A Novel Plasminogen Activator from Snake Venom

Abstract

A novel plasminogen activator from Trimeresurus stejnegeri venom (TSV-PA) has been identified and purified to homogeneity. It is a single chain glycoprotein with an apparent molecular weight of 33,000 and an isoelectric point of pH 5.2. It specifically activates plasminogen through an enzymatic reaction. The activation of human native Glu-plasminogen by TSV-PA is due to a single cleavage of the molecule at the peptide bond Arg561-Val562. Purified TSV-PA, which catalyzes the hydrolysis of several tripeptide p-nitroanilide substrates, does not activate nor degrade prothrombin, factor X, or protein C and does not clot fibrinogen nor show fibrino(geno)lytic activity in the absence of plasminogen. The activity of TSV-PA was readily inhibited by phenylmethanesulfonyl fluoride and by p-nitrophenyl-p-guanidinobenzoate. Oligonucleotide primers designed on the basis of the N-terminal and the internal peptide sequences of TSV-PA were used for the amplification of cDNA fragments by polymerase chain reaction. This allowed the cloning of a full-length cDNA encoding TSV-PA from a cDNA library prepared from the venom glands. The deduced complete amino acid sequence of TSV-PA indicates that the mature TSV-PA protein is composed of 234 amino acids and contains a single potential N-glycosylation site at Asn161. The sequence of TSV-PA exhibits a high degree of sequence identity with other snake venom proteases: 66% with the protein C activator from Agkistrodon contortrix contortrix venom, 63% with batroxobin, and 60% with the factor V activator from Russell's viper venom. On the other hand, TSV-PA shows only 21-23% sequence similarity with the catalytic domains of u-PA and t-PA. Furthermore, TSV-PA lacks the sequence site that has been demonstrated to be responsible for the interaction of t-PA (KHRR) and u-PA (RRHR) with plasminogen activator inhibitor type 1.

Purification and Characterization of a Fibrinolytic Protease from a Culture Supernatant ofFlammulina velutipesMycelia

“…29,[31][32][33][34] In recent years, however, fibrinolytic enzymes have been discovered in a variety of organisms. Currently, relatively few reports focus on the fibrinolytic enzymes in mushrooms.…”

Thermodynamic Analysis Reveals Structural Rearrangement during the Acylation Step in Human Trypsin 4 on 4-Methylumbelliferyl 4-Guanidinobenzoate Substrate Analogue

“…Other instances also exist when the non-glycine-possessing enzymes at position 193 are not to be considered as mutant proteins, but these variants represent the native enzymes; Trimeresurus stejnejeri plasminogen activator (11) and Dermasterias imbricata trypsin 1 (12) belong to this group of site 193 variants. Naturally occurring 193 mutants are reported among coagulation factors, including Factor XI (13), Factor IX (14), and Factor VII as well (15).…”

Crystal Structures and Amidolytic Activities of Two Glycosylated Snake Venom Serine Proteinases

“…Wide variance of the sequences and enzymatic properties of these proteinases, which is the feature of SV-SPs (8,9), requires more structural models to annotate the detailed relationship. In particular, although the carbohydrates in native TSV-PA were documented to have little influence on its amidolytic activity and plasminogen activation property (3,4), most of SV-SPs contain carbohydrates at variant glycosylation sites, which tempts us to explore whether all of these proteinases match TSV-PA.…”

“…Most biochemical properties, biological roles, and structure of SV-SPs are mainly characterized by using TSV-PA, a plasminogen activator from Trimeresurus stejnegeri venom, with the structure of SV-SPs as the only available model (2)(3)(4)(5)(6)(7). Wide variance of the sequences and enzymatic properties of these proteinases, which is the feature of SV-SPs (8,9), requires more structural models to annotate the detailed relationship.…”