Purification, characterization and seasonal variation of mannose-binding C-type lectin in Ictalurid catfish

Abstract

Mannose-binding C-type lectin (MBL) was purified by mannan-agarose affinity chromatography from blue catfish D + B ( Ictalurus furcatus) and channel catfish NWAC 103 ( Ictalurus punctatus) sera. The MBLs of blue catfish D + B and channel catfish NWAC 103 strains have not been purified by affinity chromatography, characterized and quantified. The affinity-purified and 2-ME reduced catfish MBLs showed one band of 66 kDa for blue catfish D + B, and one band of 63 kDa for channel catfish NWAC 103 by SDS-PAGE and Western blotting. Amino acid composition analysis (mol%) of the isolated catfish MBLs was done. The amount of methionine-S present in blue catfish D + B (0.78 mol%) was 2.2-times greater when compared with channel catfish NWAC 103 (0.35 mol%). The amount of proline present was 1.9-times greater in channel catfish NWAC 103 (8.0 mol%) when compared with blue catfish D + B (4.3 mol%). Tyrosine (2.5 mol%) was present in blue catfish D + B but not detected in channel catfish NWAC 103. N-terminal amino acid sequencing by Edman degradation for the first 10 residues gave two amino acid sequences of XRQPESFRIV and XXEDGYGKMF that were identical for both blue catfish D + B and channel catfish NWAC 103. This could represent the presence of two different isoforms of catfish MBL. Blue catfish D + B had 21.3 μg of MBL/ml of serum. Channel catfish NWAC 103 (#1 serum pool) had 15.1 μg of MBL/ml of serum (obtained in late Fall season), and channel catfish NWAC 103 (#2 serum pool) had 10.2 μg of MBL/ml of serum (obtained in Spring season). A seasonal variation in the channel catfish serum MBL was therefore seen. A standard curve for μg/ml of MBL in catfish serum was constructed with these data. The presence of MBL in blue catfish D + B and channel catfish NWAC 103 sera should be important in their innate immunity and resistance to Edwardsiella ictaluri and other microbial infections. This study describes the presence of MBL in these two Ictalurus species and evidence for a C-type lectin complement pathway of innate immunity. The channel catfish is used in aquaculture in the Southeast USA and is susceptible to bacterial infections acquired from its pond environment.