Characterization of a B lymphoblastoid cell line mutant that secretes HLA-A2

HLA-A2 antigen mutants were obtained previously from the B lymphoblastoid cell line T5-1 by mutagenesis followed by immunoselection. Here we present biochemical studies of one particular mutant, clone 8.14.1. These cells synthesize two forms of HLA-A2: a minor form, which remains cell-associated at all times, and an abundant form, which is secreted. The former appears by SDS-PAGE to be slightly larger than T5-1 HLA-A2, whereas the latter appears to be 4000 to 5000 daltons smaller. In vitro translation and in vivo pulse-chase studies suggest that these species are not related to each other by post-translational processing. Proteolytic digestion studies localize the resulting structural alteration in the mobility difference between wild-type and secreted HLA-A2 to a region near the carboxy terminus of the HLA-A2 heavy chain; however, their extreme carboxy termini appear similar, if not identical. We suggest that the secreted form may result from a pattern of RNA splicing in which the exon encoding the hydrophobic, membrane-spanning region is frequently deleted.     

Alternative splicing generates a secreted form of N-CAM in muscle and brain

A number of different membrane associated isoforms of the neural cell adhesion molecule (N-CAM) have previously been identified. Here the structure of a novel secreted isoform of N-CAM is established by analysis of a cDNA corresponding to an N-CAM mRNA from human skeletal muscle. The mRNA incorporates a novel sequence block into the extracellular domain, which introduces an in-frame stop codon and thus prematurely terminates the coding sequence, generating a truncated N-CAM polypeptide. Analysis of genomic clones indicates that the inserted sequence is present as a discrete exon within the human N-CAM gene, and Northern analysis shows it to be associated specifically with a 5.2 kb mRNA species from skeletal muscle and brain. Stable transfectants expressing the secreted isoform accumulate it in the cytoplasm and release it to the culture medium. In contrast, cells transfected with cDNA encoding lipid-tailed N-CAM express it predominantly at the cell surface. The existence of a secreted isoform may further expand the spectrum of N-CAM function beyond its known involvement in intercellular adhesion to extracellular matrix interactions.     

Alternative RNA splicing generates a glycosylphosphatidylinositol-anchored form of ceruloplasmin in mammalian brain

Ceruloplasmin is a copper-containing ferroxidase that is essential for normal iron homeostasis. Whereas ceruloplasmin in plasma is produced and secreted by hepatocytes, in the brain a glycosylphosphatidylinositol (GPI)-anchored form of ceruloplasmin is expressed on the surface of astrocytes. By using a cDNA cloning approach, we have now determined that the GPI-anchored form of ceruloplasmin is generated by alternative RNA splicing. The splicing occurs downstream of exon 18 and replaces the C-terminal 5 amino acids of the secreted form with an alternative 30 amino acids that signal GPI anchor addition. RNase protection analysis demonstrates that the GPI-anchored form is the major form in the brain, whereas the secreted form predominates in the liver. Individuals with aceruloplasminemia, a hereditary deficiency of ceruloplasmin, have severe iron deposition in a number of organs, including the brain where it results in neurodegeneration. Therefore, this novel GPI-anchored form of ceruloplasmin is likely to play an important role in iron metabolism in the central nervous system.     

Isolation and characterization of B cell differentiation factor (BCDF) secreted from a human B lymphoblastoid cell line

A subclone of a human B lymphoblastoid cell line, CESS-2, spontaneously secreted a kind of BCDF (B-BCDF) in their culture supernatant without any stimulation. B-BCDF induced IgG and IgM secretions in human B lymphoblastoid cell lines, CESS cells and CL-4 cells, respectively. BCDF-responsive CESS cells expressed IgG on their surface, whereas CESS-2, which were able to secrete B-BCDF, did not express surface IgG. B-BCDF could induce Ig-secretion in SAC-stimulated low density peripheral B cells, but did not induce Ig secretion in nonstimulated B cells. B-BCDF did not show any IL 2, BCGF, or gamma-interferon activities. B-BCDF was highly purified by gel filtration on an AcA-34 column, by chromatofocusing and ion exchange chromatography on an HPLC system, and by gel filtration on an HPLC column. Highly purified preparations showed a single protein band in SDS-PAGE analysis. The m.w. and isoelectric points of the factor were 20,000 and pH 5.1 to 5.2, respectively. The minimum protein amount required for Ig induction in B cell lines was 16 ng/ml.     

