Is C-type natriuretic peptide regulated by a feedback loop? A study on systemic and local autoregulatory effect.

C-type natriuretic peptide (CNP) is a pivotal enhancer of endochondral bone growth and is expected to be a therapeutic reagent for impaired skeletal growth. Although we showed that CNP stimulates bone growth as a local regulator in the growth plate via the autocrine/paracrine system, CNP is abundantly produced in other various tissues and its blood concentration is reported to correlate positively with growth velocity. Therefore we investigated the systemic regulation of CNP levels using rodent models. In order to examine whether CNP undergoes systemic feedback regulation, we investigated blood CNP levels and local CNP expression in various tissues, including cartilage, of 4-week-old rats after systemic administration of sufficient amounts of exogenous CNP (0.5 mg/kg/day) for 3 days. This CNP administration did not alter blood NT-proCNP levels in male rats but decreased mRNA expression only in tissue that included cartilage. Decrease in expression and blood NT-proCNP were greater in female rats. To analyze the existence of direct autoregulation of CNP in the periphery as an autocrine/paracrine system, we estimated the effect of exogenous supplementation of CNP on the expression of endogenous CNP itself in the growth plate cartilage of extracted fetal murine tibias and in ATDC5, a chondrogenic cell line. We found no alteration of endogenous CNP expression after incubation with adequate concentrations of exogenous CNP for 4 and 24 hours, which were chosen to observe primary and later transcriptional effects, respectively. These results indicate that CNP is not directly autoregulated but indirectly autoregulated in cartilage tissue. A feedback system is crucial for homeostatic regulation and further studies are needed to elucidate the regulatory system of CNP production and function.

Fluorescent turn-on assay of C-type natriuretic peptide using a molecularly imprinted ratiometric fluorescent probe with high selectivity and sensitivity.

A novel molecularly imprinted ratiometric fluorescent probe was fabricated by simple sol-gel polymerization for selective and sensitive assay of C-type natriuretic peptide (CNP) in biosamples. Both the nitrobenzoxadiazole (NBD) and carbon dots (CDs) were located on the surface of silica, used as the detection signal and reference signal, respectively. For the turn-on-based probe, the fluorescence intensity of NBD could be quantitatively enhanced by CNP based on the strategy of photo-induced electron transfer (PET), while the fluorescence of CDs remained unchanged. The obtained probe exhibited excellent recognition selectivity and fast kinetics to CNP templates, and also showed good stability. The linear range of CNP determination was 5-80 pg mL-1 with a low detection limit of 2.87 pg mL-1. Finally, the probe was successfully applied to determine CNP in human serum samples and attained high recoveries between 97.3 and 104% with precisions below 4.7%. The result indicates that the proposed method has promising potential for the assay of trace peptides in complex matrices. Schematic illustration for the formation and determination mechanism of the probe.

Circulating products of C-type natriuretic peptide and links with organ function in health and disease.

Paracrine actions of CNP and rapid degradation at source severely limit study of CNP's many roles in vivo. However provided sensitive and validated assays are used, there is increasing evidence that low concentrations of bioactive CNP in plasma, and the readily detectable concentrations of the bio-inactive processed product of proCNP (aminoterminal proCNP), can be used to advance understanding of the hormone's role in pathophysiology. Provided renal function is normal, concordant changes in both CNP and NTproCNP reflect change in tissue production of proCNP whereas change in CNP alone results from altered rates of bioactive CNP degradation and are reflected in the ratio of NTproCNP to CNP. As already shown in juveniles, where plasma concentration of CNP products are higher and are associated with concurrent endochondral bone growth, measurements of plasma CNP products in mature adults have potential to clarify organ response to stress and injury. Excepting the role of CNP in fetal-maternal welfare, this review examines evidence linking plasma CNP products with function of a wide range of tissues in adults, including the impact of extraneous factors such as nutrients, hormone therapy and exercise.

ASB20123: A novel C-type natriuretic peptide derivative for treatment of growth failure and dwarfism.

