Mutant Chinese Hamster Ovary Research Articles

Amyloid Precursor Protein Processing in Sterol Regulatory Element-binding Protein Site 2 Protease-deficient Chinese Hamster Ovary Cells

Amyloid peptides of 39-43 amino acids (Abeta) are the major constituents of amyloid plaques present in the brains of Alzheimer's (AD) patients. Proteolytic processing of the amyloid precursor protein (APP) by the yet unidentified beta- and gamma-secretases leads to the generation of the amyloidogenic Abeta peptides. Recent data suggest that all of the known mutations leading to early onset familial AD alter the processing of APP such that increased amounts of the 42-amino acid form of Abeta are generated by a gamma-secretase activity. Identification of the beta- and/or gamma-secretases is a major goal of current AD research, as they are prime targets for therapeutic intervention in AD. It has been suggested that the sterol regulatory element-binding protein site 2 protease (S2P) may be identical to the long sought gamma-secretase. We have directly tested this hypothesis using over-expression of the S2P cDNA in cells expressing APP and by characterizing APP processing in mutant Chinese hamster ovary cells that are deficient in S2P activity and expression. The data demonstrate that S2P does not play an essential role in the generation or secretion of Abeta peptides from cells, thus it is unlikely to be a gamma-secretase.

Mechanisms determining sensitivity to cisplatin in three mutant Chinese hamster ovary cell lines

To gain insight into factors determining the response of tumours to cisplatin, we studied pathways involved in resistance to cisplatin: drug uptake, cytoplasmic detoxification and DNA repair, in three cisplatin-sensitive Chinese hamster ovary (CHO)2 mutant cell lines. The mutant lines, CHO-MMC6, CHO-MMC1, CHO-MMS2, displayed inherent sensitivity to cisplatin (2.2, 4.1 and 10.6-fold, respectively) compared to the CHO-K1 line from which they were derived. CHO-MMS2 was the only mutant to show sensitivity to UV and this was slight (<2-fold). None of the mutants displayed increased sensitivity to X-irradiation. The CHO-MMS2 cell line appeared to have multiple mechanisms involved in its sensitivity to cisplatin, including increased drug accumulation, decreased levels of glutathione and a decreased capacity for DNA repair. The CHO-MMC1 mutant demonstrated reduced ability for DNA repair in a host cell reactivation assay, but no difference in drug accumulation or glutathione levels compared to the parent. The CHO-MMC6 cell line was not defective in any of the mechanisms studied. These three mutant cell lines demonstrate that similar mechanisms may account for inherent sensitivity or resistance to cisplatin, and suggest that multiple mechanisms may determine the sensitivity of human tumours to cisplatin.

Lack of β-amyloid production in M19 cells deficient in site 2 processing of the sterol regulatory element binding proteins

The processing of the amyloid precursor protein (APP) and the sterol regulatory element binding protein show remarkable analogies. Following a first lumenal cleavage, both proteins undergo a cleavage within the transmembrane domain by enzymatic activities named γ-secretase and S2P, respectively. We analyzed the processing of APP in the mutant Chinese hamster ovary (CHO) cell line M19 which lacks the S2P gene encoding for a putative metalloprotease. In these cells, we were not able to detect any β-amyloid production from endogenous or transiently overexpressed APP, although the transport of APP along the secretory pathway, its processing by α- and β-secretase, as well as its secretion were normal. This strongly suggests that the γ-secretase cleavage in M19 cells is severely impaired.

Differential Cellular Accumulation/Retention of Apolipoprotein E Mediated by Cell Surface Heparan Sulfate Proteoglycans: APOLIPOPROTEINS E3 AND E2 GREATER THAN E4

