ABSTRACT
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. 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 receptors--named TLRs 1-5--are probably the direct homologs of the fly 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 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, 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 beta/alpha 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.
Subject(s)
Drosophila Proteins , Drosophila/genetics , Insect Proteins/genetics , Membrane Glycoproteins/genetics , Receptors, Cell Surface/genetics , Amino Acid Sequence , Animals , Cloning, Molecular , Conserved Sequence , DNA, Complementary/analysis , DNA, Complementary/genetics , Humans , Molecular Sequence Data , Sequence Homology, Amino Acid , Toll-Like Receptor 4 , Toll-Like Receptor 5 , Toll-Like ReceptorsABSTRACT
MyD88 was first characterized as a myeloid differentiation primary response gene in mice, activated in M1 myeloleukemic cells following interleukin-6 (IL-6) induced growth arrest and terminal differentiation. Analysis of expressed sequence tags (ESTs) from activated dendritic cell libraries led to the indentification of cDNAs encoding the human homolog (hMyD88). The original description of MyD88 as a 243 aa protein may reflect a truncated mouse cDNA since the 2682 nt hMyD88 cDNA predicts a 296 aa cytoplasmic protein. Consistent with this proposal is the detection of a 33 kDa protein in human heart, kidney and liver tissue. The expression pattern of MyD88 is also more widespread than originally believed: a 2.6 kb hMyD88 mRNA species was found to be constitutively expressed in many adult human tissues; in addition MyD88 expression was observed in monocyte, T, B, NK and dendritic cells. The MyD88 protein has a modular structure composed of an N-terminal 'death domain' (DD) similar to the intracellular segments of TNF receptor 1 (TNFR1) and FAS and a C-terminal region related to the signaling domains of vertebrate interleukin-1 receptors (IL-1R) and the Drosophila morphogen Toll. This intriguing structural framework may endow MyD88 with unique signaling capabilities.
Subject(s)
Antigens, Differentiation , Apoptosis/genetics , DNA, Complementary/genetics , Proteins/genetics , Receptors, Immunologic , Adaptor Proteins, Signal Transducing , Adult , Alternative Splicing , Amino Acid Sequence , Base Sequence , Dendritic Cells/chemistry , Humans , Molecular Sequence Data , Myeloid Differentiation Factor 88 , Proteins/isolation & purification , Proteins/metabolism , Transcription, GeneticABSTRACT
Structure prediction algorithms have tagged leptin as the newest member of the haemopoietic cytokine family, a diverse class of secreted hormone-like factors with pleiotropic effects in immunity and haemopoietic development. While haemopoietic cytokines typically lack sequence similarity, they conserve a distinctive three-dimensional fold, a four-alpha-helix bundle structure that is recognized by the cognate family of haemopoietic cellular receptors. We have constructed a detailed molecular model of the human leptin helical fold that places the two cysteine residues of the leptin chain, Cys96 and Cys146, in close spatial proximity to each other. In this report, we present evidence that these cysteines are involved in an intrachain disulfide bridge that is critical for the structural integrity and stability of leptin. A leptin variant that is unable to form the disulfide link shows a reduced biological response when administered to leptin-deficient, ob/ob mice.
Subject(s)
Disulfides/chemistry , Protein Structure, Secondary , Proteins/chemistry , Algorithms , Animals , Appetite/drug effects , Circular Dichroism , Cysteine/chemistry , Electrophoresis, Polyacrylamide Gel , Female , Humans , Leptin , Mice , Mice, Obese , Models, Molecular , Molecular Structure , Protein Folding , Proteins/pharmacology , Recombinant Proteins/pharmacologyABSTRACT
Human obese (hOB) protein was recently identified as a secreted hormone-like factor that is exclusively produced by adipose tissue and appears to regulate the size of the body's fat stores. We describe a rapid and efficient repetitive extension PCR method for the construction of a 453-bp synthetic hOB gene with high-frequency codons to optimize expression in Escherichia coli. The use of a bacterial signal sequence fused to hOB together with expression of bacteriocin release protein resulted in efficient release of hOB into the culture medium. The protein was readily purified to homogeneity from the culture medium using a two-step procedure.
Subject(s)
Obesity/genetics , Proteins/genetics , Proteins/isolation & purification , Amino Acid Sequence , Base Sequence , DNA, Complementary/genetics , Escherichia coli/genetics , Gene Expression , Humans , Leptin , Molecular Sequence Data , Oligopeptides , Peptides/genetics , Polymerase Chain Reaction/methods , Protein Biosynthesis , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purificationABSTRACT
Human IL-6 has two disulfide bonds linking Cys45 to Cys51 and Cys74 to Cys84, respectively. Previous site-directed mutagenesis studies have demonstrated that the Cys74-Cys84 bond is essential for full biological and receptor binding activities. To address the structural importance of these disulfide bonds in the formation and stabilization of IL-6 secondary and tertiary structures, we have generated a panel of disulfide bond-deficient rIL-6 analogs both by chemical reduction and alkylation as well as by site-directed mutagenesis. Conformational changes affecting these rIL-6 analogs were probed by circular dichroism spectroscopy, as well as reactivity with monoclonal antibodies, and correlated with changes in biological activities. We have shown that the first disulfide bridge (Cys45-Cys51) is highly sensitive to reduction and, therefore, more solvent-exposed or less thermodynamically stable. Contrary to previous reports, this bridge contributes, although minimally, to the full biological activity of the cytokine. However, no significant changes in secondary or tertiary structures were observed upon removal of this bond. In marked contrast, analogs lacking the disulfide bridge between Cys74 and Cys84 exhibited as little as 0.5% and 0.05% wild-type biological and receptor binding activities, respectively. These dramatic changes correlated with a slight reduction in alpha-helical content and a decreased reactivity with the neutralizing monoclonal antibody mAb8 which recognizes a conformational epitope associated with the active site. Our results suggest that the second disulfide bridge plays a critical role in maintaining the spatial relationship between the putative IL-6 A and D helices.
