Identification, characterization and deduced amino acid sequence of the dominant protease from Kudoa paniformis and K. thyrsites: a unique cytoplasmic cysteine protease.
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 mechanisms controlling protease function in vivo. N-terminal sequence analysis of partially purified protease from hake muscle infected with K. paniformis and K. thyrsites revealed a 23 amino acid sequence that aligned with cysteine proteases. Enzyme inhibition assays confirmed the presence of an essential active site cysteine residue. Using the above K. paniformis amino acid sequence data, a corresponding cDNA sequence from K. thyrsites plasmodia was elucidated revealing a cathepsin L proenzyme (Kth-CL). The translated amino acid sequence lacked a signal sequence characteristic 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 degrees 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 showed stability and function over a broad pH range (pH 4.5-8.8). The pH dependent processing and function of Kth-CL was consistent with histidine residues in the proregion playing a critical role in the regulation of Kth-CL.
Funk
Valerie A
VA
BC Centre for Aquatic Health Sciences, 621 Island Hwy, PO Box 277, Campbell River, BC Canada V9W 5B1. val.funk@cahs-bc.ca
Olafson
Robert W
RW
Raap
Monique
M
Smith
Derek
D
Aitken
Laura
L
Haddow
Jody D
JD
Wang
Diana
D
Dawson-Coates
Jennifer A
JA
Burke
Robert D
RD
Miller
Kristina M
KM
D000595:Amino Acid Sequence; D000818:Animals; D056668:Cathepsin L; D002403:Cathepsins / Q000737:chemistry / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000378:metabolism*; D003593:Cytoplasm / Q000187:drug effects / Q000201:enzymology*; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D056890:Eukaryota / Q000187:drug effects / Q000201:enzymology*; D005455:Fluorescent Antibody Technique; D048388:Gadiformes / Q000469:parasitology; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000302:isolation & purification; D018482:Muscle, Skeletal / Q000187:drug effects / Q000469:parasitology; D011480:Protease Inhibitors / Q000494:pharmacology; D011529:Protozoan Infections, Animal / Q000201:enzymology / Q000469:parasitology; D020317:Salmo salar / Q000469:parasitology; D017422:Sequence Analysis, DNA
D018076:DNA, Complementary; D009124:Muscle Proteins; D011480:Protease Inhibitors; D002403:Cathepsins; D003546:Cysteine Endopeptidases; D056668:Cathepsin L