Sunghyun Kang

Characterization of three neutral proteases of Spirometra mansoni plerocercoid

In the pathogenesis of sparganosis, proteases have been considered to play important roles in tissue migration and parasite feeding. Several bands of proteolysis were observed when crude extracts of Spirometra mansoni plerocercoid (sparganum) were examined using gelatin substrate gel at neutral pH, of which two proteases of 198 and 104 kDa were purified by two chromatographic steps, and a 36 kDa protease was purified by gelatin-affinity and DEAE-anion exchange chromatography. All the purified proteases exhibited optimal activity at pH 7.5 and 0.1 M Tris-HCl. Proteolytic activities at 198 and 104 kDa were inhibited specifically by serine protease inhibitors and 4-(amidinophenyl)methanesulfonyl fluoride (APMSF, 0.5 mM) and N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK, 1 mM), which strongly suggested that these two proteases were trypsin-like proteases. The activity of the 36 kDa protease was inhibited by N-tosyl-L-phenylalanine chloromethyl ketone (TPCK, 1 mM) and chymostatin (0.1 mM), and was potentiated in 10 mM Ca2+ which showed that the protease had a chymotrypsin-like property. All the proteases were Schiff (PAS) positive. Proteases of 198 and 104 kDa degraded collagen completely within 24 h. The 36 kDa enzyme cleaved human recombinant interferon-gamma (rIFN gamma) and bovine myelin basic protein. In addition, all the purified proteins elicited strong antibody responses in the infected patients.

A 78 kDa glucose-regulated protein gene of Spirometra erinacei plerocercoid induced by chemical and physiological stresses.

To adapt to different environmental conditions between poikilothermic and homeothermic hosts, the plerocercoid of Spirometra erinacei (sparganum) might express a variety of biologically active molecules. We have identified a 78 kDa glucose-regulated protein of the sparganum (SpGrp78) by differential display of mRNA, employing RNAs each from sparganum adjusted at 9 degrees C and 37 degrees C. A full-length cDNA of 2148 bp encodes for a protein of 651 amino acids with a predicted molecular mass of 71 610 Da and shares molecular characteristics with heat-shock protein 70, including a putative ATP binding site, signal peptide cleavage site and endoplasmic reticulum retention signal. Phylogenetic analysis revealed that SpGrp78 was mostly related to those of Echinococcus multilocularis and E. granulosus. Expression of SpGrp78 mRNA increased approximately 7-fold by inhibition of glycosylation by tunicamycin, 2-fold by temperature-shift from 9 degrees C to 37 degrees C and slightly by pH-shift to 4.0 or 5.5. These results suggested that induction of SpGrp78 mRNA is related to the functional role of SpGrp78 as a molecular chaperone when the parasite adapts to a new host environment.