Erythrocyte membrane inhibitor of reactive lysis: effects of phosphatidylinositol-specific phospholipase C on the isolated and cell- associated protein

Abstract

The erythrocyte membrane inhibitor of reactive lysis (MIRL) is an 18-Kd protein that controls complement-mediated hemolysis by restricting the activity of the membrane attack complex. MIRL expression on the erythrocytes of paroxysmal nocturnal hemoglobinuria (PNH) is abnormally low, and the greater susceptibility of PNH erythrocytes to complement is causally related to this deficiency. Inasmuch as other proteins that are deficient in PNH are anchored to the membrane through a glycosyl phosphatidylinositol moiety, studies were undertaken to determine if MIRL shares this structural feature. Normal human erythrocytes that had been radiolabeled with 125I were incubated with phosphatidylinositol- specific phospholipase C (PIPLC), and the supernate and the solubilized membrane proteins were immunoprecipitated using anti-MIRL antiserum. The MIRL that was specifically released into the supernate had an Mr of 19 Kd, while the MIRL that remained bound to the membrane had an Mr of 18 Kd. A quantitative assay showed that approximately 10% of erythrocyte MIRL was susceptible to PIPLC; however, treatment with PIPLC had no effect on either the electrophoretic mobility or the functional activity of purified MIRL. These studies show that the effects of PIPLC on MIRL are similar to those observed for other human erythrocyte membrane proteins that are anchored by a glycosyl phosphatidylinositol moiety.

Structural properties of the glycoplasmanylinositol anchor phospholipid of the complement membrane attack complex inhibitor CD59

“…The reason in their studies Ibr the apparent discrepancy between the PI-PLC-sensilivities of in situ and purified CD59 protein is unclear, but selective loss of unacylated molecules during purification couid lead lo a higher proportion of molecules with aeylaled GPI-anchors in the latter case. Our observation that PI-PLC-cleaved CD59 molecules exhibit decreased SDS-PAGE mobility can also account for previous observations [25] that (i) MIRL released from erythrocytes by the en/yme displayed a slightly greater apparent A/r ihan MIRL immunoprecipitated from intact celis, whereas (ii) when isolated, Pl-PLC-treated MIRL showed the same apparent Mr as untreated MIRL. Previous findings that GPl-anchor removal from VSGs.…”

“…acylalion accounts for the previously reported variability in CD59's PI-PLC sensitivity. Our finding thai 84 90% of CD59 molecules in erythrocytes contain GPI-anchors that exhibit acylation can additionally explain observations by Ilolguin et al [25] that PI-PLC treatment of purified erythrocyte MIRLdid not prevent its incorporation into PNH erylhrocytes or alter its ability to inhibit C9 polymerization in the reconstituted PNH cells. The reason in their studies Ibr the apparent discrepancy between the PI-PLC-sensilivities of in situ and purified CD59 protein is unclear, but selective loss of unacylated molecules during purification couid lead lo a higher proportion of molecules with aeylaled GPI-anchors in the latter case.…”

“…the similarities with CD59 have suggested that it is anchored by an equivalent structure. In ditTerent studies, however, it exhibited variable sensitivity lo PI-PLC-mediated cleavage, whether studied /H.v/fM [ 1.2.4,6,9] or purified [25]. Additionally, in PNH a subset of patients exists whose erythrocytes (type II) [26.27] exhibit only partially enhanced ('^ 5-as opposed to ^ 25-foId greater than nntnial) sensitivity to autologous complement-mediated injury and lack DAF [13,28], but not the activity attributed to CD59 [13].…”

Paroxysmal Nocturnal Hemoglobinuria and Glycosyl Phosphatidylinositol Anchored Proteins That Regulate Complement

“…Further, it was shown that by using an antibody to block the function of MIRL, normal erythrocytes could be rendered susceptible to CVFBb-initiated hemolysis. As anticipated, PNH erythrocytes were found to be deficient in MIRL Subsequent studies showed that MIRL also belongs to the family of GPI-linked proteins (38).…”

Functional and antigenic similarities between a 94-kD protein of Schistosoma mansoni (SCIP-1) and human CD59.

“…In this report we describe a schistosome C inhibitory protein type I (SCIP-1) that is a 94-kD protein functionally and antigenically related to human CD59. We demonstrate that several properties of CD59, such as the capacity to inhibit formation of C MAC and the binding of human C8 and C9 components (23,29,30) are manifested by SCIP-1 as well, and that both molecules are, apparently, external glycosyl phosphatidylinositol-anchored membrane proteins (31). Furthermore, antibodies directed to human CD59 bind to SCIP-1 and render the parasite susceptible to C-mediated killing.…”