help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2007-0838
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Biswas, N.
Right arrow Articles by Mahata, S. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Biswas, N.
Right arrow Articles by Mahata, S. K.
Endocrinology Vol. 149, No. 2 749-757
Copyright © 2008 by The Endocrine Society

Proteolytic Cleavage of Human Chromogranin A Containing Naturally Occurring Catestatin Variants: Differential Processing at Catestatin Region by Plasmin

Nilima Biswas, Sucheta M. Vaingankar, Manjula Mahata, Madhusudan Das, Jiaur R. Gayen, Laurent Taupenot, Justin W. Torpey, Daniel T. O’Connor and Sushil K. Mahata

Departments of Medicine (N.B., S.M.V., M.M., M.D., J.R.G., L.T., D.T.O’C., S.K.M.), of Chemistry and Biochemistry (J.W.T.), and of Molecular Genetics (D.T.O’C.), University of California, San Diego, and Veteran’s Affairs San Diego Healthcare System (D.T.O., S.K.M.), La Jolla, California 92093-0838

Address all correspondence and requests for reprints to: Sushil K. Mahata, Ph.D., Department of Medicine (0838), University of California, San Diego School of Medicine and Veteran’s Affairs San Diego Healthcare System, 9500 Gilman Drive, La Jolla, California 92093-0838. E-mail: smahata{at}ucsd.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The plasma level of chromogranin A (CgA) is elevated in genetic hypertension. Conversely, the plasma level of the CgA peptide catestatin is diminished in individuals with established hypertension and those with a genetic risk of this disease. Resequencing of the human CHGA gene identified three naturally occurring variants of catestatin (Gly364Ser, Pro370Leu, and Arg374Gln) that exhibit different potencies in inhibiting catecholamine secretion. Here, we have examined whether there is any differential processing of the three CHGA variants to catestatin by the endoproteolytic enzyme plasmin. Plasmin digestion of the purified CgA proteins generated a stable biologically active 14-amino acid peptide (human CgA360–373) from the wild-type, Gly364Ser, and Arg374Gln proteins despite the disruption of the dibasic site (Arg373Arg374) in the Arg374Gln variant. Unexpectedly, the action of plasmin in generating the catestatin peptide from the Pro370Leu protein was less efficient. The efficiency of cleavage at the dibasic Arg373{downarrow}Arg374 site in synthetic human CgA360–380 was 3- to 4-fold less in Pro370Leu CgA, compared with the wild type. Circular dichroism of the synthetic CgA352–372 suggested a difference in the amount of {alpha}-helix and β-sheet between the wild-type and Pro370Leu CgA peptides. Because the Pro370 residue is in the P4 position, the local secondary structure in the vicinity of the cleavage site may enforce the specificity or accessibility to plasmin. The less efficient proteolytic processing of the Pro370Leu protein by plasmin, coupled with the strong association of this variant with ethnicity, suggests that the Pro370Leu CHGA gene variant may contribute to the differential prevalence of cardiovascular disease across ethnic groups.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CHROMOGRANIN A (CgA), a 48-kDa acidic secretory protein, is the major soluble protein in the catecholamine storage vesicles, and is widely distributed in endocrine cells and neurons (1, 2). It is coreleased with catecholamines, neuropeptides, and ATP by exocytosis from chromaffin cells of the adrenal medulla as well as from sympathetic neurons (3). Intracellular functions of CgA include binding of Ca2+ (4, 5) and catecholamines in the catecholamine storage vesicles, regulation of vesicular biogenesis (6, 7), and trafficking of proteins to regulated secretory pathway (8, 9). Extracellular functions of CgA include generation of biologically active peptides, including the dysglycemic hormone pancreastatin (human CgA250–301), which impairs glucose tolerance by inhibiting glucose-stimulated insulin release and by triggering hepatic glycogenolysis (10, 11, 12), the vasodilator (vascular smooth muscle relaxing) vasostatin (human CgA1–76) (13), and the catecholamine release inhibitory peptide catestatin (human CgA352–372) (14, 15, 16, 17, 18) by proteolytic cleavage. The latter contributes to an autocrine negative feedback mechanism on the regulation of catecholamine secretion.

Although CgA is overexpressed in human essential (hereditary) hypertension (19), the plasma concentration of catestatin is lower in both established cases and in normotensive subjects with a family history of the disease. There are three naturally occurring human CgA variants: CgA-Gly364Ser, CgA-Pro370Leu, and CgA-Arg374Gln. Unexpectedly, we found that proteolytic processing of CgA-Pro370Leu by plasmin differed markedly when compared with the wild-type (WT) or Gly364Ser and Arg374Gln variant proteins. These findings may have implications in the risk of development of hypertension.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Construction of prokaryotic expression plasmids for human CgA and its variants: pET-(human CgA-Gly364Ser-6His), pET-(human CgA-Pro370Leu-6His), and pET-(human CgA-Arg374Gln-6His)
The pET21a(+)-human CHGA-His construct was made in a similar way as described previously (20). This construct was used as a template for the generation of catestatin variants using the QuickChangeTM Site-Directed Mutagenesis kit from Stratagene (La Jolla, CA). All mutations were confirmed by sequencing across the variant base.

Overexpression and purification of recombinant proteins
BL21(DE3)pLysS (Novagen, Madison, WI) competent cells were transformed with the respective plasmids. Cells were induced with IPTG (isopropyl β-D-thiogalactopyranoside; 1 mM), and the recombinant proteins were purified as described previously using a 6-His affinity tag (21). Protein was loaded onto an anion exchange Mono Q HR 5/5 column (Amersham Pharmacia Biotech, Uppsala, Sweden) connected to a fast protein liquid chromatography separation system. The proteins were eluted with a linear gradient (0–0.6 M) of NaCl. The peak containing human CgA fractions were pooled and further purified on a Superdex 75 HR 10/30 fast protein liquid chromatography gel filtration column (10 x 300 mm, 24 ml bed volume; Amersham Pharmacia Biotech). Protein concentration was determined using Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hercules, CA). Aliquotted protein was stored at –70 C until further use.

Immunoblot analysis
Proteins were separated in a 10% SDS-PAGE (Novex precast gel; Invitrogen, San Diego, CA) gel and electrophoretically transferred to nitrocellulose membrane (Protran, BA85; Whatman Inc., Florham Park, NJ). The membrane was blocked with 5% (wt/vol) dry milk in Tris-buffered saline with 0.1% Tween 20. After incubation with primary antibodies (goat anti-CgA; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and mouse anti-His (Invitrogen), the membrane was washed and incubated with secondary antibody (horseradish peroxidase-conjugated donkey antigoat or goat antimouse). The membrane was then developed by the Supersignal west pico-chemiluminescent substrate (Pierce, Rockford, IL), according to the manufacturer’s protocol.

