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Processing of pro-b-type natriuretic peptide: furin and corin as candidate convertases.

B-type natriuretic peptide (BNP) (5) is a cardiac peptide hormone mainly expressed by ventricular myocardium in response to volume overload and increased filling pressure (1,2). An active BNP hormone comprising 32 amino acid residues (AARs), along with a physiologically inactive N-terminal fragment (NT-proBNP) (76 AAR), is formed from the precursor molecule, proBNP (108 AAR), by specific enzyme cleavage. BNP and NT-proBNP are currently established biomarkers of heart failure (HF) (3, 4) and are routinely used by clinicians for the exclusion of HF and risk assessment in patients with acute coronary syndromes (5, 6). In spite of the great interest in the products of proBNP cleavage as HF biomarkers, the mechanism of proBNP processing itself, as well as the enzymes responsible for the conversion of BNP precursor molecules, has not been well characterized. However, the comprehensive assessment of proBNP processing mechanisms could be of value for better understanding HF development and reliable interpretation of the results of BNP, NT-proBNP, and proBNP measurements.

Several recent studies demonstrated that endogenous proBNP and NT-proBNP are O-glycosylated (7-9). As follows from our previous data (10), O-glycosylation of the threonine 71 residue, located close to the proBNP cleavage site, has a pronounced suppressive effect on the processing efficiency and should be considered when studying proBNP processing (Fig. 1). Two proprotein convertases, furin and corin, are considered the most likely proBNP processing enzymes. Furin is a ubiquitously expressed proprotein convertase mainly located in the trans-Golgi network (11). A variety of proproteins have been shown to be the substrates of furin (12, 13); however, the concept of furin involved in the processing of human proBNP is currently based on a set of indirect observations (14, 15). The other candidate enzyme, corin, is a recently discovered membrane-bound serine protease mainly produced in heart (16). In cell-based experiments, corin has been shown to be a specific enzyme responsible for processing of the A-type natriuretic peptide precursor (17, 18). Corin has also been suggested to be responsible for proBNP processing (19), but its potential involvement in this process has not been supported by convincing experimental data. Thus, the role of both candidate enzymes, furin and corin, in proBNP processing should be validated. Moreover, no reported study has elucidated exactly the specificity of the proBNP cleavage by the convertases mentioned above. Therefore, we designed a study to investigate the role of both furin and corin in human proBNP processing and characterize the cleavage specificity of these convertases.


Materials and Methods

The study was conducted in accordance with the current revision of the Helsinki Declaration. Venous blood from HF patients was collected into EDTA-containing vacuette tubes (Greiner Bio-One) and centrifuged at 3000g for 15 min at 4[degrees]C. The diagnosis of HF was based on the presence of suggestive symptoms and signs and was confirmed by echocardiography studies.

All monoclonal antibodies (mAbs) specific to human proBNP were from HyTest. The mAb epitopes, corresponding to different fragments of proBNP, are indicated by subscripts (e.g., 24C587-93). We used 2 mAbs specific to the BNP-part of proBNP molecule, 24C587-93 and 50E1102-108, for preparing the BNP-specific affinity matrix. We used mAb [24E11.sub.67-76] for preparing the affinity matrix specific to the 67-76 region of proBNP. Selected mAbs were coupled with a CNBr-activated Sepharose CL 4B (GE Healthcare). We performed affinity extraction as described (8), as was sandwich 2-step immunofluorescent assay (IFA) (8). The recombinant proBNP expressed in Escherichia coli (HyTest) was used as a calibrator for all proBNP, NT-proBNP, and BNP-specific assays.

The plasmids expressing the wild type (WT) and the mutant form, with a substitution of alanine for threonine 71 (T71A), of human proBNP were described previously (10). The plasmids expressing human furin and corin were purchased from Genecopeia.

We obtained HEK 293 (human embryonic kidney) and LoVo (human colon carcinoma) cell lines from ATCC. Cells were cultured in DMEM (HEK 293) or F12-K medium (LoVo) supplemented with 100 mL/L fetal bovine serum, at 37[degrees]C in an atmosphere of 5% C[O.sub.2] in air. We performed transfection using Unifectin-56 reagent (Unifect Group).

