ISOLATION AND CHARACTERIZATION OF RHAMNOSE-BINDING LECTINS FROM EGGS OF STEELHEAD TROUT (ONCORHYNCHUS-MYKISS) HOMOLOGOUS TO LOW-DENSITY-LIPOPROTEIN RECEPTOR SUPERFAMILY

Two L-rhamnose-binding lectins named STL1 and STL2 were isolated from eggs of steelhead trout (Oncorhynchus mykiss) by affinity chromatograp hy and ion exchange chromatography. The apparent molecular masses of p urified STL1 and STL2 were estimated to be 84 and 68 kDa, respectively , by gel filtration chromatography. Sodium dodecyl sulfate polyacrylam ide gel electrophoresis and matrix-assisted laser desorption ionizatio n time of flight mass spectrometry of these lectins revealed that STL1 was composed of noncovalently linked trimer of 31.4-kDa subunits, and STL2 was noncovalently linked trimer of 21.5-kDa subunits. The minimu m concentrations of STL1, a major component, and STL2, a minor compone nt, needed to agglutinate rabbit erythrocytes were 9 and 0.2 mu g/ml, respectively. The most effective saccharide in the hemagglutination in hibition assay for both STL1 and STL2 was L-rhamnose. Saccharides poss essing the same configuration of hydroxyl groups at C2 and C4 as that in L-rhamnose, such as L-arabinose and D-galactose, also inhibited. Th e amino acid sequence of STL2 was determined by analysis of peptides g enerated by digestion of the S-carboxamidomethylated protein with Achr omobacter protease I or Staphylococcus aureus VS protease. The STL2 su bunit of 195 amino acid residues proved to have a unique polypeptide a rchitecture; that is, it was composed of two tandemly repeated homolog ous domains (STL2-N and STL2-C) with 52% internal homology. These two domains showed a sequence homology to the subunit (105 amino acid resi dues) of D-galactoside-specific sea urchin (Anthocidaris crassispina) egg lectin (37% for STL2-N and 46% for STL2-C, respectively). The N te rminus of the STL1 subunit was blocked with an acetyl group. However, a partial amino acid sequence of the subunit showed a sequence similar ity to STL2. Moreover, STL2 also showed a sequence homology to the lig and binding domain of the vitellogenin receptor. We have also employed surface plasmon resonance biosensor methodology to investigate the in teractions between STL2 and major egg yolk proteins from steelhead tro ut, lipo-vitellin, and beta'-component, which are known as vitellogeni n digests. Interestingly, STL2 showed distinct interactions with both egg yolk proteins. The estimated values for the affinity constant (K-a lpha) of STL2 to lipo-vitellin and beta' component were 3.44 x 10(6) a nd 4.99 x 10(6), respectively. These results suggest that the fish egg lectins belong to a new family of animal lectin structurally related to the low density lipoprotein receptor superfamily.