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Isolation and characterization of a 40 kDa cysteine protease from Gymnophalloides seoi adult worms
M H Choi,*1W J Park,2Y K Park,1J Y Chai,1 and S H Lee1
1Department of Parasitology, Seoul National University College of Medicine, Seoul 110-799, Korea.
Received April 06, 1998; Accepted May 12, 1998.
Abstract
A 40 kDa cysteine protease was purified from the crude extract of adult worms of Gymnophalloides seoi by two consecutive steps: Sephacryl S-200 HR and DEAE-Sephacel chromatography. Enzyme activities were completely inhibited by cysteine protease inhibitors. L-trans-epoxysuccinylleucylamido (4-guanidino) butane (E-64) and iodoacetic acid, strongly suggesting that the purified enzyme belongs to the cysteine family of proteases. The enzyme was maximally active at pH 4.5 in 0.1 M of buffer, and its activity was greatly potentiated in the presence of 5 mM dithiothreitol. The protease degraded macromolecules with differential capabilities; it degraded extracellular matrix proteins, such as collagen and fibronectin, with a stronger activity against collagen than fibronectin. However, the enzyme digested hemoglobin and human immunoglobulins only slightly, leaving them nearly intact after an overnight reaction. Our results suggest that the cysteine protease of G. seoi adults is potentially significant in the nutrient uptake from the host intestine.
Figures
Fig. 1 Elution profile of protease during purification by Sephacryl S-200 HR (A) and DEAE-Sephacel chromatography (B). Fractions were asayed for activity on CBZ-phe-arg-MNA (○) and monitored for protein content (•) at 280 nm. Fractions with high enzyme activities were pooled, as shown by the bar (-). Vertical arrows in Fig. 1B indicate stepped salt gradients.
Fig. 2 SDS-PAGE analysis of proteins purified from crude extract to cysteine protease by sequential chromatographic steps. 1, crude extract; 2, active fractions containing enzyme activity from Sephacryl S-200 HR; 3, the purified enzyme from DEAE-Sephacel.
Fig. 3 Effects of pH (A) and molar concentration (B) on proteolytic activity of the cysteine protease. Mean ±SD, n = 3.
Fig. 4 Cleaving activity of the cysteine protease against collagen (A), fibronectin (B), and hemoglobin (C). C, macromolecules only ; 1-4, macromolecules with the enzyme for 1, 3, and 5 hr, and overnight at 37℃.
Fig. 5 Degradation of IgG2a (A), and sIgA (B) by cysteine protease. No digestive products are shown in comparison with the control. C, immunoglobulins only; 1-4, immunoglobulins with the enzyme for 1, 3, and 5 hr, and overnight at 37℃.
Tables
Table 1 Summary of the purification of a cysteine protease from the crude extract of Gymnophalloides seoi adults
Table 2 Modulatory effects of various effectors on the enzyme activity
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