Differences in cysteine protease activity in Schistosoma mansoni-resistant and -susceptible Biomphalaria glabrata and characterization of the hepatopancreas cathepsin B Full-length cDNA.
Biomphalaria glabrata snails are known to display a wide range of susceptibility phenotypes to Schistosoma mansoni infection depending on the genetics of both the snail and the invading parasite. Evidence exists for a role of hydrolytic enzymes in the defense of molluscs against invading parasites. To elucidate the role of these enzymes in the outcome of infection in the snail, proteolysis was examined in parasite-resistant and -susceptible snails. Zymographs of extracts from the whole snail or hepatopancreas indicated higher proteolytic activity in resistant, compared with susceptible, snails. Lytic activity coincided with a high-molecular-weight smear (220 to 66 kDa) that was abrogated by the cysteine protease inhibitor trans-epoxysuccinyl-l-leucylamido-(4-guanidino)butane. Quantitative flourimetric assays showed 3.5-fold higher activity in resistant than in susceptible snails. From a hepatopancreas cDNA library, several cysteine protease encoding expressed sequence tags including the full-length cDNA for cathepsin B were identified. Sequence analysis revealed that this cathepsin B belonged to the C1A family of peptidases characterized by the presence of the catalytic cysteine-histidine dyad, the "occluding loop," signal sequence, and cleavage sites for the prepro and propeptides. Quantitative real-time reverse transcriptase-polymerase chain reaction showed higher up-regulation of cathepsin B transcript in resistant than in the susceptible snail after parasite exposure.
Myers
Jocelyn
J
Department of Biology, Howard University, NW, Washington, DC 20059, USA.
Ittiprasert
Wannaporn
W
Raghavan
Nithya
N
Miller
André
A
Knight
Matty
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001700:Biomphalaria / Q000201:enzymology* / Q000276:immunology / Q000469:parasitology*; D002401:Cathepsin B / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003374:Coumarins / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D018076:DNA, Complementary / Q000737:chemistry / Q000302:isolation & purification; D004151:Dipeptides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D020224:Expressed Sequence Tags; D005456:Fluorescent Dyes / Q000378:metabolism; D005470:Fluorometry; D015723:Gene Library; D043143:Hepatopancreas / Q000201:enzymology; D006790:Host-Parasite Interactions / Q000502:physiology; D007113:Immunity, Innate / Q000502:physiology; D008969:Molecular Sequence Data; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012550:Schistosoma mansoni / Q000276:immunology*
D003374:Coumarins; D015853:Cysteine Proteinase Inhibitors; D018076:DNA, Complementary; D004151:Dipeptides; D005456:Fluorescent Dyes; C021159:benzyloxycarbonyl-phenylalanylarginine-4-methylcoumaryl-7-amide; D003546:Cysteine Endopeptidases; D002401:Cathepsin B