Plasmin digestion of human CgA
Purified WT and mutant proteins (10 µM/reaction) were incubated either in the presence or absence of plasmin (2–0.004 µM as mentioned in each figure) in digestion buffer [10 mM Tris-Cl (pH 8.0) 0.15 M NaCl] at 37 C for 5 min. The reaction was terminated by the addition of aprotinin (2.5 µM), and the digestion mixture was analyzed by SDS-PAGE or matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS).

MALDI-TOF MS analysis
For MALDI-TOF, the plasmin digests were acidified in 0.1% trifluoroacetic acid, and purified using a Zip Tip (Millipore, Billerica, MA). One microliter of each sample was mixed with 4 µl {alpha}-cyano-4-hydroxycinnamic acid (Agilent Technologies, Inc, Santa Clara, CA), and 1 µl from that mixture was spotted onto a MALDI plate and then air dried. Mass spectra were acquired on a PE Biosystems Voyager DeSTR MALDI-TOF mass spectrometer (Applied Biosystems, Foster City, CA) with a nitrogen laser, operating in delayed extraction and reflectron mode. The two common peaks identified by MS/MS analysis were used to calibrate all MALDI spectra. Data Explorer software (Applied Biosystems) was used to make figures. The mass error of this method is characteristically less than 0.1% (i.e. within ± 50 ppm, parts per million). Resulting peptide masses obtained from the MALDI analyses were analyzed in the program Protein Prospector (http://prospector.ucsf.edu) to identify the possible fragments of the respective proteins.

MALDI-TOF tandem MS
Tandem mass analysis (MS/MS) was performed on a 4800 MALDI TOF-TOF mass spectrometer (Applied Biosystems), operated in 2-kV positive mode with collision-induced dissociation on. Samples were prepared as previously described and data acquired with 2000 shots at R200. Global Proteomics Server 3.1 (Applied Biosystems) and Mascot 2.1 (Matrix Science Inc., Boston, MA) were used to perform a database search of MS/MS spectra against a custom database containing 71 proteins, including human CgA. The MS/MS ion search parameters or missed cleavages and mass tolerances were set at 0.2 Da for both peptide mass tolerance and fragment ion mass tolerance. Common nonspecific variable modifications were included in the search parameters [deamidation (NQ), oxidation (M), and pyro-glu (N-term Q)], although none were found. Mascot results were used to elucidate a probable sequence, which was confirmed by subsequent sequencing.

Peptide synthesis
Peptides were synthesized by the solid-phase method using 9-fluorenylmethoxycarbonyl protection chemistry as described previously (16).

Secretagogue-stimulated catecholamine release
Norepinephrine secretion was assayed as described previously (22). Briefly, PC12 cells were plated on poly-L-lysine coated 12-well dishes, and labeled with L-[3H]norepinephrine (PerkinElmer Life Sciences, Waltham, MA) at 1 µCi/ml in the culture medium for 3 h at 37 C. Cells were then washed twice with basal medium and then with secretion buffer [150 mM NaCl, 5 mM KCl, 2 mM CaCl2, and 10 mM HEPES (pH 7.0)] for 15 min. The cells were treated with nicotine (60 µM) in secretion medium, either alone or in combination with five ascending doses (0.1, 0.33, 1, 3.3, and 10 µM) of each peptide for 30 min at 37 C. The supernatant was collected, and the cells were lysed with secretion buffer containing 0.1% Triton X-100 for 10 min. The amounts of 3[H]norepinephrine in the supernatant and cell lysates were measured by liquid scintillation counting, and the results were expressed as percent (%) secretion: [amount released/(amount released + amount in cell lysate)] x 100. Net secretion is secretagogue-stimulated release minus basal release.

Circular dichroism (CD) spectroscopy
CD spectra on human CgA synthetic peptides (CgA352–372) were obtained at 25 C in a 2-mm path length cuvette over 190–260 nm using a Aviv model 400 CD Spectrometer (Aviv Biomedical, Lakewood, NJ), with signal averaging over 10 sec/0.5-nm interval. WT and mutant synthetic catestatin peptides were present at a concentration 0.125 mg/ml in 98% 2,2,2-trifluoroethanol; the high concentration of 2,2,2-trifluoroethanol was used to accentuate secondary structure in these small peptides by decreasing the dielectric constant of the solution. The baselines were deduced from the buffer solutions alone in which peptide was suspended. Two repeat scans were obtained for each sample, and the baseline spectrum was subtracted from the average. The experiment was repeated three times, and the data were averaged. From the ellipticity data, the percent contribution of {alpha}-helix, strand/β-sheet, and random coil was computed using the neural network K2d program (http://www.embl-heidelberg.de/~andrade/k2d).

Human subjects and genotyping
A total of 3911 human subjects was recruited. Ethnicities were divided among these ancestry groups (by self-identification): white, Black, Hispanic, East Asian, South Asian, or mixed. Genomic DNA was prepared from blood leukocytes as previously described (23). Single nucleotide polymorphisms were scored at human CgA-Pro370Leu (C9578T, exon 7) by pyrosequencing (24).

Plasma catestatin and CgA measurement in humans
To measure the plasma concentrations of CgA (epitope: human CgA116–439) and catestatin (epitope: human CgA361–371) in EDTA-anticoagulated plasma, we used RIA, as previously described (25). To estimate the extent of proteolytic processing of CgA to catestatin, we then calculated the ratio of CgA to catestatin as an inverse index of the extent of processing of precursor to product.

Statistical analysis
Hardy-Weinberg equilibrium was calculated for human CgA Pro370Leu diploid genotypes in each population in which the variant occurred.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression and purification of recombinant WT and mutant human CgA
Purification of human recombinant CgA was accomplished using the published procedure with minor modifications (26). Approximately 2-mg each of the purified proteins (CgA-WT, CgA-Gly364Ser, or CgA-Arg374Gln) was obtained from 1-liter bacterial culture after specific elution from the affinity column. CgA-Pro370Leu consistently yielded approximately 5-fold less protein compared with the other variants. The final step of the purification included gel filtration chromatography to remove smaller CgA-derived peptides that may be produced by nonspecific processing from enzymes in the bacterial cytosol. Consistent with previous observations (27, 28, 29), all four variant proteins were eluted in the void volume from a column with a 3,000–70,000 separation limit, suggesting either an extended conformation or the formation of higher oligomeric structures of CgA. The resultant proteins appeared to be more than 95% pure with an expected molecular mass of 70 kDa as judged by SDS-PAGE (Fig. 1AGo). Immunoblot analysis using two antibodies detected one single band (Fig. 1BGo). Recombinant proteins were further subjected to MALDI MS, encompassing the molecular mass range between 1,000 and 80,000 Da, and showing only singly and doubly charge signals. These data were consistent with the molecular mass calculated from the primary sequence of each protein (results not shown). We used these highly purified protein substrates in our subsequent proteolysis studies.


