BIOLOGICAL OVERVIEW

18 wheeler, so named because its segmental expression pattern resembles that of the segment polarity genes, and as well, the tied down, seemingly segmented tarpaulin over the rear half of an 18 wheeled semi (a large tractor-trailor truck), was characterized in two laboratories at the same time. In the Bellen lab (Eldon, 1994), 18w was characterized in a search for full length cDNAs of the couch potato gene (Bellen, 1992). In the Beachy lab, 18w was characterized because of the segmentally reiterated transverse stripes of beta-galactosidase expression in a P-element insertion line (Chiang, 1994).

18 wheeler is expressed early in head segments and in an alternating segment polarity pattern corresponding to the domains of wingless and engrailed expression. A comparison of the late expression patterns of 18w and Toll, coding for another leucine rich repeat protein, indicates that the genes are coexpressed in many tissues: the tracheal placodes, some midline cells, the developing salivary glands, the epidermis at the intersegmental furrows, the pharynx, the cells that form the dorsal vessel, the Malpighian tubules, and the hindgut. Although the expression patterns show differences, the genes are frequently coexpressed in regions undergoing extensive cell movements. Both proteins are approximately the same size and share similar motifs throughout most of the protein, including Interleukin 1 receptor homology in the cytoplasmic domain. Both proteins behave as heterophilic cell adhesion molecules when tested for adhesion in Schneider 2 cells, suggesting that they must be able to interact with one or more proteins that are constitutively expressed by these cultured cells. In spite of these similarities, there are no obvious genetic interactions between various mutant alleles of 18w and Toll (Eldon, 1994)

18-Wheeler (18W), is a critical component of the humoral immune response. 18-wheeler is expressed in the larval fat body, the primary organ of antimicrobial peptide synthesis. 18W is also detected in the lymph gland, garland cells and salivary glands. Transcript levels of 18w increase after bacterial infection. In the absence of the 18W receptor, larvae are more susceptible to bacterial infection. Nuclear translocation of the Rel protein Dorsal-like immunity factor (Dif) is inhibited, though nuclear translocation of another Rel protein, Dorsal, is unaffected. Induction of several antibacterial genes is reduced following infection, relative to wild-type: attacin is reduced by 95%, cecropin by 65% and diptericin by 12% (Williams, 1997).

To date, no null alleles of 18w have been isolated, and this makes it difficult to appraise its developmental role. What are the effects of 18w on segmentation? The 18w stripes during segmentation correspond to the location of parasegmental grooves, and expression at later times occurs at many sites destined for invaginations involving multiple cells. These sites include the segmental gooves, the tracheal pits including anterior and posterior spiracles and the wing and haltere imaginal discs (Eldon, 1994 and Chiang, 1995). As 18W protein appears to serve an adhesive function, knowledge of its ligands would be helpful in assessing 18W's role in organogenesis. Expression in the CNS, suggests a role in neural guidance, similar to that found for Toll. Does 18W function downstream of Dorsal or Dorsal immune factor as does Toll? The story here is far from complete.

GENE STRUCTURE

After bacterial infection four novel forms of 18w transcripts are detected in addition to the expected 5.6 kb transcript. A 2.3 kb transcript is more rapidly induced than the others. Four novel 18W protein forms can be detected by Western analysis (Williams, 1997).

Bases in 5' UTR - 374

Bases in 3' UTR - 898

PROTEIN STRUCTURE

Amino Acids - 1389

Structural Domains

Two hydrophobic regions are apparent: one corresponding to a signal peptide and the second to a transmembrane domain. The putative extracellular region is unusally rich in leucine residues (17% of 971 amino acids) and contains 16 putative N-glycosylation sites and a single glycosaminoglycan site at amino acids 50-54 (Eldon, 1994).

There is a large domain with 22 consecutive 24-amino acid leucine-rich repeats (LRRs). In Drosophila, Toll, Slit, Chaoptin, Tartan and Connectin all contain these motifs. The 22 LRR repeats are terminated by a domain that contains cysteines. This domain is also found in a number of LRR-containing proteins: e.g. Slit, Toll, glycoprotein 1b and Glycoprotein IX. The C-terminal domain is followed by a second cysteine rich domain named the amino-flanking domain since it precedes the next run of LRRs. This domain is also found in Slit, GPIb, GPIX, Decorin, Biglycan, Fibromodulin (a collagen-binding protein) and oligodendrocyte-myelin glycoprotein. Finally, the extracellular moiety contains an additional five LRR followed by a carboxy flanking domain (Eldon, 1994 and Chiang, 1995).

The intracellular moiety of 18w is about 380 aa long, and consists of two domains. The first domain shows significant homology to the cytoplasmic domain of the interleukin-1-receptor. IL-1, Toll and 18w share six stretches of amino acids that are more conserved than the remaining amino acids. These regions probably correspond to functional domains since point mutations in some of these conserved regions lead to non-functional proteins. The second domain consists of a glutamine-rich OPA repeat (Eldon, 1994 and Chiang, 1995).

The discovery of sequence homology between the cytoplasmic domains of Drosophila Toll and human interleukin 1 receptors has sown the conviction that both molecules trigger related signaling pathways tied to the nuclear translocation of Rel-type transcription factors. This conserved signaling scheme governs an evolutionarily ancient immune response in both insects and vertebrates. The molecular cloning of a class of putative human receptors is reported, with a protein architecture that is similar to Drosophila Toll in both intra- and extracellular segments. Five human Toll-like receptors (TLRs 1-5) are probably the direct homologs of the fly molecule; as such, they could constitute an important and unrecognized component of innate immunity in humans. Intriguingly, the evolutionary retention of TLRs in vertebrates may indicate another role (akin to Toll in the dorsoventralization of the Drosophila embryo) as regulators of early morphogenetic patterning. Multiple tissue mRNA blots indicate markedly different patterns of expression for the human TLRs. By using fluorescence in situ hybridization and sequence-tagged site database analyses, it is shown that the cognate Tlr genes reside on chromosomes 4 (TLRs 1, 2, and 3), 9 (TLR4), and 1 (TLR5). Structure prediction of the aligned Toll-homology domains from varied insect and human TLRs, vertebrate interleukin 1 receptors, MyD88 factors, and plant disease-resistance proteins recognizes a parallel/fold with an acidic active site. A similar structure notably recurs in a class of response regulators broadly involved in transducing sensory information in bacteria (Rock, 1998).

The 22- and 31-LRR ectodomains of Toll and 18 Wheeler, respectively, are most closely related to the comparable 18-, 19-, 24- and 22-LRR arrays of TLRs 1-4. However, a striking difference in the human TLR chains is the common loss of an approximately 90-residue cysteine-rich region that is variably embedded in the ectodomains of Toll and 18 Wheeler. These cysteine clusters are bipartite, with distinct top (ending in an LRR) and bottom (stacked atop an LRR) halves; the top module recurs in Drosophila and human TLRs as a conserved juxtamembrane spacer. It is suggested that the flexibly located cysteine clusters in Drosophila receptors (when mated top to bottom) form a compact module with paired termini that can be inserted between any pair of LRRs without altering the overall fold of TLR ectodomains (Rock, 19980

date revised: 10 December 97  

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