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A Short-Form C-Type Lectin from Amphioxus Acts as a Direct Microbial Killing Protein via Interaction with Peptidoglycan and Glucan¹

State Key Laboratory of Biocontrol, Open Laboratory for Marine Functional Genomics of the State High-Tech Development Program, Guangdong Province Key Laboratory for Pharmaceutical Functional Genes, College of Life Sciences, Sun Yat-Sen (Zhongshan) University, Guangzhou, People’s Republic of China

To investigate the evolution and immune function of C-type lectin in amphioxus, the primitive representative of the chordate phylum, we identified three C-type lectins consisting solely of a carbohydrate recognition domain and N-terminal signal peptide and found that they had distinct express patterns in special tissues and immune response to stimulations analyzed by quantitative real-time PCR. We characterized the biochemical and biological properties of AmphiCTL1, which was dramatically up-regulated in amphioxus challenged with Staphylococcus aureus, Saccharomyces cerevisiae, and zymosan. Immunohistochemistry demonstrated that the localization of AmphiCTL1 protein was exclusively detected in the inner folding tissues of the hepatic diverticulum. Recombinant AmphiCTL1 was characterized as a typical Ca²⁺-dependent carbohydrate-binding protein possessing hemagglutinating activity, preferentially bound to all examined four Gram-positive bacteria and two yeast strains, but had little binding activity toward four Gram-negative bacteria we tested. It aggregated S. aureus and S. cerevisiae in a Ca²⁺-dependent manner and specifically bound to insoluble peptidoglycan and glucan, but not to LPS, lipoteichoic acid, and mannan. Calcium increased the intensity of the interaction between AmphiCTL1 and those components, but was not essential. This lectin directly killed S. aureus and S. cerevisiae in a Ca²⁺-independent fashion, and its binding to microorganism cell wall polysaccharides such as peptidoglycan and glucan preceded microbial killing activity. These findings suggested that AmphiCTL1 acted as a direct microbial killing C-type lectin through binding microbial targets via interaction with peptidoglycan and glucan. Thus, AmphiCTL1 may be an evolutionarily primitive form of antimicrobial protein involved in lectin-mediated innate immunity.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by Project 2007CB815800 of the National Basic Research Program (973) and Project 2006AA09Z433 of the State High-Tech Development Project (863) from the Ministry of Science and Technology of China, Project 2007DFA30840 of International S&T Cooperation Program of China, Key Project (0107) from the Ministry of Education, and Key Projects of Commission of Science and Technology of Guangdong Province and Guangzhou City. Xu A. is a recipient of Outstanding Young Scientist Award of National Natural Science Foundation of China.

² Address correspondence and reprint requests to Dr. Anlong Xu, Department of Biochemistry, College of Life Sciences, Sun Yat-Sen (Zhongshan) University, 135 Xingangxi Road, 510275, Guangzhou, People’s Republic of China. E-mail address: lssxal{at}mail.sysu.edu.cn

³ Abbreviations used in this paper: CRD, carbohydrate recognition domain; CTLD, C-type lectin domain; AmphiCTL, amphioxus C-type lectin; PAMP, pathogen-associated molecular pattern; DAB, diaminobenzidine; LB, Luria-Bertani; TRX, thioredoxin; LTA, lipoteichoic acid; PGN, peptidoglycan; EST, expressed sequence tag; HA, hemagglutinating activity; Q-PCR, quantitative real-time PCR; HIP, hepatocarcinomanin-intestine pancreas; PAP, pancreatitis-associated protein.

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