Partial primary structure of a fibrinogenase from the venom of the snake Lachesis stenophrys
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 Mr 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.
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Materials
LSF was purified from L. stenophrys venom, as described [18]. Sephacryl S-200 HR was from Pharmacia Biotech (Uppsala, Sweden), formic acid from Merck (Darmstadt, Germany) and acetonitrile from Rathburn (Walkerburn, UK). CNBr was purchased from Pierce (Rockford, USA), 4-vinylpyridine and trypsin (EC 3.4.21.4.) were from Sigma (St. Louis, MO, USA). Staphylococcus aureus, strain V8, proteinase (EC 3.4.21.19) was from Miles (Naperville, UK). Sequencing reagents were obtained from PE Applied
Sequence determination
We have determined almost complete amino acid sequence of a non-haemorrhagic metalloproteinase from L. stenophrys venom, LSF. The native LSF was resistant to Edman degradation, indicating that the amino terminus of the protein is blocked, as reported for some other snake venom proteinases [1], [4], [9], [17]. The basic set of peptides was generated by CNBr cleavage of pyridylethylated LSF (peptides CN2–CN8 in Fig. 1). CNBr peptides were properly ordered after sequencing the overlapping peptides
Conclusions
We report the partial amino acid sequence of a non-haemorrhagic snake metalloproteinase, LSF, from L. stenophrys venom. Structural characteristics, e.g., zinc-binding site and Met-turn place LSF in the reprolysin family of zinc-metalloproteinases. The high level of sequence identity between non-haemorrhagic LSF and haemorrhagic metalloproteinase LHF-II from L. muta muta venom, suggests that they differ in their affinities for binding matrix proteins as a consequence of structural differences at
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