LMP299Y1 Lecture Notes - Lecture 8: Crystallography, Alanine, Acylation

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Published on 6 May 2014
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LMP402 - Fall 2013 - Lecture 8: TLR4, LPS
LBP-/- mice susceptible to infection
MD2 -/- mice susceptible to infection
WHEN infect with Salmonella, and count number of surviving mice
TLR4 surface expression requires MD3 - fluorescent labelling of TLR4
Alanine scan of MD2 - where every amino acid mutated to alanine, then measure
responsiveness to LPS
Graph LPS responsiveness versus cell surface expression - should expect a rising slanted line
with slope of 1, expected if MD2 only has functions of surface expression
However, have mutants above and below this expected line, suggesting MD2 mutations may
diminish binding to ligands or impair signalling
Crystallography determined direct binding of LPS to MD2
MD2 function in cell surface TLR4 localization and LPS Response
TLR4 forms horseshoe shape, with MD2 in horseshoe
LPS forms bridge that lins the 2 protein complexes
Number of acyl chains important for whether LPS is detected or not by TLR4
Not enough chains, won't form bridge, no detection
Bacteria can modulate LPS structure
Pseudomonas aeuringinosa - 5 chains in lab strain; 6 chains in human CF patient
Human cells are defective at detecting penta-acylated LPS
Human cells and lab-adapted LPS - no TNF production
Human cells and precursor lipid A - no TNF production
Human cells and CF LPS - TNF production
Mouse cells plus any one of the above three - TNF production
Therefore CF pateints acquire P. aeuringinosa from environment - penta-acylated
LPS poorly recognized by human TLR4 therefore no recognition, faciliate colonization
VERSUS mice with CFTR mutations that do not develop chronic infections because respond to
penta-acylated LPS
Yersinia pestis LPS inhibits TLR4 activation, as measured by TNF production levels
26 degrees C LPS activates TLR4 - room temperature
37 degrees C LPS inhibits TLR4 - human body temperature
26 and 37 degrees LPS leads to decreased TNF production
LPS acylation depends on temperature
At 26 degrees, Y pestis has hexa-acylated LPS
At 37 degrees, Y pestis has penta-acylated LPS
LPxL, LpxM, LpxP are acyltransferases that modify the tetra-acylated lipid A precursor
LpxL missing and LpxP active only at low temperatures in Yersinia
Engineered Yersinia strain that has hex-acylated LPS at 26 degrees and 37 degrees
Yersinia that express LpxL is avirulent (100 percent survival rate of infected mice) because
inflammation is induced
TLR4 signalling
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