Differential splicing generates a nervous system-specific form of Drosophila neuroglian.

We recently described the characterization and cloning of Drosophila neuroglian, a member of the immunoglobulin superfamily. Neuroglian contains six immunoglobulin-like domains and five fibronectin type III domains and shows strong sequence homology to the mouse neural cell adhesion molecule L1. Here we show that the neuroglian gene generates at least two different protein products by tissue-specific alternative splicing. The two protein forms differ in their cytoplasmic domains. The long form is restricted to the surface of neurons in the CNS and neurons and some support cells in the PNS; in contrast, the short form is expressed on a wide range of other cells and tissues. Thus, whereas the mouse L1 gene appears to encode only one protein that functions largely as a neural cell adhesion molecule, its Drosophila homolog, the neuroglian gene, encodes at least two protein forms that may play two different roles, one as a neural cell adhesion molecule and the other as a more general cell adhesion molecule involved in other tissues and imaginal disc morphogenesis.     

Alternative splicing generates a family of putative secreted and membrane-associated MUC4 mucins.

The MUC4 mucin gene encodes a putative membrane-anchored mucin with predicted size of 930 kDa, for its 26.5-kb allele. It is composed of two regions, the 850-kDa mucin-type subunit MUC4alpha and the 80-kDa membrane-associated subunit MUC4beta. In this study, we cloned and characterized unique MUC4 cDNA sequences that differ from the originally published sequence. Eight alternative splice events located downstream of the central large tandem repeat array generated eight new, distinct cDNAs. The deduced sequences of these MUC4 cDNAs (sv1-MUC4 to sv8-MUC4, the full length cDNA being called sv0-MUC4) provided seven distinct variants, five secreted forms and two membrane-associated forms. Furthermore, two other alternative splicing events located on both sides of the tandem repeat array created two variants, MUC4/Y and MUC4/X, both lacking the central tandem repeat. Therefore, MUC4 can be expressed in three distinct forms, one membrane-bound, one secreted, and one lacking the hallmark feature of mucin, the tandem repeat array. Although no specific function has yet been discovered for the family of proteins putatively produced from the unique MUC4 gene, we suspect that the MUC4 proteins may be implicated in the integrity and renewal of the epithelium.     

High-level expression of functional platelet-activating factor receptors on a human B lymphoblastoid cell line.

Binding of platelet-activating factor (PAF) was characterized in a human b lymphoblastoid cell line, ASK.0. [3H]PAF binding to these cells was time-dependent, reaching equilibrium at 60 minutes, and saturable. Scatchard analyses of saturation binding experiments revealed a single class of PAF binding sites (108,000 +/- 17,000 per cell) with a KD of 2.16 +/- 0.41 nM. That the binding sites were specific for PAF was demonstrated by competition studies. PAF was shown to increase the intracellular calcium concentrations of ASK.0 cells in a dose-dependent manner with an EC50 of 7 nM. We have, therefore, identified a B cell line expressing large numbers of functional PAF receptors.     

