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. Author manuscript; available in PMC: 2012 Aug 5.
Published in final edited form as: Biochem Biophys Res Commun. 2011 Jul 6;411(3):593–598. doi: 10.1016/j.bbrc.2011.06.192

Glycosylation and Processing of Pro-B-type Natriuretic Peptide in Cardiomyocytes

Jianhao Peng a, Jingjing Jiang a, Wei Wang a, Xiaofei Qi a,b, Xue-Long Sun c, Qingyu Wu a,b,c
PMCID: PMC3152652  NIHMSID: NIHMS310455  PMID: 21763278

Abstract

B-type natriuretic peptide (BNP) and its related peptides are biomarkers for the diagnosis of heart failure. Recent studies identified several O-glycosylation sites, including Thr-71, on human pro-BNP but the functional significance was unclear. In this study, we analyzed glycosylation and proteolytic processing of pro-BNP in cardiomyocytes. Human pro-BNP wild-type (WT) and mutants were expressed in HEK 293 cells and murine HL-1 cardiomyocytes. Pro-BNP and BNP were analyzed by immunoprecipitation and Western blotting. Glycosidases and glycosylation inhibitors were used to examine carbohydrates on pro-BNP. The effects of furin and corin expression on pro-BNP processing in cells also were examined. We found that in HEK 293 cells, recombinant pro-BNP contained significant amounts of O-glycans with terminal oligosialic acids. Mutation at Thr-71 reduced O-glycans on pro-BNP and increased pro-BNP processing. In HL-1 cardiomyocytes, residue Thr-71 contained little O-glycans, and pro-BNP WT and T71A mutant were processed similarly. In HEK 293 cells, pro-BNP was processed by furin. Mutations at Arg-73 and Arg-76, but not Lys-79, prevented pro-BNP processing. In HL-1 cardiomyocytes, which express furin and corin, single or double mutations at Arg-73, Arg-76 and Lys-79 did not prevent pro-BNP processing. Only when all these three residues were mutated, was pro-BNP processing completely blocked. Our data indicate that pro-BNP glycosylation in cardiomyocytes differed significantly from that in HEK 293 cells. In HEK 293 cells, furin cleaved pro-BNP at Arg-76 whereas in cardiomyocytes corin cleaved pro-BNP at multiple residues including Arg-73, Arg-76 and Lys-79.

Keywords: BNP, pro-BNP, corin, furin, glycosylation, cardiomyocytes

1. Introduction

B-type, or brain, natriuretic peptide (BNP) is a cardiac hormone that regulates blood pressure by promoting natriuresis, diauresis and vasodilation [1-3]. As a compensatory mechanism, BNP production is elevated in hypertrophic and failing hearts. Such a pathophysiologic response has been exploited to use BNP and its related peptides as biomarkers for the diagnosis of heart failure (HF) [4].

In cardiomyocytes, human BNP is made as a prepropeptide of 134 amino acids [5]. Removal of the 26-amino acid signal peptide generates pro-BNP of 108 amino acids, which is further cleaved to produce the C-terminal BNP 1-32 that is biologically active. To date, several proteases such as furin [6, 7] and corin [7-9] have been identified to process pro-BNP. Furin is a proprotein convertase in the Golgi of many cell types [10]. Corin is a cardiac protease that also activates atrial natriuretic peptide (ANP) [11-13]. In addition, dipeptidyl peptidase (DPP) IV has been shown to remove two N-terminal residues from BNP 1-32 to produce BNP 3-32 [14, 15].

In addition to proteolytic cleavage, pro-BNP undergoes other posttranslational modifications. Recent studies show that human plasma-derived pro-BNP and recombinant pro-BNP from mammalian cells contain significant amounts of O-glycans with terminal sialic acids [16-21]. T h e sialylated O-glycans protected pro-BNP from O-glycosidase digestion and stabilized pro-BNP in cell culture [17]. The O-glycans may also influence pro-BNP processing in cells. O-glycans on pro-BNP residue Thr-71 was reported to inhibit the propeptide processing in human embryonic kidney (HEK) 293 cells [21]. It was unknown if O-glycans have a similar inhibitory effect in cardiomyocytes.

