TREM 2 shedding by cleavage at the H157‐S158 bond is accelerated for the Alzheimer's disease‐associated H157Y variant

Research Article30 August 2017Open Access Source DataTransparent process TREM2 shedding by cleavage at the H157-S158 bond is accelerated for the Alzheimer's disease-associated H157Y variant Peter Thornton Peter Thornton Neuroscience, Innovative Medicines and Early Development, AstraZeneca, Granta Park, Cambridge, UK Search for more papers by this author Jean Sevalle Jean Sevalle Tanz Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, ON, Canada Search for more papers by this author Michael J Deery Michael J Deery Cambridge Centre for Proteomics, University of Cambridge, Cambridge, UKCorrection added on 2 October 2017 after first online publication: "Mike J Deery" was corrected to "Michael J Deery". Search for more papers by this author Graham Fraser Graham Fraser Neuroscience, Innovative Medicines and Early Development, AstraZeneca, Granta Park, Cambridge, UK Search for more papers by this author Ye Zhou Ye Zhou Tanz Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, ON, Canada Search for more papers by this author Sara Ståhl Sara Ståhl AstraZeneca Translational Sciences Centre, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Elske H Franssen Elske H Franssen Neuroscience, Innovative Medicines and Early Development, AstraZeneca, Granta Park, Cambridge, UK Search for more papers by this author Roger B Dodd Roger B Dodd Department of Clinical Neurosciences, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK MedImmune Limited, Granta Park, Cambridge, UK Search for more papers by this author Seema Qamar Seema Qamar Department of Clinical Neurosciences, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK Search for more papers by this author Beatriz Gomez Perez-Nievas Beatriz Gomez Perez-Nievas Neuroscience, Innovative Medicines and Early Development, AstraZeneca, Granta Park, Cambridge, UK Search for more papers by this author Louise SC Nicol Louise SC Nicol MedImmune Limited, Granta Park, Cambridge, UK Search for more papers by this author Susanna Eketjäll Susanna Eketjäll Cardiovascular and Metabolic Diseases, Innovative Medicines and Early Development, AstraZeneca, ICMC, Huddinge, Sweden Search for more papers by this author Jefferson Revell Jefferson Revell MedImmune Limited, Granta Park, Cambridge, UK Search for more papers by this author Clare Jones Clare Jones MedImmune Limited, Granta Park, Cambridge, UK Search for more papers by this author Andrew Billinton Andrew Billinton Neuroscience, Innovative Medicines and Early Development, AstraZeneca, Granta Park, Cambridge, UK Search for more papers by this author Peter H St George-Hyslop Peter H St George-Hyslop Tanz Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, ON, Canada Department of Clinical Neurosciences, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK Search for more papers by this author Iain Chessell Iain Chessell Neuroscience, Innovative Medicines and Early Development, AstraZeneca, Granta Park, Cambridge, UK Search for more papers by this author Damian C Crowther Corresponding Author Damian C Crowther [email protected] orcid.org/0000-0001-7791-1396 Neuroscience, Innovative Medicines and Early Development, AstraZeneca, Granta Park, Cambridge, UK Search for more papers by this author Peter Thornton Peter Thornton Neuroscience, Innovative Medicines and Early Development, AstraZeneca, Granta Park, Cambridge, UK Search for more papers by this author Jean Sevalle Jean Sevalle Tanz Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, ON, Canada Search for more papers by this author Michael J Deery Michael J Deery Cambridge Centre for Proteomics, University of Cambridge, Cambridge, UKCorrection added on 2 October 2017 after first online publication: "Mike J Deery" was corrected to "Michael J Deery". Search for more papers by this author Graham Fraser Graham Fraser Neuroscience, Innovative Medicines and Early Development, AstraZeneca, Granta Park, Cambridge, UK Search for more papers by this author Ye Zhou Ye Zhou Tanz Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, ON, Canada Search for more papers by this author Sara Ståhl Sara