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A new strategy to clone a gene involved in glycosylphosphatidylinositol anchor biosynthesis
19 EFFECTS OF TOTAL EXCHANGE TRANSFUSION USING ERYTHROCYTES WITH FUNCTIONAL CR1 ON HIV PRODUCTIONAND INFLAIffdATION PHASE IN HIV DISEASE.
CHARACTERIZATION OF A C1Q HUMAN GLIOMA CELL LINES.
Y. Inada, S. Moriya, X.H. Zhang, H. Matsumoto, K. Miyoraoto, and M. Lange. S t . L u k e ' s - R o o s e v e l t Hospital Center, Columbia University, New York, NY USA
A n d r e e v 2, T.D. Smirnova 2, a n d M. Fontaine 1.
The presence of HIV-onti-HIV irrmune complexes on e r y t h r o c y t e surfaces (E-HIV-IC) are i n d i c a t i v e of HIV antigen released into the c i r c u l a t i o n and of formation of HIV-IC and of s a t u r a t i o n of E-CRI IC c l e a r i n g s y s t e m W a i c h may r e s u l t in r e a c t i v a t i o n HIV. The removal of HIV-IC by E-CR1 appears to be c r i t i c a l in preventing HIV r e la te d pathogenesis. We evaluated the p o t e n t i a l b e n e f i t of total exchange transfusion (Ex-Tx) of E with high CRI function on HIV-IC formation and inflaraatiun phase of HIV related pathogenesis. Antibody (Ab) t i t e r s to HIV isolated from E-HIV ICwere greater than 1:32 in 81% of 21 AIDS, in 19% of 21 ARC and in 12~ of 34 asyrnptomatie (AS) seroHIV(+) individuals. The mean levels of CRP for AIDS, ARC, AS and control were averages of 48, 263, 6,089, 5,496 and 675 nglml respectively. These data suggest that worsening HIV disease is associated with progressive saturation of E-CRI with HIV-IC and chronical l y elevated production of CRP. In 4 patients with ARCIAIDS, we performed 3 total Ex-Tx followed by regular Tx. E-HIV-IC t i t e r s were reduced to less than 1:32 following 3 Ex-Tx in all patients. In 1 of the 4, 25 of 27 E specimens collected during 78 weeks f o l l o ~ u p had less than I: 32 Ab t i t e r s . These observations were accompanied by a decrease of CRP levels. Based on this preliminary study, we suggest that Ex-Tx performed every 2-4 months may possibly interrupt the IC mediated chronic inflenrnation phase associated with advanced HIV infection.
RECEPTOR ON
A.M, Ischenko 1,2, P. Gasclue 1, M.T. Schouff 1, S.V
1) Inserm U78, BP 73, Bols Guillaume cedex, France. 2) Research Institute of Highly Pure Biopreporatlons, St Petersbourg, Russia,
Human
glioma cells have been
shown to
secrete
complement components of the classical and alternative pathways. The presence of a receptor for Clq (ClqR) on glioma cells was investigated by binding of 1251labelled Clq. Binding of C l q on these cells was ionic strength dependent, that is a characteristic of Clq-ClqR interaction,
More effective
binding was achieved at low ionic strength (ie 60-70mM NaCl). The Binding of 1251-Clq was time-dependent, reversible and saturable, fulfilling criteria of receptor-ligand interaction, Scatchard analysis indicated that, for the two glioma cell lines studied (U118MG and T193), the kd was 2.10-8M with a number of binding sites ranging from 105 to 2.105. ClqR was purified by affinity chromatography on C l q sepharose from triton Xl00 cell lysates, previously absorbed onto IgG- and BSA-sepharose, On SDS-PAGE, glial ClqR migrated as a doublet of 17-20 kDa.
A NEW S T R A T E G Y TO C L O N E A GENE I N V O L V E D IN G L Y C O S Y L P H O S P H A T I D Y L I N O S I T O L A N C H O R BIOSYNTHESIS
CONTRIBUTION OF TIlE SERINE-TIIREONINEENRICIIED DOMAINS TO THE COMPLEMENT R E G U L A T O R Y F U N C T I O N OF MEMBRANE COFACTOR PROTEIN (MCP)
