Mutations in C-natriuretic peptide (NPPC): a novel cause of autosomal dominant short stature.

PurposeC-type natriuretic peptide (CNP) and its principal receptor, natriuretic peptide receptor B (NPR-B), have been shown to be important in skeletal development. CNP and NPR-B are encoded by natriuretic peptide precursor-C (NPPC) and natriuretic peptide receptor 2 (NPR2) genes, respectively. While NPR2 mutations have been described in patients with skeletal dysplasias and idiopathic short stature (ISS), and several Npr2 and Nppc skeletal dysplasia mouse models exist, no mutations in NPPC have been described in patients to date.MethodsNPPC was screened in 668 patients (357 with disproportionate short stature and 311 with autosomal dominant ISS) and 29 additional ISS families in an ongoing whole-exome sequencing study.ResultsTwo heterozygous NPPC mutations, located in the highly conserved CNP ring, were identified. Both showed significant reductions in cyclic guanosine monophosphate synthesis, confirming their pathogenicity. Interestingly, one has been previously linked to skeletal abnormalities in the spontaneous Nppc mouse long-bone abnormality (lbab) mutant.ConclusionsOur results demonstrate, for the first time, that NPPC mutations cause autosomal dominant short stature in humans. The NPPC mutations cosegregated with a short stature and small hands phenotype. A CNP analog, which is currently in clinical trials for the treatment of achondroplasia, seems a promising therapeutic approach, since it directly replaces the defective protein.

C-type natriuretic peptide-modified lipid vesicles: fabrication and use for the treatment of brain glioma.

Chemotherapy of brain glioma faces a major obstacle owing to the inability of drug transport across the blood-brain barrier (BBB). Besides, neovasculatures in brain glioma site result in a rapid infiltration, making complete surgical removal virtually impossible. Herein, we reported a novel kind of C-type natriuretic peptide (CNP) modified vinorelbine lipid vesicles for transferring drug across the BBB, and for treating brain glioma along with disrupting neovasculatures. The studies were performed on brain glioma U87-MG cells in vitro and on glioma-bearing nude mice in vivo. The results showed that the CNP-modified vinorelbine lipid vesicles could transport vinorelbine across the BBB, kill the brain glioma, and destroy neovasculatures effectively. The above mechanisms could be associated with the following aspects, namely, long circulation in the blood; drug transport across the BBB via natriuretic peptide receptor B (NPRB)-mediated transcytosis; elimination of brain glioma cells and disruption of neovasculatures by targeting uptake and cytotoxic injury. Besides, CNP-modified vinorelbine lipid vesicles could induce apoptosis of the glioma cells. The mechanisms could be related to the activations of caspase 8, caspase 3, p53, and reactive oxygen species (ROS), and inhibition of survivin. Hence, CNP-modified lipid vesicles could be used as a carrier material for treating brain glioma and disabling glioma neovasculatures.

Paradoxical expressions of natriuretic peptide receptor-C and neutral endopeptidase account for C-type natriuretic peptide decline during the progression of experimental obstructive nephropathy.

INTRODUCTION: C-type natriuretic peptide (CNP) selectively binds to the guanylyl cyclase coupled natriuretic peptide receptor (NPR)-B and exerts more potent antihypertrophic and antifibrotic properties. Elimination of CNP occurs mainly by neutral endopeptidase (NEP) and NPR-C. METHODS: We established a rat model of unilateral ureteral obstruction (UUO) to examine the continuous change of the CNP expression and to assess the correlations of NPR-B, NPR-C, NEP with CNP in the obstructed kidneys. RESULTS: The expressions of CNP mRNA and protein in the obstructed kidneys tended to be higher immediately after ligation and declined at later time points compared to sham-operated rats, measured by real-time polymerase chain reaction (PCR) and western blot analysis. Subsequent correlation analysis indicated that CNP mRNA was positively correlated with NPR-B mRNA (r=+0.673, p<0.05). In addition, the increased expression of NPR-C (r=-0.943 and -0.837 for mRNA and protein respectively, p<0.05) and NEP (r=-0.687 and -0.823 for mRNA and protein respectively, p<0.05) were accompanied by a significant decline in CNP. CONCLUSIONS: A high level of CNP may contribute to the elevated expression of NPR-B in the early phase of UUO. More interestingly, paradoxical expressions of NPR-C and NEP may account for the decline of CNP in the obstructed kidneys.

