The sequence variation and functional differentiation of CRDs in a scallop multiple CRDs containing lectin

https://doi.org/10.1016/j.dci.2016.08.019Get rights and content

Highlights

  • The sequences of Ca2+-binding site 2 in CRDs of CfLec-4 were diversified.

  • Functional differentiation was observed among the CRDs of CfLec-4.

  • CRD3 and CRD4 were the dominant CRDs for CfLec-4's PAMP recognition.

  • CRD1, CRD3 and CRD4 contributed mainly for opsonization triggered by CfLec-4.

Abstract

A C-type lectin of multiple CRDs (CfLec-4) from Chlamys farreri was selected to investigate the sequence variation and functional differentiation of its CRDs. Its four CRDs with EPD/LSD, EPN/FAD, EPN/LND and EPN/YND key motifs were recombined separately. The recombinant proteins of CRD1 and CRD2 (designated as rCRD1 and rCRD2) could bind LPS and mannan, while the recombinant proteins of CRD3 and CRD4 (designated as rCRD3 and rCRD4) could bind LPS, PGN, mannan and glucan. Moreover, rCRD3 displayed broad microbe binding spectrum towards Gram-positive bacteria Staphylococcus aureus and Micrococcus luteus, Gram-negative bacteria Escherichia coli and Vibrio anguillarum, as well as fungi Pichia pastoris and Yarrowia lipolytica. These results indicated CRD3 contributed more to CfLec-4's nonself-recognition ability. Furthermore, CRD1, CRD3 and CRD4 functioned as opsonin participating in the clearance against invaders in scallops. The sequence variation in Ca2+ binding site 2 among CRDs was suspected to be associated with such functional differentiation.

Introduction

C-type lectins are a superfamily of Ca2+-dependent carbohydrate-recognition proteins. They function as PRR to discriminate self and nonself by recognizing and binding the terminal sugars from various microbes (Weis et al., 1998, Zelensky and Gready, 2005). The carbohydrate recognition domains (CRDs) endow C-type lectins with activities of nonself-recognition and clearance of invaders. Recently, C-type lectins have been assorted into 17 subgroups in vertebrates according to their structural and functional similarities (Zelensky and Gready, 2005), in which collectin is one subgroup serving as PRR to discriminate self and nonself (Cambi and Figdor, 2003, Medzhitov and Janeway, 2002), while selectin mainly mediates cellular adhesion to activate encapsulation and inflammation (Patel et al., 2002). For instance, mannose-binding lectin (MBL) belonging to collectin subgroup can recognize and bind terminal mannose residues and initiate the lectin pathway of complement system (Endo et al., 2006, Fujita, 2002). And P-selectin in selectin subgroup can attract and enrich leukocytes to the site of infection to quickly eliminate the pathogenic bacteria.

There are 1–4 Ca2+ binding sites in each CRD, among which Ca2+ binding site 2 has been proved to determine the binding specificity of C-type lectins in vertebrates (Weis et al., 1991, Weis et al., 1998, Weis and Drickamer, 1996, Zelensky and Gready, 2005). Briefly, there are two motifs existing in Ca2+ binding site 2. The first motif is EPN (Glu-Pro-Asn) or QPD (Gln-Pro-Asp), which is experimentally verified to determine the binding specificity of C-type lectin. The second motif is WND (Trp-Asn-Asp) which is identified to cooperate with the first motif in binding process against carbohydrates (Cambi et al., 2005, Weis and Drickamer, 1996, Weis et al., 1998). Moreover, intensive researches demonstrated that C-type lectins with EPN/WND motifs bind d-mannose or similar sugar, while C-type lectins with QPD/WND motifs bind d-galactose or similar sugar (Wang et al., 2011a, Zelensky and Gready, 2005). However, the two motifs are of great diversity of characteristics in invertebrate, and various kinds of amino acid sequence have been found in the corresponding sites of two specific motifs. For instance, EPD (Glu-Pro-Asp), QPG (Gln-Pro-Gly), QPS (Gln-Pro-Ser), YPG (Tyr-Pro-Gly), and YPT (Tyr-Pro-Thr) were found in the first motif, at the same time WID (Trp-Ile-Asp), WSD (Trp-Ser-Asp), WHD (Trp-His-Asp), FSD (Phe-Ser-Asp) and LSD (Leu-Ser-Asp) were found in the second motif (Wang et al., 2011a).

