Identification, characterization and deduced amino acid sequence of thedominant protease from Kudoa paniformis and K. thyrsites: A uniquecytoplasmic cysteine protease

摘要

Kudoa paniformis and Kudoa thyrsites (Myxozoa: Myxosporea) infections are associated with severe proteolysis of host muscle tissue post-mortem. The present study was undertaken to identify and characterize the protease responsible for myoliquefaction and determine mechanismscontrolling protease function in vivo. N-terminal sequence analysis of partially purified protease from hake muscle infected with K. paniformisand K. thyrsites revealed a 23 amino acid sequence that aligned with cysteine proteases. Enzyme inhibition assays confirmed the presence of anessential active site cysteine residue. Using the above K. paniformis amino acid sequence data, a corresponding cDNA sequence from K. thyrsitesplasmodia was elucidated revealing a cathepsin L proenzyme (Kth-CL). The translated amino acid sequence lacked a signal sequencecharacteristic of lysosomal and secreted proteins suggesting a unique cytoplasmic location. Only the proenzyme form of Kth-CL was present in Atlantic salmon muscle anti-mortem but this form became processed in vivo when infected muscle was stored at 4 °C. The proenzyme of Kth-CL showed uninhibited activity at pH 6.0, negligible activity at pH 6.5 and no measurable activity at pH 7.0 whilst the processed protease showedstability and function over a broad pH range (pH 4.5-8.8). The pH dependent processing and function of Kth-CL was consistent with histidineresidues in the proregion playing a critical role in the regulation of Kth-CL.