Ligand specificities and structural requirements of two Tachypleus plasma lectins for bacterial trapping.
TPL (Tachypleus plasma lectin)-1 was purified by using a Sepharose column and TPL-2 was purified from an LPS-Sepharose (LPS coupled to Sepharose matrix) affinity column, as described previously [Chiou, Chen, Y.-W., Chen, S.-C., Chao and Liu (2000) J. Biol. Chem. 275, 1630-1634] and the corresponding genes were cloned [Chen, Yen, Yeh, Huang and Liu (2001) J. Biol. Chem. 276, 9631-9639]. In the present study, TPL-1 and -2 were produced in yeast, and the recombinant proteins secreted into the media were purified and characterized. The proteins show specific PGN (peptidoglycan)- and LPS-binding activity, suggesting a role in trapping Gram-positive and Gram-negative bacteria respectively in innate immunity. Using BIAcore assays, the dissociation constant for the TPL-1-PGN complex was measured as 8x10(-8) M. Replacement of Asn74, the N-glycosylation site of TPL-1, with Asp abolishes the PGN-binding affinity, whereas the unglycosylated TPL-2 N3D mutant retains LPS-binding activity. DTT (dithiothreitol) treatment to break disulphide linkages abrogates TPL-2 activity but does not interfere with TPL-1 function. Cys4 in TPL-2 may form an intermolecular disulphide bond, which is essential for activity. As a result, the TPL-2 C4S mutant is inactive and is eluted as a monomer on a non-reducing gel. TPL-2 C6S is active and forms a non-covalently linked dimer. A model describing TPL-2 binding with LPS is proposed. These two plasma lectins that have different ligand specificities can be used for the detection and discrimination of bacteria and removal of endotoxins.
Kuo
Tun-Hsun
TH
Institute of Biochemical Sciences, National Taiwan University, Taipei 106, Taiwan.
Chuang
Shiao-Cheng
SC
Chang
Sing-Yang
SY
Liang
Po-Huang
PH
D000595:Amino Acid Sequence; D000818:Animals; D001412:Bacillus subtilis / Q000276:immunology / Q000378:metabolism; D001419:Bacteria / Q000276:immunology* / Q000378:metabolism*; D004926:Escherichia coli / Q000276:immunology / Q000378:metabolism; D015870:Gene Expression; D006031:Glycosylation; D006737:Horseshoe Crabs; D007113:Immunity, Innate; D037102:Lectins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D008024:Ligands; D008969:Molecular Sequence Data; D009154:Mutation; D010457:Peptidoglycan / Q000378:metabolism; D010843:Pichia / Q000378:metabolism; D011485:Protein Binding; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D037102:Lectins; D008024:Ligands; D010457:Peptidoglycan; C425303:Tachypleus plasma lectin-1; C425304:Tachypleus plasma lectin-2