HEK 293 cells were treated with a furin inhibitor, decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone (DecRVKR-CMK) (Enzo Life Sciences), or cotransfected with a furin-specific small interfering RNA (siRNA) (Santa Cruz Biotechnology) along with proBNP transfection. The furin inhibitor was added to the medium to obtain a final concentration of 60 jumol/L. Transfection with the furin-specific siRNA was performed twice, 24 h before and 24 h after the transfection of proBNP-expressing plasmids. The final concentration of siRNA was 50 nmol/L.

The proBNP processing rate was calculated as the ratio of the NT-proBNP molar concentration to the total [proBNP + NT-proBNP] molar concentration. To quantify both proBNP and NT-proBNP, we used the in-house sandwich IFA [21E3.sub.13-24][-29D1.sub.5-12] (capture mAb-detection mAb). The assay recognizes both proteins equally. We measured the NT-proBNP concentration by use of the same assay in the samples after proBNP was removed by passing through the BNP-specific affinity matrix, which extracted 99% of the proBNP.

We separated proteins from the conditioned media of HEK 293 cells transfected with the plasmid expressing proBNP-T71A by size-exclusion chromatography using a Superdex 75 10/300 GL column (GE Healthcare) as described (10). We analyzed BNP immunoreactivity in fractions by use of the in-house sandwich IFA 24C5-Ab-BNP2 that recognizes BNP and proBNP with the same efficiency (20). This assay consists of capture mAb 24C5 specific for BNP peptide 11-17 (the region 87-93 within proBNP sequence) and detection mAb Ab-BNP2 that recognizes only the immune complex of mAb 24C5 with BNP/proBNP.

Cleavage reaction was performed as described (10). Briefly, 40 ng recombinant proBNPs or 4 ng endogenous proBNP were incubated with 1.5 U recombinant human furin (Sigma) for 3.5 h at 37[degrees]C. We evaluated cleavage efficiency using the following formula: [1 - (proBNP concentration in the furin-treated sample/proBNP concentration in the control sample)] X 100%. The concentration of proBNP in the samples was estimated by use of the in-house sandwich IFA [50E1.sub.102-108[]-16F3.sub.13-20.]

Mass spectrometry (MS) was performed on an Ultraflex II MALDI-TOF mass spectrometer (Bruker Daltonik). Before MALDI-TOF MS analysis, we extracted BNP-related peptides from the samples using the BNP-specific affinity matrix. Correspondence of the found masses in MALDI-TOF experiments to the BNP peptides (21, 22) was interpreted with the help of GPMAW 8.0 software (Lighthouse Data) using 0.01%-0.02% precision as a criterion. For calculations, we assumed an intact disulfide bond between Cys-10 and Cys-26 of BNP.

HEK 293 cells transiently transfected with the corin-expressing plasmid were used for the experiments on corin-mediated cleavage of proBNP. Recombinant proBNP (E. coli), the affinity-purified recombinant proBNPs expressed in HEK 293 (WT and the T71A form), or synthetic BNP (Peptide Institute) were separately dissolved in DMEM medium containing 5 g/L BSA to obtain a final concentration of 500 [micro]g/L (proBNPs) or 150 [micro]g/L (BNP). HEK 293 cells transfected with the corin-expressing plasmid were washed 5 times with DMEM medium containing 5 g/L bovine serum albumin, and peptide solutions were added and incubated with cells for 3 h at 37[degrees]C.Weperformed experiments with HEK 293 cells transfected with the furin-expressing plasmid only with proBNP (E. coli)in the same conditions. We used a Superdex Peptide column (GE Healthcare) to separate proteins from the samples obtained after the incubation of proBNP (E. coli) with HEK 293 cells (nontransfected or transfected with the corin- or furin-expressing plasmids). Separation was performed as described (20).

All data are described as mean (SD) (n = 3) and statistically analyzed using an unpaired Student's t-test. We considered a value of P < 0.01 to be statistically significant.



As recently demonstrated (10), the processing of human proBNP is inhibited by O-glycosylation of the threonine 71 residue located close to the cleavage site. Based on this phenomenon, we chose to use the proBNP-T71A variant in the experiments because the processing efficiency of this mutant form is not affected by O-glycosylation.