Here we examined glycosylation and processing of pro-BNP in HEK 293 cells and cardiomyocytes. Our results showed that glycosylation and processing of pro-BNP in cardiomyocytes differed significantly from that in HEK 293 cells, and that in cardiomyocytes pro-BNP can be processed by corin at several different sites.

2. Materials and methods

2.1. Cell culture

HEK 293 cells were grown in 6-well plates in DMEM medium containing 10% fetal bovine serum (FBS). Murine atrial HL-1 cardiomyocytes from Dr. William Claycomb (Louisiana State University Medical Center) [22] were cultured in Claycomb medium (Sigma) with 10% FBS, 100 μM norepinephrine, and 4 mM L-glutamine. The cells were grown at 37°C in humidified incubators with 5% CO2 and 95% air.

2.2. Expression plasmids

Plasmids expressing human pro-BNP, corin and furin were reported previously [8, 23]. Plasmids expressing pro-BNP mutants T71A, R73A, R76A, K79A, R73A/R76A, R76A/K79A and R73A/R76A/K79A, in which residues Thr, Arg or Lys were replaced by Ala, were constructed by site-directed mutagenesis. All constructs were verified by DNA sequencing. Recombinant pro-BNPs encoded by these plasmids contained a V5 tag at their C-termini to facilitate protein detection.

2.3. Transfection, immunoprecipitation and Western blotting

Plasmids were transfected into HEK 293 and HL-1 cells using FuGENE (Roche Diagnostics) or Lipofectamine 2000 (Invitrogen) reagents. Conditioned medium from the transfected cells was collected and recombinant proteins were immunoprecipitated by an anti-V5 antibody. The cells were washed with a buffer and lysed in a solution containing 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40 (v/v), and a protease inhibitor cocktail (1:100 dilution, Sigma). Pro-BNP and BNP in the conditioned medium and cell lysate were analyzed by Western blotting using an anti-V5 antibody (Invitrogen), as described previously [24]. To quantify pro-BNP processing, the optical density of bands on X-ray films was measured densitometrically, and the percentage of pro-BNP to BNP conversion was calculated using computer software, as described previously [25].

2.4. Inhibition of O-glycosylatioin in cultured cells

To inhibit O-glycosylation on pro-BNP, we used benzyl 2-acetamido-2-deoxy-α-D-galactopyranoside (Ben-gal) (Sigma), which inhibits UDP-GlcNAc:GalNAc-β1,3-N-acetylglucosaminyl-transferase activity [26]. HEK 293 and HL-1 cells expressing recombinant pro-BNP were grown in the presence of Ben-gal (0.7 mmol/L for HEK 293 cells and 4 mmol/L for HL-1 cells) or vehicle control (DMSO) at 37°C overnight. The conditioned medium was collected and pro-BNP and BNP were analyzed by immunoprecipitation and Western blotting.

2.5. Glycosidase digestion

Glycosidases including PNGase F from Chryseobacterium meningosepticum, O-glycosidase from Streptococcus pneumonia and α(2-3,6,8,9) neuraminidase (also called sialidase A) from Arthrobacter ureafaciens (Prozyme) were used to analyze the carbohydrate contents on pro-BNP. Additional sialidases from Streptococcus pneumoniae, Clostridium perfringens, and Vibrio cholerae (Prozyme) that cleave α(2-3)-, α(2-3,6)- or α(2-3,6,8)-linked sialic acids, respectively, were used to predict sialic acid structures on pro-BNP. The conditioned medium containing pro-BNP from transfected HEK 293 and HL-1 cells was treated with glycosidases, either individually or in combination, at 37°C for 3 h. Proteins were analyzed by Western blotting using an anti-V5-HRP antibody.

