Identification of novel loci affecting circulating chromogranins and related peptides

Abstract

Chromogranins are pro-hormone secretory proteins released from neuroendocrine cells, with effects on control of blood pressure. We conducted a genome-wide association study for plasma catestatin, the catecholamine release inhibitory peptide derived from chromogranin A (CHGA), and other CHGA- or chromogranin B (CHGB)-related peptides, in 545 US and 1252 Australian subjects. This identified loci on chromosomes 4q35 and 5q34 affecting catestatin concentration (P = 3.40 × 10-30 for rs4253311 and 1.85 × 10-19 for rs2731672, respectively). Genes in these regions include the proteolytic enzymes kallikrein (KLKB1) and Factor XII (F12). In chromaffin cells, CHGA and KLKB1 proteins co-localized in catecholamine storage granules. In vitro, kallikrein cleaved recombinant human CHGA to catestatin, verified by mass spectrometry. The peptide identified from this digestion (CHGA360-373) selectively inhibited nicotinic cholinergic stimulated catecholamine release from chromaffin cells. A proteolytic cascade involving kallikrein and Factor XII cleaves chromogranins to active compounds both in vivo and in vitro.

Figure 1

Discovery of novel loci on chromosome 4q35 (KLKB1, Fletcher factor) and chromosome 5 (F12, Hageman factor) that influence plasma concentrations of CHGA fragments. Results from meta-analysis of UCSD and QIMR GWAS results for CHGA 361–372 (catestatin).

Figure 2

Regional plots for the chromosome 4 and chromosome 5 loci; combined data from UCSD and QIMR for CHGA 361–372 (catestatin).

Figure 3

Sub-cellular co-localization of CHGA and KLKB1 in catecholamine storage vesicles of chromaffin cells. Experiments were conducted in PC12 cells, with immuno-staining of KLKB1 (green conjugate) and CHGA (red conjugate). A series of x/y optical sections along z-axis were acquired with increments of 0.2 µm. Data were processed to generate pseudo-three-dimensional (3D) or representative x/y sections. Co-localization of KLKB1 (green) and CHGA (red) was shown by yellow fluorescence, with a Pearson coefficient of overlap = 0.67.

Figure 4

Catestatin derived by KLKB1 digestion of recombinant human CHGA: Synthesis potency and selective effects on catecholamine secretion triggered by the nicotinic cholinergic pathway in chromaffin cells. PC12 cells were labelled with [³H]-norepinephrine and then incubated with either 60 µM nicotine, or 55 mM KCl (for membrane depolarization), or vehicle. Secretory stimulation occurred either alone or in combination with ascending doses (0.1, 1 and 10 µM) of each catestatin peptide, either the KLKB1-derived (and then synthesized) version ARAYGFRGPGPQLR (hCgA_360–373), or the usual longer version (hCgA_352–372). Control (100%) net norepinephrine release represents the release in the presence of nicotine or KCl (without inhibitor).

Figure 5

Differential control of expression of Chga versus Klkb1 mRNA in the adrenal gland for two rodent models of human essential (genetic) hypertension: rat SHR (Spontaneously Hypertensive rat, versus its WKY [Wistar-Kyoto] control); and mouse BPH (Blood Pressure High, versus its BPL [Blood Pressure Low] control).

Figure 6

Proposed schema for the effects of functional genetic variation at sequential serine proteases F12 (Hageman Factor; 5’-UTR C46T, rs2731672) and KLKB1 (Fletcher factor; Asn124Ser, rs3733402), on formation of active peptides by proteolytic cleavage of CHGA or CHGB.