HEK 293 cells that normally express functional furin (13,23) were transfected with the plasmids encoding proBNP-T71A or proBNP-WT and treated with a furin inhibitor, Dec-RVKR-CMK, or cotransfected with a furin-specific siRNA. As shown in Fig. 2A, the rate of proBNP-T71A processing was 6.4% (0.9%) in HEK 293 cells treated with Dec-RVKR CMK and 16.0% (1.1%) in cells treated with the furin-specific siRNA, compared with 62.4% (1.2%) in nontreated cells. The suppression effect of both the furin inhibitor and the furin-specific siRNA on the processing of proBNP-WT was also evident, but not as prominent as that of proBNP-T71A. The reduction of proBNP-T71A processing efficiency caused by furin suppression was confirmed by gel filtration. As shown in Fig. 2B, BNP production was markedly reduced by both the Dec-RVKR-CMK and the furin-specific siRNA treatment.


We transfected furin-deficient LoVo cells (24) with the plasmid expressing proBNP-T71A alone or along with the furin-expressing plasmid and analyzed proBNP processing. The processing rate of the proBNP-T71A expressed alone was extremely low [5.8% (1.1%)], whereas in the case of coexpression with functional furin it was significantly higher, up to 63.5% (3.4%) (Fig. 3).


Both T71A and WT forms of recombinant proBNP were affinity-purified from the conditioned media of transfected LoVo cells and incubated with purified furin. Nonglycosylated recombinant proBNP (E. coli) was also treated with furin under the same conditions (positive control). We estimated the cleavage efficiency by measuring the proBNP concentration in both treated and nontreated samples. In the presence of furin, proBNP-T71A expressed in LoVo cells was effectively cleaved, similarly to the nonglycosylated peptide (E. coli) [91.6% (2.0%) and 98.2% (1.3%), respectively] (Fig. 4A). WT form was barely susceptible to furin-mediated cleavage [6.0% (3.5%)], confirming our previous results regarding the inhibitory effect of the threonine 71-bound O-glycans on proBNP processing (10). We analyzed the products of in vitro proBNP cleavage using the MALDI-TOF MS technique. The furin-mediated cleavage of recombinant proBNP-T71A expressed in LoVo cells led to the specific formation of a unique BNP form comprising 32 AAR (BNP 1-32) (Fig. 4B). The same result was obtained for recombinant proBNP (E. coli) (data not shown).

We added a nonglycosylated (E. coli) and 2 glycosylated (proBNP-T71A and proBNP-WT expressed in HEK 293 cells) peptides to HEK 293 cells transfected with the plasmid expressing human corin and estimated the rate of proBNP processing. Control experiments were performed with nontransfected cells. As shown in Fig. 5A, corin was able to process the original nonglycosylated proBNP [40.7% (1.1%)] and proBNP-T71A [11.7% (1.3)%], whereas proBNP-WT glycosylated in the cleavage site region (10) was unsusceptible to corin-mediated cleavage. Gel filtration studies demonstrated the presence of the truncated BNP form in the case of the corin-mediated cleavage of recombinant proBNP (E. coli) (Fig. 5B). MS analysis revealed that this form corresponds to BNP 4-32 (Fig. 5C). The ability of corin to produce BNP 4-32 from the BNP 1-32 molecule was also tested. We added synthetic BNP 1-32 to the corin-transfected HEK 293 cells and analyzed the final products with MALDI-TOF MS. No detectable BNP 4-32 formation from BNP 1-32 was observed in this case. HEK 293 cells transfected with the plasmid expressing furin were also able to process nonglycosylated proBNP (E. coli)at the cell surface, confirmed by gel filtration studies (Fig. 5B).


We analyzed 23 plasma samples from HF patients to assess the glycosylation status of endogenous proBNP in the cleavage site region using 2 sandwich IFAs. Assay 50E1102-108-16F313-20 was chosen as a reference assay for measuring total proBNP, because it is not affected by proBNP glycosylation (25). We used assay 21E667-7324C587-93 for quantifying the proBNP nonglycosylated in the cleavage site region because it uses the mAb 21E667-73, which is sensitive to the proBNP glycosylation in that region (10). The proBNP nonglycosylated in the cleavage site region was present in concentrations that were 33.7% (11.3%) of total proBNP in all tested samples (Fig. 6A).

