title: Four N-linked glycosylation sites in human toll-like receptor 2 cooperate to direct efficient biosynthesis and secretion.
abstract: Most higher organisms have a system of innate immune defense that is mediated by a group of evolutionarily related, germ line-encoded receptors, so-called Toll-like receptors. In mammals Toll-like receptors signal in response to pathogen-associated microbial structures. For example, Toll-like receptor 2 appears to mediate responses to bacterial peptidoglycan and acylated lipoproteins and Toll-like receptor 4 to bacterial lipopolysaccharide. However, the structural principles that underlie recognition of these structures are poorly understood. Toll-like receptors have leucine-rich repeats in their extracellular domains and are thus believed to adopt solenoid structures, similar to that found in platelet glycoprotein Ib. Additionally, all Toll-like receptors contain N-linked glycosylation consensus sites, and Toll-like receptor 4 requires glycosylation for function. Toll-like receptor glycosylation is also likely to influence receptor surface representation, trafficking, and pattern recognition. Using circular dichroism spectroscopy, we show here that purified human Toll-like receptor 2 and 4 proteins have secondary structure contents similar to glycoprotein Ib. We have also analyzed where consensus glycosylation sites are located in the extracellular domains of other human Toll-like receptors. We found that there are significant differences in the location and degree of conservation between sites in different Toll-like receptors. Using site-directed mutagenesis, we have found that in Toll-like receptor 2 extracellular domain all four predicted glycosylation sites are substituted, although one site is inefficiently core-glycosylated and its removal drastically affects secretion. The remaining Toll-like receptor 2 glycosylation sites also contribute to efficient protein secretion, albeit to a lesser degree.
authors.affiliation: RA Pharmacology, RI CEDD, GlaxoSmithKline Medicines Research Centre, Stevenage SG1 2NY, United Kingdom.
authors.forename: Alexander N R
authors.initials: AN
authors.lastname: Weber
authors.forename: Mary A
authors.initials: MA
authors.lastname: Morse
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Gay
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002942:Circular Dichroism; D019295:Computational Biology; D019992:Databases as Topic; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007313:Insecta; D007930:Leucine / Q000737:chemistry; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000096:biosynthesis* / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014162:Transfection
chemical_list: D008562:Membrane Glycoproteins; D019038:Platelet Glycoprotein GPIb-IX Complex; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C495342:TLR2 protein, human; C495345:TLR4 protein, human; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D006026:Glycoside Hydrolases; D007930:Leucine