Full length articleMolecular cloning, characterization and expression profiles of CD2AP in Nile tilapia (Oreochromis niloticus) responding to Streptococcus agalactiae infection and interaction with CD2 cytoplasmic segment
Introduction
T cells activation that is a complicated process of multiple molecular interactions involves two kinds of signals. The first of activation signal initiates when T cell receptors (TCRs) on T cells engaged by the peptides-major histocompatibility complex (p-MHC) from antigen presenting cells (APCs). The second signals or co-stimulatory signals result from the combination of co-stimulatory receptors on T cells with their ligands on APCs [[1], [2], [3], [4]]. As one of the most important co-stimulatory signaling molecules, CD2 is an adhesion molecule on the membrane of almost T cells and natural killer (NK) cells [[5], [6], [7]]. It believes that the interaction of CD2 and its ligand (CD58 in humans and CD48 in rodents) facilitates the contact between T cells and APC cells that provides optimal spacing for antigen recognition [[8], [9], [10]]. In our previously study [11], two IgSF domains, four cysteine residues and a proline-rich cytoplasmic tail have been found in CD2 of Nile tilapia. However, there are significant CD2 family expansion in lower vertebrates, which remains challenges to research. At least seventy CD2-like genes have been found in Xenopodinae amphibians [12], multiple CD2 family receptors in cyprinid fish and zebra fish [13], and more than nine CD2-like genes in Nile tilapia genome. As a trans-membrane protein, CD2 transmits extracellular signals to the intracellular through its cytoplasmic segments. And many proteins interacting with CD2 cytoplasmic segments (CD2C) have been found, such as CD2 antigen cytoplasmic tail-binding protein 1 (CD2BP1) [7], CD2BP2 [14], CD2BP3 [15] and CD2AP [16]. However, immune-receptor tyrosine switch motifs (ITSM) that plays key roles in protein interaction is no found in the tilapia CD2C, suggesting there are likely supplements of ITSM function by other CD2 like genes.
Of the CD2C interacting proteins, CD2AP plays important role in all stages of thymocyte development [6,17]. Structurally, CD2AP is characterized by three highly conserved N-terminal SH3 domains, a proline-rich region and a C-terminal coiled coil (CC) region. The SH3 domain is a eukaryotic protein domain, which can closely bind to receptor tyrosine kinase phosphorylation residues [18], thus regulating the signal transduction pathways [19]. C-terminal half of CD2AP can effectively bind to filamentous actin (F-actin) [20]: The proline-rich region of CD2AP is considered as the main binding sites for SH3 domains, and coiled-coil region is believed to facilitate protein-protein interactions [21]. Gabriel Gaidos et el. have proposed a model that CD2AP requires at least two independent actin-binding surfaces to bind and bundle actin filaments. In detail, proline-rich region of CD2AP directly binds to SH3 domain of other CD2AP, and the individual actin filaments that tightly combine to the CC domain of CD2AP can be bundled [20]. In addition, CD2AP not only forms stress fibers by interacting with dendrin, synaptopodin and dynamin but also participates in actin capping modification by interacting with CAPZ proteins [22,23].
The roles of CD2AP in regulation of essential cellular process are well supported by CD2AP deficient mice. CD2AP knockout (KO) mice perinatally deadly 6–7 weeks after birth as a result of proteinuria and renal failure, because deficiency of CD2AP leads to deposition of renal immunoglobulin due to the decline of podocytes clearance function [24]. In addition, CD2AP has been presented to media the integrity blood-brain barrier [25,26]. The impaired blood–brain barrier was found in CD2AP knockout mice [25]. In cerebrovascular endothelial cells of healthy mice, high expression of CD2AP was found [27,28], which help maintain the integrity of adheres junctions in blood-brain barrier [26].
