Tracing the evolution of novel features of human Toll‐like receptor 4

Abstract

Toll-like receptor 4 (TLR4) is a critical innate immune protein that activates inflammation in response to extracellular cues. Much of the work to understand how the protein works in humans has been done using mouse models. Although human and mouse TLR4 have many shared features, they have also diverged significantly since their last common ancestor, acquiring 277 sequence differences. Functional differences include the extent of ligand-independent activation, whether lipid IVa acts as an antagonist or agonist, and the relative species cross-compatibility of their MD-2 cofactor. We set out to understand the evolutionary origins for these functional differences between human and mouse TLR4. Using a combination of phylogenetics, ancestral sequence reconstruction, and functional characterization, we found that evolutionary changes to the human TLR4, rather than changes to the mouse TLR4, were largely responsible for these functional changes. Human TLR4 repressed ancestral ligand-independent activity and gained antagonism to lipid IVa. Additionally, mutations to the human TLR4 cofactor MD-2 led to lineage-specific incompatibility between human and opossum TLR4 complex members. These results were surprising, as mouse TLR4 has acquired many more mutations than human TLR4 since their last common ancestor. Our work has polarized this set of transitions and sets up work to study the mechanistic underpinnings for the evolution of new functions in TLR4.Additional Supporting Information may be found in the online version of this article. Jeremy A. Anderson and Andrea N. Loes contributed equally to this work. Significance statement:We used an evolutionary lens to compare human and mouse innate immune proteins. We found several ways that both the human and mouse proteins have changed significantly since their last common ancestor. This provides context for interpreting studies in mice to understand human biology and health.

Identification and Characterization of Zebrafish Tlr4 Coreceptor Md-2

“…All the while, mutations to Md-2 changed its specificity for LPS and its chemical analogs (73). For example, humans acquired unique lipid IVa antagonism sometime after the divergence of humans and mice (74,75).…”

Evolution of multifunctionality through a pleiotropic substitution in the innate immune protein S100A9

“…We then tested modern and ancestral S100s for activity against human TLR4. Following previous work, 45,64,67,68 we transiently transfected HEK293T cells with plasmids encoding TLR4 and its species-matched cofactors MD-2 and CD14, added purified S100 proteins to the growth media, and then measured output of luciferase under control of an NF-B promoter. Consistent with previous results, 64 we found that human A9 potently activated human TLR4, resulting in high levels of NF-B production (Figure 2b, Figure 2 -supplemental figures 1-2).…”

“…Our findings suggest new directions for understanding how A9 potently activates TLR4. TLR4driven inflammation has been the focus of intense study for over 20 years, 19,[23][24][25]27,67,[76][77][78][79] and the structural basis of TLR4 activation by exogenous agonists, such as the bacterial cell wall component lipopolysaccharide (LPS), is well understood. 80 In contrast, little is known about how A9 activates TLR4.…”

“…All plasmids, cell culture conditions, and transfections for measuring the activity of S100s against TLR4s were identical to those previously described. 38,3964,67 species and normalized to the activity of LPS alone for each TLR4 species to normalize between plates.…”

“Multiplex” rheostat positions cluster around allosterically critical regions of the lactose repressor protein

“…(2)(3)(4)(5)(6)) and upon ancestral reconstruction from sequence alignments (e.g. (7)(8)(9)(10)(11)(12)(13)). As an alternative approach, we agnostically, but systematically, substituted nonconserved positions with a wide variety of amino acids.…”