Phosphorylation and O-glycosylation sites of human chromogranin A (CGA(79-439)) from urine of patients with carcinoid tumors

Because of their water-soluble properties, chromogranins (CGs) and chromogr anin-derived fragments are released together with catecholamines from adren al chromaffin cells during stress situations and can be detected in the blo od by radiochemical and enzyme assays. It is well known that chromogranins can serve as immunocytochemical markers for neuroendocrine tissues and as a diagnostic tool for neuroendocrine tumors. In 1993, large CGA-derived frag ments have been shown to be excreted into the urine in patients with carcin oid tumors and the present study deals with three characterization of the p ost-translational modifications (phosphorylation and O-glycosylation) locat ed along the largest natural CGA-derived fragment CGA(79-439). Using mild p roteolysis of peptidic material, high performance liquid chromatography, se quencing, and mass spectrometry analysis, six post-translational modificati ons were detected along the C-terminal CGA-derived fragment CGA(79-439). Th ree O-linked glycosylation sites were located in the core of the protein on Thr(163), Thr(165), and Thr(233), consisting in di-, tri-, and tetrasaccha rides. Three phosphorylation sites were located in the middle and C-termina l domain, on serine residues Ser(200), Ser(252), and Ser(315). These modifi ed sites were compared with sequences of others species and discussed in re lation with the post-translational modifications that we: have reported pre viously for bovine CGA.