A novel C1q-domain-containing protein from Zhikong scallop Chlamys farreri with lipopolysaccharide binding activity
Introduction
The C1q-domain-containing (C1qDC) proteins are a family of proteins characterized by a globular C1q (gC1q) domain in their C-terminus [1]. The canonical gC1q signature domain is ∼140 amino acid residues long with a compact jelly-roll β-sandwich fold and eight highly conserved residues (positions F124, F142, N142, F160, G166, Y168, F242 and G244 in human C1q B chain) packed in the hydrophobic core of the globular structure [1], [2]. This domain is often situated at the C-terminus of a collagen stalk, by which three protomers trimerize to form a collagen triple-helix and further multimerize to form a bouquet structure.
Recently, C1qDC proteins have been widely found in vertebrates, including complement component C1q, adiponectin, precerebellin, hibernation protein, multimerin, elastin microfibril interface-located (EMILIN) protein and so on [2]. They have been demonstrated to be involved in various processes of host defense, apoptosis, autoimmunity, phagocytosis, neurogenesis, inflammation, organogenesis, hibernation and energy homeostasis [3], [4], [5], [6], [7], [8], [9].
Among all the C1qDC proteins, C1q is the most prototypical and representative one. As a subcomponent of the complement C1 complex, it plays a crucial role in the recognition of microbial surfaces and antibody-antigen complexes to initiate the classical complement pathway and mediate adaptive immunity [10]. In addition, C1q is also considered to be a versatile pattern recognition receptor (PRR) in innate immunity, because it can recognize and bind pathogen-associated molecular patterns (PAMPs) and then trigger rapid enhanced phagocytosis resulting in efficient containment of pathogens [11], [12]. C1q possesses powerful capability of binding to a wide variety of ligands besides PAMPs, including envelope proteins of certain retroviruses, lipopolysaccharides (LPS), β-amyloid fibrils, porins from Gram-negative bacteria, phospholipids, apoptotic cells and even some acute phase reactants [12], [13], [14]. The majority of C1q ligands are recognized via the heterotrimeric gC1q, which is an extremely efficient and versatile charge pattern recognition domain [14].
In invertebrates, researches on C1qDC proteins is sparse. So far, only a few C1qDC-encoding sequences from invertebrates have been deposited in the NCBI database, such as sea urchin corticle vesicle proteins (AAK11302, AAK11303, AAK11305, AAK11309 and AAG15425) and sialic acid binding lectin from Helix pomatia (ABF00124) and Cepaea hortensis (CAD83837) [1], [15], and the exact function of C1qDC proteins in invertebrates is still unclear. Because the C1q found in the most primitive vertebrate Lampetra japonicus acted as a lectin in innate immunity, it was supposed that the ancient C1q molecules in invertebrates should also function as a PRR (e.g. lectin) and bind relatively broad categories of molecules as PAMPs via the gC1q-domains [12], [16], [17], [18]. But no report has focused on the PRR roles of these proteins in invertebrates, especially their binding capability to PAMPs such as the main component on Gram-negative bacteria, LPS.
Zhikong scallop Chlamys farreri (Mollusca; Bivalvia; Lamellibranchia) is an important bivalve cultured widely in the northern coastal provinces of China. Investigation of the roles of C1qDC protein in the C. farreri immune responses to invading microorganisms and its binding activity to PAMPs would provide new insights into the function of this important, widespread and functionally diverse family of proteins and give new evidence for further discussion about the primary complement-like molecules in invertebrates. The main objectives of the present study were: (1) to clone the full-length cDNA of the C1qDC protein from scallop C. farreri (designated as CfC1qDC); (2) to investigate the expression pattern of CfC1qDC mRNA in different tissues and its temporal response to LPS treatment in primary cultured hemocytes; and (3) to characterize the activity of recombinant CfC1qDC protein, especially the LPS-binding activity.
Section snippets
Scallops
Scallops Chlamys farreri (shell length 5–10 cm) were purchased from culture farms in Qingdao, Shandong Province, China, and maintained in aerated seawater at 18–20 °C for a week before processing.
cDNA library construction and EST analysis
A cDNA library was constructed from the whole body of a scallop challenged by Listonella anguillarum, using the ZAP-cDNA synthesis kit and ZAP-cDNA GigapackIII Gold cloning kit (Stratagene). Random sequencing of the library using T3 primer yielded 6935 successful sequencing reactions. BLAST analysis of
cDNA cloning of CfC1qDC
Sequencing of Zhikong Scallop cDNA library with T3 primer yielded 6935 EST sequences, which were clustered into 689 contigs and 2191 singletons. Blastx analysis revealed that a 434 bp fragment (clone No. rscag0_004065) was similar to C1q-related factors identified previously. Based on the sequence of this EST, two gene specific-primers (P1 and P2) were designed to clone the full-length cDNA of CfC1qDC, and two fragments of 674 bp and 325 bp were amplified by 3′RACE and 5′RACE respectively. A 777 bp
Discussion
In recent years, C1q-globular (gC1q) domain has been widely found in a diverse range of proteins particularly in vertebrates and distinguishes the C1q-domain-containing (C1qDC) protein family [1]. However, the investigation of invertebrate C1qDC proteins remains deficient. The characterization of proteins with gC1q-domain from scallop would provide further insights into the functional and evolutionary research of this protein family.
In the present study, the full-length cDNA sequence of CfC1qDC
Acknowledgements
The authors were grateful to all the laboratory members for continuous technical advice and helpful discussions. This research was supported by 973 National Key Fundamental Research Program (No. 2006CB101806) and 863 High Technology Project (No. 20060110A402) from the Chinese Ministry of Science and Technology, and a grant (No. 30730070) from NSFC to Dr. Linsheng Song.
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