Characterization of a phospholipase C-resistant inositol containing glycolipid from Trypanosoma cruzi

Since glycosylphosphatidylinositol is the most common form of attachment of proteins to membranes in T. cruzi, and that this parasite depends on surface-mediated interactions for survival within the vector and mammalian host, it is probable that a drug which interfers with the metabolism of glycosylphosphatidylinositol (GPI) could be successfully employed in chemotherapy. Over the last few years several groups have been characterizing this mode of attachment in T. cruzi and more recently we have been concentrating our efforts on the identification of candidate precursors for protein anchors in metacyclic trypomastigotes. Previously detected GPI heterogeneity regarding solubilization of a major stage-specific antigen (1G7-Ag) by phospholipase C led us to investigate whether biosynthetic precursors with similar properties could also be identified. Two glycolipid species whose migration properties resemble glycolipids A and C of T. brucei were amenable to biosynthetic radiolabelling with palmitic acid, inositol, ethanolamine, glucosamine and mannose. Following purification, these species were submitted to classical GPI diagnostic treatments. In both cases digestion with GPI-specific phospholipase D (GPIPLD) produced phosphatidic acid and treatment with either mild base or phospholipase A2 (PLA2) produced free fatty acid, indicating an acylation at least at position 2 of the glycerol. The glycolipid A-like species proved to be susceptible to solubilization by PIPLC of B. thuriengiensis and by GPIPLC of T. brucei and the glycolipid C-like material proved to be fully resistant to both lipases. Although the glycolipid A-like species indeed presents these and other properties compatible with a precursor for the chemically characterized 1G7-Ag anchor, the PLC-resistant species which is completely insensitive to nitrous acid deamination might be an exception to the general finding of a non-acetylated glucosamine in the GPI moieties so far described

Further characterization of the acidic GPI-hydrolyzing phospholipase present in human sera

A phospholipase from human serum capable of hydrolyzing glycosylphosphatidylinositol membrane anchors was described and partially characterized by our group some years ago. This activity presented a pH optimum between 5.0 and 6.0 and was inhibited by EDTA, EGTA and 1,10-phenanthroline. Partial purification showed that the enzyme was a glycoprotein with an apparent molecular weight of 140 kDa as judged by gel filtration. Other investigators characterized at the same time a phospholipase D with similar properties but with a pH optimum near 7.5. We now confirm that the human serum enzyme is indeed a phospholipase D capable of hydrolyzing mfVSG and glycolipids A and C from T. brucei. Isoelectric focusing of whole sera suggests the presence of two isoforms, one with a pI of 4.7 which was the form previously purified by our group, and others with pI from 6.2 to 7.4