Isoform-specific effects of apolipoprotein E (apoE) on neurite outgrowth and the cytoskeleton are associated with higher intracellular levels of apoE3 than apoE4 in cultured neurons. The current studies, designed to determine the mechanism for the differential intracellular accumulation or retention of apoE, demonstrate that apoE3- and apoE4-containing beta-very low density lipoproteins (beta-VLDL) possess similar cell binding and internalization and delivery of cholesterol to the cells. However, as assessed by immunocytochemistry, analysis of extracted cellular proteins, or quantitation of 125I-apoE-enriched beta-VLDL, there was a 2-3-fold greater accumulation of apoE3 than apoE4 in Neuro-2a cells, fibroblasts, and hepatocytes (HepG2) after 1-2 h, and this differential was maintained for up to 48 h. ApoE2 also accumulated in Neuro-2a cells to a greater extent than apoE4. The differential effect was mediated by the apoE-enriched beta-VLDL and not by free apoE. Neither the low density lipoprotein receptor nor the low density lipoprotein receptor-related protein was responsible for the differential accumulation of apoE3 and apoE4, since cells deficient in either or both of these receptors also displayed the differential accumulation. The effect appears to be mediated primarily by cell surface heparan sulfate proteoglycans (HSPG). The retention of both apoE3 and apoE4 was markedly reduced, and the differential accumulation of apoE3 and apoE4 was eliminated both in mutant Chinese hamster ovary cells that did not express HSPG and in HSPG-expressing cells treated with heparinase. The data suggest that cell surface HSPG directly mediate the uptake of apoE-containing lipoproteins, that the differential accumulation/retention of apoE by cells is mediated via HSPG, and that there is a differential intracellular handling of the specific apoE isoforms.

The carboxyl-terminal region is a determinant for the intracellular behavior of the chorionic gonadotropin beta subunit: effects on the processing of the Asn-linked oligosaccharides.

The placental hormone human CG (hCG) consists of two noncovalently linked alpha- and beta-subunits similar to the other glycoprotein hormones LH, FSH, and TSH. These heterodimers share a common alpha subunit but differ in their structurally distinct beta subunits. The CGbeta subunit is distinguished among the beta 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 CGbeta subunit (CGbeta114) with the alpha-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 CGbeta and CGbeta114 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.

Structural and functional analysis of the protein products derived from mutant fur alleles in an endoprotease-deficient Chinese hamster ovary cell strain.

The fur gene encodes the endoprotease, furin. We recently demonstrated mutations in both fur alleles in the mutant Chinese hamster ovary (CHO)-K1 strain, RPE.40, and hypothesized that these mutations were responsible for the endoprotease-deficient phenotype of these cells. We now present the structural and functional properties of three protein products derived from the mutant fur alleles. None of these protein products were able to process the precursor to von Willebrand factor, which is processed by wild-type furin. Pro-protein processing activity initially attributed to one of the mutant proteins was due to wild-type furin produced inadvertently from one of the expression constructs used in these experiments. None of the mutant proteins exhibited evidence of autocatalysis, consistent with the lack of activity versus the test substrate, and glycosylation patterns suggested at least two of them remained in the endoplasmic reticulum. These results confirm that RPE.40 cells are furin null mutants, as earlier evidence had suggested.

Cloning and Expression of Mouse Liver Phosphatidylserine Synthase-1 cDNA: OVEREXPRESSION IN RAT HEPATOMA CELLS INHIBITS THE CDP-ETHANOLAMINE PATHWAY FOR PHOSPHATIDYLETHANOLAMINE BIOSYNTHESIS

In eukaryotic cells, phosphatidylserine (PtdSer) is synthesized by two distinct synthases on the endoplasmic reticulum by a base-exchange reaction in which the polar head-group of an existing phospholipid is replaced with serine. We report the cloning and expression of a cDNA for mouse liver PtdSer synthase-1. The deduced protein sequence is >90% identical to that of PtdSer synthase-1 from Chinese hamster ovary cells and a sequence from a human myeloblast cell line. PtdSer synthase-1 cDNA was stably expressed in M.9.1.1 cells which are mutant Chinese hamster ovary cells defective in PtdSer synthase-1 activity, are ethanolamine auxotrophs, and have a reduced content of PtdSer and phosphatidylethanolamine (PtdEtn). The growth defect of M.9.1.1 cells was eliminated, and a normal phospholipid composition was restored in the absence of exogenous ethanolamine, implying that the cloned cDNA encoded PtdSer synthase. Mouse liver PtdSer synthase-1 was also expressed in McArdle 7777 rat hepatoma cells. In addition to a 3-fold higher in vitro serine-exchange activity, these cells also exhibited enhanced choline- and ethanolamine-exchange activities and incorporated more [3H]serine into PtdSer than did control cells. However, the levels of PtdSer and PtdEtn in cells overexpressing PtdSer synthase-1 activity were not increased. Excess PtdSer produced by the transfected cells was rapidly decarboxylated to PtdEtn and the degradation of PtdSer, and/or PtdEtn derived from PtdSer, was increased. Moreover, the CDP-ethanolamine pathway for PtdEtn biosynthesis was inhibited. These data suggest that (i) cellular levels of PtdSer and PtdEtn are tightly controlled, and (ii) the metabolism of PtdSer and PtdEtn is coordinately regulated to maintain phospholipid homeostasis.

