PLASMA-PROTEINS OF RAINBOW-TROUT (ONCORHYNCHUS MZYKISS) ISOLATED BY BINDING TO LIPOPOLYSACCHARIDE FROM AEROMONAS-SALMONICIDA

In an attempt to find plasma proteins that might be involved in the co nstitutive resistance of rainbow trout to furunculosis, a disease caus ed by Aeromonas salmonicida (AS), we purified serum and plasma protein s based on their calcium- and carbohydrate-dependent affinity for A. s almonicida lipopolysaccharide (LPS) coupled to an epoxy-activated synt hetic matrix (Toyopearl AF Epoxy 650M). A multimeric family of high mo lecular weight (96 to 200-kDa) LPS-binding proteins exhibiting both ca lcium and mannose dependent binding was isolated. Upon reduction the m ultimers collapsed to subunits of approximately 16-kDa as estimated by 1D-PAGE and exhibited pi values of 5.30 and 5.75 as estimated from 2D -PAGE. Their N-terminal sequences were related to rainbow trout ladder lectin (RT-LL), a Sepharose-binding protein. Polyclonal antibodies to the LPS-purified 16-kDa subunits recognized both the reduced 16-kDa su bunits and the non-reduced multimeric forms. A calcium- and N-acetylgl ucosamine (GlcNAc)-dependent LPS-binding multimeric protein (similar t o 207-kDa) composed of 34.5-kDa subunits was purified and found to be identical to trout serum amyloid P (SAP) by N-terminal sequence (DLQDL SGKVFV). A protein of 24-kDa, in reduced and non-reduced conditions, w as isolated and had N-terminal sequence identity with a known C-reacti ve protein (CRP) homologue, C-polysaccharide-binding protein 2 (TCBP2) of rainbow trout. A novel calcium-dependent LPS-binding protein was p urified and termed rainbow trout lectin 37 (RT-L37). This protein, com posed of dimers, tetramers and pentamers of 37 kDa subunits (pI 5.50-6 .10) with N-terminal sequence (IQE(D/N)GHAEAPGATTVLNEILR) showed no cl ose homology to proteins known or predicted from cDNA sequences. These findings demonstrate that rainbow trout have several blood proteins w ith lectin properties for the LPS of A. salmonicida; the biological fu nctions of these proteins in resistance to furunculosis are still unkn own. (C) 1998 Elsevier Science Inc. All rights reserved.