N. Inoue, T. Kinoshita, J. Takeda.
Kazunori Iwata, Hiroyoshi Ariga, Tsukasa Seya*, and Shigeharu Nagasawa Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo 060, Center for Adult Diseases, Osaka 537", Japan
Department of Immunoregulation, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan It is known that two of the human complement regulators, DAF and CD59, are anchored to the membrane by glycosylphosphatidylinositol (GPI). A number of mutant cells that are abnormal in GPI anchor biosynthesis have been established and classified into several complementation classes by somatic cell fusion analysis. Little is known, however, about genes encoding proteins involved in biosynthesis of the GPI anchor. In this study, we report a new method of expression cloning that is applicable to many mutant rodent cell lines. We transfect cDNA library containing polyoma virus origin of replication in a vector into mutant rodent cells together with a plasmid encoding polyoma virus large T antigen to allow replication of the former plasmids. We utilize this method to clone a human cDNA termed PIG-F (for Phosphatidyl-Inositol-Glycanclass E) using a Thy-l-negative thymoma cell line of complementation class F. PIG-F takes a part in the step of transfer of ethanolamine phosphate to the GPI intermediate containing three residues of mannose.This expression cloning strategy is applicable to clone other genes involved in GPI anchor biosynthesis by using other GPI-anchor deficient mutant cells.
The extracellular portion of MCP is composed of four SCR domains followed by a serine (S)-threonine (T)-enriched domain likely to be a site of heavy O-linked glycosylation. As verified by genomic analysis, the ST region consisted of three similarly sized exons, termed STA, STB, and STc. Cell lines and peripheral blood cells were demonstrated to regularly express variable quantities of four primary isoforms; STABc, STBC, STB, and STC. However, it is not yet known concerning the relative potencies of these MCP phenotypes. Three types of the MCP eDNA (STABC, STBC and STc) were prepared from mRNA of HL-60 by RT-PCR and expressed by stable transfection in CHO cells. In addition, a MCP deletion mutant lacking STABC (AST) was created by sitedirected mutagenesis and also stably expressed in CHO. The 51Cr-labeled CHO cells expressing identical amounts of the MCP phenotypes were sensitized with rabbit anti-CHO Ab and allowed to react with human serum as a source of complement. The cytolysis of Ab-sensitized CHO cells by the classical pathway was protected by the MCP phenotypes in the following order; sTC>AST>STBC=STABC;that is, the MCP phenotype of STc is the most potent for protection of CHO from the classical pathway. In contrast to DAF, total deletion of the ST domains didn't abrogate MCP function; rather AST-MCP is more potent than STABC-MCP.Thus, it appears that the O-glycosylated ST domains are not essential for the regulatory function of MCP. The protective effects of the MCP mutants on the alternative pathway will also be reported.
CHARACTERIZATION OF A C1Q HUMAN GLIOMA CELL LINES.
Y. Inada, S. Moriya, X.H. Zhang, H. Matsumoto, K. Miyoraoto, and M. Lange. S t . L u k e ' s - R o o s e v e l t Hospital Center, Columbia University, New York, NY USA
A n d r e e v 2, T.D. Smirnova 2, a n d M. Fontaine 1.
The presence of HIV-onti-HIV irrmune complexes on e r y t h r o c y t e surfaces (E-HIV-IC) are i n d i c a t i v e of HIV antigen released into the c i r c u l a t i o n and of formation of HIV-IC and of s a t u r a t i o n of E-CRI IC c l e a r i n g s y s t e m W a i c h may r e s u l t in r e a c t i v a t i o n HIV. The removal of HIV-IC by E-CR1 appears to be c r i t i c a l in preventing HIV r e la te d pathogenesis. We evaluated the p o t e n t i a l b e n e f i t of total exchange transfusion (Ex-Tx) of E with high CRI function on HIV-IC formation and inflaraatiun phase of HIV related pathogenesis. Antibody (Ab) t i t e r s to HIV isolated from E-HIV ICwere greater than 1:32 in 81% of 21 AIDS, in 19% of 21 ARC and in 12~ of 34 asyrnptomatie (AS) seroHIV(+) individuals. The mean levels of CRP for AIDS, ARC, AS and control were averages of 48, 263, 6,089, 5,496 and 675 nglml respectively. These data suggest that worsening HIV disease is associated with progressive saturation of E-CRI with HIV-IC and chronical l y elevated production of CRP. In 4 patients with ARCIAIDS, we performed 3 total Ex-Tx followed by regular Tx. E-HIV-IC t i t e r s were reduced to less than 1:32 following 3 Ex-Tx in all patients. In 1 of the 4, 25 of 27 E specimens collected during 78 weeks f o l l o ~ u p had less than I: 32 Ab t i t e r s . These observations were accompanied by a decrease of CRP levels. Based on this preliminary study, we suggest that Ex-Tx performed every 2-4 months may possibly interrupt the IC mediated chronic inflenrnation phase associated with advanced HIV infection.
