Cloning and characterization of a sub-family of human toll-like receptors: hTLR7, hTLR8 and hTLR9.

ARTICLE

INTRODUCTION

The importance of members of the Toll family of proteins in innate immune responses to microbial pathogens is now well appreciated as a result of studies in diverse organisms from Drosophila to man [1, 2]. In Drosophila the Toll (dToll) gene encodes a type-1 transmembrane receptor with an extracellular domain consisting multiple leucine-rich repeats (LRRs), and a cytoplasmic domain exhibiting structural and functional similarities to the cytoplasmic domain of interleukin-1 (IL-1) receptor [3, 4]. The dToll receptor controls dorsal-ventral patterning during the embryo development in Drosophila [5, 6]. In the adult fly, dToll plays a critical role in innate immune responses to microbial infections [7]. Activation of dToll rapidly induces the synthesis of anti-fungal peptides through a signaling pathway with similarities to the NF-kappaB pathway [8] Other members of the Toll protein family in Drosophila have also been identified and in some cases characterized with respect to function in both embryogenesis and innate immune responses [1, 2].

In mammalian cells a Toll-like receptor 4 (TLR4) was first identified as a homologue to dToll [9]. Subsequently, five other human TLRs (hTLRs), hTLR1-3, and hTLR5-6 were cloned and characterized [10-12]. All members of this hTLR family share two common features; an extracellular domain with 18-24 copies of LRRs, and a cytoplasmic domain of approximately 200 amino acids with homology to the cytoplasmic domain of IL-1 receptor. Otherwise, there is very low homology between the different known hTLRs except that there is approximately 70% identity between hTLR1 and hTLR6 [12]. In terms of function little is known about the ligands for the majority of the known TLRs. Biochemical and genetic evidence support the contention that hTLR2 and hTLR4 are receptors for products of microbial pathogens [13-16].

Here we describe three new members of the hTLR family. The totality of the data provided herein supports the contention that these hTLRs form a new subgroup within the known members of this family.

METHODS

Cloning of hTLR cDNAs

Three genes containing an open reading frame with significant homology to the cytosolic domain of hTLR4 were identified in a search of the human genomic DNA database with a Human Genome Blast program (http://www.ncbi.nlm.nih.gov/BLAST/). Two of them, hTLR7 and hTLR8, are localized on chromosome Xp22 with accession number NT-001458. The other one, hTLR9, is localized on chromosome 3p21.3 with accession number NT-001625. Rapid amplification of cDNA ends (RACE) method [17] was used to cloned cDNA containing 5'-end of these hTLRs from a cDNA library which was prepared from human placenta polyA⁺-mRNA using a Marathron™ cDNA amplification kit (Clontech, Palo Alto, CA). The RACE products were subcloned into a T/A cloning vector (Invitrogen, La Jolla, CA) and sequenced. To clone a full length cDNA for the hTLR7-hTLR9, primers corresponding to the sequences at both ends of the cDNAs were designed, PCR amplifications were performed using a human placenta first strand cDNA library as templates. This library was synthesized from polyA⁺ mRNA using a SuperScript™ preamplification kit (Gibco BRL, Gaithersburg, MD). The amplified full length cDNAs were subcloned into a T/A cloning vector and sequenced.

Computational analysis

Signal peptide in the hTLR7-9 proteins were predicted with a SPScan program (Genetic computer group, Madison, WI). Transmembrane domain was analyzed by a MEMSAT program (http://globin.bio.warwick.ac.uk/psipred/). Distribution of leucine-rich repeats in extracellular domains of each hTLRs were scanned using a FPScan program (http://www.bioinf.man.ac.uk/dbbrowser/). Multiple sequences alignment for the members in hTLR family was performed using a Prettybox program (Genetic computer group, Madison, Wisc). Phylogenetic tree for the hTLR families was created with a Distances program and drawn with a Growtree program using a UPGMA method (Genetic computer group, Madison, WI). The genomic structure of hTLR7 and hTLR8 gene on chromosome Xp22, and hTLR9 on 3p21.3 were derived by an alignment between the cDNA sequence and genomic DNA sequence for each hTLR genes using a Gap program (Genetic computer group, Madison, WI).

Tissue distribution of hTLR7, hTLR8, and hTLT9

Expression patterns of hTLR7-9 in different human tissues were determined using PCR amplification with gene specific primers designed for each hTLR, and using first strand cDNA libraries prepared with polyA⁺mRNA from different tissues as template. The gene specific primers for hTLR7 are: 5'-GCTCCC CTGAAATATGGGCAA TCTC-3'and 5'-CTGCTAAAATTAGAGGAATTAG ACATC-3'; for hTLR8 are: 5'-AAAACTGTGGGCCAGCAAATGTGTTG-3'and 5'-GAAGC ACATCCC AAATGAAGCATTCC-3'; for hTLR9 are: 5'-GCTCTGTGTCAGGTGTGGGG TGAG-3'and 5'-CAAGTGTCCCAGCAGCTCTGC-3'. First strand cDNA libraries (human multiple tissue cDNA panel I, and human immune system multiple tissue cDNA panel) were purchased from Clontech laboratories, Inc. (Palo Alto, CA). A pair of primers specific to human glyceraldehydes-3-phosphate (G3PDH) gene were used to control the using equal amount of cDNAs in each PCR amplifications.

