Abstract
We have characterized pufflectin, a novel mannose-specific lectin, from the skin mucus of the pufferfish, Fugu rubripes. Molecular mass estimations by gel filtration and matrix-assisted laser desorption ionization time-of-flight mass spectrometry and the SDS-PAGE pattern suggest that pufflectin is a homodimer composed of non-covalently associated subunits of 13 kDa. The full-length pufflectin cDNA consists of 527 bp, with 116 amino acid residues deduced from the open reading frame. The amino acid sequence of pufflectin shows no homology with any known animal lectin. Surprisingly, pufflectin shares sequence homology with mannose-binding lectins of monocotyledonous plants and has conserved two of three carbohydrate recognition domains of these plant lectins. The pufflectin gene is expressed in gills, oral cavity wall, esophagus, and skin. In addition, an isoform occurs exclusively in the intestine. Pufflectin differs from mannose-binding lectins purified from the blood plasma of Fugu. Whereas pufflectin did not agglutinate five bacterial species tested, it was demonstrated to bind to the parasitic trematode, Heterobothrium okamotoi. This finding suggests that pufflectin contributes to the parasite-defense system in Fugu.
Highlights
The integument of fishes is covered with mucus [1], just as the inner surface of the mammalian gut
Pufflectin interacts with D-mannose, whereas other fish skin mucus lectins characterized so far mostly are specific for either D-galactose or lactose [33]
The amino acid sequence of pufflectin does not resemble that of any known animal protein but shows relatively high homology to mannosebinding lectins of monocotyledonous plants
Summary
Preparation of Skin Mucus Extract—Fugu were supplied by Fukui Prefecture Fisheries Experimental Station (Japan). Partial Purification by Ion-exchange Column Chromatography—The crude skin mucus extract was analyzed by fast protein liquid chromatography (FPLC, Amersham Biosciences) on a Poros HQ/M (4.8 ϫ 100 cm, PerSeptive Biosystems) ion-exchange column with a linear gradient of 0 –1 M NaCl in 20 mM Tris-HCl buffer (pH 8.0) at a flow rate of 1 ml/min. Molecular Mass Determination—The molecular mass of intact lectin was determined by gel filtration and carried out by FPLC on a column (1 ϫ 30 cm) of Superose 6 (Amersham Biosciences) with 20 mM Tris-HCl buffer containing 150 mM NaCl (pH 8.0). To confirm independently the sequence of the 5Ј-untranslated region, which had been found to lack a signal peptide, first strand cDNA was synthesized from 1 g of skin total RNA of another individual using the GeneRacerTM kit (Invitrogen) for 5Ј-RACE. Sizes of RNA bands were estimated by comparison to a standard 0.16 –1.77-kb RNA ladder (Invitrogen)
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