Chromogranin A from bovine adrenal medulla: Molecular characterization of glycosylations, phosphorylations, and sequence heterogeneities by mass spectrometry

Chromogranin A (CGA) is a member of a family of acidic glycoproteins presen t in endocrine and neuroendocrine tissues. One of its suggested physiologic al roles is being a precursor molecule for several peptide hormons, Further interest in this protein has recently originated from its potential role i n pathophysiological processes of Alzheimer's disease. The concentration of CGA in the brain has been used for diagnosis of this disease, and CGA as a n insoluble deposit has been found in the extracellular P-amyloid plaques, By developing a new purification procedure we were able to isolate abundant CGA in high purity from bovine chromaffin cells, A MALDI-MS analysis of th e intact protein revealed a heterogeneous molecular mass of ca. 50 kDa, ind icating several structure modifications. By use of several subsequent prote olytic/chemical cleavage steps, HPLC isolation, a newly developed deglycosy lation procedure, and several MS and MS-MS fragmentation approaches, the co mplete primary structure of CGA including four sequence heterogeneities, tw o O-glycosylations, five phosphorylations, and one disulfide bridge could b e characterized. For both glycans six different forms could be identified, Ser167 was found to be mainly glycosylated by a trisaccharide, and Thr231 w as found to be mainly glycosylated by a tetrasaccharide. Ser81, Ser124, and Ser297 residues were partially phosphorylated, whereas Ser372 and Ser377 w ere found completely phosphorylated. Sequence heterogeneities were identifi ed in positions 293 (H/R), 301 (K/E), and 373 (Q/R) and at the partly missi ng C-terminal residue. Furthermore, a disulfide bridge between Cys17 and Cy s38 was ascertained. (C) 1999 Academic Press.