Functional Expression of the Chicken Low Density Lipoprotein Receptor-related Protein in a Mutant Chinese Hamster Ovary Cell Line Restores Toxicity of Pseudomonas Exotoxin A and Degradation of α2-Macroglobulin

The low density lipoprotein receptor-related protein (LRP) is responsible for the clearance of several physiological ligands including a complex of proteinase and alpha2-macroglobulin (alpha2M) and for the entrance of Pseudomonas exotoxin A (PEA) into cells. We have prepared expression plasmids for the full-length chicken LRP (designated LRP100) and two intermediates encoding 25 and 67% of the receptor (designated LRP25 and LRP67, respectively) using overlapping cDNA fragments. LRP25 and LRP67 encode the N-terminal 22 and 64%, respectively, of LRP100 plus the transmembrane and intracellular domains. Transient transfection of these plasmids into COS-7 cells yielded recombinant proteins of expected molecular mass and immunoreactivity. However, LRP100 was incompletely processed into alpha- (515-kDa) and beta- (85-kDa) chains and was poorly transported from the endoplasmic reticulum to the Golgi compartment. Stable transformants of LRP100, LRP67, and LRP25 were generated in a mutant Chinese hamster ovary cell line that lacked expression of endogenous LRP and was resistant to PEA. All forms of recombinant LRP proteins were transported from the endoplasmic reticulum to the Golgi apparatus in Chinese hamster ovary cells as shown by their sensitivity to endoglycosidase H and resistance to neuraminidase. Cell surface iodination and subcellular fractionation studies indicated that all three LRP variants were expressed on the plasma membrane. Furthermore, expression of the three LRP variants restored, to various degrees, sensitivity to PEA and the ability to degrade methylamine-activated alpha2M (alpha2M*). These data suggest that deletion of large internal portions of LRP, including the processing site, does not prevent transport of LRP to the plasma membrane, nor does it abolish the interaction of LRP with alpha2M* or PEA. This LRP expression system may allow for the characterization of domains within LRP responsible for its multifunctionality.

Biased short tract repair of palindromic loop mismatches in mammalian cells.

Mismatch repair of palindromic loops in the presence or absence of single-base mismatches was investigated in wild-type and mismatch-binding defective mutant Chinese hamster ovary cells. Recombination intermediates with a maximum heteroduplex DNA (hDNA) region of 697 bp contained a centrally located, phenotypically silent 12-base palindromic loop mismatch, and/or five single-base mismatches. In wild-type cells, both loops and single-base mismatches were efficiently repaired (80-100%). When no other mismatches were present in hDNA, loops were retained with a 1.6-1.9:1 bias. However, this bias was eliminated when single-base mismatches were present, perhaps because single-base mismatches signal nick-directed repair. In the multiple marker crosses, most repair tracts were long and continuous, with preferential loss of markers in cis to proximal nicks, consistent with nicks directing most repair in this situation. However, approximately 25% of repair tracts were discontinuous as a result of loop-specific repair, or from segregation or short tract repair of single-base mismatches. In mutant cells, single-base mismatches were repaired less frequently, but the loop was still repaired efficiently and with bias toward loop retention, indicating that the defect in these cells does not affect loop-specific repair. Repair tracts in products from mutant cells showed a wide variety of mosaic patterns reflecting short regions of repair and segregation consistent with reduced nick-directed repair. In mutant cells, single-base mismatches were repaired more efficiently in the presence of the loop than in its absence, a likely consequence of corepair initiated at the loop.

