A Boophilus microplus vitellin-degrading cysteine endopeptidase

Abstract

Here we describe the purification and characterization of a vitellin (VT) degrading cysteine endopeptidase (VTDCE) from eggs of the hard tick Boophilus microplus. A homogeneous enzyme preparation was obtained by chromatographic fractionation on ion-exchange and gel filtration columns and an autolysis step. This step consisted of incubation of a semipurified enzyme (after the first ion-exchange chromatography) at pH 4.0 that dissociated the enzyme from VT, to which VTDCE is naturally tightly associated. The enzyme purity was confirmed by capillary and native gel electrophoresis, and SDS–PAGEsuggested the enzyme is a dimer of 17 and 22 kDa.VTDCEwas active upon several synthetic substrates, with a preference for a hydrophobic or a basic residue in P1, and a hydrophobic residue in P2. VTDCE also hydrolysed haemoglobin, albumin, gelatin and vitellin.VTDCEis inactive in the absence ofDTTand was totally inhibited by E-64, indicating it is a cysteine endopeptidase. Our results suggest that VTDCE is a major enzyme involved in yolk processing during B. microplus embryogenesis.