Partial primary structure of a fibrinogenase from the venom of the snake Lachesis stenophrys

Abstract

The partial primary structure of an M_r 24 000 non-haemorrhagic metalloproteinase isolated from the venom of the snake Lachesis stenophrys has been determined. The native proteinase was resistant to Edman degradation exhibiting the N-terminal blockade. The pyridylethylated or native proteinase was chemically and enzymatically fragmented and the obtained peptides were separated by gel or reversed-phase chromatography, and sequenced. The metalloproteinase from Lachesis stenophrys contains a putative zinc-chelating sequence HELGHNLGMKH, characteristic for the reprolysin family of zinc-metalloproteinases. It contains six cysteine residues in the standard positions for this group of proteins suggesting the same disulfide bonding. Interestingly, it has almost identical sequence as the metalloproteinase from Lachesis muta muta, LHF-II, which is, however, haemorrhagic. The main structural differences between the two molecules were found in their N-terminal parts and in glycosylation. As the substrate-binding regions of both proteinases are practically identical, we suggest that the absence of haemorrhagicity in Lachesis stenophrys enzyme is due to its lower affinity for the matrix proteins and not due to different substrate specificity.

Introduction

Snake venom contains a large number of zinc metalloproteinases, which exhibit various pathological effects on blood coagulation, fibrinolysis and complement system [1]. Despite differences in their molecular masses and activities, they all appear to be related through a single ancestral gene, as observed by the comparison of their primary structures [2]. They are synthesized in the venom gland as zymogens and subsequently processed to the active forms. These proteinases are divided into four classes, P-I to P-IV, according to their domain structure [3]. The proteinase domains in all classes are structurally homologous. One of the most characteristic structural elements is the extended zinc-binding sequence HExxHxxGxxH. The three-dimensional structures of three zinc-metalloproteinases, adamalysin II [4], atrolysin C [5], and H₂-proteinase [6] revealed another conserved, five amino acid long methionine-containing structural motif, the Met-turn. The high-molecular-mass proteinases have, in addition to proteinase domain, 1–3 non-enzymatic domains located C-terminally to the proteinase domain.

To date, many snake venom metalloproteinases have been characterized and their amino acid sequences determined in order to elucidate the structural basis of their pathopharmacological actions [1], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17]. To provide further information about the structural elements which determine the haemorrhagicity of some of them we have attempted to solve the amino acid sequence of an M_r 24 000 fibrinogenase from snake Lachesis stenophrys (LSF). LSF is one of the major metalloproteinases found in L. stenophrys venom. The proteinase cleaves Aα and Bβ chains of fibrinogen, and has no haemorrhagic activity [18]. It is glycosylated and belongs to the P-I class of snake venom metalloproteinases.