Evidence for a Cholesterol Transport Pathway from Lysosomes to Endoplasmic Reticulum That Is Independent of the Plasma Membrane

We have studied the movement of low density lipoprotein (LDL)-derived cholesterol in cultured Chinese hamster ovary cells. Our hypothesis is that when LDL cholesterol is effluxed from lysosomes, the bulk of LDL cholesterol is mobilized to the plasma membrane, while another pathway delivers LDL cholesterol from lysosomes to acyl-CoA/cholesterol acyltransferase (ACAT) in the endoplasmic reticulum. Three lines of evidence support this model. First, LDL cholesterol transport to ACAT can be blocked without inhibiting the movement of cholesterol from lysosomes to plasma membrane or from plasma membrane to endoplasmic reticulum. Second, LDL cholesterol transport to ACAT is normal in a Chinese hamster ovary mutant with defective plasma membrane-to-ACAT movement. Third, LDL cholesterol is not diluted by the plasma membrane cholesterol pool before reaching ACAT. Our evidence supports a vesicular model of cholesterol transport from lysosomes to the endoplasmic reticulum that is independent of the plasma membrane.

Expression Cloning of a Human Sulfotransferase That Directs the Synthesis of the HNK-1 Glycan on the Neural Cell Adhesion Molecule and Glycolipids

The HNK-1 carbohydrate is expressed on various adhesion molecules in the nervous system and is suggested to play a role in cell-cell and cell-substratum interactions. Here we describe the isolation and functional expression of a cDNA encoding a human sulfotransferase that synthesizes the HNK-1 carbohydrate epitope. A mutant Chinese hamster ovary cell line, Lec2, which stably expresses human neural cell adhesion molecule (N-CAM) (Lec2-NCAM), was first established. Lec2-NCAM was co-transfected with a human fetal brain cDNA library, a cDNA encoding the rat glucuronyltransferase that forms a precursor of the HNK-1 carbohydrate, and a vector encoding the polyoma large T antigen. The transfected Lec2-NCAM cells expressing the HNK-1 glycan were enriched by fluorescence-activated cell sorting. Sibling selection of recovered plasmids resulted in a cDNA encoding a sulfotransferase, HNK-1ST, that directs the expression of the HNK-1 carbohydrate epitope on the cell surface. The deduced amino acid sequence indicates that the enzyme is a type II membrane protein. Sequence analysis revealed that there is a short amino acid sequence in the presumed catalytic domain, which is highly homologous to the corresponding sequence in other Golgi-associated sulfotransferases so far cloned. The amount of HNK-1ST transcript is high in fetal brain compared with fetal lung, kidney, and liver. Expression of HNK-1ST resulted in the formation of the HNK-1 epitope on N-CAM and a soluble chimeric form of HNK-1ST was shown to add a sulfate group to a precursor, GlcAbeta1-->3Galbeta1-->4GlcNAcbeta1-->R, forming sulfo-->3GlcAbeta1-->3Galbeta1-->4GlcNAcbeta1-->R. The results combined together indicate that the cloned HNK-1ST directs the synthesis of the HNK-1 carbohydrate epitope on both glycoproteins and glycolipids in the nervous tissues.

Binding of nuclear proteins associated with mammalian DNA repair to the mitomycin C-DNA interstrand crosslink

Mitomycin C (MMC) is a DNA crosslinking agent that is used in cancer chemotherapy. Unlike the DNA crosslinks formed by cisplatin or psoralen, which significantly distort the DNA helix, the MMC crosslink does not significantly disturb the B-DNA helical structure. Nonetheless, MMC interstrand crosslinks and total MMC adducts are rapidly removed in vivo. We investigated whether mammalian nuclear proteins can recognize and bind to a model 23 bp DNA duplex containing a single MMC lesion. Electrophoretic mobility shift assays identified two complexes in nuclear extracts from rodent cell lines and three complexes in human cell lines, containing proteins that appeared to specifically recognize the MMC interstrand crosslink. Nuclear extracts from normal and excision repair-defective mutant Chinese hamster ovary (CHO) cell lines, from human Xeroderma Pigmentosum (XP) complementation group A and E cell lines, and a Fanconi's Anemia cell line were also examined. The UV-20 CHO line, defective in ERCC-1, was missing one of the two rodent complexes. Two of the three human complexes were also absent in the XPA human cell line and the intensity of the third complex was significantly diminished. Based on these results, a model for MMC crosslink recognition is proposed in which ERCC-1 and XPA each participate in formation of one or more multimeric complexes on the crosslinked DNA and XPA also aids in the formation, but is not a component of a higher molecularweight multimeric complex that may contain ERCC-1.