Evaluation of the therapeutic potential of a CNP analog in a Fgfr3 mouse model recapitulating achondroplasia.

Achondroplasia (ACH), the most common form of dwarfism, is an inherited autosomal-dominant chondrodysplasia caused by a gain-of-function mutation in fibroblast-growth-factor-receptor 3 (FGFR3). C-type natriuretic peptide (CNP) antagonizes FGFR3 downstream signaling by inhibiting the pathway of mitogen-activated protein kinase (MAPK). Here, we report the pharmacological activity of a 39 amino acid CNP analog (BMN 111) with an extended plasma half-life due to its resistance to neutral-endopeptidase (NEP) digestion. In ACH human growth-plate chondrocytes, we demonstrated a decrease in the phosphorylation of extracellular-signal-regulated kinases 1 and 2, confirming that this CNP analog inhibits fibroblast-growth-factor-mediated MAPK activation. Concomitantly, we analyzed the phenotype of Fgfr3(Y367C/+) mice and showed the presence of ACH-related clinical features in this mouse model. We found that in Fgfr3(Y367C/+) mice, treatment with this CNP analog led to a significant recovery of bone growth. We observed an increase in the axial and appendicular skeleton lengths, and improvements in dwarfism-related clinical features included flattening of the skull, reduced crossbite, straightening of the tibias and femurs, and correction of the growth-plate defect. Thus, our results provide the proof of concept that BMN 111, a NEP-resistant CNP analog, might benefit individuals with ACH and hypochondroplasia.

Natriuretic peptide drug leads from snake venom.

Natriuretic peptides are body fluid volume modulators, termed natriuretic peptides due to a role in natriuresis and diuresis. The three mammalian NPs, atrial natriuretic peptide (ANP), brain or b-type natriuretic peptide (BNP) and c-type natriuretic peptide (CNP), have been extensively investigated for their use as therapeutic agents for the treatment of cardiovascular diseases. Although effective, short half-lives and renal side effects limit their use. In approximately 30 years of research, NPs have been discovered in many vertebrates including mammals, amphibians, reptiles and fish, with plants and, more recently, bacteria also being found to possess NPs. Reptiles have produced some of the more interesting NPs, with dendroaspis natriuretic peptide (DNP), which was isolated from the venom of the green mamba (Dendroaspis angusticeps), having greater potency and increased stability as compared to the mammalian family members, and taipan natriuretic peptide c (TNPc), which was isolated from the venom of the inland taipan (Oxyuranus microlepidotus) displaying similar activity to ANP and DNP at rat natriuretic peptide receptor A. Although promising, more research is required in this field to develop therapeutics that overcome receptor-mediated clearance, and potential toxicity issues. This review investigates the use of snake venom NPs as therapeutic drug leads.

C-type natriuretic peptide: Structural studies, fragment synthesis, and preliminary biological evaluation in human osteosarcoma cell lines.

Natriuretic peptides (NP) are a family of structurally related but genetically distinct hormones/paracrine factors that regulate blood volume, blood pressure, ventricular hypertrophy, pulmonary hypertension, fat metabolism, and long bone growth. In this work we present computational structural analysis of the three human NP in solution, the synthesis and preliminary biological assays of a short fragment of CNP, I(14)GSM(17), together with one small mimetic, GGSM. Synthetic peptides IGSM, GGSM, and full length CNP were preliminary tested for their ability to influence cell growth of three human osteosarcoma cell lines. Synthetic peptides were shown to successfully mimic the biological activity of the full length natural peptide: their effects, although different upon the cell types used, are in accordance with the current literature, designating a possible role for CNP, and its derivatives, in skeletogenesis.

Duck-billed platypus venom peptides induce Ca2+ influx in neuroblastoma cells.

The duck-billed platypus (Ornithorhynchus anatinus) is one of the few venomous Australian mammals. We previously found that its crude venom potently induces Ca(2+) influx in human neuroblastoma IMR-32 cells. Guided by this bioassay, we identified 11 novel peptides, including the heptapeptide H-His-Asp-His-Pro-Asn-Pro-Arg-OH (1). Compounds 1-4 and 5-11 coincided with the 6-9 N-terminal residues of Ornithorhynchus venom C-type natriuretic peptide (OvCNP) and the 132-150 part of OvCNP precursor peptide, respectively. Heptapeptide 1, which is one of the primary components of the venom fluid (approximately 200 ng/microL), induced a significant increase in [Ca(2+)](i) in IMR-32 cells at 75 microM. To the best of our knowledge, this is the first example of the isolation of the N-terminal linear fragments of CNPs in any mammal.