C-type natriuretic peptide (CNP) and its receptor natriuretic peptide receptor B (NPR-B) are physiological potent positive regulators of endochondral bone growth; therefore, the CNP/NPR-B signaling pathway is one of the most promising therapeutic targets for treating growth failure and dwarfism. In this article, we summarized the pharmacological properties of a novel CNP analog peptide ASB20123 as a therapeutic agent for short stature. ASB20123, one of the CNP/ghrelin chimeric peptides, is composed of CNP(1-22) and human ghrelin(12-28, E17D). Compared to CNP(1-22), ASB20123 showed similar agonist activity for NPR-B and improved biokinetics with a longer plasma half-life in rats. In addition, the distribution of ASB20123 to the cartilage was higher than that of CNP(1-22) after single subcutaneous (sc) injection to mice. These results suggested that the C-terminal part of ghrelin, which has clusters of basic amino acid residues and a BX7B motif, might contribute to the retention of ASB20123 in the extracellular matrix of the growth plate. Multiple sc doses of ASB20123 potently stimulated skeletal growth in rats in a dose-dependent manner, and sc infusion was more effective than bolus injection at the same dose. Our data indicated that high plasma levels of ASB20123 would not necessarily be required for bone growth acceleration. Thus, pharmaceutical formulation approaches for sustained-release dosage forms to allow chronic exposure to ASB20123 might be suitable to ensure drug effectiveness and safety.

Biophysical screening methods for extracellular domain peptide receptors, application to natriuretic peptide receptor C ligands.

Endothelium-derived C-type natriuretic peptide possesses cytoprotective and anti-atherogenic functions that regulate vascular homeostasis. The vasoprotective effects of C-type natriuretic peptide are somewhat mediated by the natriuretic peptide receptor C, suggesting that this receptor represents a novel therapeutic target for the treatment of cardiovascular diseases. In order to facilitate our drug discovery efforts, we have optimized an array of biophysical methods including surface plasmon resonance, fluorescence polarization and thermal shift assays to aid in the design, assessment and characterization of small molecule agonist interactions with natriuretic peptide receptors. Assay conditions are investigated to explore the feasibility and dynamic range of each method, and peptide-based agonists and antagonists are used as controls to validate these conditions. Once established, each technique was compared and contrasted with respect to their drug discovery utility. We foresee that such techniques will facilitate the discovery and development of potential therapeutic agents for NPR-C and other large extracellular domain membrane receptors.

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.

Cenderitide: structural requirements for the creation of a novel dual particulate guanylyl cyclase receptor agonist with renal-enhancing in vivo and ex vivo actions.

AIMS: Cenderitide is a novel dual natriuretic peptide (NP) receptor chimeric peptide activator, which targets the particulate guanylyl cyclase B (pGC-B) receptor and pGC-A unlike native NPs. Cenderitide was engineered to retain the anti-fibrotic properties of C-type natriuretic peptide (CNP)/pGC-B with renal-enhancing actions facilitated by fusion to the carboxyl terminus of Dendroaspis NP (DNP), a pGC-A agonist, to CNP. Here, we address significance of the DNP carboxyl terminus in dual pGC receptor activation and actions of cenderitide compared with CNP on renal function and cyclic guanosine monophosphate (cGMP) in vivo and ex vivo in normal canines. METHODS AND RESULTS: In vitro, only cenderitide and not CNP or three CNP-based variants was a potent dual pGC-A/pGC-B activator of cGMP production (from 5 to 237 pmol/mL) in human embryonic kidney (HEK) 293 cells overexpressing human pGC-A while in pGC-B overexpressing cells cenderitide increased cGMP production (from 4 to 321 pmol/mL) while the three CNP-based variants were weak agonists. Based upon our finding that the DNP carboxyl terminus is a key structural requirement for dual pGC-A/pGC-B activation, we defined in vivo the renal-enhancing actions of cenderitide compared with CNP. Cenderitide increased urinary cGMP excretion (from 989 to 5977 pmol/mL), net generation of renal cGMP (821-4124 pmol/min), natriuresis (12-242 µEq/min), and glomerular filtration rate (GFR) (37-51 mL/min) while CNP did not. We then demonstrated the transformation of CNP ex vivo into a renal cGMP-activating peptide which increased cGMP in freshly isolated glomeruli eight-fold greater than CNP. CONCLUSION: The current study establishes that dual pGC-A and pGC-B activation with CNP requires the specific carboxyl terminus of DNP. In normal canines in vivo and in glomeruli ex vivo, the carboxyl terminus of DNP transforms CNP into a natriuretic and GFR-enhancing peptide. Future studies of cenderitide are warranted in cardiorenal disease states to explore its efficacy in overall cardiorenal homeostasis.

[C-TYPE NATRIURETIC PEP- TIDE: WHAT, WHERE AND WHAT FOR IS THIS?].

The up-to-day world-wide data about the structure, distribution, and physiological effects of the most poorly known among the natriuretic peptides--the C-type (CNP)--are summarized in the review. Despite its name, this peptide does not stimulate sodium excretion but shares the prominent vasodilating and antyproliferating effects in different organs and tissues. The special emphasis is attended to CNP functions in central nervous system. The information about the peptide molecular biology, including intracellular processing, blood peptide concentration, specific receptors structure, and signaling pathways in target cells is presented.

