Phosphorylation and O-glycosylation sites of bovine chromogranin a from adrenal medullary chromaffin granules and their relationship with biological activities

Bovine adrenal medullary chromogranin A, the major soluble component of chromaffin granules, is a phosphorylated glycoprotein. In the present work, phosphorylation and glycosylation sites were determined using mild proteolysis, peptide separation, microsequencing, and mass analysis by electrospray and matrix-assisted laser desorption ionization time-of-flight techniques. Seven post-translational modification sites were detected. Two O-linked glycosylation sites, each consisting of the trisaccharide NeuAc alpha 2-3Gal beta 1-3GalNA alpha 1, were located in the middle part of the protein, on Ser(186) and on Thr(231). The former residue is present in the antibacterial peptide named chromacin. Four phosphorylation sites were located on serine residues at positions Ser(81) in the N-terminal region of the protein and Ser(307), Ser(372), and Ser(376) in the C-terminal end. One additional phosphorylation site was found on the tyrosine residue at position Tyr(173), the N-terminal amino acid of chromacin, With the exception of the phosphorylation on Tyr(173), all of the other post-translational modifications are located on highly conserved chromogranin A regions, implying some biological importance.