with leucine rich repeats (TRIL)
Pietretti, D., Spaink, H.P., Falco, A., Forlenza, M., and Wiegertjes, G.F.
Molecular Immunology 56 (2013), 745-756
Chapter 7
ABSTRACT
The biosynthesis and activation of Toll-like receptors (TLRs) requires accessory proteins.
In mammals, a number of accessory proteins have been characterized, that can be classified based on their function as ligand-recognition and delivery cofactors, chaperones and trafficking proteins. We identified the homologs in teleost fish genomes of mammalian accessory molecules and show their expression in transcriptome data sets. Further, we annotate in detail tlr4 interactor with leucine-rich repeats (tril) in zebrafish (Danio rerio) and in common carp (Cyprinus carpio).
In mammals, TRIL is a functional component of the TLR4 complex and is important for TLR3 signaling, and is mainly expressed in the brain. In fish, the Tril molecule has many conserved features of mouse and human TRIL, containing 13 leucine-rich repeat domains, a fibronectin and a transmembrane domain. Zebrafish tril could not be detected in the latest assembly of the zebrafish genome (Zv9) and required manual annotation based on genome and transcriptome shotgun sequencing data sets. Carp tril was found in two copies in the draft genome. Both copies of carp tril are constitutively expressed in several organs, with the highest gene expression in muscle, skin and brain. In carp, the tril gene is expressed at high levels in endothelial cells and thrombocytes. We discuss the implication of the presence of most, but not all, accessory molecules for the biosynthesis and activation of tlr molecules in fish.
INTRODUCTION
Toll-like receptors (TLRs) constitute an important class of pattern-recognition receptors (PRRs) that recognize a multitude of pathogen-associated molecular patterns, or (PAMPs) [1].
TLRs are type I transmembrane proteins consisting of three domains: an extracellular ectodomain containing tandem arrays of leucine-rich repeats (LRR) that bind to PAMPs and define the specific-ity of the TLR, a transmembrane region and an intracellular Toll/IL-1 receptor (TIR) domain, in-volved in downstream signaling cascades [2]. In general and probably true for most animal species, TLR receptors recognize and respond to a wide range of exogenous and endogenous ligands [3], either at the plasma membrane (e.g. human TLR1, TLR2, TLR4, TLR5, TLR6, TLR10) or intracel-lularly (e.g. human TLR3, TLR7, TLR8, TLR9). The number of TLR genes can vary among organ-isms. For example, ten functional TLRs are expressed in human, whereas the murine genome shows the presence of three additional TLRs, i.e. TLR11, TLR12 and TLR13, but not TLR10 [4, 5]. Thus far, homologs of TLR6 and TLR10 have not been identified in any of the teleost genomes [6-8] but several TLRs additional to the ones found in mammalian vertebrates have been described [6-15].
In mammals, several accessory proteins have been characterized that are required for the biosyn-thesis and activation of the different Toll-like receptors or required for proper TLR folding in the endoplasmatic reticulum [16]. To our knowledge, the presence and conservation of TLR accessory proteins have not been studied extensively in fish. In general, accessory molecules can be defined as required for TLR function whereby they facilitate interaction with other TLRs or with TLR ligands.
Accessory molecules can be broadly divided into i) mediators of ligand delivery and/or recognition, ii) TLR chaperones, iii) trafficking factors and iv) TLR processing factors [16].
i) Well-known examples of mediators of ligand delivery and/or recognition include LBP, CD14,
7
MD2 and CD36. LBP (lipopolysaccharide (LPS)-binding protein) is an acute phase protein that mediates innate immune responses to PAMPs from both positive and Gram-negative bacteria by facilitating their presentation to CD14 [17, 18]. CD14, a GPI-linked protein found on the surface of many TLR4 expressing cells [19], binds directly to LPS [19]
and is known to lead LPS molecules to the TLR4-MD2 signaling complex [20-22]. MD2 (Myeloid Differentiation factor-2) and TLR4 bind to LPS and initiate downstream signaling [23]. Neither TLR4−/− nor MD2−/− knockout mice respond to LPS, indicating that both members of the TLR4/MD2 complex are essential for LPS responses [24] [25]. CD36 is a scavenger receptor of the class B family and fine-tunes TLR assembly and responses to ligands, especially some TLR2-TLR6 ligands [26].
