28th ANNUAL EASTERN FISH HEALTH WORKSHOP April 21-25, 2003 N-Terminal Amino Acid Sequences Of Novel Fucolectins Of Oreochromis Niloticus Reveal Its Suspected Role In Innate Immunity Anacleto   M. Argayosa 1, Hao-Chia Chen2, Noriko Suzuki3, Reiko T. Lee3 and Yuan Chuan Lee3 1Institute of Biology, University of the Philippines, Diliman Quezon City Philippines 1101; 2National Institute of Child Health and Human Development, National Institutes of  Health,  Bethesda, Maryland, 20892; 3 Biology Department, Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218 Four carbohydrate-binding proteins (CBP) were isolated from serum of tilapia (Oreochromis niloticus).  Tilapia mannose-binding proteins (TMBP), fucose-binding proteins (TFBP), glucose-binding proteins (TGlcBP), and galactose-binding proteins (TGalBP) were purified by affinity chromatography. The highest yield was the fucose-binding protein with 23-ug/ml serum. Non-reducing SDS-PAGE analysis showed a multimeric structure of over 200 kDa. Reducing SDS-PAGE showed a large 90 kDa subunit and three to four smaller subunits ranging from 20, 30, nd 40 kDa.  All four subunits of the fucose-binding proteins stained by ruthenium red indicating a requirement for calcium ions. The subunits of the TFBP, TMBP, TGlcBP were subjected to N-terminal amino acid sequencing.  The 30kDa TFBP N-terminal sequence shared a peptide sequence PLIQT similar to the 30 kDa TGlcBP.  Three N-terminal amino acid sequences of fucose-binding lectin subunits   and two N-terminal amino acid sequences of glucose-binding lectin subunits were searched for homologies with the Takifugu rubripes (puffer fish) and Danio rerio (zebra fish) protein databases using BLAST. Sequence alignments of the 40-kDa fucose-binding protein (TFBP40) N-terminal sequence were made with puffer fish complement component precursor (C9), which is part of the membrane attack complex. The fucose-binding 25 kDa subunit N-terminal sequence had an interesting alignment with IFN-inducible antiviral protein Mx in puffer fish and cell adhesion protein in zebra fish. The 30 kDa glucose-binding protein N-terminal sequence matched lymphocyte enhancer binding factor of the zebrafish. The immunoglobulin light chain, fibronectin and collagen type X1 also gave significant alignments.  The predicted secondary structures of the N-terminal amino acid sequence and their homologies to the proteins formed either loop or alpha helices from among the three-dimensional structures searched.   Significant alignments were on the surface loop region of the natural killer (NK) cell receptor in Mus musculus.  Alignments were discussed based on sugar recognition function of the lectin and its relevance to fish health. Return to 28th Annual Eastern Fish Health Workshop Return to Leetown Science Center Home Page