Plasmids construction, cell culture and NF-kappaB reporter assay

cDNA encoding transmembrane and cytoplasmic domains of hTLR7 (aa 833-1057), hTLR8 (aa 811-1038), or hTLR9 (aa 824-1055) were ligated to a cDNA encoding extracellular domain of mouse CD4 (aa 1-388), respectively. These chimeras were subcloned into a mammalian expression vector PCDNA3.1 (Invitrogen, Carlsbad, CA), and purified with a cDNA purification kit (Qiagen, Valencia, CA). Human embryonic 293 cells were cultures in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. The cells were plated in six well plates and transfected on the following day by Lipofectamine plus reagent (Gibco BRL, Gaithersburg, MD) with indicated amount of expression vector plus 0.1 mug ELAM-1 (NF-kappaB) luciferase reporter plasmid and 0.1 mug beta-galactosidase plasmid for normalization. Twenty-fours later, the cells were lysed and luciferase activity were determined by using reagents from Promega Corp. (Madison, WI).

RESULTS AND DISCUSSION

Molecular cloning and sequence analysis of hTLR7-9

Several genes encoding open reading frames with significant homology to the cytoplasmic domain of hTLR4 were identified in a search of the human genome DNA databases with a Blast program. Further analysis revealed that three were novel genes encoding members of the hTLR family. Two of them designated hTLR7 and hTLR8 were located on chromosome Xp22, and the other one designated hTLR9 was located on chromosome 3p21.3. Rapid amplification of cDNA ends [17] was used to obtain 5'- cDNA sequence for these genes. Full length cDNAs were then cloned from a human placenta first strand cDNA library using PCR amplification with primers designed using sequences from both ends of each cDNA. Human TLR7 is a protein of 1049 amino acid residues with a calculated molecular weight of 120.9 kDa, hTLR8 and hTLR9 contains 1041, 1032 amino acid residues with a calculated molecular weight of 119.8 kDa and 115.9 kDa respectively (Figure 1).

The hTLR7-9 proteins exhibit features of type I transmembrane proteins. Each of them contains two hydrophobic regions corresponding to a transmembrane domain and a signal peptide at the amino-terminus. The transmembrane regions separate hTLR7-9 into two domains, an extracelullar domain with more than 800 amino acid residues and a cytoplasmic domain with around 200 amino acid residues. The ectodomain of hTLR7-9 contains more amino acid residues than found in hTLR1-6 (550 to 700 amino acid residues), with 27, 26, and 27 copies of LRRs separated by non-LRR region respectively. Following the LRR-flanking region is a sequence of about 60 amino acid containing 3-4 cysteine residues. This cysteine-rich sequence is conserved in hTLR1-9 (Figure 2A), as well as in several other proteins such as platelet glycoprotein IX [18], platelet glycoprotein 1b-alpha (Gp1b-alpha), Gp1b-beta [19], and serum leucine-rich glycoprotein [20]. Like TLR1-6 the cytoplasmic domains of hTLR7-9 share sequence homology with the human IL-1 receptor (IL-1R). Sequence alignments of the cytoplasmic domain of hTLR1-9 is shown in Figure 2B. Several amino acid residues shown to be critical in IL-1 R mediated NF-kappaB activation are conserved in the members of hTLR family although there is some variation within these regions (Figure 2B). Previous studies have shown that hTLR2, hTLR4,and hTLR5-6 mediate NF-kappaB activation [11, 12]. Except for hTLR1 and hTLR6 which have a 69% overall amino acid identity, the identities among different members of the hTLR family is less than 30%. A phylogenetic analysis supports the contention that hTLR7-9 are members of a new sub-family of the hTLRs (Figure 2C).

Genomic structures of hTLR7-9 and alternative splicing forms of hTLR9

The chromosomal loci of hTLR1-6 genes have been determined. Human TLR1 and TLR6 genes are located on chromosome 4p14 [10, 12]. Human TLR2-5 genes have been assigned to loci on chromosomes 4q32, 4q35, 9q32-33, and 1q33.3 respectively [10, 11]. When we searched the human genome databases for the homologues of hTLR4, we identified the two genes hTLR7 and hTLR8 from the sequences of chromosomal loci Xp22, and hTLR9 from 3p21.3. After cloning the cDNA of hTLR7-9, the cDNA sequences were aligned with genomic DNA sequences of these hTLRs to determine their genomic structures. As shown in Figure 3A, hTLR7 is located on a proximal region and hTLR8 is located on more distal region of the chromosomal locus Xp22. The cDNA sequence of hTLR7 that we isolated is covered by three exons, and the cDNA sequence of hTLR8 is covered by two exons. In both hTLR7 and hTLR8, all the amino acid residues except the first methionine residue are encoded by a single major exon (exon III for hTLR7, exon II for hTLR8). In hTLR9, similar situation was observed, all the amino acid residues are encoded by the major exon IV, except the first methionine is contributed by exon III (Figure 3B). In addition to the hTLR9, cDNAs for a non-splicing form and two alternative-splicing forms of hTLR9 were also isolated. These have been termed hTLR9.0, hTLR9.1 and hTLR9.2 respectively (Figure 3B). We sequenced 12 independent clones for hTLR9 cDNAs; nine of them encoded hTLR9, two hTLR9.1, one TLR9.2, but none was the non-splicing form, hTLR9.0, However a cDNA for this non-splicing form can be directly PCR amplified form a human placenta first strand cDNA library with primers designed base on the sequences from both ends. The non-splicing and alternative-splicing created multiple transcripts of hTLR9s with diverse amino-termini. Of them hTLR9 was the most abundant form, and contained a signal peptide with highest probability score when compared with the other hTLR9s. We therefore considered hTLR9 as the major form of these different transcripts.

