A novel LRR and Ig domain-containing protein could function as an immune effector in Crassostrea gigas

doi:10.1016/j.fsi.2019.03.003

Fish & Shellfish Immunology

Volume 88, May 2019, Pages 318-327

https://doi.org/10.1016/j.fsi.2019.03.003 Get rights and content

Highlights

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ALRR and Ig domain containing protein was identified from Crassostrea gigas.
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It was expressed in all the tissues and highest expressed in hepatopancreas.
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It could bind PAMPs and respond to PAMPs.
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It could induce the expression of cytokines and phagocytosis in hemocytes.
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It specifically expressed in a sub-population of hemocytes.

Abstract

A variety of combinations of leucine-rich repeat (LRR) and immunoglobulin-like (Ig) domains have been found and discovered in invertebrates and vertebrates, but the functions remain largely unexplored. In the present study, a novel LRR and Ig domain-containing protein (LRRIG), CgLRRIG-3, was identified and characterized from oyster Crassostrea gigas. It contained two typical LRR motifs, a LRRNT motif and an Ig domain and PSI-BALST and phylogeny analysis revealed that the sequence of CgLRRIG-3 was most related with leucine-rich repeat neuronal 1 proteins from vertebrate. Its mRNA transcripts were constitutively expressed in muscle, gill, hepatopancreas, mantle, gonad and hemocytes with the highest level in hepatopancreas. The mRNA expression level of CgLRRIG-3 in hemocytes could respond to the stimulations of variety PAMPs including lipopolysaccharide (LPS), peptidoglycan (PGN), glucan (GLU) and polyinosinic-polycytidylic acid (poly I:C). The recombinant proteins exhibited a wide PAMP binding repertoire to four typical PAMPs and could significantly induce the expression of CgTNF-1 and CgIL17-5 as well as increase phagocytosis in primary cultured oyster hemocytes. In hepatopancreas, CgLRRIG-3 was mainly distributed in the basolateral membrane of digestive tubule and the hemocoel sinusoid between the digestive tubules. And in hemocytes, the positive signal was mainly distributed in a special group of granulocytes. These results collectively indicated that CgLRRIG-3 could not only function as an immune effector.

Introduction

The innate immune system is the first line of defense against pathogens and it entails sensing various pathogen associated molecular patterns (PAMPs) via different pattern recognition receptors (PRRs), which then initiate downstream signal pathways [1,2]. Various combinations of protein domains constitute to the receptors of innate immunity including leucine-rich repeat (LRR) domain, immunoglobulin-like (Ig) domain, caspase-recruitment domain, lectin domain and so on [[3], [4], [5], [6]]. LRR domain and Ig domain have been found in a diverse set of proteins, which could mediate ligand recognition and induce immune responses against pathogens [1,2].

LRR domain usually contains 2–45 tandem LRR motifs, each of which is 20–29 residues in length, containing a motif of LxxLxLxxN/CxL [3,7]. These repeats could form a characteristic horse-shoe structure, providing molecular basis for binding to various PRRs [3]. For both vertebrates and invertebrates, LRR containing proteins have been reported to mediate protein-protein or protein-ligand interaction and play important roles in immune responses [8,9]. For example, Toll-like receptors (TLRs) are characterized by extracellular LRR domain and intracellular Toll/IL-1 receptor (TIR) domain, and mediate the TLR signaling pathways in innate immune defense [10]. Cytoplasmic nucleotide-binding oligomerization domain-like receptors (NLRs) family is also featured with C-terminal LRR domain and plays key roles in PAMP recognition and downstream signal transduction [11]. In recent years, the immune functions of proteins containing merely LRR domain (LRR-only proteins) have also been verified in a variety of invertebrates such as scallop, amphioxus and so on [[12], [13], [14]]. Two LRR-only proteins from scallop Chlamys farreri (CfLRRop1 and CfLRRop3) could function as PRRs and were demonstrated to bind various PAMPs including LPS, PGN, GLU and poly I:C [12,13]. Ig domain containing proteins serve extensive functions in direct recognition of ligands, cell adhesion, and signal transduction [15,16]. In vertebrates, the diversified forms of Ig protein are the central structural features of adaptive immune responses [15]. In invertebrates, Ig superfamily (IgSF) could combine with a series of domains and participate in various immune defenses. For example, one or two N-terminal IgSF domains and a C terminal fibrinogen (FBG) domain could form a diverse family of fibrinogen related proteins (FREPs) involved in internal defense in snail [17]. In Crassostrea gigas, one N-terminal V-set Ig domain, C2-set Ig domains and cytosolic immunoreceptor tyrosine-based inhibitory motifs (ITIMs) composed sialic acid binding immunoglobulin-type lectin (siglec), which modulated the immune responses such as cell apoptosis and phagocytosis [18].