Mutagenic radioadaptation in a human lymphoblastoid cell line

We investigated the mutagenic radioadaptive response of human lymphoblastoid TK6 cells by pretreating them with a low dose (5 cGy) of X-rays followed by a high (2 Gy) dose 6h later. Pretreatment reduced the 2-Gy-induced mutation frequency (MF) of the thymidine kinase (TK) gene (18.3 x 10(-6)) to 62% of the original level (11.4 x 10(-6)). A loss of heterozygosity (LOH) detection analysis applied to the isolated TK(-) mutants revealed the mutational events as non-LOH (resulting mostly from a point mutation in the TK gene), hemizygous LOH (resulting from a chromosomal deletion), or homozygous LOH (resulting from homologous recombination (HR) between chromosomes). For non-LOH events, pretreatment decreased the frequency to 27% of the original level (from 7.1 x 10(-6) to 1.9 x 10(-6)). cDNAs prepared from the non-LOH mutants revealed that the decrease was due mainly to the repression of base substitutions. The frequency of hemizygous LOH events, however, was not significantly altered by pretreatment. Mapping analysis of chromosome 17 demonstrated that the distribution and the extent of hemizygous LOH events were also not significantly influenced by pretreatment. For homozygous LOH events, pretreatment reduced the frequency to 61% of the original level (from 5.1 x 10(-6) to 3.1 x 10(-6)), reflecting an enhancement in HR repair of DNA double-strand breaks. Our findings suggest that the radioadaptive response in TK6 cells follows mainly from mutations at the base-sequence level, not the chromosome level.     

Modulation of a human lymphoblastoid B cell line by cyclic AMP. Ig secretion and phosphatidylcholine metabolism

A transformed human B cell line, LA350, was found to be sensitive to cAMP-elevating agents by responding with rapid (0 to 2 h) severalfold elevations of intracellular cAMP to treatment with cholera toxin, isobutylmethylxanthine (IBMX), forskolin, and dibutyryl cAMP (all p less than 0.001). These cAMP-elevating agents also produced significant inhibitions of subsequent (48 to 72 h) Ig secretion by the same B cells as measured by a reverse hemolytic plaque assay and an enzyme-linked immunoadsorbent assay for IgM (both p less than 0.001). PMA- and IBMX-treated cells were particularly responsive to the effects of cholera toxin, showing a doubling of cAMP content and profound decrease in Ig production (p less than 0.001). Because our previous studies had correlated activation of the metabolic turnover of the phosphatidylcholine (PC) fraction of membrane phospholipids with enhanced Ig secretion, we examined the sensitivity of PC metabolism to cAMP in control and PMA-stimulated cells. Formation of PC was found to be inhibited by forskolin and IBMX (both p less than 0.002) but breakdown of PC was stimulated (p less than 0.001). These findings imply that as the enzymatic products of PC, choline phosphate and diacylglycerol, are depleted due to the combined effects of cAMP upon synthesis and turnover of PC, there is a decrease in Ig secretion. Since diacylglycerol activates protein kinase C, it appears reasonable that Ig secretion is at least partially regulated by cAMP-responsive alterations in PC metabolism produced by protein kinase C-induced phosphorylation. We conclude that the early cAMP-sensitive changes in PC metabolism in this activated B cell line may signal for subsequent alterations in Ig secretion.     

Unusual N-glycosylation of a recombinant human erythropoietin expressed in a human lymphoblastoid cell line does not alter its biological properties

Erythropoietin (Epo) is a 166 amino acids protein containing three N-glycosylation sites (Asn-24, Asn-38, and Asn-83) and 1 O- glycosylation site (Ser-126) and involved in the regulation of the level of red blood cells. Today, only one recombinant human Epo (rHuEpo), produced in CHO cell line, is extensively used in therapy to cure severe anemia. The structure of the glycan chains of this rHuEpo slightly differ of those of the urinary human Epo (uHuEpo), considered as the natural Epo molecule. In an attempt to produce a rHuEpo as close as possible to the uHuEpo, Epo gene was expressed in a human lymphoblastoid cell line, named RPMI 1788. In order to fully characterize the Epo-RPMI, structural characterizations of the protein skeleton as well as glycan chains were undergone. As expected, the amino acid sequence of the Epo-RPMI conformed to that of uHuEpo. Surprisingly, the structure of some N-glycan chains, as mainly determined by ESI-MS, revealed some unusual characteristics. Thus, 80% of N-glycans possess a bisecting GlcNAc residue, 25% bear a second fucose residue which is present, in a large part, in a sialyl Le(x)motif, and 13% contain more than three LacNAc repeats (up to five per molecule). Despite these unusual structural characteristics, the data concerning the in vitro and in vivo biological activities were not impaired when compared to Epo-CHO and uHuEpo.     