Total proBNP was purified from a pooled plasma sample (12 HF patients with the highest proBNP concentration) using the BNP-specific affinity matrix. We used the affinity matrix containing the mAb 24E11 specific to the 67-76 region of proBNP molecule to extract the proBNP nonglycosylated in the cleavage site region from the total proBNP fraction, since this mAb cannot recognize the proBNP molecules of which the region 67-76 is glycosylated (10). About one third (approximately 30%) of total proBNP was extracted by this matrix. We extracted the proBNP glycosylated in this region from the flow-through using the BNP-specific affinity matrix.

Three fractions of endogenous proBNP (total, nonglycosylated, and glycosylated in the cleavage site region) were incubated with purified furin, and we estimated their cleavage efficiency. Endogenous proBNP glycosylated in the cleavage site region was hardly susceptible to furin-mediated cleavage, whereas up to 91% (1.5%) of the proBNP nonglycosylated in the designated region was cleaved by furin (Fig. 6B). The total fraction of endogenous proBNP was susceptible to furin-mediated cleavage [28% (1.0%)], which was in good agreement with our estimation of the content of the proBNP nonglycosylated in the cleavage site region. MALDI-TOF MS analysis revealed that the furin-mediated cleavage of endogenous proBNP nonglycosylated in the cleavage site region resulted in the BNP 1-32 formation.



Despite numerous studies devoted to the assessment of the clinical significance and therapeutic potential of proBNP-derived peptides, little is known about the mechanisms of proBNP processing itself. This is likely due to the limitations of human-based studies. On the other hand, exogenous protein expression in model cell lines has proven to be useful for investigating the processing mechanisms of a variety of precursor proteins (13, 23, 26). Because of the inhibitory effect of O-glycans bound to threonine 71 on proBNP processing (10), we used the proBNP-T71A variant, along with the recombinant WT and endogenous forms of the protein, in the experiments on proBNP processing. Of note, the proBNP-T71A variant has no O-glycans linked to threonine 71, because the substitution of alanine for threonine prevents O-glycosylation at this site.



The requisite condition of furin-mediated cleavage is the presence of a substrate sequence -Arg-X-X Arg [down arrow] X- (27). Human proBNP has the appropriate sequence, [-Arg.sub.73][-Ala.sub.74][-Pro.sub.75][-Arg.sub.76] [down arrow] [Ser.sub.77]-, in the cleavage site region, suggesting a high possibility of furin involvement in proBNP processing. The treatment of HEK 293 cells (normally expressing furin) with the irreversible furin inhibitor, Dec-RVKR-CMK, significantly reduced the efficiency of proBNP processing. However, the inhibitory effect of Dec-RVKR-CMK could not be specific for furin because the compound is also known to inhibit other furin-like processing enzymes (28). To block furin activity more specifically, we applied an RNA interference strategy, which is known to be an effective tool for specific inhibition of protein expression (29). The treatment of HEK 293 cells with the furin-specific siRNA led to a prominent (3.9-fold) reduction of the proBNP-T71A processing rate, suggesting that proBNP processing could be attributed to furin activity. The inhibitory effect was not complete, most likely because of the incomplete interference offurin expression bythis siRNA. On the other hand, certain minor activityofother furin-like convertases cannot be excluded.

Owing to the suppressive effect of O-glycans attached to threonine 71 (10), the processing rate of proBNP-WT was 9.8-fold lower than that of proBNP T71A. However, the evident inhibitory effect of both the Dec-RVKR-CMK and the furin-specific siRNA treatment on proBNP-WT processing efficiency was also observed. The involvement of furin in proBNP processing was further supported by additional studies using furin-deficient LoVo cells. These cells do not express functional furin due to mutations in both alleles of the furin gene (24, 30). There was no detectable processing of recombinant proBNP-T71A in LoVo cells, although the extracted protein was effectively cleaved by purified furin to form BNP 1-32 molecule, as confirmed by MS studies. In addition, transfection with the plasmid expressing functional furin provides LoVo cells with the ability to process proBNP.

