Effects of single nucleotide polymorphisms on Toll-like receptor 3 activity and expression in cultured cells.
Recognition of double-stranded RNA by Toll-like receptor 3 (TLR3) will increase the production of cytokines and chemokines through transcriptional activation by the NF-kappaB protein. Over 136 single-nucleotide polymorphisms (SNPs) in TLR3 have been identified in the human population. Of these, four alter the sequence of the TLR3 protein. Molecular modeling suggests that two of the SNPs, N284I and L412F, could affect the packing of the leucine-rich repeating units in TLR3. Notably, L412F is reported to be present in 20% of the population and is higher in the asthmatic population. To examine whether the four SNPs affect TLR3 function, each were cloned and tested for their ability to activate the expression of TLR3-dependent reporter constructs. SNP N284I was nearly completely defective for activating reporter activity, and L412F was reduced in activity. These two SNPs did not obviously affect the level of TLR3 expression or their intracellular location in vesicles. However, N284I and L412F were underrepresented on the cell surface, as determined by flow cytometry analysis, and were not efficiently secreted into the culture medium when expressed as the soluble ectodomain. They were also reduced in their ability to act in a dominant negative fashion on the wild type TLR3 allele. These observations suggest that N284I and L412F affect the activities of TLR3 needed for proper signaling.
Ranjith-Kumar
C T
CT
Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843, USA.
Miller
William
W
Sun
Jingchuan
J
Xiong
Jin
J
Santos
Jon
J
Yarbrough
Ian
I
Lamb
Roberta J
RJ
Mills
Juliane
J
Duffy
Karen E
KE
Hoose
Scott
S
Cunningham
Mark
M
Holzenburg
Andreas
A
Mbow
M Lamine
ML
Sarisky
Robert T
RT
Kao
C Cheng
CC
D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D001249:Asthma / Q000378:metabolism; D002460:Cell Line; D019143:Evolution, Molecular; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020641:Polymorphism, Single Nucleotide; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D051196:Toll-Like Receptor 3 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C495344:TLR3 protein, human; D051196:Toll-Like Receptor 3