A typical LRR and Ig domain containing-protein (LRRIG) consists an N-terminal LRR region, one or more central Ig domains, a transmembrane region, and a C-terminal cytoplasmic tail [14]. In recent years, a series of LRRIGs have been identified and some of them were found to be functioned. For example, the human leucine-rich repeats and immunoglobulin-like domains (LRIG, named by Gene Nomenclature Committee) gene family were illustrated to contain three members, LRIG1, LRIG2, and LRIG3, and they were suggested to play important functions in many cell types and organs [19,20]. In invertebrates, approximate 20 LRRIG models could be found in the sea urchin genome and 240 LRRIG models in the amphioxus genome, demonstrating the prevalence of LRRIGs [14]. In Drosophila, some LRRIGs named kekkon were revealed to be regulated by epidermal growth factor receptor (EGFR) [21,22]. However, compared to the large amount of LRRIG proteins in invertebrates, the functions of LRRIGs in invertebrates still remain largely unexplored. As both LRR and Ig motifs are competent immune recognition modules, the biological activities, cell distribution and immunological relevance of LRRIGs are worth investigating.

The Pacific oyster C. gigas is one of the most important species worldwide and considerable attention has been paid on its immune system against complex environment in the estuaries and intertidal zones [23]. In the course of long-term evolution, oysters have developed diversified PRRs, including peptidoglycan recognition proteins (PGRPs), TLRs, lectins, gram-negative binding proteins (GNBPs), fibrinogen-related proteins (FREPs) and so on [24]. In our previous research, two novel LRRIGs, CgLRRIG-1 and CgLRRIG-2, which are of the same origin, was identified in C. gigas [25]. Our results demonstrated that these two proteins could be regarded as a new type of PRR in oyster. However, compared to other oyster PRRs, more information are still needed to illustrate the exact roles of LRRIGs and deepen our understanding about the immune system of oyster. In the present study, a novel LRRIG (designated as CgLRRIG-3) has been identified in C. gigas, with the main purposes to, 1) investigate the response pattern of CgLRRIG-3 mRNA in hemocytes post different PAMPs stimulation, 2) study the PAMP binding activity and possible pro-inflammatory activity of its recombinant proteins, 3) characterize its distribution in hemocytes and explore its potential immunological function in innate immunity, hopefully to make contributions to the further understanding the cellular immunity and humoral immunity in oyster.

Section snippets

Oysters, immune stimulation and sample collection

Adult oysters C. gigas with an average 13.0 cm in shell length were collected from a local farm in Qingdao, China and were pre-punched by the side of shell and then maintained at 20 °C in the aerated seawater for one week. Approximately 300 oysters were employed for PAMPs stimulation assay. Four different PAMPs including PGN (77140, Fluck, USA), LPS (L2630, Sigma Aldrich, USA), GLU (G5011, Sigma Aldrich, USA), and poly I:C (P1530, Sigma Aldrich, USA) were used for immune stimulation. The

Sequence features and phylogeny of CgLRRIG-3

The full-length cDNA sequence of CgLRRIG-3 was retrieved from NCBI in the C. gigas genome with GenBank accession number of XP_011429625.1. An open reading frame (ORF) of 1716 bp which encoded a polypeptide of 571 amino acids could be identified. The theoretical isoelectric point was 5.59 and the predicted molecular mass was about 65.65 kD. As shown in Fig. 1, two LRR motifs (L₁₅-N₃₉) and (M₄₀-P₆₃), a LRRNT motif (N₇₅ single bond P₁₂₇), an Ig domain (Q₁₃₅-D₂₅₉) and a transmembrane domain (F₂₇₂-A₂₉₄) were

Discussion

The LRR domain and Ig domain are extremely important molecular constituents of innate immune system from invertebrates to vertebrates [3,15,18,31]. In the past decades, a variety of proteins containing both LRR and Ig domains have been discovered and the functions of some have been verified in a series of signal pathways [19]. In invertebrates, a large number of LRRIGs have been discovered but the functions remain largely unexplored. In the present study, CgLRRIG-3 was identified from oyster

Acknowledgement

This study was supported by a grant from National Natural Science Foundation of China (No. 31800414), Open Fund of Key Laboratory of Experimental Marine Biology, Chinese Academy of Sciences (No. KF2018NO1), and Research Initial Funding Project for Doctors in Qingdao University of Science and Technology (No. 010022908).