2.6. Statistical analysis

Statistical analysis was done using Student's t-test. Data were presented as means ± S.D. A p value of <0.05 was considered to be statistically significant.

3. Results

3.1. Human pro-BNP WT and T71A mutant expressed in HEK 293 and HL-1 cells

Previous studies identified several O-glycosylation sites, including Thr-71, in human recombinant pro-BNP from HEK 293 cells (Fig. 1A). To examine the importance of pro-BNP residue Thr-71 in glycosylation in cardiomyocytes, we expressed pro-BNP WT and T71A mutant in HEK 293 and HL-1 cells. In the conditioned medium from HEK 293 cells (Fig. 1B, top left), WT pro-BNP and BNP with a C-terminal V5 tag were detected on Western blots as ~26- and ~12-kDa bands, respectively. In the cell lysate (Fig. 1B, bottom left), two bands of pro-BNP, representing glycosylated (~26 kDa) and non-glycosylated (~20 kDa) forms, and a band of BNP (~12 kDa) were detected. Pro-BNP T71A from HEK 293 cell conditioned medium appeared smaller than pro-BNP WT in molecular mass (Fig. 1B, arrow in top left), indicating that the mutation prevented glycosylation at Thr-71. In HEK 293 cell lysate (Fig. 1B, bottom left), the non-glycosylated pro-BNP and mature BNP, but not the glycosylated pro-BNP, were detected in samples with pro-BNP T71A, indicating that most of the glycosylated pro-BNP T71A was secreted rapidly from the cells.

Fig. 1. Expression of pro-BNP and T71A mutant in HEK 293 and HL-1 cells.

Fig. 1

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(A) Schematic presentation of pro-BNP and BNP. Reported O-glycosylated residues are indicated by circle-and-line symbols. (B) Plasmids expressing pro-BNP WT and T71A mutant or a control vector were transfected into HEK 293 (left) or HL-1 (right) cells. Pro-BNP and BNP in the conditioned medium (top) and cell lysate (bottom) were analyzed by Western blotting. An arrow (top left) indicates the difference in molecular mass between pro-BNP WT and T71A. Data were representative of 4 independent experiments. (C) Quantitative analysis of pro-BNP processing. Percentage of pro-BNP processing was calculated by densitometric analysis of Western blots. Data are mean ± S.D. from 4 independent experiments. n.s., not statistically significant.

In HL-1 cells (Fig 1B, right), pro-BNP and BNP bands of similar sizes from WT and T71A mutant were detected in the conditioned medium (~24 kDa for pro-BNP and ~12 kDa for BNP) and cell lysate (~20 kDa for pro-BNP and ~12 kDa for BNP), suggesting that glycosylation at Thr-71 may not be significant in cardiomyocytes.

By densitometric analysis of the Western blots, the percentage of pro-BNP to BNP conversion for WT was much less than T71A mutant in HEK 293 cells (Fig. 1C, left), but similar to that of T71A mutant in HL-1 cells (Fig. 1C, right).

3.2. Glycosylation on pro-BNP T71A in HEK 293 and HL-1 cells

To examine O-glycosylation on pro-BNP, we treated HEK 293 and HL-1 cells expressing pro-BNP WT and T71A mutant with an O-glycosylation inhibitor, Ben-gal. The presence of Ben-gal in the culture medium reduced the apparent molecular mass of pro-BNP WT and T71A in HEK 293 (Fig. 2A, left) and HL-1 (Fig. 2A, right) cells, which was consistent with the previous finding of O-glycosylation in pro-BNP and indicated that mutation at Thr-71 did not prevent O-glycosylation at other pro-BNP residues.

Fig. 2. O-glycosylation in pro-BNP WT and T71A.