Figure 1
View larger version (61K):
[in this window]
[in a new window]

  		FIG. 1. Analysis of purified WT and mutant recombinant human CgA proteins by SDS-PAGE. A, The purified proteins (4 µg/lane) were subjected to SDS-PAGE and stained with Coomassie blue. Molecular size markers (precision plus protein unstained standards, kDa) were used in the lane marked M. B, Immunoblot analysis of purified protein: 0.1-µg each protein was loaded and probed with either anti-CgA or mouse anti-6-His antibody. Native and mutant polypeptide (human CgA340–372) flanking by the dibasic processing sites [KR, RQ, RR] are shown. The amino acids at positions variant in human catestatin are shown in bold, and the catestatin region (human CgA352–372) is shown as underlined residues. {alpha}-CgA, Goat antihuman CgA; {alpha}-6-His, mouse anti-6-His epitope.

 
Identification of different catestatin region peptides generated by plasmin digestion of WT CgA
We have previously shown that digestion of recombinant WT CgA by plasmin generates a stable 14-amino acid catestatin peptide, human CgA360–373 (20). To verify whether plasmin digestion generates the full-length 21-amino acid catestatin (human CgA352–372), we used different concentrations of plasmin (i.e. enzyme to protein ratios from 1:10 to 1:3000) to digest WT CgA protein, followed by analysis of the peptides by MALDI MS. The observed mass to charge ratio (m/z) was compared with the theoretical m/z in the Protein Prospector program. Fragments with m/z that matched the theoretical m/z within ± 50 ppm were considered and listed in Table 1Go. The plasmin generated a peptide with a m/z of 1545 (the longest catestatin fragment) at all enzyme concentrations tested. MS/MS was used to obtain a primary sequence identification of the catestatin-region peptides, resulting in the identification of the following peptides: m/z 1318.7 (AYGFRGPGPQLR; human CgA362–373) (a list of the peptides can be found in supplemental Fig. 1A, which is published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org); 1389.7 (ARAYGFRGPGPQL; human CgA360–372) (supplemental Fig. 1B); 1545.8 (ARAYGFRGPGPQLR; human CgA360–373) (supplemental Fig. 1C); and 1701.9 (ARAYGFRGPGPQLRR; human CgA360–374) (supplemental Fig. 1D).


View this table:
[in this window]
[in a new window]

  		TABLE 1. Catestatin region peptides from human CgA digestion by plasmin, identified by MALDI-TOF MS

 
Several peptides were also generated outside the catestatin region by plasmin and are listed in Table 2Go. It is noteworthy that plasmin cleavage occurs at the carboxyl terminus of the pancreastatin peptide (human CgA250–301).


View this table:
[in this window]
[in a new window]

  		TABLE 2. Common peptides generated in noncatestatin regions from WT and variant proteins by plasmin digestion of human CgA

 
Proteolysis of WT and mutant human CgA proteins with higher (1–2 µM) concentrations of plasmin
Two micromolars of plasmin (Fig. 2AGo, left panel).
Purified CgA-WT, CgA-Gly364Ser, CgA-Pro370Leu, and CgA-Arg374Gln proteins were subjected to proteolytic digestion by plasmin at the physiological concentration of 2 µM (20, 30). The resultant peptides then were analyzed by MALDI-MS (Fig. 2AGo).


Figure 2
View larger version (22K):
[in this window]
[in a new window]

  		FIG. 2. MALDI-TOF of recombinant WT and mutant human CgA proteins digested with plasmin. Purified protein (10 µM/reaction) was incubated for 15 min with: 2 µM plasmin (A, left panel) and 1 µM plasmin (A, right panel); and 0.2 µM (B, left panel) and 0.02 µM (B, right panel) at 37 C. C, Dose-dependent generation of the major CgA360–373 fragment (arbitrary values from the MALDI spectra) by plasmin from the WT and CgA-Pro370Leu proteins.

 
In the WT protein, after 15-min incubation, the plasmin generated a major catestatin peptide (m/z 1389.7), corresponding to human CgA360–372 (Fig. 2AGo, left panel). In the same run, another catestatin peptide, human CgA360–373 (m/z 1545.8), also was observed, albeit to a lesser extent. A similar digestion trend was noticed for the CgA-Gly364Ser, producing fragments of m/z 1419.8 (human CgA360–372) and 1575.9 (human CgA360–373). The difference in the molecular masses in the WT and the CgA-Gly364Ser variant is due to the change of Gly residue with Ser. Therefore, these two proteins underwent proteolytic cleavage at the identical site.

In the CgA-Pro370Leu variant, plasmin failed to generate the aforementioned peptides (expected peptides masses of 1405 and 1561, corresponding to human CgA360–372 or human CgA360–373) (Fig. 2AGo). In the CgA-Arg374Gln variant, analogous peptides corresponding to human CgA360–372 and human CgA360–373 were observed (Fig. 2AGo, left panel).

One micromolar of plasmin (Fig. 2AGo, right panel).
Plasmin at 1 µM preferentially generated a peptide corresponding to human CgA360–373 with m/z 1545.8 (1575.9 in CgA-Gly364Ser) after 15-min incubation from CgA-WT, CgA-Gly364Ser, or CgA-Arg374Gln. A peptide corresponding to human CgA360–372 with a m/z of 1389.7 in CgA-WT, CgA-Arg374Gln, and 1419.8 in CgA-Gly364Ser was preferentially generated after 2-h incubation with plasmin (results not shown).

Consistent with the processing of CgA-WT (Table 1Go), CgA-Gly364Ser and CgA-Arg374Gln, plasmin cleaved the CgA-Pro370Leu protein to generate peptides with m/z values of 840.4 (human CgA360–366) and 2051.9 (human CgA339–355) but did not yield peptides corresponding to human CgA360–373 and human CgA360–372 (Fig. 2Go and Table 1Go). Plasmin also generated peptides outside the catestatin domain, as summarized in Table 2Go. Among these peptides, two peptides with a m/z of 1035 (CgA429–437) and 1191 (CgA429–438) were generated from the carboxyl-terminal region of CgA. A peptide with a m/z of 1296 was also produced from the carboxyl-terminal region but contained the adventitious 6-His affinity tag. Two peptides were identified in the amino-terminal vasostatin region with m/z values of 1171 and 1919. Another two peptides with m/z values of 2464 and 2706 were generated from the region just at the carboxyl terminal to the pancreastatin (human CgA250–301) domain (Table 2Go).

