Antibacterial Activity of Glycosylated and Phosphorylated Chromogranin A-derived Peptide 173-194 from Bovine Adrenal Medullary Chromaffin Granules

Jean-Marc Strub,Yannick Goumon,Karine Lugardon,Calliope Capon,Michel Lopez,Marc Moniatte,Alain Van Dorsselaer,Dominique Aunis,Marie-Hélène Metz-Boutigue

doi:10.1074/jbc.271.45.28533

Abstract

Recently, we have isolated from bovine chromaffin granules and identified two natural peptides possessing antibacterial activity: secretolytin (chromogranin B 614-626) and enkelytin (proenkephalin-A 209-237). Here, we characterize a large natural fragment, corresponding to chromogranin A 79-431, that inhibits growth of both Gram-positive and Gram-negative bacteria. The aim of the present work was to determine the shortest active peptide located in the 79-431 chromogranin A region. Three peptides, which shared the same 173-194 chromogranin A sequence (YPGPQAKEDSEGPSQGPASREK) but differed in post-translational modifications, including O-glycosylation and tyrosine phosphorylation, were isolated. A detailed study using microsequencing and mass spectrometry allowed us to correlate their antibacterial activity with these post-translational modifications. The chromogranin A precursor fragment (79-431) and the active glycosylated and phosphorylated peptides were, respectively, named prochromacin and chromacin (P, G, and PG for phosphorylated, glycosylated, and phosphorylated-glycosylated form).

Highlights

Secretory granules from bovine adrenal medullary chromaffin cells contain a complex mixture of secretory products that include low molecular mass constituents such as catecholamines, ascorbate, nucleotides, calcium, enkephalins, and several water-soluble proteins
As a continuation of these studies, the present paper reports the antibacterial activity of a large chromogranin A (CGA) fragment generated by natural cleavage at the previously described site 78 –79 [6]
When the soluble intragranular material or the secreted fragments from Kϩ-depolarized chromaffin cells were separated by HPLC and their effects on the growth of M. luteus (Gram-positive bacteria) and E. coli (Gram-negative bacteria) tested, antibacterial activity was detected in several fractions

Summary

Introduction

Secretory granules from bovine adrenal medullary chromaffin cells contain a complex mixture of secretory products that include low molecular mass constituents such as catecholamines, ascorbate, nucleotides, calcium, enkephalins, and several water-soluble proteins. Among the latter, dopamine-␤hydroxylase, proenkephalin-A, and the chromogranins/secretogranins have been extensively studied. As a continuation of these studies, the present paper reports the antibacterial activity of a large CGA fragment (residues 79 – 431) generated by natural cleavage at the previously described site 78 –79 [6] This CGA fragment was detectable in the intragranular matrix, was released during exocytosis and inhibited the growth of both Gram-positive (Micrococcus luteus) and Gram-negative (Escherichia coli) bacteria. Structural features and more posttranslational modifications are discussed in relation with their antibacterial activity

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Conclusion