Other examples of mediators of ligand delivery and/or recognition include (pro)granulin, HMGB1, LL37 and TRIL. Granulin is produced as a result of the proteolytic processing of its percursor progranulin by serine proteases, binds to oligonucleotides and facilitates the delivery of CpG DNA to TLR9 [27]. HMGB1 (high-mobility group box 1) is a nuclear protein that binds to DNA and displays pro-inflammatory functions once released by the cell.
HMGB1 binds to both DNA (through TLR9) and RNA (through TLR7 and TLR8) [28]. LL37 is a 37 amino acid amphipathic peptide that is activated through the cleavage of its precursor, the antimicrobial peptide cathelicidin, by a serine protease. LL37 may serve mostly as a DNA-delivery molecule in situations of cell injury [29]. TRIL (TLR4 interactor with leucine-rich repeats) is a recently described mediator of ligand delivery which is highly expressed in brain and facilitates recognition of LPS and poly(I:C) [30, 31]. Knockdown experiments demonstrated that TRIL mediates TLR4 and TLR3, but not TLR2 and TLR9 signaling [30, 31]
ii) Examples of TLR chaperones include Gp96 and PRAT4. Gp96 (also known as GRP94, HSP90b1) is a member of the heat shock protein 90 family and functions as a chaperone for TLR1, TLR2, TLR4, TLR5, TLR7 and TLR9 [32]. Macrophages deficient for Gp96 show a defective cytokine production in response to signaling via most TLRs [33]. PRAT4 (protein associated with TLR4) associates with TLR4 and TLR9 and is required for the trafficking of these TLRs to the plasma membrane and endolysosome, respectively [34].
iii) Examples of TLR trafficking factors include UNC93B1 and AP3. UNC93B1 (uncoordinated 93 homolog B1) is responsible for the translocation of TLR7 and TLR9 from the ER in unstimulated cells to lysosomes after ligand stimulation [35]. UNC93B1 -/- knockout mice show defects in cytokine production and upregulation of costimulatory molecules in response to ligands of TLR7, TLR9 as well as TLR3 and are more susceptible to viral and bacterial infection [36]. UNC93B1 specifically binds to the transmembrane region of TLR3, TLR7 and TLR9 in the ER [33]. AP3 (adaptor protein 3) is a tetrameric complex involved in protein trafficking from the endosomes to the lysosomes [37] and is a required component of the trafficking machinery of TLR9 [38].
iv) TLR-processing enzymes include cathepsins and AEP. Cathepsins are important for the cleavage of TLR9, an event required for optimal signaling [39]E. This proteolytic process has also been reported for TLR3 and TLR7 and may be a general event for endosomal TLR activation [40]. AEP (asparagine endopeptidase) is a lysosomal protein that cleaves asparagine residues; AEP has been shown to cleave TLR9 and mediate its activation in dendritic cells [41].
In this manuscript we identify the presence in teleost fish genomes of the above-described accessory molecules defined as required for TLR function and analyze their expression in
transcriptome data sets. We characterize in detail, TLR4 interactor with leucine-rich repeats (tril) in zebrafish (Danio rerio) and common carp (Cyprinus carpio). In mammals, only recently, TRIL has been identified as functional component of TLR4 and TLR3 signaling. Fish Tril has many of the conserved features of mammalian TRIL containing a 13 leucine-rich repeat domain, a fibronectin domain and a short transmembrane domain. Common carp tril is constitutively expressed in a large number of organs, with highest gene expression in muscle, skin and brain tissue. The screening of a cDNA library made from different cell types of common carp showed that carp tril is expressed at high levels in endothelial cells and thrombocytes.
Studies on teleost Tlrs, aimed at the characterization of their biological activity, are frequently hampered by the lack of suitable cell lines that could act as expression systems. One of the reasons that, for example, mammalian cell lines may not always support biological activity of fish Tlrs could be that not all accessory molecules required for function of fish Tlrs would be present. The identification of TLR accessory molecules may help refine studies on the biological activity of Tlrs in fish and will be further discussed.