A Mammalian Homolog of the Yeast LCB1 Encodes a Component of Serine Palmitoyltransferase, the Enzyme Catalyzing the First Step in Sphingolipid Synthesis

Serine palmitoyltransferase (SPT; EC 2.3.1.50) catalyzes the initial step dedicated to sphingolipid biosynthesis and is thought to be a key enzyme for regulating cellular sphingolipid content. For SPT activity, the yeast Saccharomyces cerevisiae requires two genes, LCB1 and LCB2. We isolated mammalian LCB1 cDNA homologs from mouse and Chinese hamster ovary (CHO) cells and an LCB2 cDNA homolog from CHO cells. The mammalian LCB1 proteins are predicted to have about 35% amino acid identity to the yeast Lcb1 protein, whereas the CHO LCB2 protein is predicted to have about 40% amino acid identity to the yeast Lcb2 protein. Northern blot analysis of mRNA isolated from various mouse tissues revealed that the tissue distribution of both LCB1 and LCB2 messengers followed a similar pattern. Transfection of an SPT-defective CHO mutant strain with a CHO LCB1-expressing plasmid restored both SPT activity and de novo sphingolipid synthesis to the wild type levels, whereas transfection of the mutant strain with a CHO LCB2-expressing plasmid did not exhibit any recovery effects, indicating that the SPT defect in the mutant cells is specifically complemented by the CHO LCB1 homolog. Furthermore, when the SPT-defective mutant cells were transfected with a plasmid encoding a His6-tagged CHO LCB1 protein, SPT activity bound to a Ni2+-immobilized resin. These results indicate that the CHO LCB1 homolog encodes a component of SPT.

The human XRCC9 gene corrects chromosomal instability and mutagen sensitivities in CHO UV40 cells.

The Chinese hamster ovary (CHO) mutant UV40 cell line is hypersensitive to UV and ionizing radiation, simple alkylating agents, and DNA cross-linking agents. The mutant cells also have a high level of spontaneous chromosomal aberrations and 3-fold elevated sister chromatid exchange. We cloned and sequenced a human cDNA, designated XRCC9, that partially corrected the hypersensitivity of UV40 to mitomycin C, cisplatin, ethyl methanesulfonate, UV, and gamma-radiation. The spontaneous chromosomal aberrations in XRCC9 cDNA transformants were almost fully corrected whereas sister chromatid exchanges were unchanged. The XRCC9 genomic sequence was cloned and mapped to chromosome 9p13. The translated XRCC9 sequence of 622 amino acids has no similarity with known proteins. The 2.5-kb XRCC9 mRNA seen in the parental cells was undetectable in UV40 cells. The mRNA levels in testis were up to 10-fold higher compared with other human tissues and up to 100-fold higher compared with other baboon tissues. XRCC9 is a candidate tumor suppressor gene that might operate in a postreplication repair or a cell cycle checkpoint function.

Cell Adhesion to a Motif Shared by the Malaria Circumsporozoite Protein and Thrombospondin Is Mediated by Its Glycosaminoglycan-binding Region and Not by CSVTCG

