Genes from the medicinal leech (Hirudo medicinalis) coding for unusual enzymes that specifically cleave endo-ɛ(γ-Glu)-Lys isopeptide bonds and help to dissolve blood clots

Abstract

We previously detected in salivary gland secretions of the medicinal leech (Hirudo medicinalis) a novel enzymatic activity, endo-epsilon(gamma-Glu)-Lys isopeptidase, which cleaves isopeptide bonds formed by transglutaminase (Factor XIIIa) between glutamine gamma-carboxamide and the epsilon-amino group of lysine. Such isopeptide bonds, either within or between protein polypeptide chains are formed in many biological processes. However, before we started our work no enzymes were known to be capable of specifically splitting isopeptide bonds in proteins. The isopeptidase activity we detected was specific for isopeptide bonds. The enzyme was termed destabilase. Here we report the first purification of destabilase, part of its amino acid sequence isolation and sequencing of two related cDNAs derived from the gene family that encodes destabilase proteins, and the detection of isopeptidase activity encoded by one of these cDNAs cloned in a baculovirus expression vector. The deduced mature protein products of these cDNAs contain 115 and 116 amino acid residues, including 14 highly conserved Cys residues, and are formed from precursors containing specific leader peptides. No homologous sequences were found in public databases.

Enzyme from the medicinal leech (Hirudo medicinalis) that specifically splits endo‐ϵ(‐γ‐Glu)‐Lys isopeptide bonds: cDNA cloning and protein primary structure

“…To complete sequencing the longest BrCN peptide, 2 ( Fig. 1B), started in [10], it was digested with Staphylococcus aureus glutamic acid specific protease V-8 ('Sigma') by the method described in [15]. Vertical arrows in Fig.…”

“…1B indicate positions of the splits. The shorter peptides were sequenced as described [10]. The sequence of 10 C-terminal amino acids of BrCN peptide 4 (Fig.…”

“…The sequence of 10 C-terminal amino acids of BrCN peptide 4 (Fig. 1B) was also determined as described in [10].…”

“…Due to its ability to dissolve stabilized fibrin in vitro [8] ~,nd thus to reverse the critical final step in blood coagulation [~)], destabilase is a potential thrombolytic agent. In our prex ious work [10] the corresponding protein was purified, its partial amino acid sequence determined, and two cDNAs ent oding closely related but still different proteins were cloned and sequenced, demonstrating the existence of the gene family l)r destabilase related proteins. The structural homology cor-J elated with functional similarity of the destabilase and the t,roduct of one of the cDNAs cloned into a baculovirus ext,ression vector and expressed in insect cells.…”

“…However, the polypeptides encoded by these two cDNAs were only partially *Corresponding author. Fax: (7) homologous to the independently isolated protein [10]. Here we describe the identification and sequence of a cDNA coding for the third member of the family with precisely the same sequence as the destabilase protein.…”