Humanized TLR1 Variants Have Differential

Responses to Pam3CSK4
Elizabeth Chang, Susanna Harju-Baker, Mark M. Wurfel
Division of Pulmonary and Critical Care, University of Washington

Background
Toll-like receptor (TLR) proteins are
expressed on cells of the innate immune
system and recognize the structural
motifs on microbial pathogens.
Pathogen recognition by TLRs activates
an intracellular signaling cascade and
activation of NF-kB, a transcription
factor that is critical for expression of
inflammatory mediators.
Previous studies from our laboratory
have shown that single nucleotide
variations present in human TLR1 lead to
altered expression of TLR1 and are
associated with increased risk of death
in patients with sepsis.

TLR1 Variant Constructs

The TLR1/2 Signaling
Pathway

SNP

pUNO-mTLR1
DNA

pUNO-mTLR Human
AA
Variant

Mouse
Variants

rs4833095

AAT (1321..1323) ->

AGT
Asn251
CTC (2383..2385)
Leu605

Asn -> Ser

Asn251Ser

rs5743618

Asn248Ser

Leu -> Ser or Ile Ile602Ser

Results continued

Leu605Ser/Ile

Descriptions of the SNPs, their locations, and protein variations in

humans and mice. Mutagenesis of the TLR1 construct was performed in
pUNO-mTLR1 construct (Invivogen). cDNAs of the mutagenized constructs
were subcloned into pCIneo for ease of plasmid growth with carbenicillin as
the selection reagent instead of blasticidin.
(Ref 2)

Human TLR1 variants confer

increased Pam3CSK4-induced
responses. Cells were transfected
with human TLR2 and various
TLR1 constructs and incubated
with Pam3CSK4 or IL-1 (Ref 1).

Results

Leu605Ile and Leu605Ser variants showed effects on activity

relative to the wild type consistent with other experiments.
Tlr1 with a Ser allele at position 251 showed decreased
activity compared to Asn, which differs from the result of the
previous experiment. Elucidating the specific effect will
require further investigation.

Hypothesis: Variations of mouse Tlr1which correspond to

functional variants in human TLR1 result in comparable changes in
TLR1 activity.

Conclusions

Methods

Site-directed mutagenesis was used to introduce coding variations

in theTlr1 cDNA. cDNA of the constructs was subcloned into the
pCIneo vector from pUNO. After transfecting the variants into
cultured human cells, they were stimulated with Pam3CSK4, a
triacylated lipoprotein known to be recognized by the TLR1/TLR2
complex. We then used a bioluminescent luciferase reporter system
to measure the levels of NF-kB transcriptional activity.

Variations in the transmembrane domain of mouse TLR1 result in

alterations in function comparable to that of human variants in the
same region.
The variations may affect responses by altering cell surface
expression.
Models may be used to study TLR1 variants effect on the immune
response in humans.
This could lead to a better understanding of the mechanisms
leading to poor clinical outcomes for patients with sepsis and
other inflammatory diseases.

Tlr1 with an Ile allele at position 605 showed increased activity

relative to a Leu allele.
Tlr1 with a Ser allele at position 605 showed increased activity
compared to Leu, but to a lesser degree than the Ile allele.
Tlr1 with a Ser allele at position 251 showed increased activity
compared to Asn.
Expression levels from pUNO constructs were higher compared to
pCI-neo, but plasmid growth in pCI-neo was higher and allowed
for easier analysis.
The mouse variants show similar effects to previously studied
human variants.

Acknowledgements
This work was supported by NIH NWRCE grant 5U54AI057141-6248
to M.M.Wurfel.
Thanks to Osamu Kajikawa for performing the site-directed
mutagenesis at Tom Martins lab.
Thank you to Susanna Harju-Baker for mentoring and encouraging me
throughout my time at HMC.

References
1. Wurfel M. et al., Am J Respir Crit Care Med. 2008 Oct
1;178(7):710-20
2. Hennessy E.J., et al, Nature Reviews Drug Discovery. 2010 Apr; 9:
293-307