The C‐terminal Bisphosphorylated proenkephalin‐A‐(209–237)‐peptide from Adrenal Medullary Chromaffin Granules Possesses Antibacterial Activity

Abstract

The chromaffin granules have been shown to be an excellciit model to study the processing of proenkephalin-A and chromogranins. Recently, we reported a study dealing with the processing of chromogranin B/secretogranin 1 and the occurrence of the C-tertninal chromogranin B-derivcd peptide 614-626 which was shown to have antibacterial iictivity [Strub, J. M., Garcia-Sablonc, P., Looning, K., Taupenol, L., Hubert, P., Van Riochem. 229,. We also observed that this new antibacterial activity prcsent in chromaffin granules was associated with other endogenous protein-derived fragments yet to be characterizcd. The present study reports the isolation rind characterization of a pcptide which possesses antibacterial activity and which corresponds to the C-tcrniinal 209-237 sequence ol' procnkephalin-A. A detailcd study using niicroscquciicing and matrix-assisted-laser-desorptioii time-of-tlight Inass spectrometry (MALD-TOF MS) allowed us to correlate the antibacterial activity of this peptide named enkelytin (FAEPTBSEEEGE-SYSKEVPEMEKRYGGFM) with post-translational modifications. Endogenouc bisphosphorylated proenkephalin-A-(209 -237) was activc on Mirrocwcc.i~,r luteus and Bocillms negaterium killing bacteria in the 0.2-0.4 pM range but was inactive in similar conditions towards Excherichia coli. Eiikelytiii shares sequence and structiiral similarities with thc antibacterial C-terminal domain o l diazcpam-binding inhihitor. According to this similarity, a prediction of secondary structure is proposed for enkelylin and discussed in relation to its biological activity.KtJywor~1,s: bovinc adrenal medulla; chromaffin granules ; proenkephalin-A-derived fragments ; diazepamhinding inhibitor; antibacterial peptides.A large number of biologically active peptides are synthe-chrornaffin granules, these organelles havc been shown to reprcsized as part of much larger precursor inolecules (Mains ct al., sent iin excellent model LO study thc intragranular processing of 1983) and the conversion of thesc prohormones to active pep-thesc protcins. tides requires transfer from the rough endoplasmic reticulum to Chroiiiograniiis are widely distributed in endocrine cells and the Golgi apparatus, packaging into secretory granules (Gainer in neurons (Simon and Aunis, 1989). The function of each of the et d., 1985), limited proteolysis by highly specific proreascs different members is still elusive but thcy are iiow considered to &oh et al., 198s) and further dcgradation in the extracellular be precursor molecules (Dillen ct al., 1993). In bovine adrenal space after exocytotic rcleasc. The secretory granules of the bo-medulla, the major component, chrornogranin A represents 40%) vine adrenal medullary chromaffin cell contain a complex mix-of total soluble granulc proteins. Examination of the cDNA seture of secretory products which includc low-molecular-mass quence of human and bovine chromogranin A revcals thc presconstituents such as catecholamines, ascorbnte, nucltotidt.s, ence (if conservccl pairs of basic amino acids which represent ca...

Gambicin: A novel immune responsive antimicrobial peptide from the malaria vector Anopheles gambiae

“…The results obtained by enzymatic digestion and mass spectrometry indicate that methionine at position 1 is the best candidate for oxidation. A similar posttranslational oxidation of methionine residues has been described for a fragment of proenkephalin A, a protein with antibacterial activity isolated from bovine chromaffin granules (36). Oxidation of gambicin but not of proenkephalin A influences antimicrobial activity.…”

Neuropeptide-Derived Antimicrobial Peptides from Invertebrates for Biomedical Applications

“…Relatively large amounts of pro-enkephalin-A and chromogranin-derived peptides are also found. MetzBoutique's group has shown in bovine that antibacterial activity is present within the intragranular chromaffin granule matrix and the extracellular medium following exocytosis [22][23][24][25]. These peptides inhibit the growth of Gram-positive bacteria (Micrococcus luteus and Bacillus megaterium) at micromolar concentrations.…”

“…The use of these informations coming from invertebrates can be useful tools for treating septic diseases like Heart failure, the Crohn's disease or reactional arthritis [1][2][3][4][5][6]. Moreover, since these last 15 years, several antibacterial peptides have also been identified in mammals, particularly in secretions of immune cells or genital, digestive and intestinal epithelia tracts and skin of human [19][20][21] Secretory granules from adrenal medullary chromaffin cells contain a complex mixture of low molecular mass constituents, such as catecholamines, ascorbate, nucleotides, calcium and several water-soluble peptides and proteins [22][23][24][25]. These components are released into the circulation in response to splanchnic nerve stimulation.…”

Processing of Proenkephalin-A in Bovine Chromaffin Cells

“…Since 1995, we have shown that antibacterial activities are present within the intragranular chromaffin granule matrix and are recovered in the extracellular medium following secretion. This activity has been assigned to chromogranin A and B fragments and PEAPs (4,(12)(13)(14)(15)(16)(17). Among the intragranular matrix components, enkelytin, an antibacterial peptide corresponding to the bisphosphorylated fragment PEAP209 -237, was identified (14).…”

“…This activity has been assigned to chromogranin A and B fragments and PEAPs (4,(12)(13)(14)(15)(16)(17). Among the intragranular matrix components, enkelytin, an antibacterial peptide corresponding to the bisphosphorylated fragment PEAP209 -237, was identified (14). The antibacterial spectrum of enkelytin shows that this peptide is active in the micromolar range against several Gram-positive bacteria including Staphylococcus aureus, but it is not able to inhibit Gram-negative bacterial growth nor to lyse erythrocytes (15).…”