Affected paroxysmal nocturnal hemoglobinuria T lymphocytes harbor a common defect in assembly of N-acetyl-D-glucosamine inositol phospholipid corresponding to that in class A Thy-1- murine lymphoma mutants.

C Armstrong,J Schubert,E Ueda,J.J Knez,D Gelperin,S Hirose,R Silber,S Hollan,R.E Schmidt,M.E Medof

doi:10.1016/s0021-9258(19)74046-0

C Armstrong, J Schubert + Show 8 more

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https://doi.org/10.1016/s0021-9258(19)74046-0

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Abstract

Deficient expression of glycoinositol phospholipid (GPI) anchored proteins in affected paroxysmal nocturnal hemoglobinuria (PNH) cells has been traced to a defect in GPI anchor assembly. In a previous study (Schubert, J., Schmidt, R. E., and Medof, M. E. (1993) J. Biol. Chem., in press) we characterized the biosynthesis of putative Man-containing GPI anchor precursors in normal peripheral blood lymphocytes and investigated assembly of these intracellular GPI intermediates in CD48- affected and CD48+ unaffected T and natural killer cell lines of PNH patients. We found that affected T cells from five patients exhibited a uniform defect in which dolichol-phosphoryl-Man was synthesized but no GPI mannolipids were expressed. In this study, membranes of patients' affected T cells were labeled with UDP-[3H]GlcNAc to evaluate earlier steps in GPI synthesis, and intact cells were fused to Thy-1- murine lymphoma mutants harboring different defects in early GPI assembly to test for the presence of corresponding or complementary lesions. In all cases, affected cell membranes failed to assemble GlcNAc-inositol phospholipid, the initial precursor of GPI anchor structures, and the intact cells failed to complement class A mutants while complementing other classes. Affected polymorphonuclear leukocytes from three additional patients of different origin were then labeled with [3H]Man and the labeling patterns found to correspond to those obtained with the T lymphocytes. Taken together the data indicate that the genetic lesion in PNH cells resides in a DNA element which: 1) encodes a product required for the synthesis of GlcNAc-inositol phospholipid, 2) corresponds to that altered in class A Thy-1- murine lymphoma mutants, and 3) is commonly affected in different patients.

Highlights

From the $Institute of Pathology, Case Western Reserve University, Cleveland, Ohio 44106, the SAbteilung Irnmunologie und Transfusionsmedizin, Medizinische Hochschule,0-3000 Hannover 61, Germany, TFukuoka UniversitySchool of Medicine, Fukuoka 814-01, Japan, IINew York University Medical Center, New York, New York 10003, and the **National Institute of Haematology and Blood Transfusion, H-I 113 Budapest, Hungary
Chem., in press) we characterized the biosyn- includedecay-accelerating factor(DAF)and CD59, which thesis of putative Man-containing GPI anchor precur- render the affected cells sensitive to autologous complementsors in normal peripheral blood lymphocytes and in- mediated injury, aswell as alkaline phosphatase andFc,RIII
Were labeled with UDP-[3H]GlcNActo evaluate earlier In aprevious study (7), affected GPI-anchor defective steps in GPI synthesis, and intact cells were fused to CD48- and unaffected GPI-anchor sufficient CD48+ T cell

Summary

Deficientexpression of glycoinositolphospholipid

In paroxysmal nocturnal hemoglobinuria (PNH),’ somatic (GPI) anchored proteins in affected paroxysmal noc- mutation of one or moremarrow progenitors gives rise to turnal hemoglobinuria (PNH) cells has been traced to subpopulations of circulating blood elements which fail to a defect in GPI anchor assembly. N"-p-tosyl-L-lysine chloromethyl ketone (TLCK) and0.1 pg/ml leupeptin/lO' cells) for 5min on ice. An equal volume of 100mM HEPES mannolipid GPI production in affected T cells of the other patients and exclude possible differences in assembly of the two intermediates between patients' T cell lines and control PHA blasts, hypotonic lysatesof CD48- cells from a second (pH 7.4) buffer After thawing and ultracentrifugationat 189,000 X g for 20 min at 4 'C, pelleted membranes resuspended in 200 pl of 50 mM HEPES (pH 7.4) buffer supplemented with 25 mM KCI, 5 mM MgC12,0.1 M TLCK, 1pg/ml leupeptin, 5 mM MnClZ,[1] mM ATP, 0.5 mM dithiothreitol,and 0.2 pg/pl tunicamycin were various samples anfdrom PHA blastswere labeled with UDP[3H]GlcNAc,this timefor a longer periodof 15min, and GPI products were isolated and analyzed. 1640 supplemented with 10%dialyzed fetal bovine serum, 10 pg/ml Complementation Analysesbetween Affected PNH Cells and

EBV B cells

CLASS A

DISCUSSION