Structural and functional analyses of the human Toll-like receptor 3. Role of glycosylation.
Toll-like receptors (TLRs) play critical roles in bridging the innate and adaptive immune responses. The human TLR3 recognizes foreign-derived double-stranded RNA and endogenous necrotic cell RNA as ligands. Herein we characterized the contribution of glycosylation to TLR3 structure and function. Exogenous addition of purified extracellular domain of TLR3 (hTLR3 ECD) expressed in human embryonic kidney cells was found to inhibit TLR3-dependent signaling, thus providing a reagent for structural and functional characterization. Approximately 35% of the mass of the hTLR3 ECD was due to posttranslational modification, with N-linked glycosyl groups contributing substantially to the additional mass. Cells treated with tunicamycin, an inhibitor of glycosylation, prevented TLR3-induced NF-kappaB activation, confirming that N-linked glycosylation is required for bioactivity of this receptor. Further, mutations in two of these predicted glycosylation sites impaired TLR3 signaling without obviously affecting the expression of the protein. Single-particle structures reconstructed from electron microscopy images and two-dimensional crystallization revealed that hTLR3 ECD forms a horseshoe structure similar to the recently elucidated x-ray structure of the protein expressed in insect cells using baculovirus vectors (Choe, J., Kelker, M. S., and Wilson, I. A. (2005) Science 309, 581-585 and Bell, J. K., Botos, I., Hall, P. R., Askins, J., Shiloach, J., Segal, D. M., and Davies, D. R. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 10976-10980). There are, however, notable differences between the human cell-derived and insect cell-derived structures, including features attributable to glycosylation.
Department of Biology, Texas A&M University, College Station 77843, USA.
Jingchuan
J
Sun
Karen E
KE
Duffy
C T
CT
Ranjith-Kumar
Jin
J
Xiong
Roberta J
RJ
Lamb
Jon
J
Santos
Hema
H
Masarapu
Mark
M
Cunningham
Andreas
A
Holzenburg
Robert T
RT
Sarisky
M Lamine
ML
Mbow
Cheng
C
Kao
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002460:Cell Line; D002469:Cell Separation; D018360:Crystallography, X-Ray; D004252:DNA Mutational Analysis; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D005822:Genetic Vectors; D006031:Glycosylation; D006801:Humans; D007091:Image Processing, Computer-Assisted; D008024:Ligands; D013058:Mass Spectrometry; D008854:Microscopy, Electron; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D016328:NF-kappa B / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012330:RNA, Double-Stranded / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D051196:Toll-Like Receptor 3 / Q000378:metabolism / Q000502:physiology*; D014415:Tunicamycin / Q000494:pharmacology
D018076:DNA, Complementary; D008024:Ligands; D016328:NF-kappa B; D012330:RNA, Double-Stranded; D011994:Recombinant Proteins; D051196:Toll-Like Receptor 3; D014415:Tunicamycin