Immune function of CD2AP has been of great concerns since it was identified in a yeast two hybrid screen as the interacting protein of CD2 [29]. The interaction between CD2 and CD2AP provides key functions for T cells activation, which has been well demonstrated in mammals [30]. CD2AP can bind and cluster CD2 in T-cells to induce cell polarization [16]. CD2AP is found to interact to proline-rich motifs of CD2 through its SH3 domains [31], which would promote actin polymerization required for synapse formation [32]. Subsequently, CD2 clustering results in formation of immunological synapse that allows the rare specific p-MHC complex to be effectively linked to multiple TCRs [33]. Finally, engagement of TCR by p-MHC complex subsequently induce a chain of reactions to activate T cells. Interestingly, recent studies suggest that CD2AP may also involve in degradation of T cells surface antigen recognition receptors preventing the excessive immune response. The naïve T cells isolated from CD2AP deficient mice presented retardancy of TCR degradation, prolongation of TCR activation and improvement of cytokine production in vitro after p-MHC stimulation [34]. TCR signaling lengthening was also observed in the CD2AP −/−CD4 T cells particularly under TH1status in vitro [35].In addition, ICAM-1 (intercellular adhesion molecule-1) clustering that is key to neutrophil adhesion and trans-cellular transmigration can be promoted in the case of CD2AP deficiency [36]. With further studies of CD2AP, more and more attention has been paid to its function in immunity. However, there has been little study on CD2AP in bony fish since firstly identified in Oplegnathus fasciatus [37].
Oreochromis niloticus is one of most popular commercial fishes in the world. The study of mechanism of fish T cells activation is rather limited. Our previous studies have described several T cell activation relative molecules from Nile tilapia [11,38,39], suggesting mechanism of T cells activation is possibly conserved in teleost. In this work, a new CD2AP gene (On-CD2AP) was identified in Nile tilapia, O.niloticus, and its tissues distribution and mRNA expression model with S.agalactiae infection were detected. And we also found the directly interaction between On-CD2AP and the On-CD2C through yeast two-hybrid and pull-down assay. The present results can help to further study on the process of T cells activation in teleosts.
Section snippets
Fish culture, tissue RNA extraction and cDNA synthesis
Healthy Nile tilapia weighting 100 ± 10 g were bought from a commercial fish farm in Zhanjiang, Guangdong province, China and were acclimated under 28 ± 2 °C. They were fed twice daily with commercial feed. After 4 weeks nine kinds of tissues (muscle, brain, thymus, head kidney, intestine, gill, liver, spleen, and skin) were collected from five healthy Nile tilapia after anaesthetization and immediately collected and frozen by liquid nitrogen. The challenge experiment was also carried out. The
Molecular cloning, bioinformatic analysis and sub-cellular localization of On-CD2AP
The cDNA of On-CD2AP ORF consists of 1941 bp nucleotide sequence and encode a protein of 646 amino acids with a calculated molecular weight of 80.8 kDa and a theoretical isoelectric point of 5.76 (Fig. 1A). Three key structural features of the known CD2AP proteins were found in On-CD2AP, including SH3 domain, proline-rich motif, and coiled coil region at the C terminus (Fig. 1B). The predicted secondary structure of On-CD2AP protein contains random curl (68.89%), alpha-helix (19.20%) and
Discussion
CD2AP has been recognized as an adapt protein that playing crucial roles in various cellular process. One of the best studied is its role in immunity: the interaction between CD2 and CD2AP provides key functions for T cells activation [30].However, the information about the functions of CD2AP remains limited in no-mammals. In rock bream (Oplegnathus fasciatus) CD2AP was found to involved in immune response to different pathogens infection [37].To better understand the function of CD2AP in fish,
Funding
This work was supported by the National Natural Science Foundation of China (Grant No. 31702386), International Cooperation Science & Technology Planning Project of Guangdong Province of China (Grant No. 2017A050501037), Shenzhen Science and Technology Project (JCYJ20180306173022502).
