The N-terminus moiety of the cystatin SmCys from Schistosoma mansoni regulates its inhibitory activity in vitro and in vivo.
The complete sequence of SmCys, a cystatin expressed by Schistosoma mansoni, was obtained. Constructs of SmCys consisting of deletions of 10 and 20 amino acid residues from the N-terminal of the full length recombinant protein, were cloned in the pQE-30 vector, expressed in Escherichia coli and assayed for inhibitory activity against papain. Kinetic analysis showed that SmCys -10 and SmCys -20 had K(i) values of 0.7391 and 4.9154, respectively, as compared to 0.0647, displayed by the full length recombinant. Protease inhibition by SmCys was also observed in vivo. When the recombinant products were incubated during 7 days with live schistosomula in the presence of red blood cells, only the full length product could completely inhibit the formation of haemozoin, a dark pigment formed as a by-product of haemoglobin digestion. The sequence data of the recombinant SmCys proteins were used for the construction of molecular models, which were then subjected to molecular dynamics for 2ns. In comparison to the full length, the models corresponding to the truncated constructs, showed a distinctive change on the surface charge distribution. This parameter was more pronounced in SmCys -20, which also displayed a significant displacement of the inhibitory domain, a result which could explain the kinetic data in terms of the loss of attachment sites. These changes correlated well with the progressive lack of inhibition observed for the recombinant deletion constructs, in vitro and in vivo.
Morales
Fabiana Carvalho
FC
Departamento de Bioquímica Médica, ICB/CCS, Universidade Federal do io de Janeiro, Ilha do Fundão, CEP 21941-590, Rio de Janeiro, Brazil.
Furtado
Daniel Rodrigues
DR
Rumjanek
Franklin David
FD
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003198:Computer Simulation; D015891:Cystatins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D018509:DNA, Helminth / Q000737:chemistry / Q000302:isolation & purification; D015801:Helminth Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology; D006420:Hemeproteins / Q000378:metabolism; D006454:Hemoglobins / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010206:Papain / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D012550:Schistosoma mansoni / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D018509:DNA, Helminth; D015801:Helminth Proteins; D006420:Hemeproteins; D006454:Hemoglobins; D011994:Recombinant Proteins; C020753:hemozoin; D010206:Papain