Maculatin 2.1

Many parasites and pathogens suppress host immunity to maintain infection or initiate disease. On the skin of many amphibians, defensive peptides are active against the fungus Batrachochytrium dendrobatidis (Bd), the causative agent of the emerging infectious disease chytridiomycosis. We tested the hypothesis that infection with the fungus may be linked to lower levels of defensive peptides. We sampled both ambient (or constitutive) skin peptides on the ventral surface immediately upon capture, and stored skin peptides induced from granular glands by norepinephrine administration of Australian green-eyed treefrogs, Litoria serrata. Upon capture, uninfected frogs expressed an array of antimicrobial peptides on their ventral surface, whereas infected frogs had reduced skin peptide expression. Expression of ambient skin peptides differed with infection status, and antimicrobial peptides maculatin 1.1 and 2.1 were on average three times lower on infected frogs. However, the repertoire of skin peptides stored in granular glands did not differ with infection status; on average equal quantities were recovered from infected and from uninfected frogs. Our results could have at least two causes: (1) frogs with reduced peptide expression are more likely to become infected; (2) Bd infection interferes with defence peptides by inhibiting release or causing selective degradation of peptides on the skin surface. Immune evasion therefore may contribute to the pathogenesis of chytridiomycosis and a mechanistic understanding of this fungal strategy may lead to improved methods of disease control.

Anuran peptides which contain His, including caerin 1.8 (GLFKVLGSVAKHLLPHVVPVIAEKL-NH(2)), caerin 1.2 (GLLGVLGSVAKHVLPHVVPVIAEHL-NH(2)), Ala(15) maculatin 1.1 (GLFGVLAKVAAHVVAIEHF-NH(2)), fallaxidin 4.1 (GLLSFLPKVIGHLIHPPS-OH), riparin 5.1 (IVSYPDDAGEHAHKMG-NH(2)) and signiferin 2.1 (IIGHLIKTALGMLGL-NH(2)), all form MMet(2+) and (M + Met(2+)-2H(+))(2+) cluster ions (where Met is Cu, Mg and Zn) following electrospray ionisation (ESI) in a Waters QTOF 2 mass spectrometer. Peaks due to Cu(II) complexes are always the most abundant relative to other metal complexes. Information concerning metal(2+) connectivity in a complex has been obtained (at least in part) using b and y fragmentation data from ESI collision-induced dissociation tandem mass spectrometry (CID MS/MS). Theoretical calculations, using AMBER version 10, show that MCu(2+) complexes with the membrane active caerin 1.8, Ala(15) maculatin 1.1 and fallaxidin 4.1 are four-coordinate and approximating square planar, with ligands including His and Lys, together with the carbonyl oxygens of particular backbone amide groups. When binding can occur through two His, or one His and one Lys, the His/Lys ligand structure is the more stable for the studied systems. The three-dimensional (3D) structures of the complexes are always different from the previously determined structures of the uncomplexed model peptides (using 2D nuclear magnetic resonance (NMR) spectroscopy in membrane-mimicking solvents like trifluoroethanol/water).

A novel linker for the synthesis of C-terminal acetylene-functionalized protected peptides is described. This SAM1 linker is applied in the manual Fmoc-based solid-phase peptide synthesis of Leu-enkephalin and in microwave-assisted automated synthesis of Maculatin 2.1, an antibacterial peptide that contains 18 amino acid residues. For the cleavage, treatment with tetramethylammonium fluoride results in protected acetylene-derivatized peptides. Alternatively, a one-pot cleavage-click procedure affords the protected 1,2,3-triazole conjugate in high yields after purification.