhesion receptors CSA, CD36, or ICAM-1. However, we found that CHO-745-selected parasitized erythrocytes bind normal human IgM and that adhesion to CHO-745 cells is inhibited by protein A in the presence of serum, but not in its absence, indicating a non-specific inhibitory effect. Thus, protein A, which has been used as an inhibitor for a recently described interaction between infected erythrocytes and the placenta, may not be an appropriate in vitro inhibitor for understanding in vivo adhesive interactions.

Mammalian PIG-X and Yeast Pbn1p Are the Essential Components of Glycosylphosphatidylinositol-Mannosyltransferase I

Within the endoplasmic reticulum (ER), mannoses and glucoses, donated from dolichol-phosphate-mannose and -glucose, are transferred to N-glycan and GPI-anchor precursors, and serine/threonine residues in many proteins. Glycosyltransferases that mediate these reactions are ER-resident multitransmembrane proteins with common characteristics, forming a superfamily of >10 enzymes. Here, we report an essential component of glycosylphosphatidylinositol-mannosyltransferase I (GPI-MT-I), which transfers the first of the four mannoses in the GPI-anchor precursors. We isolated a Chinese hamster ovary (CHO) cell mutant defective in GPI-MT-I but not its catalytic component PIG-M. The mutant gene, termed phosphatidylinositolglycan-class X (PIG-X), encoded a 252-amino acid ER-resident type I transmembrane protein with a large lumenal domain. PIG-X and PIG-M formed a complex, and PIG-M expression was <10% in the absence of PIG-X, indicating that PIG-X stabilizes PIG-M. We found that Saccharomyces cerevisiae Pbn1p/YCL052Cp, which was previously reported to be involved in autoprocessing of proproteinase B, is the functional homologue of PIG-X; Pbn1p is critical for Gpi14p/YJR013Wp function, the yeast homologue of PIG-M. This is the first report of an essential subcomponent of glycosyltransferases using dolichol-phosphate-monosaccharide.

A Fluorescent Probe of Polyamine Transport Accumulates into Intracellular Acidic Vesicles via a Two-step Mechanism

Mammalian polyamine carriers have not yet been molecularly identified. The fluoroprobe Spd-C2-BODIPY faithfully reports polyamine transport and accumulates almost exclusively in polyamine-sequestering vesicles (PSVs). Polyamines might thus be imported first by a plasma membrane carrier and then sequestered into pre-existing PSVs (model A), or be directly captured by polyamine receptors undergoing endocytosis (model B). Spd-C2-BODIPY uptake was unaffected in receptor-mediated endocytosis-deficient Chinese hamster ovary cell mutants. PSVs strongly colocalized with acidic vesicles of the late endocytic compartment and the trans Golgi. Virtually perfect colocalization between PSVs and acidic vesicles was found in Chinese hamster ovary cell mutants that are blocked either in the late endosome/lysosome fusion process or in the maturation of multivesicular bodies. Prior inhibition of the V-ATPase dramatically decreased total Spd-C2-BODIPY accumulation while increasing cytosolic fluorescence. Conversely, cells pre-loaded with the probe slowly released it from PSVs upon V-ATPase inhibition. The present data thus support model A, and indicate that polyamine accumulation is primarily driven by the activity of a vesicular H+:polyamine carrier.

Functional Sialylated O-Glycan to Platelet Aggregation on Aggrus (T1α/Podoplanin) Molecules Expressed in Chinese Hamster Ovary Cells

Aggrus, also called T1alpha and podoplanin, is a novel platelet aggregation-inducing factor that is expressed in various carcinoma cells. Aggrus/T1alpha/podoplanin is known to be expressed in lung type I alveolar cells or lymphatic endothelial cells. However, its physiological role has not been clarified. To assess the attribution of glycosylation to Aggrus platelet aggregation activity, recombinant molecules were stably expressed in a series of Chinese hamster ovary (CHO) cell mutants, N-glycan-deficient Lec1, CMP-sialic acid transporter-deficient Lec2, and UDP-galactose transporter-deficient Lec8. A new anti-human Aggrus monoclonal antibody, YM-1, was established to detect the expression of human Aggrus on these CHO cell mutants. Aggrus on Lec1 cells induced platelet aggregation, but those on Lec2 and Lec8 cells did not. Further, the glycans on Aggrus were analyzed by lectin blotting. Aggrus expressed in CHO and Lec1 cells showed Wheat-germ agglutinin, Jacalin, and Vicia villosa lectin bindings. Lectin blotting results indicated that sialylated core 1 structures, sialic acid plus Galbeta1,3GalNAc-Ser/Thr, were critical for the platelet aggregation activity. This oligosaccharide structure is known as tumor-associated antigen, which is potentially related to the metastasis process of cancer cells.