The other candidate enzyme, corin, is located on the plasma membrane and has been shown to cleave the precursors upon secretion (18). The potential ability of corin to cleave proBNP has been described (19), whereas both the influence of proBNP glycosylation on corin-mediated processing and the specificity of cleavage have not been previously shown. Experiments on the corin-mediated processing of proBNP were performed using HEK 293 cells transfected with the corin-expressing plasmid, because soluble corin was not available for the analysis. We observed that corin was able to cleave nonglycosylated proBNP (E. coli), as well as the proBNP nonglycosylated at threonine 71 (proBNP-T71A), but failed to process the proBNP-WT modified by O-glycans at threonine 71. In the case of proBNP-T71A, however, the efficiency of corin-mediated proBNP processing was markedlylower than that of nonglycosylated proBNP (E. coli), possibly due to the influence of O-glycans at other sites of the proBNP molecule. Interestingly, HEK 293 cells transfected with the furin-expressing plasmid were also able to process exogenous proBNP (E. coli). This observation suggests the possibility of furin-mediated processing of proBNP at the cell surface.

Corin is thought to cleave potential substrates with a preference for Arg Lys residues at the P1 position (toward the N-terminus from the cleavage site), Pro Phe/Gly at the P2 position, and small neutral amino acids at the P3 position (31). The appropriate substrate profile is presented twice in human proBNP sequence close to the known cleavage site, [-Ala.sub.74][-Pro.sub.75][-Arg.sub.76] [down arrow] [Ser.sub.77]- and [-Ser.sub.77][-Pro.sub.78][-Lys.sub.79] [down arrow] [Met.sub.80]-. The cleavage between [Arg.sub.76] and [Ser.sub.77] is similar to furin-like activity and would give rise to BNP 1-32, whereas the cleavage between [Lys.sub.79] and [Met.sub.80] should be uniquely attributed to the action of corin and is expected to result in BNP 4-32 production. Gel filtration studies revealed that corin-mediated cleavage led to the formation of a BNP form, shorter than the synthetic one or the one generated byfurin-mediated cleavage. According to MS analysis, the proBNP cleavage by corin resulted in BNP 4-32 formation, suggesting corin's preference for the [-Ser.sub.77][-Pro.sub.78][-Lys.sub.79] [down arrow] [Met.sub.80]- profile. Of note, this form was observed only in the case of corin expressing cells and never in nontransfected cells or furin-transfected cells. Corin was not able to process BNP 1-32 to BNP 4-32, possibly because of the absence of additional residues before the N-terminal serine in BNP 1-32 that could be important for substrate recognition by the enzyme.

The presence of N-terminal truncated BNP forms, including BNP 3-32, BNP 4-32, and BNP 5-32, in HF patients has been reported recently (32). The formation of BNP 3-32 is attributed to the action of dipeptidyl peptidases (33), whereas the mechanisms that lead to the formation of other truncated forms has not been characterized. In this study, we observed the formation of BNP 4-32 due to the specific processing activity of corin. Hereby, we can speculate that the presence of BNP 4-32 with possibly reduced hormonal activity (34) in plasma of HF patients could be partially explained by corin-mediated processing activity.

It has recently been shown that proBNP represents a major BNP-immunoreactive plasma component in HF patients (25, 35). Unprocessed proBNP has markedly reduced, if any, hormonal activity (35, 36). The presence of unprocessed prohormone in the blood of HF patients suffering from insufficient natriuresis and diuresis remains an unexplained phenomenon. Unprocessed proBNP in circulation would suggest that either proBNP in the blood of HF patients is protected from processing or HF patients have impaired proBNP processing. The recent data suggest that both hypotheses could be correct. The inhibitory effect of Oglycosylation on the processing efficiency of proBNP (10) can explain the presence of unprocessed proBNP glycosylated in the region located close to the cleavage site. In the current study, we have demonstrated that a considerable amount (about 30%) of total proBNP in the plasma of HF patients is not glycosylated in the designated region and could be processed by furin to form mature BNP 1-32. These data could indicate that the mechanism of proBNP processing is impaired in HF patients.