Fig. 2

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(A) HEK 293 (left) and HL-1 (right) cells expressing pro-BNP WT or T71A mutant were cultured with (+) or without (-) Ben-gal for 24 h. Pro-BNP and BNP in the conditioned medium were analyzed by Western blotting. Data were representative for at least three independent experiments. (B) Pro-BNP WT (top) and T71A mutant (bottom) from HEK 293 (left) or HL-1 (right) cells were digested with PNGase F (F), O-glycosidase (O), and sialidase A (S), individually or in combination, and analyzed by Western blotting. Data were representative for at least three independent experiments.

3.3. Treatment of pro-BNP T71A with glycosidases

To verify this result, we incubated pro-BNP WT and T71A mutant from HEK 293 and HL-1 cells with PNGase F, O-glycosidase, and sialidase A, individually or in combination. As shown in Fig. 2B, treatment of PNGase F or O-glycosidase alone did not alter the apparent molecular mass of pro-BNP WT (top) and T71A (bottom). When pro-BNPs were treated with sialidase A, alone or with O-glycosidase, the apparent molecular mass was reduced, indicating that sialylated O-glycans were present on pro-BNP WT and T71A mutant. PNGase F in combination with sialidase A and O-glycosidase showed no further reduction of the apparent mass of pro-BNP WT and T71A mutant, confirming that pro-BNP WT and T71A mutant contained significant amounts of sialylated O-glycans but little N-glycans.

To gain insights into sialic acid structures on pro-BNP, we treated pro-BNP WT and T71A mutant with sialidases that favor specific linkages. When pro-BNP WT and T71A from HEK 293 and HL-1 cells were treated with sialidases that cleave α(2-3,6,8,9)- and α(2-3,6,8)-linkages, the apparent molecular mass of pro-BNP WT and T71A mutant was significantly reduced to a similar degree (Fig. 3). Treatment of sialidase that cleaves α(2-3)-linked sialic acids caused a smaller reduction in pro-BNP molecular mass, which was similar to the effect of sialidase that cleaves α(2-3,6)-linkages, indicating that most sialic acids on pro-BNP existed in the forms of either α(2-8)- or α(2-3)-linked moieties.

Fig. 3. Pro-BNP WT and T71A mutant digested with different sialidases.

Fig. 3

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Pro-BNP WT (top) and T71A mutant (bottom) from HEK 293 (left) or HL-1 (right) cells were digested with O-glycosidase with (+) or without (-) sialidases that favor specific linkages. Pro-BNP and BNP were analyzed by Western blotting. Data were representative for at least three independent experiments.

3.4. Processing of pro-BNP WT and T71A in HEK 293 and HL-1 cells

We examined the processing of pro-BNP WT and T71A mutant. When pro-BNP WT was expressed in HEK 293 or HL-1 cells, a significant portion of pro-BNP in the conditioned medium remained unprocessed (Fig. 4A, left). When recombinant furin or corin was co-expressed in these cells, pro-BNP processing was mostly completed in HL-1 cells (Fig. 4A, bottom left) but there were still significant portions of unprocessed pro-BNP in HEK 293 cells (Fig. 4A, top left). Similarly, significant portions of unprocessed pro-BNP T71A were present in the conditioned medium from HEK 293 and HL-1 cells (Fig. 4A, right). However, when recombinant furin or corin was co-expressed in these cells, pro-BNP T71A processing was mostly completed. The results showed that O-glycosylation at Thr-71 inhibited pro-BNP processing in HEK 293 cells, and that Thr-71 residue, which was not O-glycosylated in HL-1 cells, had little effect on pro-BNP processing in these cardiomyocytes.

Fig. 4. Processing of pro-BNP WT and mutants in HEK 293 and HL-1 cells.

Fig. 4

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(A) Pro-BNP WT (left) and T71A mutant (right) were expressed in HEK 293 (top) and HL-1 (bottom) cells that were either parental or co-transfected with plasmids expressing furin or corin, or a control vector. Pro-BNP and BNP in the conditioned medium were analyzed by Western blotting. (B) Schematic presentation of human pro-BNP with O-glycosylated residues and the cleavage site. (C) Pro-BNP WT and mutants with single, double or triple mutations were expressed in HEK 293 (top) or HL-1 (bottom) cells. Pro-BNP and BNP in the conditioned medium were analyzed by Western blotting. Data were representative for at least three independent experiments.

