Isolation and characterization of novel glycoproteins from fish epidermal mucus: correlation between their pore-forming properties and their antibacterial activities

In fish, a layer of mucus covers the external body surface contributing the refore, among other important biological functions, to the defense system o f fish. The prevention of colonization by aquatic parasites, bacteria and f ungi is mediated both by immune system compounds (IgM, lysozyme, etc.) and by antibacterial peptides and polypeptides. We have recently shown that onl y the hydrophobic components of crude epidermal mucus of fresh water and se a water fish exhibit strong pore-forming properties, which were well correl ated with antibacterial activity [N. Ebran, S. Julien, N. Orange, P. Saglio , C, Lemaitre, G, Molle, Comp. Biochem. Physiol. 122 (1999)]. Here, we have isolated novel glycosylated proteins from the hydrophobic supernatant of t ench (Tinca tinca), eel (Anguilla anguilla) and rainbow trout (Oncorhynchus mykiss) mucus. The study of their secondary structure was performed by cir cular dichroism and revealed structures in random coil and a-helix in the s ame proportions. When reconstituted in planar lipid bilayer, they induced t he formation of ion channels. This pore-forming activity was well correlate d with a strong antibacterial activity (minimal inhibitory concentration <1 mu M for the three proteins) against both Gram-negative and Gram-positive bacteria. Our results suggest that fish secrete antibacterial glycoproteins able to kill bacteria by forming large pores (several hundreds to thousand s of pS) in the target membrane. (C) 2000 Elsevier Science B.V. All rights reserved.