A novel type of limulus lectin-L6. Purification, primary structure, and antibacterial activity.
Lipopolysaccharide (LPS)-binding protein(s) was first screened in the detergent extract of horseshoe crab (limulus) hemocytes using LPS-immobilized agarose. A protein, designated L6 (M(r) = 27,000), was found to bind to LPS-agarose and was eluted with EDTA or o-phenanthroline. The L6 protein, however, did not inhibit the LPS-mediated activation of a limulus serine protease zymogen factor C. L6 had an affinity to the matrix of Sepharose CL-6B itself, and it could be eluted with high concentrations of monosaccharides (0.5-1.0 M), such as glucose, mannose, and galactose, suggesting a lectin-like nature. The entire amino acid sequence of L6 was determined by sequencing peptides derived from CNBr and enzymatic cleavages. L6 contained 7 half-cystines, and 1 cysteine residue at position 201 had a free SH-group. In addition, positions of the remaining three intrachain disulfide bonds were assigned by amino acid and sequence analyses of three cystinyl peptides produced by lysyl endopeptidase digestion. These results indicated that the entire sequence of L6 consisted of 221 residues with no N-linked sugar and was composed of six tandem repeats, each consisting of 33-38 amino acid residues. Inductively coupled plasma spectrometry of L6 indicated the presence of 0.75 mol zinc/mol of protein. No significant sequence homology was observed between L6 and other proteins, including various animal lectins and LPS-binding proteins. However, L6 showed agglutinating activity on LPS-coated sheep erythrocytes and Gram-negative and Gram-positive bacteria, it inhibited the growth of Gram-negative bacteria, and thus it presumably recognizes carbohydrate components in the cell wall of bacteria.
Saito
T
T
Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka, Japan.
Kawabata
S
S
Hirata
M
M
Iwanaga
S
S
D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006737:Horseshoe Crabs; D037102:Lectins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008070:Lipopolysaccharides / Q000378:metabolism; D008670:Metals / Q000032:analysis; D008969:Molecular Sequence Data; D011485:Protein Binding; D012756:Sheep; D013347:Subcellular Fractions / Q000378:metabolism
D000900:Anti-Bacterial Agents; D004220:Disulfides; D037102:Lectins; D008070:Lipopolysaccharides; D008670:Metals; C425303:Tachypleus plasma lectin-1; C094130:lectin L6, Tachypleus tridentatus