Novel bifunctional natriuretic peptides as potential therapeutics.

Synthetic atrial natriuretic peptide (carperitide) and B-type natriuretic peptide (BNP; nesiritide) are used to treat congestive heart failure. However, despite beneficial cardiac unloading properties, reductions in renal perfusion pressures limit their clinical effectiveness. Recently, CD-NP, a chimeric peptide composed of C-type natriuretic peptide (CNP) fused to the C-terminal tail of Dendroaspis natriuretic peptide (DNP), was shown to be more glomerular filtration rate-enhancing than BNP in dogs. However, the molecular basis for the increased responsiveness was not determined. Here, we show that the DNP tail has a striking effect on CNP, converting it from a non-agonist to a partial agonist of natriuretic peptide receptor (NPR)-A while maintaining the ability to activate NPR-B. This effect is specific for human receptors because CD-NP was only a slightly better activator of rat NPR-A due to the promiscuous nature of CNP in this species. Interesting, the DNP tail alone had no effect on any NPR even though it is effective in vivo. To further increase the potency of CD-NP for NPR-A, we converted two different triplet sequences within the CNP ring to their corresponding residues in BNP. Both variants demonstrated increased affinity and full agonist activity for NPR-A, whereas one was as potent as any NPR-A activator known. In contrast to a previous report, we found that DNP binds the natriuretic peptide clearance receptor (NPR-C). However, none of the chimeric peptides bound NPR-C with significantly higher affinity than endogenous ligands. We suggest that bifunctional chimeric peptides represent a new generation of natriuretic peptide therapeutics.

Characterization and isolation of L-to-D-amino-acid-residue isomerase from platypus venom.

Platypus venom contains an isomerase that reversibly interconverts the second amino-acid residue in some peptides between the L-form and the D-form. The enzyme acts on the natriuretic peptides OvCNPa and OvCNPb, and on the defensin-like peptides DLP-2 and DLP-4, but it does not act on DLP-1. While the isomerization of DLP-2 to DLP-4 is inhibited by the amino-peptidase inhibitor amastatin, it is not affected by the leucine amino-peptidase inhibitor bestatin. The enzyme, that is only present in minute quantities in an extract of the venom gland, is thermally stable up to 55 degrees C, and it was found by anion-exchange chromatography to be acidic. Isolation of the isomerase was carried out by combined ion-exchange chromatography and reverse-phase high performance liquid chromatography (HPLC).

C-type natriuretic peptide and heart failure.

C-type natriuretic peptide (CNP) is a peptide produced by the vascular endothelium with vasodilative properties. It shares structural and physiological properties with the atrial and brain natriuretic peptides (ANP and BNP), whose central role in the pathophysiology of heart failure (CHF) is firmly established. The role of CNP, first isolated from porcine brain, has not been yet completely determined. The transcription of the gene, that in man is located on chromosome 2, is regulated by factors such as tumor necrosis factor and interleukin-1. Two mature forms of the peptide exist: CNP-53, that predominates in tissues and CNP-22, found mainly in plasma. As recently found, CNP is produced directly in the myocardium and an increase in plasma levels of this peptide and of its precursor was observed in CHF. The aim of this review was to examine the current literature relating to cardiovascular functions of CNP and in particular to its role in CHF. In fact, CNP may represent an important new local autocrine and endocrine mediator in CHF although further evaluations are required to define its full pathophysiological role in this disease.

C-type natriuretic peptide in growth: a new paradigm.

C-type natriuretic peptide (CNP), acting through its receptor, natriuretic peptide receptor-B (NPR-B), plays a critical role in linear growth. Knockout mice for CNP and NPR-B are dwarfed, and transgenic mice overexpressing CNP are overgrown. CNP has a direct regulatory effect on growth plate chondrocytes, acting primarily to promote terminal differentiation and hypertrophy. In humans, homozygous NPR-B mutations are the cause of acromesomelic dysplasia, Maroteaux type (AMDM), a severe form of disproportionate dwarfism. A patient with AMDM and the NPR-B knockout mouse both have low insulin-like growth factor I (IGF-I) levels, suggesting an interaction between these regulatory systems. Heterozygous carriers of NPR-B mutations also have reduced stature, but no other abnormalities. Hence, heterozygous NPR-B mutations are another cause of "idiopathic" short stature. The CNP-NPR-B system has only recently been found to be an important regulator of human growth, and abnormalities in this system have clinical implications. Considerable work is needed to further understand this new paradigm of human growth regulation.

