Purification and characterization of a saccharide-binding protein from penetration glands of Diplostomum pseudospathaceum--a bifunctional molecule with cysteine protease activity.
A beta-1,3-glucan-binding lectin from the penetration glands of Diplostomum pseudospathaceum cercariae was isolated by affinity chromatography using yeast glucan and curdlan as affinity matrices. Further purification to homogeneity was performed by cation-exchange chromatography. The protein migrated as a double band around 24 kDa in gels after SDS-PAGE. The protein is of strongly basic nature--its pI shown by native IEF was around 10. The mass of the protein determined by MALDI-TOF mass spectrometry was 23.9 kDa. N-terminal sequence as well as some internal sequences showed significant alignments with several cysteine protease sequences found in databases. The protein bound a biotinylated synthetic analogue of the irreversible inhibitor of cysteine proteases, E-64 and, moreover, its proteolytic activity was demonstrated in substrate gels. The enzymatic activity could be inhibited by the cysteine protease inhibitor E-64; therefore, the investigated protein was considered to be a bifunctional molecule possessing both lectin and enzyme activities. Glycanohydrolytic activity was not proved. The detected characters of this molecule lead to a hypothesis on its role in penetration of Diplostomum cercariae into fish hosts--that of binding to the carbohydrates of fish mucus and concurrent cleaving of protein components of the mucus and skin.
Mikes
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Department of Parasitology, Charles University, Vinicná 7, 12844 Prague 2, Czech Republic. mikes@natur.cuni.cz
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003546:Cysteine Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005088:Exocrine Glands / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D011956:Receptors, Cell Surface; D016415:Sequence Alignment; D012908:Snails / Q000469:parasitology; D014200:Trematoda / Q000737:chemistry / Q000201:enzymology* / Q000254:growth & development
D002352:Carrier Proteins; D011956:Receptors, Cell Surface; C055213:saccharide-binding proteins; D003546:Cysteine Endopeptidases