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Localization of the human CD59 gene on chromosome region 11p13
14 STRUCTURE AND FUNCTION OF SOLUBLE FORMS OF MCP (CD46) T. Hara, S. Kuriyama*, S. Miyagawa, M. Hatanaka, M. Matsumoto, Y. Nagase°, and T. Seya. Center for Adult Diseases Osaka, Osaka, and *Bioscience Laboratory, Mochida Co., Tokyo Japan. Blood plasma (bp) and seminal plasma (sp) contain ~50 ng/ml and 500 ng/ml of soluble MCP, respectively. These soluble forms of MCP were purified by immune-affinity method, bpMCP was composed of three bands of Mr 60, 55, and 30 kDa on SDS-PAGE/immunob[otting, whereas spMCP was a single band of Mr 65 kDa. The 65 kDaspMCP, and the 60 and 55 kDa bpMCP were O-glycosylated, the deglycosylated forms being 38-42 kDa. In 0.02°/o NP40 fluid phase, the soluble forms served as a cofactor for factor I-mediated cleavage of C3b/C4b as efficiently as solubilized membrane forms of MCP. The soluble forms were also active even without NP40. The membrane MCP expressed on CHO cells protected host cells from human C3 deposition and Cmediated cytolysis by antibody activation of the alternative pathway. Likewise, exogenously added spMCP protected untransfected CHO cells; however, its potency was less than that of the endogenous membrane form. In the same CHO cell system, the soluble forms barely blocked classical pathwaymediated C3 depesition/cytolysis, while the membrane form moderately blocked. To confirm and develop these findings, recombinant soluble forms were prepared from the supernatant of CHO cell transfactants. The soluble form MCP may be useful for suppression of C activation in vivo.
MECHANISMS INVOLVED IN LOSS OF PIANCHORED COMPLEMENT REGULATORY PROTEINS (DAF AND CD59) IN HUMAN LEUKEMIA CELL LINES. M. Hatanaka1,2), T. Hara2), M. Nonaka3), J. Takeda4), T. Seya2), and A. Shimizu 1) 1)Osaka Medical College, Osaka, 2)Center for Adult Diseases Osaka, Osaka, 3)Nagoya City University, Nagoya, 4)Osaka university, Osaka, Japan. DAF and CD59 are PI-anchored proteins that protect host cells from autologous complement, and become deficient in pathological conditions such as paroxysmal nocturnal hemoglobinuria (PNH) or some malignancies. The mechanism of DAF/CD59 deficiency in leukemia remains unknown, although pathogenesis of PNH is shown as a defect of a protein participating in PI-anchor formation. We found by flow cytometry and western blotting that several human leukemia cell lines lack DAF and/or CD59:U937 (DAF+/CD59-), CEM (DAF-/CD59+), TALL (DAF-/CD59-), and Ramos-(DAF/CD59-). No gene abnormality was as-yet detected by southern blotting analysis. Unlike PNH cells, DAF/ CD59 mRNA was negative by northern blotting in these cells, except for Ramo8-that expressed the normal sizes of DAF/CD59 mRNA. In most cases this was confirmed by RT-PCR. Thus, the mechanisms of the defect of DAF/CD59 in leukemia are not uniform and in most cases are different from that proposed in PNH. Further studies including analyses of the promoter region and testing the ability to form PI-anchor are in progress to clarify the DAF/CD59 abnormality in leukemia.
PROTEIN TYROSINE PHOSPHORYLATION INDUCED BY M O N O C L O N A L ANTIBODIES AGAINST MCP (CD46) K. Hazeki, O. Hazeki, T. Seya', and M. Ui Department of Pharmaceutical Sciences, University of Tokyo, Tokyo, and *Center for Adult Diseases Osaka, Osaka, Japan MCP is a widely-distributed factor I-cofactor. Its major role is to inactivate C3b/C4b deposited on the same cell membranes and to protect host cells from homologous C attack. Protein tyrosine phosphorylation (PTP) was found to be induced in human myeleid, T, and B cell lines by treatment with anti-MCP and second cross-linking Ab. PTP was induced rapidly, within 30 sec. after the second Ab treatment. M177 and M75, being capable of blocking cofactor activity of MCP, induced PTP, while M160, having minimal blocking activity, did not. Phospholyrated bands of multiple 55-70 kDa, an 85 kDa, and a 110 kDa proteins were specifically observed regardless of cell species by SDSPAGE/immnoblotting using anti-phosphotyrosine. Similar results were obtained with the CHO cells transfected with MCP cDNA and human cell lines lacking DAF and CD59. What happens by interaction of MCP with natural ligand C3b/C4b is being examined. Although identification of these phospholyrated proteins and of final responses are in progress, we speculate that MCP is engaged in signal transduction in association with C3b/C4b regulatory function.