Secreted and cellular proteochondroitin sulfates of a human B lymphoblastoid cell line contain different protein cores.

Proteoglycans of the human B lymphoblastoid cell line LICR-LON-HMy2 were metabolically labeled with [35S]sulfate. High-density fractions of 35S-labeled material separated by CsCl gradient ultracentrifugation were further purified by anion exchange chromatography and gel filtration. Two proteoglycans, isolated from cell lysates and culture supernatants, were characterized by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in combination with enzymatic degradation. Treatment with chondroitinase AC completely degraded the glycosaminoglycan moiety of the proteoglycans. Three to 4 chondroitin sulfate chains (average molecular mass = 26 kDa) were estimated for each of the two proteoglycans. Differences between the proteochondroitin sulfates (CSPG) were observed in the content of N-linked oligosaccharides. After chondroitinase AC treatment the resulting band in SDS-PAGE of the secreted CSPG was sensitive to treatment with endoglycosidase F (Endo F) which further reduced the molecular mass from 30 to 21.5 kDa, whereas the band of the cellular CSPG after chondroitinase AC treatment (molecular mass = 30 kDa) remained resistant to Endo F treatment. The composition of amino acids was different in the protein cores, suggesting differences in the primary structure. Both CSPG contained a high percentage of glycine and serine. For both CSPG a molecular mass of approximately 135 kDa was deduced from the hydrodynamic sizes of the glycosaminoglycan chains obtained after alkaline/borohydride treatment and the migration of the protein/oligosaccharide complexes in SDS-PAGE. 75% of all [35S]sulfate-labeled molecules were found in the culture supernatant and 25% in the cellular fraction. 35S-Labeled material in the culture supernatant consisted exclusively of intact CSPG, whereas 35S-Labeled molecules in the cellular preparation consisted largely of free chondroitin sulfate chains. Only 8.3% of the cellular material, isolated from the microsomal fraction, was intact CSPG. In pulse-chase experiments maximal secretion of CSPG was found after 4 h, comprising approximately 40% of totally synthesized CSPG. From these experiments we tentatively conclude that a small proportion of CSPG synthesized by LICR-LON-HMy2 cells is membrane-associated, a larger portion is secreted, and another portion is intracellularly degraded.     

Xenobiotic metabolism and mutation in a human lymphoblastoid cell line

Aryl hydrocarbon hydroxylase-1 (AHH-1) cells are a human lymphoblastoid cell line competent in some aspects of xenobiotic metabolism. This cell line contains stable mixed function oxidase activity which is inducible by polycyclic aromatic hydrocarbons (PAHs) but not by phenobarbital or Arochlor 1254. Two substrates for the cellular mixed function oxidase activity, benzo[a]pyrene (B[a]P) and 7-ethoxyresorufin, have been examined. The basal and induced activities have different kinetic parameters toward these two substrates. In contrast, basal and induced activities had similar sensitivities to two cytochrome P-450 suicide substrates. B[a]P metabolism and mutagenicity were studied in this cell line. AHH-1 cells were found to produce predominantly B[a]P phenols and quinones. The major phenol metabolite cochromatographed with authentic 9-hydroxy B[a]P. AHH-1 cells were capable of forming glucuronic acid conjugates of B[a]P phenols; the major product after hydrolysis cochromatographed with 3-hydroxy B[a]P standard. AHH-1 cells did not contain detectable epoxide hydrolase activity using B[a]P-4,5-oxide as substrate. This observation is consistent with the absence of trans-dihydrodiol B[a]P metabolites in the metabolic profile. B[a]P-induced mutagenicity at the hypoxanthine guanine phosphoribosyl transferase (hgprt) locus in AHH-1 cells was found to be linearly related to phenol production during treatment and inhibited by alpha-naphthoflavone (ANF).     