Identification of snake bradykinin-potentiating peptides (BPPs)-simile sequences in rat brain--Potential BPP-like precursor protein?

Bradykinin-potentiating peptides (BPPs) from the South American pit viper snake venom were the first natural inhibitors of the human angiotensin I-converting enzyme (ACE) described. The pioneer characterization of the BPPs precursor from the snake venom glands by our group showed for the first time the presence of the C-type natriuretic peptide (CNP) in this same viper precursor protein. The confirmation of the BPP/CNP expression in snake brain regions correlated with neuroendocrine functions stimulated us to pursue the physiological correlates of these vasoactive peptides in mammals. Notably, several snake toxins were shown to have endogenous physiological correlates in mammals. In the present work, we expressed in bacteria the BPPs domain of the snake venom gland precursor protein, and this purified recombinant protein was used to raise specific polyclonal anti-BPPs antibodies. The correspondent single protein band immune-recognized in adult rat brain cytosol was isolated by 2D-SDS/PAGE and/or HPLC, before characterization by MS fingerprint analysis, which identified this protein as superoxide dismutase (SOD, EC 1.15.1.1), a classically known enzyme with antioxidant activity and important roles in the blood pressure modulation. In silico analysis showed the exposition of the BPP-like peptide sequences on the surface of the 3D structure of rat SOD. These peptides were chemically synthesized to show the BPP-like biological activities in ex vivo and in vivo pharmacological bioassays. Taken together, our data suggest that SOD protein have the potential to be a source for putative BPP-like bioactive peptides, which once released may contribute to the blood pressure control in mammals.

Controlled release of C-type natriuretic peptide by microencapsulation dampens proinflammatory effects induced by IL-1ß in cartilage explants.

C-type natriuretic peptide (CNP) exhibits potent anti-inflammatory effects in chondrocytes that have the potential to repair cartilage damage observed in osteoarthritis (OA). However, treatments for OA have been challenging due to poor targeting and delivery of therapeutics. The present study fabricated polyelectrolyte microcapsules loaded with CNP and examined whether the layer-by-layer (LbL) approach could have protective effects in cartilage explants treated with the pro-inflammatory cytokine, interleukin-1ß (IL-1ß). SEM showed uniform, 2 to 3 µm spherical microcapsules with morphological characteristic similar to templates loaded with or without CNP. The protein was localized around the external surface of the microcapsules with encapsulation efficiencies >82.9%. CNP release profiles were broadly similar following 9 days of culture. The presence of CNP microcapsules did not significantly affect cell viability (80%) with DNA values that remained stable throughout the culture conditions. Confocal imaging showed clustering of microcapsules in chondrocytes to natriuretic peptide receptor (Npr) 2 and 3. Treatment of cartilage explants with CNP microcapsules led to concentration-dependent inhibition of NO release in response to IL-1ß and restoration of matrix synthesis. In summary, we demonstrate controlled delivery of CNP to dampen pro-inflammatory effects induced by IL-1ß in cartilage explants. The LbL approach has the potential to promote cartilage repair in vivo.

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.

Isolation, homology modeling and renal effects of a C-type natriuretic peptide from the venom of the Brazilian yellow scorpion (Tityus serrulatus).

Mammalian natriuretic peptides (NPs) have been extensively investigated for use as therapeutic agents in the treatment of cardiovascular diseases. Here, we describe the isolation, sequencing and tridimensional homology modeling of the first C-type natriuretic peptide isolated from scorpion venom. In addition, its effects on the renal function of rats and on the mRNA expression of natriuretic peptide receptors in the kidneys are delineated. Fractionation of Tityus serrulatus venom using chromatographic techniques yielded a peptide with a molecular mass of 2190.64 Da, which exhibited the pattern of disulfide bridges that is characteristic of a C-type NP (TsNP, T. serrulatus Natriuretic Peptide). In the isolated perfused rat kidney assay, treatment with two concentrations of TsNP (0.03 and 0.1 µg/mL) increased the perfusion pressure, glomerular filtration rate and urinary flow. After 60 min of treatment at both concentrations, the percentages of sodium, potassium and chloride transport were decreased, and the urinary cGMP concentration was elevated. Natriuretic peptide receptor-A (NPR-A) mRNA expression was down regulated in the kidneys treated with both concentrations of TsNP, whereas NPR-B, NPR-C and CG-C mRNAs were up regulated at the 0.1 µg/mL concentration. In conclusion, this work describes the isolation and modeling of the first natriuretic peptide isolated from scorpion venom. In addition, examinations of the renal actions of TsNP indicate that its effects may be related to the activation of NPR-B, NPR-C and GC-C.

CD-NP: a novel engineered dual guanylyl cyclase activator with anti-fibrotic actions in the heart.

