Protein and lipid interactions of mammalian antibacterial peptides

Gene-encoded antibacterial peptides are multifunctional effector molecules and play an important role in host innate immunity. Upon stimulation, the mature active peptides are released from inactive precursors. Cathelicidins constitute a family of antibacterial peptides, which share a conserved N-terminal cathelin-like region followed by a variable C-terminal antibacterial domain.

In addition to its antibacterial activity, LL-37, the only cathelicidin found in human, is cytotoxic at concentrations higher than the bactericidal concentration. An LL-37 binding protein was purified from human plasma and identified as apolipoprotein A-I (apoA-I). A KD value of 0.6-2.4 µM for the interaction between LL-37 and apoA-1 was determined, and this translates to approximately 90% scavenging of LL-37 at physiological apoA-I plasma concentrations. In an inhibition zone assay, apoA-I gave 50 % of the inhibition attained by plasma, and addition of anti-apoA- I IgG to plasma partly restored the LL-37 antibacterial activity. This suggested that apoA-I can modulate the concentration of free LL- 37, thereby preventing cytotoxic effects.

Porcine cathelicidin prophenin (PF) and an 18-residue fragment thereof (PF-18) were found in organic extracts of lung tissues and also in a porcine surfactant preparation. Structure analysis by mass spectrometry revealed a mixture of variant forms of prophenin, including 79-residue PF-1, 80-residue PF-2 with N-terminal Gln and 80residue PF-2 with N-terminal pyroglutamic acid.

The antibacterial activity of LL-37 and PF were affected by different factors. PF was more active at lower salt concentrations. In the presence of surfactant preparations, the activity of LL-37 and PF-18 against group B stretptoccoci (GBS) were inhibited in a dose-dependent manner, which was likely to be caused by peptide binding to surfactantlipids.

From human granulocytes obtained after treatment with granulocyte-colony stimulating factor (G-CSF) and cortisol, several histone fragments were isolated which exhibited activity against B. megateriumin Bm11 Among these, two Cterminal histone H1A fragments were characterised, corresponding to Lys152-Lys222 and Lys167-Lys222, and both were modified by conjugation of the C-terminal carboxyl group of the tripeptide Arg-Gly-Gly to the side chain amino group of Lys222 via an isopeptide bond. The C-terminal tripeptide Arg- Gly-Gly corresponds uniquely to the three C-terminal residues of ubiquitin. This is the first evidence for ubiquitination of histone H1A.

The interpretation of the tandem mass spectra leading to characterisation of the histone H1A modification was aided by a knowledge of the gas-phase ion chemistry leading to the proline effect. From studies of proline rich PF it was suggested that cleavage of Pro-Pro and Pro- Xxx amide bonds proceeds with the formation of (protonated) substituted diketopiperazines or cyclic peptides as the N-terminal fragments and (protonated) peptides as the C-terminal fragments.

List of scientific papers

I. Wang Y, Agerberth B, Lothgren A, Almstedt A, Johansson J (1998). "Apolipoprotein A-I binds and inhibits the human antibacterial/cytotoxic peptide LL-37." J Biol Chem 273(50): 33115-33118
https://pubmed.ncbi.nlm.nih.gov/9837875

II. Wang Y, Griffiths WJ, Curstedt T, Johansson J (1999). "Porcine pulmonary surfactant preparations contain the antibacterial peptide prophenin and a C-terminal 18-residue fragment thereof." FEBS Lett 460(2): 257-262
https://pubmed.ncbi.nlm.nih.gov/10544246

III. Wang Y, Johansson J, Griffiths WJ (2000). "Characterisation of variant forms of prophenin: mechanistic aspects of the fragmentation of proline-rich peptides." Rapid Commun Mass Spectrom 14(23): 2182-2202
https://pubmed.ncbi.nlm.nih.gov/11114029

IV. Wang Y, Johansson J, Griffiths WJ, (2000). "Antibacterial activitiy of the cathelicidins prophenin (62-79)and LL-37 in the presence of a lung surfactant preparation." (Manuscript)

V. Wang Y, Griffiths WJ, Jörnvall H, Agerberth B, Johansson J (2001). "Antibacterial peptides in stimulated human granulocytes: Characterisation of ubiquitinated histone H1A." (Submitted)