LOCALIZATION OF CHROMOSOME REGION
THE HUMAN Iip13
CD59
GENE
ON
B. H e c k l - O s t r e i c h e r I, S. W u d u n n 3, M. D r e c h s l e r 2, H. Scherthan 2 , B. Royer-Pokora 2 , iInstitute of Immunology, 2Institute of Human Genetics, University of Heidelberg, D-6900 Heidelberg; 3 I n s t i t u t e of P a t h o l o g y , D - 6 8 0 0 M a n n h e i m C D 5 9 is a 1 8 - 2 5 K D a h u m a n g l y c o p r o t e i n t h a t is e x p r e s s e d on t h e m e m b r a n e s of b l o o d c e l l s as w e l l as o n v a s c u l a r e n d o t h e l i a and e p i t h e l i a of v a r i o u s t i s s u e s a n d is k n o w n to i n h i b i t c e l l - l y s i s b y h o m o logous complement. In a r e c e n t s t u d y u s i n g s o m a t i c cell hybrids, the CD59 gene was assigned to t h e short a r m of c h r o m o s o m e ii. W e investigated its regional localization on chromosome ii in more detail. By chromosomal in situ hybridization we c o u l d s h o w t h a t t h e g e n e is l o c a t e d on t h e s h o r t a r m of c h r o m o s o m e ii r e g i o n l l p 1 2 - p 1 3 . To f u r t h e r e v a l u a t e its l o c a l i z a t i o n on llp13 t h e l l p l 3 - s p e c i fic p r o b e s H D - 2 and H D - 1 8 w e r e u s e d in p u l s e d f i e l d gel a n a l y s i s . H D - 2 is p o s i t i o n e d d i s t a l to t h e g e n e for Catalase (CAT), the centromeric border of llp13, and flanks the translocation breakpoint of a c u t e T c e l l l e u k e m i a (T-All) on c h r o m o s o m e ii. T h e p r o b e H D - 1 8 is l o c a t e d 200 kb d i s t a l to H D - 2 a n d a b o u t 1 5 0 0 kb p r o x i m a l to t h e l o c u s of W i l m s ' t u m o r T h e p r o b e s H D - 2 and H D - 1 8 and t h e C D 5 9 c D N A p r o b e d e t e c t t h e s a m e L A Z cell D N A f r a g m e n t s o b t a i n e d by d i g e s t i o n w i t h t h e r e s t r i c t i o n e n z y m e s Not I, M l u I a n d N r u I. We h a v e f u r t h e r e x a c t l y m a p p e d t h e 5' end of t h e gene because we could show that an oligonucleotide corresponding to the first exon hybridizes to the HD-18 probe. Therefore we localize the CD59 gene on llp13 distal to the translocation breakpoint of T - A l l and p r o x i m a l to t h e W i l m ' s t u m o r gene.
MECHANISMS INVOLVED IN LOSS OF PIANCHORED COMPLEMENT REGULATORY PROTEINS (DAF AND CD59) IN HUMAN LEUKEMIA CELL LINES. M. Hatanaka1,2), T. Hara2), M. Nonaka3), J. Takeda4), T. Seya2), and A. Shimizu 1) 1)Osaka Medical College, Osaka, 2)Center for Adult Diseases Osaka, Osaka, 3)Nagoya City University, Nagoya, 4)Osaka university, Osaka, Japan. DAF and CD59 are PI-anchored proteins that protect host cells from autologous complement, and become deficient in pathological conditions such as paroxysmal nocturnal hemoglobinuria (PNH) or some malignancies. The mechanism of DAF/CD59 deficiency in leukemia remains unknown, although pathogenesis of PNH is shown as a defect of a protein participating in PI-anchor formation. We found by flow cytometry and western blotting that several human leukemia cell lines lack DAF and/or CD59:U937 (DAF+/CD59-), CEM (DAF-/CD59+), TALL (DAF-/CD59-), and Ramos-(DAF/CD59-). No gene abnormality was as-yet detected by southern blotting analysis. Unlike PNH cells, DAF/ CD59 mRNA was negative by northern blotting in these cells, except for Ramo8-that expressed the normal sizes of DAF/CD59 mRNA. In most cases this was confirmed by RT-PCR. Thus, the mechanisms of the defect of DAF/CD59 in leukemia are not uniform and in most cases are different from that proposed in PNH. Further studies including analyses of the promoter region and testing the ability to form PI-anchor are in progress to clarify the DAF/CD59 abnormality in leukemia.
