ÁREA: Bioquímica e Biotecnologia
TÍTULO: A novel natriuretic peptide from the venom of the Crotalus oreganus abyssus (North American Grand Canyon rattlesnake)
AUTORES: DA SILVA, S.L. (UFSJ) ; MARANGONI, S. (UNICAMP)
RESUMO: New natriuretic peptides (NPs) have been isolated and characterized from the venom of
the Crotalinae family; the NP family comprises three members: ANP, BNP and CNP, and has
an important role in blood pressure regulation and electrolyte homeostasis. The aim of
the present study was to characterize a novel natriuretic-like peptide (Coa_NP2),
isolated from Crotalus Oreganus abyssus venom. The Coa_NP2 presents an average
molecular mass of 3419.88 Da and its amino acid sequence has 32 amino acids and its
complete sequence is SYGISSGCFGLKLDRIGTMSGLGCWRLLQDSP. We showed that Coa_NP2 produces
a dose-dependent decrease in mean arterial pressure in rats, followed by significant
increases in plasma nitrite levels and vasorelaxation in a thoracic aortic ring bath.
PALAVRAS CHAVES: crotalus oreganus abyssus, hypotensive agents, natriuretc peptides.
INTRODUÇÃO: Many snake venoms contain toxins that produce interesting cardiovascular effects,
such as hypotension, or bradykinin-potentiating peptides or renal effects.
Natriuretic peptides (NPs) are body fluid volume modulators, termed natriuretic
peptides, due to a role in natriuresis and dieresis. The three mammalian NPs, atrial
natriuretic peptide (ANP), brain or b-type natriuretic peptide (BNP) and cardiac or
c-type natriuretic peptide (CNP), have been extensively investigated for their use as
therapeutic agents for the treatment of cardiovascular diseases.
Human NPs form a family of structurally-similar polypeptides. They have a highly
conserved 17-residue intra-molecular disulphide loop (CFGxxxDRIxxxSGLGC), which is
important for their biological activity. Within this cyclic structure, nine amino
acids are identical in all three classes of NPs. However, they differ from each other
in that they have different numbers of amino acid residues at the N- and C-terminal
portion of the peptide. In 1992, it was identified the first venom NP from the green
mamba snake, Dendroaspis angusticeps, and named it as the Dendroaspis natriuretic
peptide (DNP). Recently, another NP isolated from the venom of the Lachesis muta
snake (Lm-CNP) was identified with a similar structure to human CNP. In addition, a
cDNA sequence of Bj-CNP from Bothrops jararaca has also been shown to encode a CNP-
like NP and seven bradykinin-potentiating peptides. On the other hand, knowledge
regarding the composition of Crotalus oreganus abyssus venom is scarce. From previous
experiments in our laboratory that have studied the effects of peptides isolated from
Crotalus oreganus abyssuss venom, we showed the presence of a natriuretic peptide in
its venom (now called Coa_NP1) that produced hypotensive and vasorelaxation effects.
MATERIAL E MÉTODOS: Crotalus oreganus abyssuss (Coa) whole venom was submitted to an FPLC chromatographic
column packed with Sephadex G-75. Only fraction CoaV presented a hypotensive effect.
Fraction CoaV was then submitted to ultra-filtration using the MidJet apparatus,
equipped with the UFP-10-C-MM01A cartridge. The filtrate was subjected to reverse
phase HPLC using an analytical C5 column. The peptides were purified using a linear
gradient of buffer B concentration (66% acetonitrile in buffer A) and the
chromatographic UV monitoring was carried out at 216 nm. The purified peptide was
dissolved in guanidine chloride solution. The reduced peptide was sequenced using an
automatic peptide sequencer. The phenylthyoidantoin (PTH) amino acids were identified
by comparing their retention times to the 20 PTH amino acid standards. The molecular
mass of isolated Coa-NP was analyzed by MALDI-TOF mass spectrometry using a Voyager-
DE PRO MALDI-TOF apparatus. Male Wistar rats were used in this study. To blood
pressure measurements male Wistar rats were anesthetized and thereafter, the right
carotid artery was cannulated with a polyethylene tube. Mean arterial pressure was
continuously recorded using a pressure transducer connected to a polygraph. Solutions
used as controls and tests (Coa_NP2) were injected through a catheter. To aortic ring
experiments male Wistar rats were killed and the descending thoracic aorta was
rapidly removed and flushed with physiological solution. 5 mm rings were obtained
from preparations of endothelium-intact and endothelium-denuded. The aortic rings
were submitted to a tension of 1g during a 60-min equilibration period and were
considered to have an intact functional endothelium when acetylcholine produced a
relaxation of more than 80%.
RESULTADOS E DISCUSSÃO: After the elution of the whole venom of Crotalus oreganus abyssus) using gel
filtration (Sephadex G-75), the filtrate obtained from ultra filtration was analyzed
by tricine SDS-PAGE electrophoresis and presented protein and peptides bands with
molecular masses of around or smaller than 10 kDa. The filtrate decreased blood
pressure and was further purified by C5-HPLC. The RP-HPLC chromatography of filtrate
demonstrated seven different peaks and one peak was presented the natriuretic
activity. The complete amino acid sequence of Coa_NP2 was carried out by Edman
degradation and average molecular mass (3419.88 Da) was confirmed by mass
spectrometry. The amino acid sequence of Coa_NP presented the loop region that is
characteristic of natriuretic peptides (NP domain consensus = CFGxxxDRIxxxSGLGC) and
presented 8 amino acid residue extensions following the NP domain in the sequence.
The amino acid sequence of Coa_NP2 was identified as:
SYGISSGCFGLKLDRIGTMSGLGCWRLLQDSP. As expected for natriuretic-like peptides, the
primary structure revealed two half cysteines, suggesting the presence of one
disulfide bridge and belongs to the ANP/BNP-like family, since the carboxyterminal
regions of C-natriuretic peptides (CNP) end in NP domains. The natriuretic peptide
isolated from Crotalus oreganus abyssus venom (Coa_NP2) caused a dose-dependent
decrease in the arterial pressure median after its intravenous infusion. We may
observe an increase in the production of nitrite after the infusion of the Coa_NP2
isolated from the Crotalus oreganus abyssus venom. Coa_NP2 induced a relaxant effect
on endothelium-intact thoracic aortic rings that were precontracted with
phenylephrine in the absence and presence of ISATIN. Coa_NP2 failed to relax
endothelium-intact aortic ring.
CONCLUSÕES: This study describes the isolation of a new natriuretic peptide (Coa_NP2), whose
primary structure was determined as SYGISSGCFGLKLDRIGTMSGLGCWRLLQDSP. We found that
the natriuretic peptide isolated from C. o. abyssus venom (Coa_NP2) presented a
homologous structure to ANP and BNP. Furthermore, the mean functional findings of
this present study were that (i) the Coa_NP2 produced a dose-dependent decrease in
the mean arterial pressure; (ii) this hypotensive effect occurred along with a
significant increase in plasma nitrite levels; and (iii) the vasorelaxation produced
by the natriuretic.
AGRADECIMENTOS: We would like to thank CAPES, CNPq, FAPEMIG and FAPESP (Brazilian agencies) for
financial support.
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