Preparation, crystallization, and preliminary crystallographic analysis of wild-type and mutant human TREM-2 ectodomains linked to neurodegenerative and inflammatory diseases
Introduction
The molecular mechanisms contributing to age-related neurodegenerative disease pathogenesis are not well understood. Genetic studies released within the last year have identified specific mutations in TREM2 that are linked to a significantly increased risk for the development of late-onset Alzheimer’s Disease (1AD) [1], [2]. The greatest risk is associated with the point mutant R47H, and is roughly equivalent to that associated with mutations in APOE4, currently the strongest known risk factor for late-onset AD [3]. This mutation has now been found in cohorts from Iceland [2], USA, Germany, Netherlands, Norway [1], Spain [4], and France [5]. Further, this mutation has recently been identified as a determinant in Parkinson’s disease (PD) patients [6], [7]. Additional variants including D87N and T96K have been linked to AD development [1] and two other point mutations in TREM-2 (Y38C and T66M) have been linked to the development of frontotemporal dementia (FTD) [8], [9], [10], [11]. These findings highlight a crucial homeostatic role for TREM2 in maintaining neuronal health and represent the first molecular link between innate inflammatory signaling and neurodegenerative diseases [12]. However, it is currently unknown how these mutations affect TREM-2 function and thereby contribute to neurodegenerative disease pathogenesis.
The TREM2 gene encodes the protein triggering receptor expressed on myeloid cells 2 (TREM-2). TREM-2 is an innate immune receptor expressed on dendritic cells, macrophages, osteoclasts, and microglia [13]. The protein has a minimal intracellular sequence and therefore traffics and signals through association with the adaptor protein DAP12 [14]. The ectodomain consists of a single Ig fold anchored to a transmembrane domain via a short stalk. The major role of the Ig domain is presumed to be ligand engagement, though the endogenous signaling ligand(s) of TREM-2 are uncertain. Several different types of ligands have been suggested, including various anionic bacterial carbohydrates [15], apoptotic cell membrane components [16], the immune receptor Plexin-A1 [17], and the chaperone Hsp60 [18]. A proper understanding of the function of TREM-2 has been constrained by a lack of structural information and the elusive nature of TREM-2 ligands.
A crucial role for TREM-2 in neuronal health was first suggested through association with neurodegenerative Nasu-Hakola disease (NHD) [19]. The major mutations associated with NHD produce premature termination of TREM-2 protein and are also phenocopied by deletion of DAP12. In contrast, all of the point mutations associated with AD, PD, and FTD are located within the Ig domain, suggesting these mutations alter function of full-length TREM-2.
Proper understanding of how the newly identified point mutations of TREM-2 could modulate function and contribute to disease requires not only functional studies but also structural and biophysical comparisons of wild-type (WT) and mutant TREM-2 ectodomains. In order to facilitate this approach, we developed a mammalian cell-based expression and purification system to address the difficulties with generating TREM-2 protein suitable for structural and biophysical studies. Indeed, we were able to identify experimental conditions that produce crystals of human TREM-2 that diffract to 3.3 Å resolution and can be phased by molecular replacement. The studies outlined here provide a methodological framework for understanding the molecular basis of TREM-2 mutations and their contribution to the development of neurodegenerative diseases.
Section snippets
Expression constructs
For expression in Escherichia coli, the Ig domains of human TREM-2 (NBCI ref NP_061838; amino acids 19–134; MWcalc[with tag] = 14387 Da; pIcalc = 8.5) and mouse TREM-2 (NCBI ref NP_112544; amino acids 19–136; MWcalc[with tag] = 14440 Da; pIcalc = 8.4) were amplified using PCR and cloned into pET23b as tagless constructs using standard molecular biology techniques. Full-length human TREM-2 in pMX-3p plasmid (a gift from Marco Colonna) and mouse Trem-2 cDNA were used as PCR templates, respectively.
TREM-2 Ig domain refolding screen
Initial experiments were directed at obtaining the large amounts of purified protein that are required for structural and biophysical studies. Structure predictions estimate that the TREM-2 ectodomain consists of a single immunoglobulin (Ig) domain followed by an approximately 40 amino acid unstructured stalk (Fig 1a). Most of the mutations linked to increased risk of neurodegenerative diseases lie in the Ig domain [12], which is most likely to be involved in ligand engagement for signaling. A
Conclusions
TREM-2 is an innate immune receptor that is implicated in regulating inflammatory responses as well as acute and chronic inflammatory diseases [34]. In this capacity it has been suggested to contribute to and/or represent a biomarker for a number of diseases including multiple sclerosis [35], [36], stroke [37], inflammatory bowel disease [38], and chronic inflammatory lung disease [39]. Recent genetics studies have uncovered point mutations in the TREM-2 Ig domain that correlate with a
Acknowledgments
We thank Marco Colonna for the kind gift of the human TREM-2 WT construct in pMX-3P plasmid. We thank Artie Romero for synthesizing PEI-TMC25. We thank Zeynep Yurtsever and Jennifer Alexander-Brett for critical reading of the manuscript. This work was supported in part by funding from NIH R01-HL119813 (T.J.B) and U19-AI070489 (M.J.H.). Results were derived from work performed at Argonne National Laboratory, which is operated by U. Chicago Argonne, LLC, for the U.S. DOE, Office of Biological and
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