Tissue distribution of hTLR7-9

Members of hTLR family have been reported to differentially expressed in different tissues. Human TLR1 is expressed ubiquitously in various tissues, hTLR2 is found in lung, heart, brain, muscle, and peripheral blood leukocytes, hTLR3 is predominantly expressed in placenta, and pancreas, hTLR4 is found in spleen, peripheral blood leukocytes, placenta and lung, hTLR5 was detected in ovary and peripheral blood leukocytes [10, 11]. Tissue distribution of hTLR6 was not reported, but murine TLR6 is detected in thymus, spleen, ovary, and lung [12]. To investigate the expression pattern of hTLR7-9, we performed PCR analysis using first strand cDNA library prepared from various different tissues and primers specific to each hTLR. The results indicate that the predominant expression of hTLR7 is in lung, placenta, and spleen, and detectable in lymph node and tonsil. Human TLR8 is more abundant in lung, peripheral blood leukocytes and detectable in placenta, spleen, lymph node and bone marrow, In contrast to all of the other hTLRs, the hTLR9 is preferentially expressed in immune-cell rich tissue including spleen, lymph node, bone marrow, and peripheral blood leukocytes (Figure 4).

Activation of NF-kappaB by hTLR7-9

Human TLR2 and 4 have been shown to active NF-kappaB and production of inflammatory cytokines through a MyD88/IRAK/TRAF6 signaling pathway [21, 22]. This signaling pathway is parallel to the Tube/ Pelle/Dorsal signaling pathway in Drosophila cells [23]. Constitutively active forms of hTLR2, 4, 5, and 6 were generated by fusing the extracellular domain of Fas (CD95) receptor (or mouse CD4 antigen) to the transmembrane and cytoplasmic domain of each of these hTLRs. These constructs were able to active NF-kappaB in different cell types [9, 11, 12]. Here we determined that over-expression of full length hTLR7-9 did not activate NF-kappaB in human embryonic kidney 293 cells (data not shown). To confirm that the hTLR7-9 are functional receptors, DNA encoding chimeric receptors comprising the extracellular domain of mouse CD4 antigen and the transmembrane and cytoplasmic domains from each of these hTLRs was generated. These constructs were transiently transfected into 293 cells along with the luciferase reporter gene for NF-kappaB. Each of these CD4-TLR chimeras constitutively activated NF-kappaB (Figure 5).

CONCLUSION

In summary, we have cloned cDNA for hTLR7, hTLR8 and hTLR9, three new members of the hTLR family. The hTLR7-9 exhibit structural features shared by all the other hTLRs, including a extracellular domain consisting multiple LLRs followed by a cysteine rich sequence, and a cytoplasmic domain with homology to that in IL-1 receptor. The hTLR7-9 differ from TLR1-6 insofar as hTLR7-9 have higher molecular weight and a longer extracellular domain. The overall amino acid identity among these hTLR7-9 are higher to each other than to the other hTLRs. Thus these data support the contention that hTLR7-9 are members of a new sub-family of the hTLRs. Recently Beutler et al. have also identified the same three members of the TLR family described herein (B. Beutler, personal communication, and submitted).

Chimeric forms of hTLR7-9 activate NF-kappaB in 293 cells. Since the known hTLR1-9, IL-1R and dToll have homologous cytoplasmic domains ([4], and Figure 2B), they may use related signal transduction pathway to activate NF-kappaB. Indeed, it has been demonstrated that the signaling pathway downstream of hTLR2, 4, and IL-1 receptor involve MyD88, IL-1 receptor associated kinase (IRAK) and another adaptor protein, TRAF6. MyD88 is a mammalian homologue of Drosophila Tube and IRAK is homologous to Drosophila Pelle [21, 22]. Drosophila homologues of TRAF6 has been identified but their involvement in dToll signaling pathway is unknown [24]. Whether or not hTLR7-9 also activate NF-kappaB through the MyD88/ IRAK/TRAF6 pathway will require further analysis.

Acknowledgements. This is publication number 13272-IMM from the Department of Immunology, The Scripps Research Institute. This work was supported by Grant-in-Aid #9960029 from the American Heart Association Western Affiliate (THC), the Arthritis Foundation Arthritis Investigator Award (THC), and by grants from the National Institutes of Health NIH AI15136 (RJU) and GM28485 (RJU).

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