Influence Of Deglycosylation On Some Biological Characteristics Of The Earthworm Fibrinolytic Enzymes

The components of earthworm fibrinolytic enzymes (EFEs) were purified from earthworm (Eisenia fetida) homogenate by affinity chromatography using SBTI as a ligand and Sepharose-4B as a carrier. Components EfP-I-1 and EfP-I-2 were purified through DEAE-Cellulose-52 chromatography and preparative electrophoresis. The fibribolytic activity was identified by the fibrin plate assay and BAEE method.All the EFE components are glycoproteins identified by Schiff's reagent. The amount of sugar contained in EFEs is 2.98% determined by sulfuric acid-phenol method. And the amount of sugar contained in EfP-I-1 and EfP-I-2 is 5.71% and 1.93% respectively。The results of dot-blotting by ConA, LCA-B and AAL-B showed that mannose residue could be detected in the end of the oligosaccharides of EfP-I-1 and EfP-I-2,and they were fucosylation on the asparagine-linked N-acetylglucosamine.After deglycosylation by trifluoromethanesulfonate (TFMS),the antisera against EFEs and deglycosylated EFEs were developed by immunizing rabbits with EFEs and deglycosylated EFEs respectively, no significant changes were detected in the immunogenicities or immunoreactivities of EFEs and deglycosylated EFEs using the method of ELISA and Western-blotting. The deglycosylated EfP-I-1 and EfP-I-2 by TFMS were no longer resistant to trypsin, however, the natural EfP-I-1 and EfP-I-2 had great resistance to the hydrolysis of trypsin. And the deglycosylated EFEs lost the fibribolytic activity completely after the deglycosylation by TFMS.After being immobilized onto Sepharose-4B-SBTI, EFEs were deglycosylated by peptide-N-glycosidase A. The results of affinity blotting with ConA-HRP and Schiff's reagent staining showed that the oligosaccharides of EFEs were removed completely, and the fibribolytic activity reduced about 30% by the fibrin plate assay.