The number and organization of CRDs determine the affinity and spectrum of lectins to recognize and bind nonself invaders. Some vertebrate C-type lectins contained several CRDs and single CRD containing C-type lectins usually tend to form polymers to perform functions. For instance, the mannose receptor is composed of eight CRDs, whereas MBL is inclined to form polymers to activate complement system (Zelensky and Gready, 2005). To our knowledge, most reported invertebrate C-type lectins containe one single CRD (Ao et al., 2007, Belogortseva et al., 1998, Huang et al., 2015, Li et al., 2015, Mu et al., 2012). Very recently, however, several invertebrate C-type lectins with double or multiple CRDs have been proved to involve in immune responses including PAMPs and microbes binding (Yang et al., 2015), bacteria agglutination (Tian et al., 2009) and opsonization (Wang et al., 2011b). Fc-Lec2 from Fenneropenaeus chinensis with two tandem CRDs and CfLec-3 from C. farreri with three dissimilar CRDs have broad and strong affinity to recognize microbes and PAMPs (Yang et al., 2015, Zhang et al., 2009). Nevertheless, the relationship between the structure and functions of multidomain C-type lectins is still defectively understood, especially in invertebrates.

In our previous study, CfLec-4 with four CRDs was demonstrated to be involved in PAMPs recognition, microbe binding and phagocytosis enhancement against bacteria. In the current study, the four CRDs of CfLec-4 were expressed in Escherichia coli separately, and their PAMP binding specificities, microbe binding spectrums as well as the phagocytosis enhancement activities were examined to comprehensively explore the role of each CRD played in immune response against invaders.

Section snippets

Scallop and microbes

Adults of scallop C. farreri with an average 55 mm of shell length were collected from a farm in Qingdao, Shandong Province, China, and maintained in the aerated seawater at 18 °C for a week before processing.

Bacteria Micrococcus luteus, Staphylococcus aureus and Escherichia coli was purchased from Microbial Culture Collection Center (Beijing, China). Vibrio anguillarum was kindly provided by Dr. Zhaolan Mo. Yarrowia lipolytica was kindly provided by Dr. Zhenming Chi. Pichia pastoris GS115 was

The sequence features and phylogeny of CRD1, CRD2, CRD3 and CRD4

Multiple sequence alignment of CRDs was developed by ClustalW to identify the signature sequences of the four CRDs (Fig. 1). Each CRD contained four cysteine residues involving in the formation of the conserved internal disulfide bridges. Besides, the two N-terminus cysteine residues in all CRDs indicated that they were of long-form (Zelensky and Gready, 2005). The key motifs of Ca2+ binding site 2 in four CRDs were EPD/LSD, EPN/FAD, EPN/LND and EPN/YND, respectively. A phylogenetic tree was

Discussion

C-type lectins play key roles in defense against pathogen infection by serving as PRR in innate immunity (Akira et al., 2006, Cambi and Figdor, 2003, Weis et al., 1998). The immune functions of C-type lectin mainly rely on its characteristic CRDs, which can bind various carbohydrates through Ca2+ binding site 2. Some multidomain C-type lectins have been reported to involve in pathogen recognition (Akira et al., 2006, Cambi et al., 2005, Geijtenbeek et al., 2004, Vasta et al., 2007, Zelensky and

Acknowledgements

The authors would thank all of the colleagues in our lab for helpful discussion and technical advice. This research was supported by National High Technology Research and Development Program (863 Program, No. 2014AA093501) from the Chinese Ministry of Science and Technology, grants (No. 31530069 to L.S., No. 41006096 to H.Z.) from National Science Foundation of China, and Dalian high level talent innovation support program (No. 2015R020). Thanks for Dr. Zhenming Chi for kindly providing

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