Telomeric associations involving Yq12 in a lymphoblastoid cell line

A number of apparent telomeric associations/fusions (TAs) involving Yq12 were observed in a lymphoblastoid cell line (GM6892A) established from a fragile-X-negative, phenotypically normal male with the fragile X gene mutation. The TAs were seen when these cells were grown for an extended period of time in medium 199, which is deficient in thymidine and folic acid. Because the low thymidine and folic acid condition of medium 199 is known to induce chromosome and chromatid gaps and breaks at folate-sensitive fragile sites, other fragile site-induction regimes were examined to determine if the TAs seen in GM6892A were due to a fragile site in the Yq12 band. No TAs were seen with any of the other regimes that were tried.     

Identification and characterization of a Ca2+-calmodulin-sensitive cyclic nucleotide phosphodiesterase in a human lymphoblastoid cell line.

This study examines the pattern and regulatory properties of cyclic nucleotide phosphodiesterases in a human lymphoblastoid B-cell line (RPMI 8392) established from a patient with acute lymphocytic leukaemia. In this cell line, phosphodiesterase activity measured at 0.25 microM-cyclic AMP is approx. 7-fold greater than that in isolated human peripheral-blood lymphocytes, and 16% of the phosphodiesterase activity in RPMI 8392 cells is associated with particulate fractions. Phosphodiesterase activity in crude fractions of this cell line is reproducibly stimulated by about 60-80% by Ca2+-calmodulin. In the presence of 20 nM-calmodulin, half-maximal stimulation occurs at 0.7 microM-Ca2+. The cytosolic phosphodiesterase activity of RPMI 8392 cells is separated into two forms by DEAE-Sephacel chromatography. The first form is eluted at approx. 0.2 M-sodium acetate, catalyses the hydrolysis of both cyclic AMP and cyclic GMP, and is stimulated 3-fold by Ca2+-calmodulin. This form exhibits non-linear kinetics for cyclic AMP in the absence of calmodulin, with extrapolated Km values of 0.8 and 4 microM, and non-linear kinetics in the presence of calmodulin, with extrapolated Km values of 0.5 and 1 microM. The Vmax. values are increased approx. 3-fold by calmodulin. The second form is eluted at approx. 0.6 M-sodium acetate, is specific for cyclic AMP, and insensitive to stimulation by Ca2+-calmodulin. The Ca2+-calmodulin-sensitive phosphodiesterase from the DEAE-Sephacel column can be adsorbed to a calmodulin-Sepharose affinity column and eluted with EGTA. This enzymic activity can also be immunoprecipitated by a monoclonal antibody directed against a calmodulin-bovine heart phosphodiesterase complex. This study documents the existence of Ca2+-calmodulin-sensitive phosphodiesterase in a cultured lymphoblastoid cell line derived from a leukaemic patient.     

Modulation of human lymphoblastoid B cell line by phorbol ester and sphingosine. A 31P-NMR study.

Changes in phospholipid and energy metabolism in Epstein-Barr Virus transformed B lymphocytes (EBV-B), induced by phorbol 12,13-dibutyrate (PD) and sphingosine (an inhibitor of protein kinase C), have been evaluated by 31P-NMR spectroscopy. The effects of PD and sphingosine on [3H]thymidine incorporation have also been studied. An increase in phosphorylcholine (PCho) levels has been observed in sphingosine and sphingosine + PD treated cells after 30 min of incubation, whereas no change was observed in lymphocytes incubated with PD during the same period. Extracellular choline levels increased in sphingosine treated cells but decreased in PD treated cells. Hence, a sphingosine-dependent hydrolysis of choline-linked phospholipids is suggested. A time-dependent reduction of PCho observed after 120 min PD incubation is consistent with an increase of the synthesis of choline-linked phospholipids.