. Like Drosophila Toll, human Toll is a type I transmembrane protein with an extracellular domain consisting of a leucine-rich repeat (LRR) domain, and a cytoplasmic domain homologous to the cytoplasmic domain of the human interleukin (IL)-1 receptor. Both Drosophila Toll and the IL-1 receptor are known to signal through the NF-kB pathway 5-7 . We show that a constitutively active mutant of human Toll transfected into human cell lines can induce the activation of NF-kB and the expression of NF-kB-controlled genes for the inflammatory cyto- kines IL-1, IL-6 and IL-8, as well as the expression of the co- stimulatory molecule B7.1, which is required for the activation of naive T cells. The Toll protein controls dorsal-ventral patterning in Drosophila embryos and activates the transcription factor Dorsal upon binding to its ligand Spatzle 8 . In adult Drosophila, the Toll/Dorsal signalling pathway participates in an anti-fungal immune response 2 . Signal- ling through Toll parallels the signalling pathway induced by the IL- 1 receptor (IL-1R) in mammalian cells: IL-1R signals through the NF-kB pathway, and Dorsal and its inhibitor Cactus are homo- logous to NF-kB and I-kB proteins, respectively 5,6 . Moreover, the cytoplasmic domain of Drosophila Toll is homologous to the cytoplasmic domain of IL-1R (ref. 9). Remarkably, the tobacco- virus-resistance gene that encodes N-protein is also similar to Toll in that it contains both a Toll signalling domain and an LRR domain 10 . It thus appears that the immune-response system mediated by Toll represents an ancient host defence mechanism 6 (Fig. 1). To inves- tigate the possibility that this pathway has been retained in the immune system of vertebrates, we used sequence and pattern searches 11 of the expressed-sequence tag (EST) database at the fragment was used to probe northern blots containing poly(A) + RNA from several organs. Most organs expressed two mRNA species: one of ,5 kilobases (kb) was predominant in most tissues except peripheral blood leukocytes (PBL), and corresponded to the length of the cDNA that we cloned. The lower band was ,4 kb long and this band was predominant in the PBL. The 4-kb band was not detectable in kidney, and liver did not contain any mRNA at all (Fig. 3). We also tested different mouse and human cell lines for expression of hToll mRNA by using PCR with reverse transcription (RT-PCR). We found mRNA for hToll in monocytes, macrophages, dendritic cells, g/d T cells, Th1 and Th2 a/b T cells, a small intestinal epithelial cell line, and a B-cell line (data not shown). The hToll gene is expressed most strongly in spleen and PBL (Fig. 3); its expression in other tissues may be due to the presence of macrophages and dendritic cells, in which it could act as an early-warning system for infection. Alternatively, hToll may be widely expressed because hToll signals through the conserved NF-kB pathway (see below) and NF- kB is a ubiquitous transcription factor. To characterize hToll functions and see whether it can induce transcription of immune response genes like dToll, we generated a dominant-positive mutant of hToll because the natural ligand of hToll is unknown. To produce a constitutively active mutant of hToll, we made use of genetic information from dToll: analysis of ventra- lizing mutants in Drosophila embryos had identified the function of the ectodomain C-flanking cysteine-rich region in dToll 16 as control- ling the activity of dToll in signal transduction. In three dominant

/pdf/a-human-homologue-of-the-drosophila-toll-protein-signals-29hti72mw2.pdf

A human homologue of the Drosophila Toll protein signals activation of adaptive immunity

Selective chemical reactions enacted within a cellular environment can be powerful tools for elucidating biological processes or engineering novel interactions. A chemical transformation that permits the selective formation of covalent adducts among richly functionalized biopolymers within a cellular context is presented. A ligation modeled after the Staudinger reaction forms an amide bond by coupling of an azide and a specifically engineered triarylphosphine. Both reactive partners are abiotic and chemically orthogonal to native cellular components. Azides installed within cell surface glycoconjugates by metabolism of a synthetic azidosugar were reacted with a biotinylated triarylphosphine to produce stable cell-surface adducts. The tremendous selectivity of the transformation should permit its execution within a cell's interior, offering new possibilities for probing intracellular interactions.

Cell Surface Engineering by a Modified Staudinger Reaction

The discovery of sequence homology between the cytoplasmic domains of Drosophila Toll and human inter- leukin 1 receptors has sown the conviction that both molecules trigger related signaling pathways tied to the nuclear trans- location of Rel-type transcription factors. This conserved signaling scheme governs an evolutionarily ancient immune response in both insects and vertebrates. We report the molecular cloning of a class of putative human receptors with a protein architecture that is similar to Drosophila Toll in both intra- and extracellular segments. Five human Toll-like re- ceptors—named TLRs 1-5—are probably the direct homologs of the f ly molecule and, as such, 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 dorsoventral- ization of the Drosophila embryo—as regulators of early morphogenetic patterning. Multiple tissue mRNA blots indi- cate markedly different patterns of expression for the human TLRs. By using f luorescence in situ hybridization and se- quence-tagged site database analyses, we also show 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 and MyD88 factors, and plant disease-resistance proteins recognizes a parallel bya 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.

