Isolation of lysozyme and an antifungal peptide from sea lamprey (Petromyzon marinus) plasma.

The sea lamprey (Petromyzon marinus) belongs to the most primitive class of fish and has only innate immunity. The innate immune factors, lysozyme and an antifungal peptide, were isolated from sea lamprey plasma. Sea lamprey plasma (40.1mg protein/ml) was assayed for lysozyme activity by gel diffusion assay. Using hen egg white lysozyme standards, plasma concentration of lamprey lysozyme was 5microg lysozyme/mg total protein. The presence of lysozyme in such high concentration in lamprey plasma could be important in their innate immunity and resistance to infection. Lysozyme and the antifungal peptide were isolated by low molecular weight gel filtration chromatography from sea lamprey plasma. Gel filtration chromatography yielded two peak tubes containing lysozyme (1microg/211microg total protein) and antifungal peptide (1microg/66microg total protein). Lysozyme and antifungal activity of each fraction were determined by well diffusion assay using Gram-negative bacteria, Gram-positive bacteria and two fungal species. The molecular weight of lamprey lysozyme was 14.3kDa. The sea lamprey lysozyme was effective against Gram-positive bacteria but not against Gram-negative bacteria or fungi. Molecular weight of the antifungal peptide was approximately 3000Da. Antifungal plasma activity was seen against Penicillium notatum and Aspergillus flavus. No plasma antibacterial peptide was found.

Isolation of mannose-binding C-type lectin from sea lamprey (Petromyzon marinus) plasma and binding to Aeromonas salmonicida.

The sea lamprey (Petromyzon marinus) is a parasitic cartilaginous fish of the North American Great Lakes and a predator of many bony fish species of commercial importance to the fishing industry. Mannose-binding C-type lectin (MBL) was isolated by mannan-agarose affinity chromatography from sea lamprey plasma. Mannose-binding lectin has not before been identified and quantitated in the plasma of this sea lamprey species. The affinity-purified and 2-ME reduced lamprey MBL showed two bands of 35kDa and 65kDa by SDS-PAGE and Western blotting using guinea pig anti-MBL IgG as the primary antibody. Amino acid composition analysis (mol%) of the purified lamprey MBL found high amounts of histidine, threonine, tyrosine and phenylalanine present when compared with three other vertebrate MBLs. N-terminal amino acid sequencing by Edman degradation for the first 10 residues gave XXXTKGCPDA. Lamprey plasma contained 261mug of MBL/ml of plasma. Plasma protein concentration was 40.1mg/ml. Lamprey MBL was present then in plasma at 6.5mug MBL/mg total protein. The sea lamprey MBL also specifically binds to mannose on the surface of the pathogen Aeromonas salmonicida. The presence of MBL in high concentration in lamprey plasma could be important in their innate immunity and resistance to infection. This study describes the presence of MBL in sea lamprey plasma and evidence for a C-type lectin complement pathway of innate immunity.