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. 2015 Sep 15;290(43):26033–26042. doi: 10.1074/jbc.M115.677286

FIGURE 5.

FIGURE 5.

The TREM2 R47H polymorphism reduces the affinity of TREM2 for ApoE. A, immunoprecipitation (IP) of macaque CSF was performed as in the legend to Fig. 1A with the addition of a TREM2-Ig bait containing the R47H mutation. CSF input (leftmost lane) and precipitates were separated by SDS-PAGE and blotted for the indicated apolipoproteins. The R47H mutation decreases, but does not abolish, the precipitation of ApoE from CSF. This experiment is representative of two. B, wild-type TREM2-Ig, mutant TREM2-Ig constructs, and CD4-Ig were used as primary immunoreagents in ELISAs against recombinant ApoE and ApoA-I (E. coli produced), ApoA-II (from human plasma), and ApoJ (of NS-20 NS0 mouse cell origin). TREM2-Ig (wild-type) bound strongly to ApoE. The R47H mutation severely impaired binding of TREM2-Ig as did similar mutations. Error bars show 95% confidence intervals for triplicate wells. C, the effects R47H and related experimental mutations on TREM2/ApoE interaction were confirmed with ApoE purified from human plasma. In this, and following experiments, the concentration of ApoE used to coat the plate was varied rather than the concentration of Ig fusion. Error bars show 95% confidence intervals of triplicate wells of one of two experiments. D, the specificity of TREM2-Ig for ApoE was confirmed via ELISA with plates coated with apoproteins E, A-I, and A-II all purified from human plasma. The dashed gray line indicates the mean level of CD4-Ig binding to the highest concentration of ApoE on the plate (mean signal from 3 wells). Error bars show 95% confidence intervals for triplicate wells from a representative experiment. E, either ApoE antiserum (AS) or control goat serum (CS) were titered onto immobilized purified plasma-derived ApoE or a BSA control ligand. ApoE antiserum, but not the control serum, blocked TREM2-Ig binding in a concentration-dependent manner. Error bars show upper (AS) or lower (CS) 95% confidence intervals for the TREM2 binding signal. BSA error bars are omitted for clarity. The serum dilution series begins at a 1:50 dilution (corresponding to a relative dilution factor of 1 on the graph). The dotted gray line shows the TREM2/ApoE binding signal in the absence of serum (mean value from 6 wells).