Amino-terminal proCNP: a putative marker of cartilage activity in postnatal growth.

Recent evidence from rodents and humans shows that C-type natriuretic peptide (CNP) plays an essential role in endochondral bone growth. We recently identified a stable product of proCNP, amino-terminal proCNP (NT-proCNP), which unlike CNP is readily measurable in human and ovine plasma. Hypothesizing that plasma NT-proCNP concentrations reflect in part CNP synthesis within growth plates of rapidly growing cartilage, we studied levels of CNP forms in both children and lambs and related these to age, growth velocity, and biochemical markers of bone turnover. Plasma NT-proCNP levels were elevated at birth and fell progressively with age. Significant associations between plasma NT-proCNP and height velocity, alkaline phosphatase, and type 1 collagen C telopeptide were identified in children (aged 5-18 y). In longitudinal animal studies, elevated plasma concentration of NT-proCNP in 1-wk-old lambs fell progressively to mature adult levels at age 27 wk. Plasma NT-proCNP showed a highly significant association with alkaline phosphatase and metacarpal growth velocity. Glucocorticoids, a treatment known to inhibit cartilage proliferation, reduced metacarpal growth elongation in 4-wk-old lambs and markedly lowered circulating NT-proCNP levels during the treatment period. In summary, NT-proCNP levels in blood show a strong association with growth velocity and markers of bone formation and may well serve as a useful marker of growth plate activity in humans and other mammals.

Functional analysis of genomic DNA, cDNA, and nucleotide sequence of the mature C-type natriuretic peptide gene in vascular cells.

OBJECTIVE: The aim of this study was to investigate the effect of various C-type natriuretic peptide (CNP) sequences (genomic DNA [CNPDNA], cDNA derived from mRNA [CNPcDNA], and sequence coding for 22 amino acids of the mature CNP [CNP22aa]) on the growth of primary porcine vascular cells. METHODS AND RESULTS: Gene transfer was performed with cationic lipid DOCSPER or linear polyethylenimine. All 3 CNP sequences led to significant inhibition of smooth muscle cell (SMC) proliferation. In contrast, significant stimulation of cell growth was observed in endothelial cells (ECs) using CNPDNA or CNPcDNA but not CNP22aa. In a porcine restenosis model, a significant reduction in neointima hyperplasia was found 3 months after application of the CNPcDNA vector compared with the control transfection. CONCLUSIONS: The results demonstrate that the first intron in the CNP sequence does not contain any additional enhancer-binding sites. However, the signal sequence is indispensable for secretion of CNP and its appropriate physiological function. Furthermore, the results show for the first time the therapeutic effect of CNP using liposome-mediated gene transfer and local adventitial delivery. Advantages of the CNP gene are its dual effects with inhibition of SMC proliferation and simultaneous promotion of EC growth. Functional analysis of various C-type natriuretic peptide (CNP) sequences on growth of vascular cells. For the first time, dual therapeutic effects of CNP with inhibition of smooth muscle cell proliferation and stimulation of re-endothelialization were demonstrated in a pig restenosis model using liposome-mediated gene transfer and local adventitial delivery.

Cardiovascular effects of natriuretic peptides and their interrelation with endothelin-1.

The natriuretic peptides are a group of structurally related but genetically distinct peptides. Four types of natriuretic peptides have been found thus far: atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP) and Dendroaspis natriuretic peptide (DNP). ANP and BNP are secreted mainly from the heart and function as hormones with vasodilatory and natriuretic effects. CNP originates mainly from endothelial cells with a paracrine effect to induce vasodilation. Other effects of natriuretic peptides including negative inotropy, antimitogenic and anticoagulation have been described. Three types of natriuretic peptide receptors mediate their functions, and among them two are cGMP-coupled. Clearance of natriuretic peptides is via its clearance receptor through the action of neutral endopeptidases. Natriuretic peptides interact with other vasoactive peptides including endothelin. The putative role of natriuretic peptides in the pathophysiology of various cardiovascular diseases including congestive heart failure, hypertension, ischemic heart disease, and cardiomyopathy are discussed. Natriuretic peptide plasma levels are used for the diagnosis and therapeutic follow-up of congestive heart failure patients. Increasing the levels of natriuretic peptides by natriuretic peptide mimetics and neutral endopeptidase inhibitors may provide a new therapeutic strategy for the treatment of cardiovascular diseases such as congestive heart failure and hypertension.