/pdf/a-family-of-human-receptors-structurally-related-to-50qmlesswg.pdf

A family of human receptors structurally related to Drosophila Toll

Changes in glycosylation are often a hallmark of disease states. For example, cancer cells frequently display glycans at different levels or with fundamentally different structures than those observed on normal cells. This phenomenon was first described in the early 1970s, but the molecular details underlying such transformations were poorly understood. In the past decade advances in genomics, proteomics and mass spectrometry have enabled the association of specific glycan structures with disease states. In some cases, the functional significance of disease-associated changes in glycosylation has been revealed. This review highlights changes in glycosylation associated with cancer and chronic inflammation and new therapeutic and diagnostic strategies that are based on the underlying glycobiology.

Glycans in cancer and inflammation — potential for therapeutics and diagnostics

▪ Abstract In “gene-for-gene” interactions between plants and their pathogens, incompatibility (no disease) requires a dominant or semidominant resistance (R) gene in the plant, and a corresponding avirulence (Avr) gene in the pathogen. Many plant/pathogen interactions are of this type. R genes are presumed to (a) enable plants to detect Avr-gene-specified pathogen molecules, (b) initiate signal transduction to activate defenses, and (c) have the capacity to evolve new R gene specificities rapidly. Isolation of R genes has revealed four main classes of R gene sequences whose products appear to activate a similar range of defense mechanisms. Discovery of the structure of R genes and R gene loci provides insight into R gene function and evolution, and should lead to novel strategies for disease control.

/pdf/plant-disease-resistance-genes-3jj1d7brmf.pdf

Plant Disease Resistance Genes

The technique of native chemical ligation has enabled the total chemical synthesis of proteins with molecular weights far in excess of those achievable by conventional stepwise solid-phase peptide synthesis. The method involves the condensation of two unprotected peptide segments, one bearing a C-terminal αthioester and the other an N-terminal cysteine residue, to afford a protein with a native amide linkage at the site of ligation. Here we report an extension of the native chemical ligation method to the total synthesis of a glycosylated protein, the antimicrobial O-linked glycoprotein diptericin. The major challenge in our synthesis was preparation of a 24-residue glycopeptide-αthioester segment, which was complicated by the incompatibility of glycosidic linkages with Boc chemistry and by the incompatibility of thioesters with Fmoc chemistry. The use of an alkanesulfonamide “safety-catch” linker circumvented this problem and permitted the solid-phase synthesis of the glycopeptide-αthioester using standa...

Fmoc-Based Synthesis of Peptide-αThioesters: Application to the Total Chemical Synthesis of a Glycoprotein by Native Chemical Ligation

A Strategy for the Chemoselective Synthesis of O-Linked Glycopeptides with Native Sugar−Peptide Linkages

Insects protect themselves against bacterial infection by secreting a battery of antimicrobial peptides into the hemolymph. Despite recent progress, important mechanistic questions, such as the precise bacterial targets, the nature of any cooperation that occurs between peptides, and the purpose of multiple peptide isoforms, remain largely unanswered. We report herein the chemical synthesis and preliminary mechanistic investigation of diptericin, an 82 residue glycopeptide that contains regions similar to two different types of antibacterial peptides. A revised, highly practical synthesis of the precursor Nα-Fmoc-Thr(Ac3-α-d-GalNAc) allowed us to produce sufficient quantities of the glycopeptide for mechanistic assays. The synthetic, full-length polypeptide proved to be active in growth inhibition assays with an IC50 of approximately 250 nM, a concentration similar to that found in the insect hemolymph. Biological analysis of diptericin fragments indicated that the main determinant of antibacterial activi...

A chemically synthesized version of the insect antibacterial glycopeptide,diptericin, disrupts bacterial membrane integrity

A microtiter plate assay for UDP-galactopyranose mutase, an essential cell wall biosynthetic enzyme of Mycobacterium tuberculosis, was developed. The assay is based on the release of tritiated formaldehyde from UDP-galactofuranose but not UDP-galactopyranose by periodate and was used to identify a uridine-based enzyme inhibitor from a chemical library.

Drug Targeting Mycobacterium tuberculosis Cell Wall Synthesis: Development of a Microtiter Plate-Based Screen for UDP-Galactopyranose Mutase and Identification of an Inhibitor from a Uridine-Based Library

Katharine A. Winans

Papers

Fmoc-Based Synthesis of Peptide-αThioesters: Application to the Total Chemical Synthesis of a Glycoprotein by Native Chemical Ligation

A Strategy for the Chemoselective Synthesis of O-Linked Glycopeptides with Native Sugar−Peptide Linkages

A chemically synthesized version of the insect antibacterial glycopeptide,diptericin, disrupts bacterial membrane integrity

Drug Targeting Mycobacterium tuberculosis Cell Wall Synthesis: Development of a Microtiter Plate-Based Screen for UDP-Galactopyranose Mutase and Identification of an Inhibitor from a Uridine-Based Library

An inhibitor of the human UDP-GlcNAc 4-epimerase identified from a uridine-based library: a strategy to inhibit O-linked glycosylation.