Our findings provide several new insights into human proBNP processing mechanisms. Our data strongly support the hypothesis of furin involvement in human proBNP processing. The corin-mediated processing of proBNP results in the formation of trun cated BNP 4-32, suggesting that corin is unlikely to be the primary candidate for the role of proBNP processing enzyme. Our data indicate that the proBNP cleavage by corin is also inhibited by O-glycosylation in the region located close to the cleavage site, similar to what was previously demonstrated for furin (10). Finally, for the first time, we report that some portion of endogenous proBNP in the plasma of HF patients is not glycosylated in the region located close to the cleavage site and is susceptible to furin-mediated processing. These findings may improve our understanding of the clinical significance of proBNP-derived peptides and highlight the importance of further investigations of proBNP processing mechanisms.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors' Disclosures of Potential Conflicts of Interest: Upon manuscript submission, all authors completed the Disclosures of Potential Conflict of Interest form. Potential conflicts of interest:

Employment or Leadership: A.G. Semenov, HyTest Ltd.; N.N. Tamm, HyTest Ltd.; K.R. Seferian, HyTest Ltd.; A.B. Postnikov, HyTest Ltd.; A.G. Katrukha, R&D Department, HyTest Ltd. Consultant or Advisory Role: None declared. Stock Ownership: A.G. Katrukha, HyTest Ltd. Honoraria: None declared.

Research Funding: This study was supported by HyTest LTD, Turku, Finland.

Expert Testimony: None declared.

Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.

Acknowledgments: We are grateful to Alexey Nikashin for his valuable advice in the preparation of the manuscript. We thank laboratory technicians Elena Akinfieva, Elena Kochkareva, Nadezhda Kuzina, and Svetlana Ovtina for expert technical assistance.


(1). Hosoda K, Nakao K, Mukoyama M, Saito Y, Jougasaki M, Shirakami G, et al. Expression of brain natriuretic peptide gene in human heart: production in the ventricle. Hypertension 1991;17:1152-5.

(2). Yasue H, Yoshimura M, Sumida H, Kikuta K, Kugiyama K, Jougasaki M, et al. Localization and mechanism of secretion of B-type natriuretic peptide in comparison with those of A-type natriuretic peptide in normal subjects and patients with heart failure. Circulation 1994;90:195-203.

(3). Clerico A, Fontana M, Zyw L, Passino C, Emdin M. Comparison of the diagnostic accuracy of brain natriuretic peptide (BNP) and the N-terminal part of the propeptide of BNP immunoassays in chronic and acute heart failure: a systematic review. Clin Chem 2007;53:813-22.

(4). Hammerer-Lercher A, Neubauer E, Muller S, Pachinger O, Puschendorf B, Mair J. Head-to-head comparison of N-terminal pro-brain natriuretic peptide, brain natriuretic peptide and N-terminal pro-atrial natriuretic peptide in diagnosing left ventricular dysfunction. Clin Chim Acta 2001;310:193-7.

(5). Hunt SA. ACC/AHA 2005 guideline update for the diagnosis and management of chronic heart failure in the adult: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (Writing Committee to Update the 2001 Guidelines for the Evaluation and Management of Heart Failure). J Am Coll Cardiol 2005;46:e1-82.

(6). Doust JA, Pietrzak E, Dobson A, Glasziou P. How well does B-type natriuretic peptide predict death and cardiac events in patients with heart failure? Systematic review. BMJ 2005;330:625.

(7). Schellenberger U, O'Rear J, Guzzetta A, Jue RA, Protter AA, Pollitt NS. The precursor to B-type natriuretic peptide is an O-linked glycoprotein. Arch Biochem Biophys 2006;451:160-6.

(8). Seferian KR, Tamm NN, Semenov AG, Tolstaya AA, Koshkina EV, Krasnoselsky MI, et al. Immunodetection of glycosylated NT-proBNP circulat ing in human blood. Clin Chem 2008;54:866-73.

(9). Hammerer-Lercher A, Halfinger B, Sarg B, Mair J, Puschendorf B, Griesmacher A, et al. Analysis of circulating forms of proBNP and NT-proBNP in patients with severe heart failure. Clin Chem 2008;54:858-65.

(10). Semenov AG, Postnikov AB, Tamm NN, Seferian KR, Karpova NS, Bloshchitsyna MN, et al. Processing of pro-brain natriuretic peptide is suppressed by O-glycosylation in the region close to the cleavage site. Clin Chem 2009;55:489-98.