A Mutation Inactivating the Mitochondrial Inner Membrane Folate Transporter Creates a Glycine Requirement for Survival of Chinese Hamster Cells

A mutant Chinese hamster ovary cell line, glyB, that required exogenous glycine for survival and growth was reported previously (Kao, F., Chasin, L., and Puck, T. T. (1969) Proc. Natl. Acad. Sci. U. S. A. 64, 1284-1291). We now report that the defect in glyB cells causative of this phenotype is a point mutation in an inner mitochondrial membrane protein required for transport of folates into mitochondria. The CHO mitochondrial folate transporter (mft) was sequenced and compared with that from glyB cells. The hamster sequence was nearly identical to that of the recently reported human mitochondrial folate transporter. The corresponding cDNA from glyB cells contained a single nucleotide change that introduced a glutamate in place of the glycine in wild-type hamster MFT at codon 192 in a predicted transmembrane domain. Transfection of the wild-type hamster cDNA into glyB cells allowed cell survival in the absence of glycine and the accumulation of folates in mitochondria, whereas transfection of the Glu-192 cDNA did not. Genomic sequence analysis and fluorescence in situ hybridization demonstrated a single mutated allele of the mft gene in glyB cells, whereas there were two alleles in CHO cells. We conclude that we have defined the cause of the glyB auxotrophy and that the glyB mft mutation identified a region of this mitochondrial folate carrier vital to its transport function.

Sialylated Complex-type N-Glycans Enhance the Signaling Activity of Soluble Intercellular Adhesion Molecule-1 in Mouse Astrocytes

Intercellular adhesion molecule-1 (ICAM-1) occurs as both a membrane and a soluble, secreted glycoprotein (sICAM-1). ICAM-1 on endothelial cells mediates leukocyte adhesion by binding to leukocyte function associated antigen-1 (LFA-1) and macrophage antigen-1 (Mac-1). Recombinant mouse sICAM-1 induces the production of macrophage inflammatory protein-2 (MIP-2) in mouse astrocytes by a novel LFA-1- and Mac-1-independent mechanism. Here we showed that N-glycan structures of sICAM-1 influence its ability to induce MIP-2 production. sICAM-1 expressed in Chinese hamster ovary (CHO) cells was a more potent inducer of MIP-2 production than sICAM-1 expressed in HEK 293 cells, suggesting that posttranslational modification of sICAM-1 could influence its signaling activity. To explore the roles of glycosylation in sICAM-1 activity, we expressed sICAM-1 in mutant CHO cell lines differing in glycosylation, including Lec2, Lec8, and Lec1 as well as in CHO cells cultured in the presence of the alpha-mannosidase-I inhibitor kifunensine. Signaling activity of sICAM-1 lacking sialic acid was reduced 3-fold compared with sICAM-1 from CHO cells. The activity of sICAM-1 lacking both sialic acid and galactose was reduced 12-fold, whereas the activity of sICAM-1 carrying only high mannose-type N-glycans was reduced 12-26-fold. sICAM-1 glycoforms carrying truncated glycans retained full ability to bind to LFA-1 on leukocytes. Thus, sialylated and galactosylated complex-type N-glycans strongly enhanced the ability of sICAM-1 to induce MIP-2 production in astrocytes but did not alter its binding to LFA-1 on leukocytes. Glycosylation could therefore serve as a means to regulate specifically the signaling function of sICAM-1 in vivo.

Regulation of group VIA phospholipase A 2 expression by sterol availability

Several lines of evidence suggest that glycerophospholipid mass is maintained through the coordinate regulation of CTP:phosphocholine cytidylyltransferase-α (CTα) and the group VIA calcium-independent phospholipase A 2 (iPLA 2). CTα expression is modulated by sterol and this is mediated in part through sterol regulatory element binding proteins (SREBP). In this report, we investigate the possibility that iPLA 2 expression is controlled in a similar manner. When Chinese hamster ovary (CHO) cells were cultured under sterol-depleted conditions, iPLA 2 catalytic activity, mRNA, and protein were induced by between two- and threefold. These inductions were suppressed when the cells were supplemented with exogenous sterols. Luciferase reporter assays indicated that sterol depletion induced transcription of iPLA 2, an analysis of the 5′ flanking region suggested that the iPLA 2 gene contained a putative sterol regulatory element (SRE), and electrophoretic mobility shift assay (EMSA) analysis indicated that this element can bind SREBP-2. Notably, a mutant CHO cell line (SRD4) that constitutively generates mature SREBP proteins exhibited increased iPLA 2 activity and expression compared to wild-type cells. These data suggest that iPLA 2 expression is regulated in a manner consistent with other important genes in sterol and glycerophospholipid metabolism. Such coordinate regulation may be essential for maintaining the lipid composition of cell membranes.