Abstract
Glycoinositol phospholipid (GPI) anchor structures derive from sequentially glycosylated inositol phospholipid precursors assembled in the endoplasmic reticulum. To characterize GPI biosynthesis in nontransformed human lymphocytes and to define the GPI synthetic defect underlying deficient expression of GPI-anchored proteins by paroxysmal nocturnal hemoglobinuria (PNH) cells, putative intracellular GPI intermediates were analyzed following [3H]Man labeling of normal and affected lymphocytes. In unstimulated normal peripheral blood lymphocytes, [3H]Man incorporation into GPIs was minimally detectable but after phytohemagglutinin (PHA), allogeneic cell, or anti-CD3 stimulation, assembly of [3H]Man-labeled GPIs markedly increased. Expression of GPIs by prestimulated quiescent PHA blasts could be efficiently induced by phorbol 12-myristate 13-acetate (PMA) and increased by the Ca2+ ionophore A23187 independently of new protein synthesis. Utilizing allogeneically stimulated cells in conjunction with PMA induction, products deriving from [3H]Man labeling of affected CD48- T and natural killer lymphocyte cell lines from five PNH patients were compared to those deriving from unaffected CD48+ cell lines from the same patients or controls. In contrast to unaffected paired control cells, affected cells of all of the patients exhibited a common abnormality in which they assembled dolichol-phosphoryl-Man but failed to express [3H]Man-containing GPIs. These data indicate that 1) significant GPI production in lymphocytes is dependent on prior stimulation of the cells, 2) exposure of lymphocytes to agents which activate protein kinase C induces GPI synthesis, and 3) in five PNH patients affected lymphocytes are uniformly defective in an early GPI biosynthetic step which undermines expression of GPI mannolipids.
Highlights
From the $Institute of Pathology, Case Western Reserve University, Cleveland,Ohio 44106 and SAbteilung Immunologie und Transfusionsrnedizin, MedizinischeHochschule, 0-3000 Hannover 61, Germany
To characterize Glycoinositol phospholipid (GPI) biosynthesis in non- chored surface proteins appear in the circulatio(nreviewed in transformed human lymphocytes and to define the GRPefI. 1).The abnormal cells arise as a consequence of expansyntheticdefectunderlyingdeficientexpression of sion of one or morebonemarrow progenitorsaltered by GPI-anchored proteinsby paroxysmal nocturnalhem- somatic mutation[2]
We further found that affected lymphocyte lines from five Paroxysmalnocturnal hemoglobinuria (PNH) patients uniformly fail to synthesize Man-containing GPI products
Summary
H2-H8 are designations of the respective species defined in studies with HeLa cell microsomes. In previous studies [17, 19],failure of expression of the most polar GPI product(s) was documented in DAFdeficient polymorphonuclear cells (PMN) from several PNH patients. Less polar GPI species corresponding to incompletely assembled intermediates were present in some patients suggesting that thebiochemical block washeterogenous. In the present study we characterized GPI synthesis in normal peripheral blood T lymphocytes in theresting state andfollowingstimulation indifferent ways. Utilizing conditions under which GPI expression was maximized, we compared GPI assembly in affected (CD48-) and unaffected (CD48+)T and NK cell lines of PNH patients [6, 7]. We further found that affected lymphocyte lines from five PNH patients uniformly fail to synthesize Man-containing GPI products
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