Proteolysis of WT and mutant human CgA proteins with lower (0.008–0.2 µM) concentrations of plasmin
Plasmin at 0.2 µM concentration generated cleaner MALDI spectra, showing a predominant peptide with a m/z of 1545.8 (1575.9 in CgA-Gly364Ser); corresponding to human CgA360–373. Plasmin at this concentration did not generate any catestatin peptide from CgA-Pro370Leu (Fig. 2BGo, left panel). At lower concentrations (0.02, 0.008, and 0.004 µM), plasmin still cleaved wild type, CgA-Gly364Ser, and CgA-Arg374Gln to generate catestatin peptide, CgA360–373 (Fig. 2Go, B, right panel, and C). The generation of the major 14-mer catestatin peptide, CgA360–373, was linear at low plasmin dose and reached maxima at 1 µM plasmin (Fig. 2CGo). At 2 µM plasmin, the cleavage occurred at {downarrow}Arg373 residue to generate predominant peptide of m/z 1389.7 (Fig. 2Go, A, left panel, and C). Plasmin with these doses failed to cleave CgA-Pro370Leu to generate any peptide (Fig. 2Go, B and C). These data indicate that CgA-Pro370Leu is more resistant to plasmin digestion.

Effect of plasmin-generated peptides on secretagogue-stimulated catecholamine secretion from PC12 cells
We synthesized peptides corresponding to the plasmin-generated products, along with the full-length catestatin, and tested their effects on nicotinic cholinergic pathway evoked catecholamine secretion from PC12 cells (Fig. 3Go). Consistent with the previous studies, we found potent inhibition by WT catestatin, human CgA352–372 (IC50 0.74 µM) (14, 17), to inhibit nicotine-stimulated catecholamine secretion.


Figure 3
View larger version (34K):
[in this window]
[in a new window]

  		FIG. 3. Effect of human catestatin synthetic peptides on secretagogue-stimulated norepinephrine secretion by PC12 cells. PC12 cells were labeled with L-[3H]norepinephrine and treated with 60 µM nicotine, either alone or in combination with varying concentrations of synthetic catestatin region peptides, corresponding to isoforms derived from plasmin cleavage of human CgA. After 30-min incubation, the cells and media were analyzed for norepinephrine secretion. In control, the net norepinephrine release (100%) was measured in the presence of 60 µM nicotine alone.

 
Intermediate-sized plasmin-generated catestatin fragments retained some potency to inhibit the nicotinic cholinergic pathway. The IC50 for CgA360–373 (peptide 4) was 2.4 µM, whereas that for CgA360–374 (peptide 5) was 3.04 µM. Deletion of a single Arg residue at the carboxyl terminal [from CgA360–373 (peptide 4) to CgA360–372 (peptide 3)] completely abolished potency (IC50 increasing to > 10 µM) (Fig. 3Go).

The small internal fragments, human CgA360–366 with a m/z of 840 (peptide 1) and human CgA362–373 with a m/z of 1318 (peptide 2), were completely ineffective at inhibiting catecholamine secretion. The extreme amino-terminal fragment of catestatin, human CgA351–359 with m/z 1070 (peptide 6), also lost most of its potency (Fig. 3Go).

Secondary structure as assessed by CD spectroscopy using catestatin synthetic peptides
Amino acid proline by virtue of its five-member pyrrolidine ring can alter the secondary structure of proteins and peptides. If the WT residue Pro370 is an important residue for determination of local secondary structure near the dibasic cleavage recognition site Arg373Arg374, it is expected that its substitution by Leu370 will change the local optical properties of the peptide. This hypothesis was supported upon comparing the CD spectra of the synthetic peptides (CgA352–372), wild type vs. Pro370Leu (Fig. 4Go), wherein there was a clear difference in spectra between the peptides, especially in the 200- to 230-nm range. Deconvolution of the spectra by K2d algorithm-predicted secondary structure, indicated 27% {alpha}-helix, 20% β-sheet, and 54% random coil in CgA-WT, compared with 11% {alpha}-helix, 41% β-sheet, and 49% random coil in the Pro370Leu peptide (Fig. 4Go). Thus, CD confirmed a marked change in the secondary structure of the Pro370Leu peptide compared with WT.


Figure 4
View larger version (41K):
[in this window]
[in a new window]

  		FIG. 4. Secondary structure: CD spectroscopy of WT vs. variant catestatin region from human CgA. CD spectra of 21-mer catestatin peptides (human CgA352–372) at 0.125 mg/ml: WT (SSMKLSFRARAYGFRGPGP370QL) or Pro370Leu (SSMKLSFRARAYGFRGPGL370QL).

 
CD spectra obtained on the full-length CgA proteins, varying at the catestatin region, were indistinguishable (data not shown).

Plasmin susceptibility of intact CgA
The CgA-Pro370Leu variant appeared to be resistant to plasmin digestion (Fig. 2Go). To compare the overall digestion pattern of CgA-Pro370Leu with the other variants, the four CgA-proteins were incubated with different doses of plasmin for 5 min, and the resulting mixtures were analyzed by SDS-PAGE (Fig. 5Go). WT-CgA and its variants were completely digested with plasmin at 0.4 µM, yielding only smaller molecular mass fragments (<10 to ~40 kDa). A lower molecular mass band at approximately 12 kDa was apparent after digestion of CgA-Pro370Leu and CgA-Arg374Gln, and this band was further cleaved in the WT and CgA-Gly364Ser proteins. The residual band migrating in the same position as the parent band (~70 kDa) in lanes 6–9 represents the plasmin band as evident in lane 10 that contained only plasmin. No difference in the proteolytic processing among all four CgA proteins was observed with 0.2 µM plasmin. The intact precursor (migrating ~70 kDa) was unchanged upon exposure to the lowest dose of plasmin (0.004 µM), suggesting less digestion.


Figure 5
View larger version (62K):
[in this window]
[in a new window]

  		FIG. 5. Overall susceptibility of WT and mutant human CgA proteins to plasmin digestion. WT and mutant proteins (10 µM/reaction) were digested with different concentrations of plasmin for 5 min at 37 C, and the reactions were terminated by addition of aprotinin. The digestion mixtures (8 µg each) were analyzed by SDS-PAGE. Lanes 1 and 11, Molecular mass standards (kDa). Lane 10, Plasmin only. Lane 20, The reaction was performed with aprotinin alone (without CgA).

