Trypanosoma cruzi: cruzipain and membrane-bound cysteine proteinase isoform(s) interacts with human α 2-macroglobulin and pregnancy zone protein

Abstract

Plasmatic levels of pregnancy zone protein (PZP) increase in children with acute Chagas disease. PZP, as well as α 2-macroglobulin (α2-M), are able to interact with Trypanosoma cruzi proteinases. The interaction of α2-M and PZP with cruzipain, the major cysteine proteinase of T. cruzi, was investigated. Several molecular changes on both α-M inhibitors under reaction with cruzipain were found. PAGE analysis showed: (i) formation of complexes of intermediate mobility and tetramerization of native α2-M and PZP, respectively; (ii) limited proteolysis of bait region in α2-M and PZP, and (iii) covalent binding of cruzipain to PZP and α 2-M. Conformational and structural changes experimented by α-Ms correlate with modifications of the enzyme electrophoretic mobility and activity. Cruzipain–α-M complexes were also detected by gelatin SDS–PAGE and immunoblotting using polyclonal anti-cruzipain antibodies. Concomitantly, α2-M and PZP impaired the activity of cruzipain towards Bz-Pro-Phe-Arg-pNA substrate. In addition, α-Ms were able to form covalent complexes with membrane isoforms of cysteine proteinases cross-reacting with cruzipain. The present study suggests that both human α-macroglobulin inhibitors could prevent or minimize harmful action of cruzipain on host's molecules and hypothetically regulate parasite functions controlled by cruzipain. Index Descriptors and Abbreviations: Human α-macroglobulins (α-Ms); α 2-Macroglobulin (α2-M); pregnancy zone protein (PZP); Chagas' disease; T. cruzi; cysteine proteinase (CP); cruzipain; Hydrophobic membrane proteins (HMP); Isoforms of cysteine proteinases; Polyacrylamide gel electrophoresis (PAGE); NADP-linked glutamate dehydrogenase (NADP-GDH); Phenylmethanesulphonyl fluoride (PMSF); N-Benzoyl-Pro-Phe-Arg- p-nitroanilide hydrochloride (Bz-Pro-Phe-Arg-pNA); Low-density lipoprotein receptor-related protein (LRP).