RECEPTOR ON
A.M, Ischenko 1,2, P. Gasclue 1, M.T. Schouff 1, S.V
1) Inserm U78, BP 73, Bols Guillaume cedex, France. 2) Research Institute of Highly Pure Biopreporatlons, St Petersbourg, Russia,
Human
glioma cells have been
shown to
secrete
complement components of the classical and alternative pathways. The presence of a receptor for Clq (ClqR) on glioma cells was investigated by binding of 1251labelled Clq. Binding of C l q on these cells was ionic strength dependent, that is a characteristic of Clq-ClqR interaction,
More effective
binding was achieved at low ionic strength (ie 60-70mM NaCl). The Binding of 1251-Clq was time-dependent, reversible and saturable, fulfilling criteria of receptor-ligand interaction, Scatchard analysis indicated that, for the two glioma cell lines studied (U118MG and T193), the kd was 2.10-8M with a number of binding sites ranging from 105 to 2.105. ClqR was purified by affinity chromatography on C l q sepharose from triton Xl00 cell lysates, previously absorbed onto IgG- and BSA-sepharose, On SDS-PAGE, glial ClqR migrated as a doublet of 17-20 kDa.
A NEW S T R A T E G Y TO C L O N E A GENE I N V O L V E D IN G L Y C O S Y L P H O S P H A T I D Y L I N O S I T O L A N C H O R BIOSYNTHESIS
CONTRIBUTION OF TIlE SERINE-TIIREONINEENRICIIED DOMAINS TO THE COMPLEMENT R E G U L A T O R Y F U N C T I O N OF MEMBRANE COFACTOR PROTEIN (MCP)
N. Inoue, T. Kinoshita, J. Takeda.
Kazunori Iwata, Hiroyoshi Ariga, Tsukasa Seya*, and Shigeharu Nagasawa Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo 060, Center for Adult Diseases, Osaka 537", Japan
Department of Immunoregulation, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan It is known that two of the human complement regulators, DAF and CD59, are anchored to the membrane by glycosylphosphatidylinositol (GPI). A number of mutant cells that are abnormal in GPI anchor biosynthesis have been established and classified into several complementation classes by somatic cell fusion analysis. Little is known, however, about genes encoding proteins involved in biosynthesis of the GPI anchor. In this study, we report a new method of expression cloning that is applicable to many mutant rodent cell lines. We transfect cDNA library containing polyoma virus origin of replication in a vector into mutant rodent cells together with a plasmid encoding polyoma virus large T antigen to allow replication of the former plasmids. We utilize this method to clone a human cDNA termed PIG-F (for Phosphatidyl-Inositol-Glycanclass E) using a Thy-l-negative thymoma cell line of complementation class F. PIG-F takes a part in the step of transfer of ethanolamine phosphate to the GPI intermediate containing three residues of mannose.This expression cloning strategy is applicable to clone other genes involved in GPI anchor biosynthesis by using other GPI-anchor deficient mutant cells.
The extracellular portion of MCP is composed of four SCR domains followed by a serine (S)-threonine (T)-enriched domain likely to be a site of heavy O-linked glycosylation. As verified by genomic analysis, the ST region consisted of three similarly sized exons, termed STA, STB, and STc. Cell lines and peripheral blood cells were demonstrated to regularly express variable quantities of four primary isoforms; STABc, STBC, STB, and STC. However, it is not yet known concerning the relative potencies of these MCP phenotypes. Three types of the MCP eDNA (STABC, STBC and STc) were prepared from mRNA of HL-60 by RT-PCR and expressed by stable transfection in CHO cells. In addition, a MCP deletion mutant lacking STABC (AST) was created by sitedirected mutagenesis and also stably expressed in CHO. The 51Cr-labeled CHO cells expressing identical amounts of the MCP phenotypes were sensitized with rabbit anti-CHO Ab and allowed to react with human serum as a source of complement. The cytolysis of Ab-sensitized CHO cells by the classical pathway was protected by the MCP phenotypes in the following order; sTC>AST>STBC=STABC;that is, the MCP phenotype of STc is the most potent for protection of CHO from the classical pathway. In contrast to DAF, total deletion of the ST domains didn't abrogate MCP function; rather AST-MCP is more potent than STABC-MCP.Thus, it appears that the O-glycosylated ST domains are not essential for the regulatory function of MCP. The protective effects of the MCP mutants on the alternative pathway will also be reported.