 
Differential cleavage of synthetic peptides (human CgA360–380) by plasmin
We have shown (Fig. 2Go) that plasmin was less effective in the generation of catestatin peptide from CgA-Pro370Leu than from the other CgA variants due to the less efficient cleavage between Arg373{downarrow}Arg374. The efficiency of plasmin to cut at the Arg373{downarrow}Arg374 site was tested by synthesizing two 21-mer peptides spanning CgA residues 360 to 380: WT (ARAYGFRGPGP370QLR373RGWRPSS) or Pro370Leu (ARAYGFRGPGL370QLR373RGWRPSS). Boldfaceindicates the site of mutation. These two peptides with varying concentrations (1, 2, 4, 10, 20, 50, 100, or 1000 µM) were incubated with a fixed amount of plasmin (0.01 µM) at 37 C for 15 min. The reactions were terminated by the addition of aprotinin, and the samples were then analyzed by MALDI-TOF. Plasmin caused differential generation of a 14-mer peptide (m/z 1545.8 in wild type and 1561.8 in Pro370Leu), corresponding to human CgA360–373 (Fig. 6AGo). In the absence of plasmin, each spectrum showed a clear peak with a m/z of either 2372 or 2389, corresponding to the uncut 21-mer (CgA360–380) in WT or Pro370Leu peptides, respectively (results not shown). At a substrate concentration of 4 µM, plasmin resulted 21% cleavage of the parent molecule to CgA360–373 in Pro370Leu compared with 72% cleavage in wild type (Fig. 6Go, A and B). At a substrate concentration of 10 µM, plasmin was less efficient in processing the Pro370Leu variant (11% of the parent molecule) compared with wild type (31% of the parent molecule). These findings suggest that plasmin is 3-fold less efficient in processing of the Pro370Leu variant at or between the Arg373{downarrow}Arg374 residues. An analogous trend was found at even higher substrate concentrations of 20 and 50 µM (results not shown).


Figure 6
View larger version (30K):
[in this window]
[in a new window]

  		FIG. 6. MALDI-TOF of synthetic catestatin isoform peptides subjected to digestion with plasmin. A, Synthetic peptides (human CgA360–380), wild type (ARAYGFRGPGP370QLRRGWRPSS; m/z = 2372.2), or Pro370Leu (ARAYGFRGPGL370QLRRGWRPSS; m/z = 2388.3) at a concentration of 4 (left panels) or 10 µM (right panels) per reaction, were incubated with 0.01 µM plasmin for 15 min at 37 C. B, Generation of the 14-mer (human CgA360–373) peptide fragment (as a percentage of its precursor value). For wild type (Pro370), the generated peptide is ARAYGFRGPGP370QLR, m/z = 1545.8. For the variant (Leu370), the generated peptide is ARAYGFRGPGL370QLR, m/z = 1561.8.

 
Association of CHGA Pro370Leu genetic polymorphism with ethnicity
During systematic polymorphism discovery at CHGA by resequencing genomic DNA from 180 individuals (2n=360 chromosomes), we identified three variants in the catestatin region: Gly364Ser, Pro370Leu, and Arg374Gln (17, 23). At the time of discovery in 2n=360 chromosomes, Pro370Leu was relatively uncommon, with a minor allele frequency of 0.6%. After we established that plasmin digestion of recombinant CgA-Pro370Leu protein failed to generate the catestatin fragment (Fig. 2Go) and given the importance of catestatin in cardiovascular physiology (7, 31), we reinvestigated this particular single nucleotide polymorphism in larger populations. In whites, the 370Leu (minor) allele was found in four heterozygotes among 2373 individuals (2n=4746 chromosomes), with a minor allele frequency of 0.084% (Table 3Go). In Blacks, the 370Leu allele was found at a higher frequency with nine heterozygotes among 1276 individuals (2n=2552 chromosomes) and a minor allele frequency of 0.35%. The 370Leu variant was not observed in individuals of Hispanic or Asian ancestry. Thus, Pro370Leu diploid genotype frequencies differed by ethnicity: Black vs. white {chi}2 = 6.73, P = 0.0095. Hardy-Weinberg equilibrium obtained in the ethnic group (Black) with the highest frequency of 370Leu ({chi}2 = 0.016; P = 0.899).


View this table:
[in this window]
[in a new window]

  		TABLE 3. CHGA Pro370Leu diploid genotypes and allelic frequencies in subjects stratified by ethnicity

 
CgA Pro370Leu and catestatin processing and secretion in vivo
We were able to obtain plasma for the measurement of the CgA precursor and the catestatin product in three heterozygous carriers of the 370Leu minor allele.

In 452 WT healthy controls, plasma catestatin concentrations were 1.47 ± 0.06 nM, whereas the CgA precursor levels were detected at 3.84 ± 0.20 nM. The ratio of CgA to catestatin (an inverse index of processing) was 4.46 ± 0.17. In the three 370Leu carriers, catestatin levels ranged from 0.62–1.64 nM, whereas CgA levels were between 2.99 and 4.71 nM, and the CgA to catestatin ratio was 2.56–7.60. In one individual carrier (subject no. 4251), catestatin level was substantially diminished (at 0.62 nM), and CgA->catestatin processing was also impaired (ratio = 7.60).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Overview: plasmin cleavage of CgA
We have previously shown that naturally occurring catestatin variants display differential potencies in inhibiting nicotine-stimulated catecholamine secretion from PC12 cells (17, 23). The Gly364Ser variant caused profound alterations in human autonomic nervous system activity and altered the risk of hypertension, especially in males (25). These studies prompted us to investigate, using both MS and biochemical studies, whether plasmin can cleave CgA variants (Gly364Ser, Pro370Leu, and Arg374Gln) with equal efficacies to generate catestatin. Although catestatin is generated by proteolytic processing of CgA (14, 21, 32, 33, 34), the mechanism and the specific proteases involved in processing of CgA to catestatin are not fully understood.

Plasmin is a major fibrinolytic enzyme and is located at specific, saturable binding to the chromaffin cell surface (35), where its activation is enhanced (36). An in situ plasminogen/plasmin/tissue plasminogen activator system may exist on chromaffin cells, in which the tissue plasminogen activator is trafficked to and coreleased from the chromaffin granules by exocytosis with CgA (37), therefore, providing a mechanism for the generation of active plasmin, which can in turn cleave CgA to catestatin (20, 35). Thus, the evidence is compelling for plasmin as a candidate enzyme for CgA processing to catestatin (20).

Plasmin dose dependently cleaved WT-CgA to catestatin (Table 1Go and Fig. 2CGo), and the peptide with a m/z of 1545.8 (human CgA360–373) appeared to be the preferred product, even at relatively low concentrations of plasmin (as low as 0.004 µM) and in most of the conditions tested (Fig. 2Go). Although human CgA360–373 was a consistent peptide product, plasmin at a very high concentration (2 µM) or with longer incubation periods (results not shown) cleaved at the Leu372{downarrow}Arg373 peptide bond, thereby removing Arg373 and yielding peptide human CgA360–372 with a m/z of 1389.7. These results suggest that the preferential (or initial) site of plasmin cleavage is toward the carboxyl terminus of CgA and is at the Arg373{downarrow}Arg374 site (Figs. 2Go and 7Go).


Figure 7
View larger version (6K):
[in this window]
[in a new window]

  		FIG. 7. Preferred cleavage sites by plasmin within the catestatin region of human CgA. The catestatin region within human CgA is shown as underlined residues human CgA352–372. All of the monobasic and paired (di-) basic sites (K, R) are shown in bold. The predicted (post-basic) cleavage sites are shown by arrows and are numbered (one to nine). *, Most preferred cleavage sites. Amino acids at positions variant in human catestatin are shown in italics.