The malaria circumsporozoite protein (CS), thrombospondin (TSP), and several other proteins including the terminal complement proteins and the neural adhesion molecules F-spondin and Unc-5, share a cell adhesive sequence. In CS this sequence is designated as region II-plus (EWSPCSVTCGNGIQVRIK) and in TSP it is found in the type I repeats. Previous studies aimed at fine mapping the amino acid residues required for cell adhesion have yielded discrepant results. Here we show in three different cell lines that the downstream basic residues are required for cell adhesion whereas the CSVTCG sequence is not. Using mutant Chinese hamster ovary cells selected for deficiencies in proteoglycan synthesis, we show that in wild type cells, heparan sulfate proteoglycans are the binding sites for this motif. This finding is supported by additional experiments with two other cell lines demonstrating that treatment with heparitinase but not chondroitinase abolishes cell adhesion to peptides representing this motif. Using Chinese hamster ovary cell mutants deficient in heparan sulfate proteoglycans but possessing chondroitin sulfate proteoglycans, we show that cell surface chondroitin sulfate proteoglycans can also mediate binding to this motif although higher concentrations of peptides are required for adhesion. Chondroitinase, but not heparitinase, treatment of these cells destroys cell surface-binding sites. Taken together, these results indicate that cell adhesion to this motif involves an interaction between the downstream positively-charged residues and the negatively charged glycosaminoglycan chains of heparan sulfate, or in some cases chondroitin sulfate, proteoglycans on the cell surface.

Substantial narrowing of the Niemann-Pick C candidate interval by yeast artificial chromosome complementation.

Niemann-Pick disease type C (NP-C) is an autosomal recessive lipidosis linked to chromosome 18q11-12, characterized by lysosomal accumulation of unesterified cholesterol and delayed induction of cholesterol-mediated homeostatic responses. This cellular phenotype is identifiable cytologically by filipin staining and biochemically by measurement of low-density lipoprotein-derived cholesterol esterification. The mutant Chinese hamster ovary cell line (CT60), which displays the NP-C cellular phenotype, was used as the recipient for a complementation assay after somatic cell fusions with normal and NP-C murine cells suggested that this Chinese hamster ovary cell line carries an alteration(s) in the hamster homolog(s) of NP-C. To narrow rapidly the candidate interval for NP-C, three overlapping yeast artificial chromosomes (YACs) spanning the 1 centimorgan human NP-C interval were introduced stably into CT60 cells and analyzed for correction of the cellular phenotype. Only YAC 911D5 complemented the NP-C phenotype, as evidenced by cytological and biochemical analyses, whereas no complementation was obtained from the other two YACs within the interval or from a YAC derived from chromosome 7. Fluorescent in situ hybridization indicated that YAC 911D5 was integrated at a single site per CT60 genome. These data substantially narrow the NP-C critical interval and should greatly simplify the identification of the gene responsible in mouse and man. This is the first demonstration of YAC complementation as a valuable adjunct strategy for positional cloning of a human gene.

Synthesis of Dolichol in a Polyprenol Reductase Mutant Is Restored by Elevation ofcis-Prenyl Transferase Activity

CHB11-1-3 is a glycosylation mutant of Chinese hamster ovary (CHO) cells, isolated by screening mutagenized cells for those with decreased intracellular lysosomal enzyme activity [C. W. Hallet al.(1986)Mol. Cell. Biochem.72, 35–45]. CHB11-1-3 synthesizes the lipid polyprenol, the metabolic precursor of dolichol, rather than dolichol, indicating a defect in polyprenol reductase. This defect was demonstrated previously in Lec9 CHO mutants, and cell fusion experiments confirmed that CHB11-1-3 is a member of this complementation group. A revertant of CHB11-1-3, CHBREV, isolated for its ability to grow at 39°C, synthesizes dolichol at near-normal levels. CHBREV is probably a second-site revertant, because it synthesizes three to four times as much polyprenol as CHB11-1-3 and exhibits a similar elevation in the specific activity ofcis-prenyl transferase. This higher activity appears to reflect an increase in enzyme molecules rather than the presence of an activator or absence of an inhibitor. These results suggest that CHB11-1-3 is a “Km” mutant, because synthesis of higher amounts of the substrate of polyprenol reductase obviates the defect.

Role of the extracellular domain of human herpesvirus 7 glycoprotein B in virus binding to cell surface heparan sulfate proteoglycans.