(11). Molloy SS, Thomas L, VanSlyke JK, Stenberg PE, Thomas G. Intracellular trafficking and activation of the furin proprotein convertase: localization to the TGN and recycling from the cell surface. EMBO J 1994;13:18-33.

(12). Nakayama K Furin: a mammalian subtilisin/ Kex2p-like endoprotease involved in processing of a wide variety of precursor proteins. Biochem J 327(Pt 3):625-35, 1997.

(13). Wu C, Wu F, Pan J, Morser J, Wu Q. Furin-mediated processing of pro-C-type natriuretic peptide. J Biol Chem 2003;278:25847-52.

(14). Sawada Y, Suda M, Yokoyama H, Kanda T, Sakamaki T, Tanaka S, et al. Stretch-induced hypertrophic growth of cardiocytes and processing of brain-type natriuretic peptide are controlled by proprotein-processing endoprotease furin. J Biol Chem 1997;272:20545-54.

(15). Sawada Y, Inoue M, Kanda T, Sakamaki T, Tanaka S, Minamino N, et al. Co-elevation of brain natriuretic peptide and proprotein-processing endoprotease furin after myocardial infarction in rats. FEBS Lett 1997;400:177-82.

(16). Yan W, Sheng N, Seto M, Morser J, Wu Q. Corin, a mosaic transmembrane serine protease encoded by a novel cDNA from human heart. J Biol Chem 1999;274:14926-35.

(17). Yan W, Wu F, Morser J, Wu Q Corin, a trans membrane cardiac serine protease, acts as a proatrial natriuretic peptide-converting enzyme. Proc Natl Acad SciUSA2000;97:8525-9.

(18). Gladysheva IP, Robinson BR, Houng AK, KovatsT, King SM. Corin is co-expressed with pro-ANP and localized on the cardiomyocyte surface in both zymogen and catalytically active forms. J Mol Cell Cardiol 2008;44:131-42.

(19). Wang W, Liao X, Fukuda K, Knappe S, Wu F, Dries DL, et al. Corin variant associated with hypertension and cardiac hypertrophy exhibits impaired zymogen activation and natriuretic peptide processing activity. Circ Res 2008;103:502-8.

(20). Tamm NN, Seferian KR, Semenov AG, Mukharyamova KS, Koshkina EV, Krasnoselsky MI, et al. Novel immunoassay for quantification of brain natriuretic peptide and its precursor in human blood. Clin Chem 2008;54:1511-8.

(21). Sudoh T, Maekawa K, Kojima M, Minamino N, Kangawa K, Matsuo H. Cloning and sequence analysis of cDNA encoding a precursor for human brain natriuretic peptide. Biochem Biophys Res Commun 1989;159:1427-34.

(22). Hino J, Tateyama H, Minamino N, Kangawa K, Matsuo H. Isolation and identification of human brain natriuretic peptides in cardiac atrium. Biochem Biophys Res Commun 1990; 167:693-700.

(23). Wang P, Tortorella M, England K, Malfait AM, Thomas G, Arner EC, Pei D. Proprotein convertase furin interacts with and cleaves pro-ADAMTS4 (Aggrecanase-1) in the trans-Golgi network. J Biol Chem 2004;279:15434-40.

(24). Takahashi S, Kasai K, Hatsuzawa K, Kitamura N, Misumi Y, Ikehara Y, et al. A mutation of furin causes the lack of precursor-processing activity in human colon carcinoma LoVo cells. Biochem Biophys Res Commun 1993;195:1019-26.

(25). Seferian KR, Tamm NN, Semenov AG, Mukharyamova KS, Tolstaya AA, Koshkina EV, et al. The brain natriuretic peptide (BNP) precursor is the major immunoreactive form of BNP in patients

with heart failure. Clin Chem 2007;53:866-73.

(26). Misumi Y, Oda K, Fujiwara T, Takami N, Tashiro K, Ikehara Y. Functional expression of furin demonstrating its intracellular localization and endoprotease activity for processing of proalbumin and complement pro-C3. J Biol Chem 1991;266: 16954-9.