References (45)

S. Akira et al.
Pathogen recognition and innate immunity
Cell
(2006)
L. Wang et al.
Research progress on the mollusc immunity in China
Dev. Comp. Immunol.
(2013)
B. Kobe et al.
The leucine-rich repeat as a protein recognition motif
Curr. Opin. Struct. Biol.
(2001)
A. Bej et al.
LRRsearch: an asynchronous server-based application for the prediction of leucine-rich repeat motifs and an integrative database of NOD-like receptors
Comput. Biol. Med.
(2014)
M. Wang et al.
An LRR-only protein representing a new type of pattern recognition receptor in Chlamys farreri
Dev. Comp. Immunol.
(2016)
M. Wang et al.
Two novel LRR-only proteins in Chlamys farreri: similar in structure, yet different in expression profile and pattern recognition
Dev. Comp. Immunol.
(2016)
J.P. Cannon et al.
Recognition of additional roles for immunoglobulin domains in immune function
Semin. Immunol.
(2010)
C. Liu et al.
A novel siglec (CgSiglec-1) from the Pacific oyster (Crassostrea gigas) with broad recognition spectrum and inhibitory activity to apoptosis, phagocytosis and cytokine release
Dev. Comp. Immunol.
(2016)
F. Mao et al.
LRIG proteins in glioma: functional roles, molecular mechanisms, and potential clinical implications
J. Neurol. Sci.
(2017)
D. Guo et al.
The LRIG gene family has three vertebrate paralogs widely expressed in human and mouse tissues and a homolog in Ascidiacea
Genomics
(2004)

M. Musacchio et al.

The Drosophila kekkon genes: novel members of both the leucine-rich repeat and immunoglobulin superfamilies expressed in the CNS

Dev. Biol.

(1996)

L. Wang et al.

The oyster immunity

Dev. Comp. Immunol.

(2018)

X. Wang et al.

Two novel LRR and Ig domain-containing proteins from oyster Crassostrea gigas function as pattern recognition receptors and induce expression of cytokines

Fish Shellfish Immunol.

(2017)

X. Wang et al.

Ocean acidification stimulates alkali signal pathway: a bicarbonate sensing soluble adenylyl cyclase from oyster Crassostrea gigas mediates physiological changes induced by CO₂ exposure

Aquat. Toxicol.

(2016)

X. Wang et al.

Soluble adenylyl cyclase mediates mitochondrial pathway of apoptosis and ATP metabolism in oyster Crassostrea gigas exposed to elevated CO₂

Fish Shellfish Immunol.

(2017)

Z. Jia et al.

An integrin from oyster Crassostrea gigas mediates the phagocytosis toward Vibrio splendidus through LPS binding activity

Dev. Comp. Immunol.

(2015)

Y. Sun et al.

The immunomodulation of a novel tumor necrosis factor (CgTNF-1) in oyster Crassostrea gigas

Dev. Comp. Immunol.

(2014)

L. Xin et al.

CgIL17-5, an ancient inflammatory cytokine in Crassostrea gigas exhibiting the heterogeneity functions compared with vertebrate interleukin17 molecules

Dev. Comp. Immunol.

(2015)

M. Wang et al.

The versatile functions of LRR-only proteins in mollusk Chlamys farreri

Dev. Comp. Immunol.

(2017)

S. Homma et al.

Expression pattern of LRR and Ig domain-containing protein (LRRIG protein) in the early mouse embryo

Gene Expr. Patterns

(2009)

K. Tossell et al.

Lrrn1 is required for formation of the midbrain–hindbrain boundary and organiser through regulation of affinity differences between midbrain and hindbrain cells in chick

Dev. Biol.

(2011)

W. Wang et al.

The granulocytes are the main immunocompetent hemocytes in Crassostrea gigas

Dev. Comp. Immunol.

(2017)

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Full length articleA novel LRR and Ig domain-containing protein could function as an immune effector in Crassostrea gigas

Highlights

Abstract

Introduction

Section snippets

Oysters, immune stimulation and sample collection

Sequence features and phylogeny of CgLRRIG-3

Discussion

Acknowledgement

Cell

Dev. Comp. Immunol.

Curr. Opin. Struct. Biol.

Comput. Biol. Med.

Dev. Comp. Immunol.

Dev. Comp. Immunol.

Semin. Immunol.

Dev. Comp. Immunol.

J. Neurol. Sci.

Genomics

Dev. Biol.

Dev. Comp. Immunol.

Fish Shellfish Immunol.

Aquat. Toxicol.

Fish Shellfish Immunol.

Dev. Comp. Immunol.

Dev. Comp. Immunol.

Dev. Comp. Immunol.

Dev. Comp. Immunol.

Gene Expr. Patterns

Dev. Biol.

Dev. Comp. Immunol.

Full length article
A novel LRR and Ig domain-containing protein could function as an immune effector in Crassostrea gigas