Ligand specificities and structural requirements of two Tachypleus plasma lectins for bacterial trapping

Tun Hsun Kuo, Shiao Cheng Chuang, Sing Yang Chang, Po Huang Liang*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

12 Scopus citations

Abstract

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 8 × 10 8 M. Replacement of Asn 74, 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.

Original languageEnglish
Pages (from-to)757-766
Number of pages10
JournalBiochemical Journal
Volume393
Issue number3
DOIs
StatePublished - 1 Feb 2006

Keywords

  • Horseshoe crab
  • Host defence
  • Innate immunity
  • Lectin
  • Lipopolysaccharide (LPS)
  • Peptidoglycan (PGN)
  • Tachypleus tridentatus

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