 
Biological activity of the catestatin peptides liberated from CgA by plasmin
To evaluate the consequences of plasmin cleavage, we synthesized and purified the pertinent peptides (Fig. 3Go). The plasmin-generated peptides displayed differential potencies toward inhibition of nicotine-evoked catecholamine secretion.

The residues at positions Arg373 and Arg374 were found to be important. Although the deletion of Arg374 (from CgA360–374 to CgA360–373) had little effect on the potency (IC50 from 3.04–2.4 µM), removal of Arg373 (from CgA360–373 to CgA360–372) resulted in a complete loss of the effect (IC50 from 2.4 to > 10 µM). The dipeptide Ala360Arg361 also played an important role. Deletion of Ala360Arg361 to yield CgA362–373 (peptide 2, Fig. 3Go) resulted in a marked decrease in activity (IC50 from 2.4 to > 10 µM).

These results define human CgA360–373 as a minimal active region of human catestatin that is generated by plasmin. We have previously identified a minimal active core of bovine catestatin that consists of 15-amino-terminal amino acids (bCgA344–358) within the catestatin (bCgA344–364) domain (16). We have also identified the major prevalent catestatin isoforms within bovine chromaffin granules as a 33-mer bCgA332–364 (cleaved between dibasic sites Lys330Arg331 and Arg365Arg366) and a 20-mer bCgA343–362 (21). The secretion media from isolated bovine chromaffin cells contained a major catestatin peptide identified as bCgA344–364 (34). However, in human pheochromocytoma chromaffin granules, the major catestatin form identified was a 32-mer, human CgA340–372, which results from cleavage between the dibasic sites Lys338Arg339 and Arg373Arg374 (21). In the present study, we were unable to detect any longer catestatin fragment(s) generated by plasmin digestion. Other proteases may act on CgA within the chromaffin granule to generate longer forms of catestatin peptide (34), which after secretion (along with the intact precursor) may be subject to further extracellular cleavage by plasmin to generate the shorter 14-mer peptide human CgA360–373 (Fig. 7Go).

Liberation of catestatin from CgA by plasmin: effect of naturally occurring genetic variation in the catestatin region
Despite the loss of a dibasic cleavage site in the Arg374Gln variant, the overall digestion pattern by plasmin was very similar to that of CgA-WT (Fig. 2Go). This suggests that plasmin preferentially cleaves a site before Arg374 (such as Arg373{downarrow}Arg374) and that the Arg374Gln replacement failed to influence processing by plasmin. Although there are nine basic residues within the catestatin region, plasmin seems to cleave preferentially at Arg359{downarrow}Ala360 and Arg373{downarrow}Arg374 sites (Fig. 7Go).

Because plasmin failed to produce the human CgA360–373 peptide from the CgA-Pro370Leu substrate, the Pro370Leu substitution probably altered the environment of the usual/preferential downstream Arg373{downarrow}Arg374 cleavage site. In addition to this dibasic site (e.g. Arg373Arg374), other features of the local substrate region determine the effectiveness of cleavage. For example, the effect of P4P3P2P1{downarrow}P1'P2'P3'P4' residues determine the cleavage where P1, P2, P3, and P4 are positions in the amino-terminal direction from the cleaved bond, and P1', P2', P3', and P4' are the residues in carboxyl terminal. Pro370Leu at residue P4 might decrease cleavage at P1{downarrow}P1' (Arg373{downarrow}Arg374) because the amino acid Pro, with its five-member ring in the peptide backbone, typically influences local secondary structural features (such as {alpha}-helix or β-sheet), and probably increases the accessibility of the local region to entry of a proteolytic enzyme. Although CD spectra of the full-length CgA variant proteins were similar to each other, the Pro370Leu change in the smaller peptides (CgA352–372) suggested differences in the local secondary structure by decreasing the amount of {alpha}-helix and increased the amount of β-sheet. It has been previously shown that the Pro residue located near the processing sites of human pro-somatostatin is important for its processing (38). Consistent with this finding in pro-somatostatin, we found a 3.4-fold reduction in proteolytic processing of the Pro370Leu mutant peptide by plasmin, compared with the WT peptide (Fig. 6Go, A and B). Plasmin was able to cleave the P1{downarrow}P1' site (at Arg373{downarrow}Arg374) more efficiently in the synthetic peptide CgA360–380 (Fig. 6Go, A and B) than in the intact protein (Fig. 2Go), suggesting the contribution of additional structural elements, to create this cleavage site specificity in this local region.

Significance of the Pro370Leu variant
Although a synthetic peptide containing the 370Leu variant (SSMKLSFRARAYGFRGPGL370QL) was even more potent than the WT version (SSMKLSFRARAYGFRGPGP370QL) in the inhibition of catecholamine secretion (17), the CgA-Pro370Leu precursor protein was found to be the most resistant to plasmin digestion in generation of the catestatin peptide fragment. If plasmin used the next dibasic sites K400{downarrow}K401 instead of R373{downarrow}R374, a longer, 39-amino acid (human CgA360–399) peptide may be generated. Thus, even though the 370Leu version of catestatin is potent in the inhibition of nicotine-stimulated catecholamine secretion, it is not formed by plasmin processing of CgA.

A diminution in the formation of catestatin has been observed not only in hypertensive subjects but also in normotensive individuals with a risk of developing hypertension as deduced by a positive family history (31). The involvement of catestatin in blood pressure regulation (7) raises the possibility that the Pro370Leu variant might be associated with hypertension. However, the variant allele (370Leu) is sufficiently uncommon (at only 0–0.35%, Table 3Go) that thousands of subjects would be required for a definitive test of this hypothesis. Nonetheless, our population study of Pro370Leu (Table 3Go) demonstrated an increased allele frequency of the 370Leu variant within the Black population ({chi}2 = 6.73; P = 0.0095), and this group is at an increased risk for the development of hypertension (39).

Finally, we have explored here the action of only a single type of protease (plasmin) in generation of catestatin from CgA-WT and its genetic variants. Future studies of additional enzymes in physiological pathways of CgA processing may yield insights into the role of the catestatin system in the development of human hypertension.


   Acknowledgments
 
We thank Professor Elizabeth A. Komives (Department of Chemistry and Biochemistry, University of California, San Diego) for helping us in matrix-assisted laser desorption/ionization time-of-flight and mass spectrometry/mass spectrometry experiments.


   Footnotes
 
This work was supported by grants from the Department of Veterans Affairs (to S.K.M. and D.T.O.) and the National Institutes of Health (R29 DA011311 and R01 DA011311 to S.K.M.; DK 60702 to D.T.O.; and P01 HL58120 and U01 HL69758 to S.K.M. and D.T.O., respectively).

Disclosure Statement: The authors have nothing to disclose.