Furin-mediated processing of Pro-C-type natriuretic peptide.

C-type natriuretic peptide (CNP) is a member of the natriuretic peptide family that is involved in a variety of homeostatic processes. Here we characterize the processing essential for the conversion of the precursor, human pro-CNP, to the biologically active hormone. In human embryonic kidney 293 and chondrosarcoma SW 1353 cells, recombinant pro-CNP was converted into a mature peptide intracellularly as detected by Western analysis. Expression of recombinant human corin, a proatrial natriuretic peptide convertase, did not enhance the processing of pro-CNP in these cells. The processing of pro-CNP was inhibited in the presence of an inhibitor of the endoprotease furin but was not affected by inhibitors of matrix metalloproteinases and tumor necrosis factor-alpha convertase. In furin-deficient human colon adenocarcinoma LoVo cells, no conversion of recombinant pro-CNP to CNP was detected. Expression of recombinant human furin in LoVo cells restored the ability of these cells to process pro-CNP. Furthermore, incubation of purified recombinant human furin with LoVo cell lysate containing pro-CNP led to the conversion of the precursor to a mature peptide. The furin-processed CNP was shown to be biologically active in a cell-based cGMP assay. These results demonstrate that furin is a critical enzyme for the processing of human pro-CNP.

Diagnostic and prognostic usefulness of natriuretic peptides in emergency department patients with dyspnea.

More than 4.5 million Americans have congestive heart failure (CHF), close to 550,000 new cases are diagnosed each year, and one third of known patients with CHF are annually admitted to the hospital. Emergency department diagnosis of CHF is often based on history and physical examination findings along with results of ancillary tests, such as chest radiography and ECG. Although signs and symptoms of fluid overload, such as lower extremity edema and dyspnea, raise the suspicion of CHF, their lack of sensitivity makes them poor screening tools. The natriuretic peptides are promising markers of myocardial dysfunction and heart failure. Because of their relationship to myocardial pressure and stretching, natriuretic peptides have been investigated over the past 5 decades as both diagnostic and prognostic markers in acute coronary syndromes and CHF. This article discusses each of the natriuretic peptides and attempts to delineate their potential diagnostic and prognostic roles in the ED.

Release of C-type natriuretic peptide accounts for the biological activity of endothelium-derived hyperpolarizing factor.

Endothelial cells in most vascular beds release a factor that hyperpolarizes the underlying smooth muscle, produces vasodilatation, and plays a fundamental role in the regulation of local blood flow and systemic blood pressure. The identity of this endothelium-derived hyperpolarizing factor (EDHF), which is neither NO nor prostacyclin, remains obscure. Herein, we demonstrate that in mesenteric resistance arteries, release of C-type natriuretic peptide (CNP) accounts for the biological activity of EDHF. Both produce identical smooth muscle hyperpolarizations that are attenuated in the presence of high [K(+)], the G(i) G protein (G(i)) inhibitor pertussis toxin, the G protein-gated inwardly rectifying K(+) channel inhibitor tertiapin, and a combination of Ba(2+) (inwardly rectifying K(+) channel blocker) plus ouabain (Na(+)K(+)-ATPase inhibitor). Responses to EDHF and CNP are unaffected by the natriuretic peptide receptor (NPR)-AB antagonist HS-142-1, but mimicked by the selective NPR-C agonist, cANF(4-23). EDHF-dependent relaxation is concomitant with liberation of endothelial CNP; in the presence of the myoendothelial gap-junction inhibitor 18alpha-glycyrrhetinic acid or after endothelial denudation, CNP release and EDHF responses are profoundly suppressed. These data demonstrate that acetylcholine-evoked release of endothelial CNP activates NPR-C on vascular smooth muscle that via a G(i) coupling promotes Ba(2+)ouabain-sensitive hyperpolarization. Thus, we have revealed the identity of EDHF and established a pivotal role for endothelial-derived CNP in the regulation of vascular tone and blood flow.