Natriuretic peptides (NPs) are cardioprotective through the activation of guanylyl cyclase (GC) receptors A and B. CD-NP, also known as cenderitide, is a novel engineered NP that was designed to uniquely serve as a first-in-class dual GC receptor agonist. Recognizing the aldosterone suppressing actions of GC-A activation and the potent inhibitory actions on collagen synthesis and fibroblast proliferation through GC-B activation, the current study was designed to establish the anti-fibrotic actions of CD-NP, administered subcutaneously, in an experimental rat model of early cardiac fibrosis induced by unilateral nephrectomy (UNX). Our results demonstrate that a two week subcutaneous infusion of CD-NP significantly suppresses left ventricular fibrosis and circulating aldosterone, while preserving both systolic and diastolic function, in UNX rats compared to vehicle treated UNX rats. Additionally we also confirmed, in vitro, that CD-NP significantly generates the second messenger, cGMP, through both the GC-A and GC-B receptors. Taken together, this novel dual GC receptor activator may represent an innovative anti-fibrotic therapeutic agent.

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.

A novel method for the large-scale production of PG-CNP37, a C-type natriuretic peptide analogue.

Achondroplasia is the most common form of human dwarfism caused by a mutation in the fibroblast growth factor receptor 3 (FGFR3), resulting in abnormal endochondral bone formation. C-type natriuretic peptide (CNP) is a potent stimulator of endochondral bone growth and represents a potential therapy for achondroplasia. We have developed a novel, simple and cost effective method to produce a CNP analogue, PG-CNP37, at a large scale from Escherichia coli. A PG-CNP37 fusion protein was over-expressed as inclusion bodies in E. coli, which were purified then cleaved by formic acid to release the PG-CNP37 peptide. Approximately 0.5g of 95% pure, soluble and active PG-CNP37 peptide was produced from 1L of culture using this method and may represent a viable means for large-scale production of other therapeutic peptides.

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.

AC-NP: a novel chimeric peptide with natriuretic and vasorelaxing actions.

The aim of this study was to evaluate the cardiovascular and renal activities of a newly designed natriuretic peptide (NP). Here, we engineered a novel 28-amino acid chimeric peptide, termed AC-NP that combined the 17-amino acid ring of C type natriuretic peptide (CNP) with the 6-amino acid N-terminus and 5-amino acid C-terminus of atrial natriuretic peptide (ANP). Both in vitro and in vivo experiments were performed to determine the actions of AC-NP. In normal rats, AC-NP proved to be more potentially diuretic, natriuretic and hypotensive compared with other NPs, such as ANP, CNP and vasonatrin peptide (VNP), which is another man-made NP. In relaxation of isolated abdominal aorta from rat, AC-NP was equally effective to ANP, CNP and VNP. Elevated levels of 3',5'-guanosine monophosphate (cGMP) in plasma and urine cGMP excretion indicated the participation of cGMP in the functions of AC-NP. Taken together, innovative designed AD-NP might be a new candidate therapeutic peptide against cardiorenal disorders.

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.

Effect of sialylated O-glycans in pro-brain natriuretic peptide stability.

BACKGROUND: Atrial, brain, and C-type natriuretic peptides (ANP, BNP, and CNP) are important in regulating a variety of cardiovascular and cellular functions. In cells, these peptides are made as proforms that are converted to mature forms. BNP and its related peptides are biomarkers for the diagnosis of heart failure. In this study, we examined glycosylation in pro-ANP, pro-BNP, and pro-CNP, which may alter their biochemical and metabolic properties. METHODS: Human pro-ANP, pro-BNP, and pro-CNP were expressed in HEK 293 cells and murine HL-1 cardiomyocytes and analyzed by immunoprecipitation and Western blotting. We used deglycosylation enzymes to determine the carbohydrate content on these peptides and examined the effects of inhibiting O-glycosylation on cellular expression and stability of the peptides. RESULTS: In HEK 293 and HL-1 cells, pro-BNP, but not pro-ANP and pro-CNP, from the culture medium had a greater molecular mass than that from cell lysate. Digestion with PNGase F, O-glycosidase, and sialidase A indicated that pro-BNP contained O-glycans but not N-glycans. The O-glycans on pro-BNP had sialic acids at their termini, protecting it from O-glycosidase digestion. In contrast, pro-ANP and pro-CNP contained no detectable amounts of N- or O-glycans. Inhibition of O-glycosylation on pro-BNP did not prevent its expression in the cells. However, partially O-glycosylated pro-BNP was much less stable than fully O- glycosylated pro-BNP. CONCLUSIONS: O-glycosylation is not necessary for pro-BNP expression but important for its stability.