(27). Molloy SS, Bresnahan PA, Leppla SH, Klimpel KR, Thomas G. Human furin is a calcium-dependent serine endoprotease that recognizes the sequence Arg-X-X-Arg and efficiently cleaves anthrax toxin protective antigen. J Biol Chem 1992; 267:16396-402.

(28). Jean F, Stella K, Thomas L, Liu G, Xiang Y, Reason AJ, Thomas G. alpha1-Antitrypsin Portland, a bioengineered serpin highly selective for furin: application as an antipathogenic agent. Proc Natl Acad SciUSA 1998;95:7293-8.

(29). Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 2001;411:494-8.

(30). Takahashi S, Nakagawa T, Kasai K, Banno T, Duguay SJ, Van de Ven WJ, et al. A second mutant allele of furin in the processing incompetent cell line, LoVo: evidence for involvement of the homo B domain in autocatalytic activation. J Biol Chem 1995;270:26565-9.

(31). Knappe S, Wu F, Masikat MR, Morser J, Wu Q. Functional analysis of the transmembrane domain and activation cleavage of human corin: design and characterization of a soluble corin. J Biol Chem 2003;278:52363-70.

(32). Niederkofler EE, Kiernan UA, O'Rear J, Menon S, Saghir S, Protter AA, et al. Detection of endogenous B-type natriuretic peptide at very low concentrations in patients with heart failure. Circ Heart Fail 2008;1:258-64.

(33). Brandt I, Lambeir AM, Ketelslegers JM, Vander heyden M, Scharpe S, De Meester I. Dipeptidylpeptidase IV converts intact B-type natriuretic peptide into its des-SerPro form. Clin Chem 2006; 52:82-7.

(34). Boerrigter G, Costello-Boerrigter LC, Harty GJ, Lapp H, Burnett JC Jr. Des-serine-proline brain natriuretic peptide 3-32 in cardiorenal regulation. Am J Physiol Regul Integr Comp Physiol 2007;292:R897-901.

(35). Liang F, O'Rear J, Schellenberger U, Tai L, Lasecki M, Schreiner GF, et al. Evidence for functional heterogeneity of circulating B-type natriuretic peptide. J Am Coll Cardiol 2007;49:1071-8.

(36). Heublein DM, Huntley BK, Boerrigter G, Cataliotti A, Sandberg SM, Redfield MM, Burnett JC Jr. Immunoreactivity and guanosine 3',5'-cyclic monophosphate activating actions of various molecular forms of human B-type natriuretic pep tide. Hypertension 2007;49:1114-9.

Alexander G. Semenov, [1] * Natalia N. Tamm, [1] Karina R. Seferian, [1] Alexander B. Postnikov, [1] Natalia S. Karpova, [2] Daria V. Serebryanaya, [2] Ekaterina V. Koshkina, [3] Mihail I. Krasnoselsky, [4] and Alexey G. Katrukha [1]

[1] HyTest Ltd., Turku, Finland; [2] Department of Biochemistry, Moscow State University, Moscow, Russia; [3] 67 City Hospital, Moscow, Russia; [4] Moscow State Medico-Stomatological University, Moscow, Russia.

* Address correspondence to this author at: HyTest Ltd., Intelligate, 6th floor, Joukahaisenkatu 6, 20520 Turku, Finland. Fax 358-25120909; e-mail

[dagger] Portions of this work were presented at AACC Annual Meeting in Chicago, IL, July 2009.

Received January 19, 2010; accepted April 28, 2010.

Previously published online at DOI: 10.1373/clinchem.2010.143883

[5] Nonstandard abbreviations: BNP, B-type natriuretic peptide; AAR, amino acid residue; NT-proBNP, N-terminal fragment of proBNP; proBNP, BNP precursor; HF, heart failure; mAb, monoclonal antibody; IFA, immunofluorescent assay; WT, wild type; Dec-RVKR-CMK, decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone; siRNA, small interfering RNA; MS, mass spectrometry.
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Title Annotation:Proteomics and Protein Markers
Author:Semenov, Alexander G.; Tamm, Natalia N.; Seferian, Karina R.; Postnikov, Alexander B.; Karpova, Nata
Publication:Clinical Chemistry
Date:Jul 1, 2010
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