CRediT authorship contribution statement
Yuan Li: Data curation, Writing - original draft. Bei Wang: Conceptualization, Writing - review & editing. Zhi-wen Wang: Visualization, Investigation. Yu Huang: Formal analysis. Ji-chang Jian: Funding acquisition. Yi-shan Lu: Supervision.
Declaration of competing interest
We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled.
References (55)
- et al.
LAT: the ZAP-70 tyrosine kinase substrate that links T cell receptor to cellular activation
Cell
(1998) - et al.
Molecular characterization and expression of CD2 in Nile tilapia (Oreochromis niloticus) in response to Streptococcus agalactiae stimulus
Fish Shellfish Immunol.
(2016) - et al.
Diversity of CD2 subfamily receptors in cyprinid fishes
Results in Immunology
(2012) - et al.
A novel adaptor protein orchestrates receptor patterning and cytoskeletal polarity in T-cell contacts
Cell
(1998) SH2/SH3 signaling proteins
Curr. Opin. Genet. Dev.
(1994)- et al.
A novel adaptor protein orchestrates receptor patterning and cytoskeletal polarity in T-cell contacts
Cell
(1998) - et al.
Linking the T cell surface protein CD2 to the actin-capping protein CAPZ via CMS and CIN85
J. Biol. Chem.
(2003) - et al.
The Wiskott-aldrich syndrome protein acts downstream of CD2 and the CD2AP and PSTPIP1 adaptors to promote formation of the immunological synapse
Immunity
(2003) - et al.
The first report of CD2 associated protein gene, in a teleost (Rock bream, Oplegnathus fasciatus ): an investigation of the immune response upon infection with several pathogens
Fish Shellfish Immunol.
(2017) - et al.
Molecular and functional characterization of CD59 from Nile tilapia (Oreochromis niloticus) involved in the immune response to Streptococcus agalactiae
Fish Shellfish Immunol.
(2015)
Generation, purification and functional characterization of three C3a anaphylatoxins in rainbow trout: role in leukocyte chemotaxis and respiratory burst
Dev. Comp. Immunol.
Conservation of structural and interactional features of CD28 and CD80/86 molecules from Nile tilapia (Oreochromis niloticus)
Fish Shellfish Immunol.
Dynamic bacterial colonization and microscopic lesions in multiple organs of tilapia infected with low and high pathogenic Streptococcus agalactiae strains
Aquaculture
Structural characterization and biological function of bivalent binding of CD2AP to intrinsically disordered domain of chikungunya virus nsP3 protein
Virology
An enigmatic tail of CD28 signaling
Cold Spring Harbor Perspectives in Biology
T cell receptor signaling precedes immunological synapse formation
Science
CD28 and CTLA-4 coreceptor expression and signal transduction
Immunol. Rev.
T cell-signaling network analysis reveals distinct differences between CD28 and CD2 costimulation responses in various subsets and in the MAPK pathway between resting and activated regulatory T cells
J. Immunol.
Molecular cloning of the CD2 antigen, the T-cell erythrocyte receptor, by a rapid immunoselection procedure
CD2BP1 modulates CD2-dependent T cell activation via linkage to protein tyrosine phosphatase (PTP)-PEST
J. Immunol.
Ligation of the CD2 co-stimulatory receptor enhances IL-2 production from first-generation chimeric antigen receptor T cells
Gene Ther.
Enhanced murine CD4+ T cell responses induced by the CD2 ligand CD48
Eur. J. Immunol.
Mechanisms of cellular avidity regulation in CD2-CD58-mediated T cell adhesion
ACS Chem. Biol.
Expansion and diversification of the signaling capabilities of the CD2/SLAM family in Xenopodinae amphibians
Immunogenetics
Identification of a proline-binding motif regulating CD2-triggered T lymphocyte activation
Procnatl Acadsciusa
CD2BP3, CIN85 and the structurally related adaptor protein CMS bind to the same CD2 cytoplasmic segment, but elicit divergent functional activities
Int. Immunol.
Alternative pathway activation of T cells by binding of CD2 to its cell-surface ligand
Nature
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