3.5. Pro-BNP processing sites in HEK 293 and HL-1 cells

Previous studies showed that both furin and corin cleaved pro-BNP. To examine the cleavage sites of furin- and corin-mediated pro-BNP processing, we tested pro-BNP mutants with altered residues at Arg-73, Arg-76 and Lys-79 around the cleavage site (Fig. 4B). In HEK 293 cells, which express furin but not corin, mutations at Arg-73, Arg-76 or Arg-73/Arg-76 prevented pro-BNP processing (Fig. 4C, top). In contrast, mutation at Lys-79 alone did not alter pro-BNP processing (Fig. 4C, top). Only when mutation at Lys-79 was combined with mutations at Arg-76 or Arg-73/Arg-76, was pro-BNP processing prevented. The results showed that Arg-73 and Arg-76, but not Lys-79, were important for furin-mediated pro-BNP processing in HEK 293 cells.

In HL-1 cells, in contrast, mutations at Arg-73 and Arg-76, alone or in combination, only slightly reduced pro-BNP processing (Fig. 4C, bottom). Nor did mutation at Lys-79, alone or in combination with Arg-76 mutation, prevent pro-BNP processing. Only when residues Arg-73, Arg-76 and Lys-79 were all mutated, was pro-BNP processing prevented (Fig. 4C, bottom), indicating that in HL-1 cells, which express both furin and corin, pro-BNP was processed at multiple sites including Arg-73, Arg-76, and Lys-79.

4. Discussion

ANP, BNP and C-type natriuretic peptide (CNP) are natriuretic peptide family members, which are made as inactive proforms and activated by proteolytic enzymes. Recent studies have identified significant amounts of O-glycans in human pro-BNP and this posttranslational modification appears to be unique for pro-BNP because no carbohydrates were detected in pro-ANP or pro-CNP [17]. In general, carbohydrates participate in protein trafficking, enzyme activation, and protein-protein interactions [25, 27, 28]. The function of O-glycans on pro-BNP is not fully understood. We showed that O-glycans increased pro-BNP stability in culture [17]. Others reported that O-glycans at residue Thr-71 markedly inhibited recombinant pro-BNP processing in HEK 293 cells [21]. This finding is intriguing, raising an inevitable question of how pro-BNP is possibly processed if similar O-glycosylation occurs in cardiomyocytes.

To address this question, we analyzed pro-BNP WT and T71A mutant in HEK 293 cells and HL-1 cardiomyocytes. We found that pro-BNP WT and T71A mutant in HEK 293 and HL-1 cells contained O-glycans with terminal sialic acids via α(2-3) and/or α(2-8) linkages (Figs. 2 and 3). The extent of pro-BNP O-glycosylation in cardiomyocytes, however, was less than that in HEK 293 cells. Consistent with the previous report of O-glycans on residue Thr-71 [20, 21], T71A mutation reduced pro-BNP glycosylation in HEK 293 cells (Fig. 1A). In these cells, pro-BNP WT, compared to T71A mutant, was more resistant to endogenous furin- and recombinant furin- or corin-mediated processing (Figs. 1C and 4A), supporting a role of O-glycans on Thr-71 in inhibiting pro-BNP processing.

Unlike in HEK 293 cells, residue Thr-71 contained little O-glycans when pro-BNP was expressed in HL-1 cardiomyocytes, as indicated by similar migration bands of pro-BNP and BNP from WT and T71A mutant on Western blots (Fig. 1B). Consistently, processing of pro-BNP WT and T71A mutant was similar in HL-1 cells, with or without furin or corin co-expression (Figs. 1C and 4A). Thus, our results indicate that O-glycosylation at pro-BNP residue Thr-71 identified in HEK 293 cells may not occur in cardiomyocytes. It is likely, therefore, that the function of O-glycans on pro-BNP may be involved primarily in peptide stability and/or extracellular distribution rather than inhibiting pro-BNP processing in cardiomyocytes.