First Published Online November 8, 2007

Abbreviations: CD, Circular dichroism; CgA, chromogranin A; MALDI, matrix-assisted laser desorption/ionization; MS, mass spectrometry; MH+, singly-charged molecular ion; m/z, mass to charge ratio; TOF, time-of-flight; WT, wild type.

Received June 25, 2007.

Accepted for publication October 26, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Winkler H, Fischer-Colbrie R 1992 The chromogranins A and B: the first 25 years and future perspectives. Neuroscience 49:497–528[CrossRef][Medline]
  2. Taupenot L, Harper KL, O’Connor DT 2003 Mechanisms of disease: the chromogranin-secretogranin family. N Engl J Med 348:1134–1149[Free Full Text]
  3. Takiyyuddin MA, Cervenka JH, Sullivan PA, Pandian MR, Parmer RJ, Barbosa JA, O’Connor DT 1990 Is physiologic sympathoadrenal catecholamine release exocytotic in humans? Circulation 81:185–195[Abstract/Free Full Text]
  4. Reiffen FU, Gratzl M 1986 Ca2+ binding to chromaffin vesicle matrix proteins: effect of pH, Mg2+, and ionic strength. Biochemistry 25:4402–4406[CrossRef][Medline]
  5. Videen JS, Mezger MS, Chang YM, O’Connor DT 1992 Calcium and catecholamine interactions with adrenal chromogranins. Comparison of driving forces in binding and aggregation. J Biol Chem 267:3066–3073[Abstract/Free Full Text]
  6. Kim T, Tao-Cheng J, Eiden LE, Loh YP 2001 Chromogranin A, an "On/Off" switch controlling dense-core secretory granule biogenesis. Cell 106:499–509[CrossRef][Medline]
  7. Mahapatra NR, O’Connor DT, Vaingankar SM, Sinha Hikim AP, Mahata M, Ray S, Staite E, Wu H, Gu Y, Dalton N, Kennedy BP, Ziegler MG, Ross Jr J, Mahata SK 2005 Hypertension from targeted ablation of chromogranin A can be rescued by the human ortholog. J Clin Invest 115:1942–1952[CrossRef][Medline]
  8. Kelly RB 1985 Pathways of protein secretion in eukaryotes. Science 230:25–32[Abstract/Free Full Text]
  9. Taupenot L, Harper KL, Mahapatra NR, Parmer RJ, Mahata SK, O’Connor DT 2002 Identification of a novel sorting determinant for the regulated pathway in the secretory protein chromogranin A. J Cell Sci 115:4827–4841[CrossRef][Medline]
  10. Tatemoto K, Efendic S, Mutt V, Makk G, Feistner GJ, Barchas JD 1986 Pancreastatin, a novel pancreatic peptide that inhibits insulin secretion. Nature 324:476–478[CrossRef][Medline]
  11. Cadman PE, Rao F, Mahata SK, O’Connor DT 2002 Studies of the dysglycemic peptide, pancreastatin, using a human forearm model. Ann NY Acad Sci 971:528–529[Medline]
  12. O’Connor DT, Cadman PE, Smiley C, Salem RM, Rao F, Smith J, Funk SD, Mahata SK, Mahata M, Wen G, Taupenot L, Gonzalez-Yanes C, Harper KL, Henry RR, Sanchez-Margalet V 2005 Pancreastatin: multiple actions on human intermediary metabolism in vivo, variation in disease, and naturally occurring functional genetic polymorphism. J Clin Endocrinol Metab 90:5414–5425[Abstract/Free Full Text]
  13. Aardal S, Helle KB, Elsayed S, Reed RK, Serck-Hanssen G 1993 Vasostatins, comprising the N-terminal domain of chromogranin A, suppress tension in isolated human blood vessel segments. J Neuroendocrinol 5:405–412[CrossRef][Medline]
  14. Mahata SK, O’Connor DT, Mahata M, Yoo SH, Taupenot L, Wu H, Gill BM, Parmer RJ 1997 Novel autocrine feedback control of catecholamine release. A discrete chromogranin A fragment is a noncompetitive nicotinic cholinergic antagonist. J Clin Invest 100:1623–1633[Medline]
  15. Mahata SK, Mahata M, Parmer RJ, O’Connor DT 1999 Desensitization of catecholamine release: the novel catecholamine release-inhibitory peptide catestatin (chromogranin A344–364) acts at the receptor to prevent nicotinic cholinergic tolerance. J Biol Chem 274:2920–2928[Abstract/Free Full Text]
  16. Mahata SK, Mahata M, Wakade AR, O’Connor DT 2000 Primary structure and function of the catecholamine release inhibitory peptide catestatin (chromogranin A344–364): identification of amino acid residues crucial for activity. Mol Endocrinol 14:1525–1535[Abstract/Free Full Text]
  17. Mahata SK, Mahata M, Wen G, Wong WB, Mahapatra NR, Hamilton BA, O’Connor DT 2004 The catecholamine release-inhibitory "catestatin" fragment of chromogranin a: naturally occurring human variants with different potencies for multiple chromaffin cell nicotinic cholinergic responses. Mol Pharmacol 66:1180–1191[Abstract/Free Full Text]
  18. Mahata SK 2004 Catestatin—the catecholamine release inhibitory peptide: a structural and functional overview. Curr Med Chem Immunol Endocr Metab Agents 4:221–234[CrossRef]
  19. O’Connor DT, Takiyyuddin MA, Printz MP, Dinh TQ, Barbosa JA, Rozansky DJ, Mahata SK, Wu H, Kennedy BP, Ziegler MG, Wright FA, Schlager G, Parmer RJ 1999 Catecholamine storage vesicle protein expression in genetic hypertension. Blood Press 8:285–295[CrossRef][Medline]
  20. Jiang Q, Taupenot L, Mahata SK, Mahata M, O’Connor DT, Miles LA, Parmer RJ 2001 Proteolytic cleavage of chromogranin A (CgA) by plasmin: selective liberation of a specific bioactive CgA fragment that regulates catecholamine release. J Biol Chem 276:25022–25029[Abstract/Free Full Text]
  21. Taylor CV, Taupenot L, Mahata SK, Mahata M, Wu H, Yasothornsrikul S, Toneff T, Caporale C, Jiang Q, Parmer RJ, Hook VY, O’Connor DT 2000 Formation of the catecholamine release-inhibitory peptide catestatin from chromogranin A. Determination of proteolytic cleavage sites in hormone storage granules. J Biol Chem 275:22905–22915[Abstract/Free Full Text]
  22. Mahata M, Mahata SK, Parmer RJ, O’Connor DT 1996 Vesicular monoamine transport inhibitors. Novel action at calcium channels to prevent catecholamine secretion. Hypertension 28:414–420[Abstract/Free Full Text]
  23. Wen G, Mahata SK, Cadman P, Mahata M, Ghosh S, Mahapatra NR, Rao F, Stridsberg M, Smith DW, Mahboubi P, Schork NJ, O’Connor DT, Hamilton BA 2004 Both rare and common polymorphisms contribute functional variation at CHGA, a regulator of catecholamine physiology. Am J Hum Genet 74:197–207[CrossRef][Medline]
  24. Ahmadian A, Ehn M, Hober S 2006 Pyrosequencing: history, biochemistry and future. Clin Chim Acta 363:83–94[CrossRef][Medline]
  25. Rao F, Wen G, Gayen JR, Das M, Vaingankar SM, Rana BK, Mahata M, Kennedy BP, Salem RM, Stridsberg M, Abel K, Smith DW, Eskin E, Schork NJ, Hamilton BA, Ziegler MG, Mahata SK, O’Connor DT 2007 Catecholamine release-inhibitory peptide catestatin (chromogranin A(352–372)): naturally occurring amino acid variant Gly364Ser causes profound changes in human autonomic activity and alters risk for hypertension. Circulation 115:2271–2281[Abstract/Free Full Text]
  26. Taupenot L, Remacle JE, Helle KB, Aunis D, Bader MF 1995 Recombinant human chromogranin A: expression, purification and characterization of the N-terminal derived peptides. Regul Pept 56:71–88[CrossRef][Medline]
  27. Smith AD, Winkler H 1967 Purification and properties of an acidic protein from chromaffin granules of bovine adrenal medulla. Biochem J 103:483–492[Medline]
  28. Yoo SH, Lewis MS 1992 Effects of pH and Ca2+ on monomer-dimer and monomer-tetramer equilibria of chromogranin A. J Biol Chem 267:11236–11241[Abstract/Free Full Text]
  29. Yoo SH, Lewis MS 1996 Effects of pH and Ca2+ on heterodimer and heterotetramer formation by chromogranin A and chromogranin B. J Biol Chem 271:17041–17046[Abstract/Free Full Text]
  30. Vassalli JD, Sappino AP, Belin D 1991 The plasminogen activator/plasmin system. J Clin Invest 88:1067–1072[Medline]
  31. O’Connor DT, Kailasam MT, Kennedy BP, Ziegler MG, Yanaihara N, Parmer RJ 2002 Early decline in the catecholamine release-inhibitory peptide catestatin in humans at genetic risk of hypertension. J Hypertens 20:1335–1345[CrossRef][Medline]
  32. Simon JP, Bader MF, Aunis D 1988 Secretion from chromaffin cells is controlled by chromogranin A-derived peptides. Proc Natl Acad Sci USA 85:1712–1716[Abstract/Free Full Text]
  33. Metz-Boutigue MH, Garcia-Sablone P, Hogue-Angeletti R, Aunis D 1993 Intracellular and extracellular processing of chromogranin A. Determination of cleavage sites. Eur J Biochem 217:247–257[Medline]
  34. Lee JC, Taylor CV, Gaucher SP, Toneff T, Taupenot L, Yasothornsrikul S, Mahata SK, Sei C, Parmer RJ, Neveu JM, Lane WS, Gibson BW, O’Connor DT, Hook VY 2003 Primary sequence characterization of catestatin intermediates and peptides defines proteolytic cleavage sites utilized for converting chromogranin a into active catestatin secreted from neuroendocrine chromaffin cells. Biochemistry 42:6938–6946[CrossRef][Medline]
  35. Parmer RJ, Mahata M, Gong Y, Mahata S, K., Jiang Q, O’Connor DT, Xi X-P, Miles LA 2000 Processing of chromogranin A by plasmin provides a novel mechanism for regulating catecholamine secretion. J Clin Invest 106:907–915[Medline]
  36. Miles LA, Andronicos NM, Baik N, Parmer RJ 2006 Cell-surface actin binds plasminogen and modulates neurotransmitter release from catecholaminergic cells. J Neurosci 26:13017–13024[Abstract/Free Full Text]
  37. Parmer RJ, Mahata M, Mahata S, Sebald MT, O’Connor DT, Miles LA 1997 Tissue plasminogen activator (t-PA) is targeted to the regulated secretory pathway. Catecholamine storage vesicles as a reservoir for the rapid release of t-PA. J Biol Chem 272:1976–1982[Abstract/Free Full Text]
  38. Brakch N, Rholam M, Boussetta H, Cohen P 1993 Role of beta-turn in proteolytic processing of peptide hormone precursors at dibasic sites. Biochemistry 32:4925–4930[CrossRef][Medline]
  39. Williams RA, Gavin 3rd JR, Phillips RA, Sumner AE, Duncan AK, Hollar D, Hennekens CH 2006 High-risk African Americans with multiple risk factors for cardiovascular disease: challenges in prevention, diagnosis, and treatment. Ethn Dis 16:633–639[Medline]