PROTEIN TYROSINE PHOSPHORYLATION INDUCED BY M O N O C L O N A L ANTIBODIES AGAINST MCP (CD46) K. Hazeki, O. Hazeki, T. Seya', and M. Ui Department of Pharmaceutical Sciences, University of Tokyo, Tokyo, and *Center for Adult Diseases Osaka, Osaka, Japan MCP is a widely-distributed factor I-cofactor. Its major role is to inactivate C3b/C4b deposited on the same cell membranes and to protect host cells from homologous C attack. Protein tyrosine phosphorylation (PTP) was found to be induced in human myeleid, T, and B cell lines by treatment with anti-MCP and second cross-linking Ab. PTP was induced rapidly, within 30 sec. after the second Ab treatment. M177 and M75, being capable of blocking cofactor activity of MCP, induced PTP, while M160, having minimal blocking activity, did not. Phospholyrated bands of multiple 55-70 kDa, an 85 kDa, and a 110 kDa proteins were specifically observed regardless of cell species by SDSPAGE/immnoblotting using anti-phosphotyrosine. Similar results were obtained with the CHO cells transfected with MCP cDNA and human cell lines lacking DAF and CD59. What happens by interaction of MCP with natural ligand C3b/C4b is being examined. Although identification of these phospholyrated proteins and of final responses are in progress, we speculate that MCP is engaged in signal transduction in association with C3b/C4b regulatory function.
LOCALIZATION OF CHROMOSOME REGION
THE HUMAN Iip13
CD59
GENE
ON
B. H e c k l - O s t r e i c h e r I, S. W u d u n n 3, M. D r e c h s l e r 2, H. Scherthan 2 , B. Royer-Pokora 2 , iInstitute of Immunology, 2Institute of Human Genetics, University of Heidelberg, D-6900 Heidelberg; 3 I n s t i t u t e of P a t h o l o g y , D - 6 8 0 0 M a n n h e i m C D 5 9 is a 1 8 - 2 5 K D a h u m a n g l y c o p r o t e i n t h a t is e x p r e s s e d on t h e m e m b r a n e s of b l o o d c e l l s as w e l l as o n v a s c u l a r e n d o t h e l i a and e p i t h e l i a of v a r i o u s t i s s u e s a n d is k n o w n to i n h i b i t c e l l - l y s i s b y h o m o logous complement. In a r e c e n t s t u d y u s i n g s o m a t i c cell hybrids, the CD59 gene was assigned to t h e short a r m of c h r o m o s o m e ii. W e investigated its regional localization on chromosome ii in more detail. By chromosomal in situ hybridization we c o u l d s h o w t h a t t h e g e n e is l o c a t e d on t h e s h o r t a r m of c h r o m o s o m e ii r e g i o n l l p 1 2 - p 1 3 . To f u r t h e r e v a l u a t e its l o c a l i z a t i o n on llp13 t h e l l p l 3 - s p e c i fic p r o b e s H D - 2 and H D - 1 8 w e r e u s e d in p u l s e d f i e l d gel a n a l y s i s . H D - 2 is p o s i t i o n e d d i s t a l to t h e g e n e for Catalase (CAT), the centromeric border of llp13, and flanks the translocation breakpoint of a c u t e T c e l l l e u k e m i a (T-All) on c h r o m o s o m e ii. T h e p r o b e H D - 1 8 is l o c a t e d 200 kb d i s t a l to H D - 2 a n d a b o u t 1 5 0 0 kb p r o x i m a l to t h e l o c u s of W i l m s ' t u m o r T h e p r o b e s H D - 2 and H D - 1 8 and t h e C D 5 9 c D N A p r o b e d e t e c t t h e s a m e L A Z cell D N A f r a g m e n t s o b t a i n e d by d i g e s t i o n w i t h t h e r e s t r i c t i o n e n z y m e s Not I, M l u I a n d N r u I. We h a v e f u r t h e r e x a c t l y m a p p e d t h e 5' end of t h e gene because we could show that an oligonucleotide corresponding to the first exon hybridizes to the HD-18 probe. Therefore we localize the CD59 gene on llp13 distal to the translocation breakpoint of T - A l l and p r o x i m a l to t h e W i l m ' s t u m o r gene.