Previously, we and others reported that furin and corin processed pro-BNP [6-9]. The sequence-specificity of furin- and corin-mediated pro-BNP processing is not well defined. Using non-glycosylated pro-BNP made in E. coli, Semenov et al. showed that furin cleaved pro-BNP at Arg-76, generating BNP 1-32, whereas corin cleaved pro-BNP at Lys-79, generating BNP 4-32 [7]. More recently, corin was shown to be shed from the cell surface and soluble corin was detected in human plasma [29-34]. Ichiki et al. reported that pro-BNP was processed by plasma soluble corin to produce BNP 1-32 and BNP 3-32 [32]. Because BNP 3-32 was derived from the cleavage between Pro-78↓Lys-79, it is unlikely that this fragment was cleaved directly by corin, which as a trypsin-like enzyme, favors basic residues [35]. Most likely, BNP 1-32 was produced first by plasma soluble corin and in turn trimmed N-terminally by DPP IV to BNP 3-32 [14, 15].

To determine the sequence-specificity of furin- and corin-mediated pro-BNP processing, we tested a series of pro-BNP mutants around the cleavage site. In HEK 293 cells expressing furin but not corin, mutations at Arg-73 and Arg-76, either alone or in combination, prevented pro-BNP processing (Fig. 4C, top). In contrast, mutation at Lys-79 had little effect on pro-BNP processing (Fig. 4C, top), indicating that furin cleaved pro-BNP at Arg-76, consistent with the furin recognition consensus sequence, RXXR↓, where the cleavage at the P1 Arg requires another basic residue at the P4 position [10].

Interestingly, in HL-1 cells expressing both furin and corin, single mutations at Arg-73, Arg-76, or Lys-79 did not prevent pro-BNP processing. Moreover, neither double mutations at Arg-73/Arg-76 nor Arg-76/Lys-79 prevented pro-BNP processing. Only when residues Arg-73, Arg-76 and Lys-79 were all mutated, was pro-BNP processing blocked (Fig. 4C, bottom). These data indicate that in HL-1 cells corin cleaved pro-BNP at multiple sites, including Arg-73, Arg-76 and Lys-79. Previous studies have shown that HL-1 cells retained all structural and functional characteristics of adult cardiomyocytes [22]. Our results suggest that in hearts corin may cleave pro-BNP at different sites. Consistent with this hypothesis, BNP fragments with different N-termini have been found in human plasma, including BNPs 1-32, 3-32 and 4-32 [36]. In principle, BNP 1-32 may be from corin- or furin-mediated cleavage, whereas BNP 4-32 may come from corin-mediated cleavage. BNP 3-32 is most likely from DPP IV-mediated cleavage of BNP 1-32 [14, 15]. Thus, our results provide new insights into the biochemical basis of pro-BNP processing. Studies have shown that corin expression is up-regulated in hypertrophic and failing hearts [37, 38]. High levels of plasma BNPs 1-32, 3-32 and 4-32 are found in patients with HF [36]. Further studies are needed to determine if the cleavage of pro-BNP by corin is regulated in the heart under physiological and pathological conditions.

Acknowledgments

We thank Dr. William Claycomb for HL-1 cells. This work was supported in part by grants from the Bakken Heart-Brain Institute, the National Institutes of Health (HL089298, HL089298-S1, HD064634), the National Natural Science Foundation of China (31070716), and the Priority Academic Program Development of Jiangsu Higher Education Institutions.

Abbreviations

ANP

atrial natriuretic peptide

BNP

B-type or brain natriuretic peptide

CNP

C-type natriuretic peptide

HEK

human embryonic kidney

HF

heart failure

NT

N-terminal

FBS

fetal bovine serum

Ben-gal

Benzyl 2-acetamido-2-deoxy-α-D-galactopyranoside

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