Identification of amino-terminal pro-C-type natriuretic peptide in human plasma.

We report the first identification of a circulating peptide from the amino-terminal end of proCNP. A specific radioimmunoassay was established based on antisera to the synthetic peptide proCNP(1-15). Extracts of plasma, drawn from patients with congestive heart failure or from sheep with experimental heart failure, were subjected to size exclusion and reverse-phase high-pressure liquid chromatography (HPLC) coupled to radioimmunoassay (RIA). These studies revealed the presence of an immunoreactive peptide with a molecular weight (M(r) approximately 5 kDa) similar to that expected for NT-proCNP(1-50), a potential fragment released during processing of pro(CNP). The same material was isolated from extracts of homogenized ovine pituitary, a tissue known to be a relatively enriched source of CNP. Plasma NT-proCNP levels in 22 patients with congestive heart failure (9.7 +/- 0.5 pmol/L, mean +/- SEM, range 5.4-13.7 pmol/L) were raised (P = 0.003) compared to those in 16 healthy volunteers (7.4 +/- 0.3 pmol/L, range 5.7-10.7 pmol/L) and were higher than levels reported for CNP in similar subjects. This first identification of circulating NT-proCNP opens the possibility of studying the factors regulating CNP production and metabolism in vivo.

cDNA cloning of bradykinin-potentiating peptides-C-type natriuretic peptide precursor, and characterization of the novel peptide Leu3-blomhotin from the venom of Agkistrodon blomhoffi.

A cDNA clone, 1.8 kb long, was isolated from a venom gland cDNA library of Agkistrodon blomhoffi that encodes a large plurifunctional precursor composed of 263 amino-acid residues. Nucleotide sequence analysis of this clone revealed that sequences which code for blomhotin and a novel peptide Leu3-blomhotin are located in the N-terminal region of the precursor polypeptide, followed by four tandemly aligned sequences which code for three types of bradykinin-potentiating peptide. In the C-terminal region, the sequence for the C-type natriuretic peptide was located along with a preceding processing signal. The deduced amino-acid sequences for the four bradykinin-potentiating peptides coincided exactly with previously known sequences for potentiator B, potentiator C and potentiator E. The actual Leu3-blomhotin peptide was subsequently isolated from the venom of A. blomhoffi and characterized. Leu3-blomhotin possesses contractile activity in isolated rat stomach fundus smooth muscle in the same manner as blomhotin. Furthermore, it was shown that blomhotin and Leu3-blomhotin retained activity to inhibit the angiotensin-converting enzyme.

Bradykinin-potentiating peptides and C-type natriuretic peptides from snake venom.

Cloning of cDNAs encoding bradykinin-potentiating peptides (BPPs)-C-type natriuretic peptide (CNP) precursor or its homologue was performed for cDNA libraries of Bothrops jararaca (South American snake), Trimeresurus flavoviridis, Trimeresurus gramineus and Agkistrodon halys blomhoffi (Asian snakes), all belonging to Crotalinae subfamily. Each cDNA library was constructed from the venom glands of a single snake to preclude ambiguity by intraspecies variation in venom components. Thirteen positive clones derived from B. jararaca were divided into two types depending on restriction sites. Differences in the nucleotide sequence arise at three locations and two of them accompanied amino acid conversions. Despite the differences, both types of cDNA clones encode the BPP-CNP precursor of 256 amino acid residues. Sequence analysis demonstrated that cDNA clones from three Asian snakes encode homologues of the BPP-CNP precursor from B. jararaca. In a precursor polypeptide, a signal sequence (approximately 25 aa) at the N-terminus is followed by sequences of BPP or the analogue (5-13 aa) with flanking spacer sequences (indefinite number of aa), an intervening linker sequence (approximately 144 aa) with unidentified function, and a CNP sequence (22 aa) with a preceding processing signal sequence (10 aa). cDNA clones from A. halys blomhoffi encode two distinct peptides in place of BPP, and T. flavoviridis and T. gramineus were shown to have considerably different sequences in the BPP domain from those known as BPP sequences. The present results provide evidence for a wide distribution of the orthologous gene expressing a series of bioactive peptides among Crotalinae subfamily.