This article has been cited by other articles:


Home page
 EndocrinologyHome page
J. R. Gayen, Y. Gu, D. T. O'Connor, and S. K. Mahata
Global Disturbances in Autonomic Function Yield Cardiovascular Instability and Hypertension in the Chromogranin A Null Mouse
Endocrinology, November 1, 2009; 150(11): 5027 - 5035.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
K. B. Helle
The chromogranin A-derived peptides vasostatin-I and catestatin as regulatory peptides for cardiovascular functions
Cardiovasc Res, August 18, 2009; (2009) cvp266v2.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
N. Biswas, J. L. Rodriguez-Flores, M. Courel, J. R. Gayen, S. M. Vaingankar, M. Mahata, J. W. Torpey, L. Taupenot, D. T. O'Connor, and S. K. Mahata
Cathepsin L Colocalizes with Chromogranin A in Chromaffin Vesicles to Generate Active Peptides
Endocrinology, August 1, 2009; 150(8): 3547 - 3557.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
H. Yuan, K. M. Vance, C. E. Junge, M. T. Geballe, J. P. Snyder, J. R. Hepler, M. Yepes, C.-M. Low, and S. F. Traynelis
The Serine Protease Plasmin Cleaves the Amino-terminal Domain of the NR2A Subunit to Relieve Zinc Inhibition of the N-Methyl-D-aspartate Receptors
J. Biol. Chem., May 8, 2009; 284(19): 12862 - 12873.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
N. R. Mahapatra
Catestatin is a novel endogenous peptide that regulates cardiac function and blood pressure
Cardiovasc Res, December 1, 2008; 80(3): 330 - 338.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
T. Angelone, A. M. Quintieri, B. K. Brar, P. T. Limchaiyawat, B. Tota, S. K. Mahata, and M. C. Cerra
The Antihypertensive Chromogranin A Peptide Catestatin Acts as a Novel Endocrine/Paracrine Modulator of Cardiac Inotropism and Lusitropism
Endocrinology, October 1, 2008; 149(10): 4780 - 4793.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Biswas, N.
Right arrow Articles by Mahata, S. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Biswas, N.
Right arrow Articles by Mahata, S. K.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals