Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2008 Feb;149(2):749-57.
doi: 10.1210/en.2007-0838. Epub 2007 Nov 8.

Proteolytic cleavage of human chromogranin a containing naturally occurring catestatin variants: differential processing at catestatin region by plasmin

Nilima Biswas  1 , Sucheta M Vaingankar, Manjula Mahata, Madhusudan Das, Jiaur R Gayen, Laurent Taupenot, Justin W Torpey, Daniel T O'Connor, Sushil K Mahata
Affiliations

Proteolytic cleavage of human chromogranin a containing naturally occurring catestatin variants: differential processing at catestatin region by plasmin

Nilima Biswas et al. Endocrinology. 2008 Feb.

Abstract

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 CgA(360-373)) from the wild-type, Gly364Ser, and Arg374Gln proteins despite the disruption of the dibasic site (Arg(373)Arg(374)) 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 Arg(373) downward arrowArg(374) site in synthetic human CgA(360-380) was 3- to 4-fold less in Pro370Leu CgA, compared with the wild type. Circular dichroism of the synthetic CgA(352-372) suggested a difference in the amount of alpha-helix and beta-sheet between the wild-type and Pro370Leu CgA peptides. Because the Pro(370) 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.

PubMed Disclaimer

Figures

Figure 1
Figure 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. α-CgA, Goat antihuman CgA; α-6-His, mouse anti-6-His epitope.
Figure 2
Figure 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.
Figure 3
Figure 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.
Figure 4
Figure 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).
Figure 5
Figure 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).
Figure 6
Figure 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.
Figure 7
Figure 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.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Winkler H, Fischer-Colbrie R 1992 The chromogranins A and B: the first 25 years and future perspectives. Neuroscience 49:497–528 - PMC - PubMed
    1. Taupenot L, Harper KL, O’Connor DT 2003 Mechanisms of disease: the chromogranin-secretogranin family. N Engl J Med 348:1134–1149 - PubMed
    1. 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 - PubMed
    1. Reiffen FU, Gratzl M 1986 Ca2+ binding to chromaffin vesicle matrix proteins: effect of pH, Mg2+, and ionic strength. Biochemistry 25:4402–4406 - PubMed
    1. 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 - PubMed

Publication types

MeSH terms