Biochemical properties and cDNa cloning of two new lectins from the plasma of Tachypleus tridentatus: Tachypleus plasma lectin 1 and 2+.
A Sepharose CL-4B-binding protein, Tachypleus plasma lectin 1 (TPL-1), and a lipopolysaccharide (LPS)-binding protein, Tachypleus plasma lectin-2 (TPL-2), have been isolated from the plasma of Tachypleus tridentatus and biochemically characterized. Each protein is coded by a homologous family of multigenes. TPL-1 binds to Sepharose CL-4B and was eluted with buffer containing 0.4 m GlcNAc. The deduced amino acid sequence of TPL-1 consisted of 232 amino acids with an N-glycosylation site, Asn-Gly-Ser at residues 74-76. It shares a 65% sequence identity and similar internal repeats of about 20 amino acid motifs with tachylectin-1. Tachylectin-1 was identified as a lipopolysaccharide-agarose binding nonglycosylated protein from the amebocytes of T. tridentatus. TPL-2 was eluted from the LPS-Sepharose CL-4B affinity column in buffer containing 0.4 m GlcNAc and 2 m KCl. The deduced amino acid sequence of TPL-2 consisted of 128 amino acids with an N-glycosylation site, Asn-Cys-Thr, at positions 3-5. It shares an 80% sequence identity with tachylectin-3, isolated from the amebocytes of T. tridentatus. TPL-2 purified by LPS-affinity column from the plasma predominantly exists as a dimer of a glycoprotein with an apparent molecular mass of 36 kDa. Tachylectin-3 is an intracellular nonglycosylated protein that also exists as a dimer in solution with an apparent molecular mass of 29 kDa. It recognizes Gram-negative bacteria through the 0-antigen of LPS. Western blot analyses showed that, in the plasma, TPL-1 and TPL-2 exist predominantly as oligomers with molecular masses above 60 kDa. They both bind to Gram-positive and Gram-negative bacteria, and this binding is inhibited by GlcNAc. Possible binding site of TPL-1 and TPL-2 to the bacteria could be at the NAc moiety of GlcNAc-MurNAc of the peptidoglycan. The physiological function of TPL-1 and TPL-2 is most likely related to their ability to form a cluster of interlocking molecules to immobilize and entrap invading organisms.
Chen
S C
SC
Institute of Biological Chemistry, Academia Sinica, Nankang Borough, Taipei 115, Taiwan, Republic of China.
Yen
C H
CH
Yeh
M S
MS
Huang
C J
CJ
Liu
T Y
TY
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019921:Biotinylation; D015153:Blotting, Western; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006031:Glycosylation; D006458:Hemolymph / Q000378:metabolism; D006737:Horseshoe Crabs / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D037102:Lectins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D019081:O Antigens / Q000378:metabolism; D010457:Peptidoglycan / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests; D011485:Protein Binding; D012685:Sepharose / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D018076:DNA, Complementary; D037102:Lectins; D019081:O Antigens; D010457:Peptidoglycan; C425303:Tachypleus plasma lectin-1; C425304:Tachypleus plasma lectin-2; C094130:lectin L6, Tachypleus tridentatus; C403522:tachylectin P, Tachypleus tridentatus; C117330:tachylectin-3 protein, Tachypleus tridentatus; D012685:Sepharose