In an attempt to identify the human herpesvirus 7 (HHV-7) envelope protein(s) involved in cell surface binding, the extracellular domain of the HHV-7 glycoprotein B (gB) homolog protein was cloned and expressed as a fusion product with the Fc domain of human immunoglobulin G heavy chain gamma1 (gB-Fc) in an eukaryotic cell system. Indirect immunofluorescence followed by flow cytometric analysis revealed specific binding of gB-Fc to the membrane of SupT1 cells but not to other CD4+ T-lymphoblastoid cell lines, such as Jurkat or PM1, clearly indicating that gB-Fc did not bind to the CD4 molecule. This was also suggested by the ability of gB-Fc to bind to CD4-negative fibroblastoid Chinese hamster ovary (CHO) cells. The binding was abrogated by enzymatic removal of cell surface heparan sulfate proteoglycans by heparinase and heparitinase but not by treatment with condroitinase ABC. In addition, binding of the gB-Fc fusion protein to CHO cells was severely impaired in the presence of soluble heparin, as well as when heparan sulfate-deficient mutant CHO cells were used. Consistent with these findings, soluble heparin was found to block HHV-7 infection and syncytium formation in the SupT1 cell line. Although the CD4 antigen is a critical component of the receptor for the T-lymphotropic HHV-7, these findings suggest that heparin-like molecules also play an important role in HHV-7-cell surface interactions required for infection and that gB represents one of the HHV-7 envelope proteins involved in the adsorption of virus-to-cell surface proteoglycans.

Extracellular Cleavage of the Vascular Endothelial Growth Factor 189-Amino Acid Form by Urokinase Is Required for Its Mitogenic Effect

Alternative splicing of vascular endothelial growth factor (VEGF) mRNA results in three distinct molecular forms of 121 or 165 (V165) amino acids that are released in the conditioned medium of cultured cells and one longer isoform of 189 amino acids (V189) that remains cell-associated. V189 has been expressed in wild type CHO-K1 cells and in glycosaminoglycan-deficient pgsA-745 Chinese hamster ovary (CHO) mutant cells. It could be released from CHO-K1 cell membranes by heparin or a synthetic peptide designed on the sequence encoded by exon 6 but was freely released from CHO mutant cells. In both cases, the immunoreactive V189 was mainly released as a 40-kDa cleaved form, provided that the serine protease urokinase, but not plasmin, was active. Recombinant V189 was purified from insect cells infected with a recombinant baculovirus as a nonmitogenic 50-kDa precursor that binds to the receptor Flt-1 but not to Flk-1. It could be matured by urokinase as a 38-kDa fragment able to bind to Flk-1 and to trigger cell proliferation. V165 and V189, however, could be cleaved by plasmin as 34-kDa fragments that exhibit a decreased mitogenic activity. These findings indicate that the carboxyl-terminal domain of V189 masks its binding domain to Flk-1.

Isolation and Characterization of Peroxisome-Deficient Chinese Hamster Ovary Cell Mutants Representing Human Complementation Group III

We made use of the 9-(1′-pyrene)nonanol/ultraviolet (P9OH/UV) method and isolated peroxisome-deficient mutant cells. TKa cells, the wild-type Chinese hamster ovary (CHO) cells, CHO-K1, that had been stably transfected with cDNA encoding Pex2p (formerly peroxisome assembly factor-1, PAF-1) were used to avoid frequent isolation of the Z65-type, PEX2-defective mutants. P9OH/UV-resistant cell colonies were examined for the intracellular location of catalase, a peroxisomal matrix enzyme, by immunofluorescence microscopy and using anti-catalase antibody. As six mutant cell clones showed cytosolic catalase, there was likely to be a deficiency in peroxisome assembly. These mutants also showed the typical peroxisome assembly-defective phenotype, including significant decrease of dihydroxyacetonephosphate acyltransferase, the first step key enzyme in plasmalogen synthesis, and loss of resistance to 12-(1′-pyrene)dodecanoic acid/UV treatment. By transfection of Pex2p and Pex6p (formerly PAF-2) cDNAs and cell fusion analysis between the CHO cell mutants, two mutants, ZP104 and ZP109, were found to belong to a novel complementation group. Further complementation analysis using fibroblasts from patients with peroxisome biogenesis disorders revealed that the mutants belonged to human complementation group III. Taken together, ZP104 and ZP109 are in a newly identified fifth complementation group in CHO mutants reported to date and represent the human complementation group III.