Ståhl AstraZeneca Translational Sciences Centre, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Elske H Franssen Elske H Franssen Neuroscience, Innovative Medicines and Early Development, AstraZeneca, Granta Park, Cambridge, UK Search for more papers by this author Roger B Dodd Roger B Dodd Department of Clinical Neurosciences, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK MedImmune Limited, Granta Park, Cambridge, UK Search for more papers by this author Seema Qamar Seema Qamar Department of Clinical Neurosciences, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK Search for more papers by this author Beatriz Gomez Perez-Nievas Beatriz Gomez Perez-Nievas Neuroscience, Innovative Medicines and Early Development, AstraZeneca, Granta Park, Cambridge, UK Search for more papers by this author Louise SC Nicol Louise SC Nicol MedImmune Limited, Granta Park, Cambridge, UK Search for more papers by this author Susanna Eketjäll Susanna Eketjäll Cardiovascular and Metabolic Diseases, Innovative Medicines and Early Development, AstraZeneca, ICMC, Huddinge, Sweden Search for more papers by this author Jefferson Revell Jefferson Revell MedImmune Limited, Granta Park, Cambridge, UK Search for more papers by this author Clare Jones Clare Jones MedImmune Limited, Granta Park, Cambridge, UK Search for more papers by this author Andrew Billinton Andrew Billinton Neuroscience, Innovative Medicines and Early Development, AstraZeneca, Granta Park, Cambridge, UK Search for more papers by this author Peter H St George-Hyslop Peter H St George-Hyslop Tanz Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, ON, Canada Department of Clinical Neurosciences, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK Search for more papers by this author Iain Chessell Iain Chessell Neuroscience, Innovative Medicines and Early Development, AstraZeneca, Granta Park, Cambridge, UK Search for more papers by this author Damian C Crowther Corresponding Author Damian C Crowther [email protected] orcid.org/0000-0001-7791-1396 Neuroscience, Innovative Medicines and Early Development, AstraZeneca, Granta Park, Cambridge, UK Search for more papers by this author Author Information Peter Thornton1,‡, Jean Sevalle2,‡, Michael J Deery3, Graham Fraser1, Ye Zhou2, Sara Ståhl4, Elske H Franssen1, Roger B Dodd5,6, Seema Qamar5, Beatriz Gomez Perez-Nievas1, Louise SC Nicol6, Susanna Eketjäll7, Jefferson Revell6, Clare Jones6, Andrew Billinton1, Peter H St George-Hyslop2,5,‡, Iain Chessell1,‡ and Damian C Crowther *,1,‡ 1Neuroscience, Innovative Medicines and Early Development, AstraZeneca, Granta Park, Cambridge, UK 2Tanz Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, ON, Canada 3Cambridge Centre for Proteomics, University of Cambridge, Cambridge, UK 4AstraZeneca Translational Sciences Centre, Karolinska Institutet, Stockholm, Sweden 5Department of Clinical Neurosciences, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK 6MedImmune Limited, Granta Park, Cambridge, UK 7Cardiovascular and Metabolic Diseases, Innovative Medicines and Early Development, AstraZeneca, ICMC, Huddinge, Sweden ‡These authors contributed equally to this work ‡These authors contributed equally to this work *Corresponding author. Tel: +44 020 3749 6149; E-mail: [email protected] EMBO Mol Med (2017)9:1366-1378https://doi.org/10.15252/emmm.201707673 See also: K Schlepckow et al (October 2017) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract We have characterised the proteolytic cleavage events responsible for the shedding of triggering receptor expressed on myeloid cells 2 (TREM2) from primary cultures of human macrophages, murine microglia and TREM2-expressing human embryonic kidney (HEK293) cells. In all cell types, a soluble 17 kDa N-terminal cleavage fragment was shed into the conditioned media in a constitutive process that is inhibited by G1254023X and metalloprotease inhibitors and siRNA targeting ADAM10. Inhibitors of serine proteases and matrix metalloproteinases 2/9, and ADAM17 siRNA did not block TREM2 shedding. Peptidomimetic protease inhibitors highlighted a possible cleavage site, and mass spectrometry confirmed that shedding occurred predominantly at the H157-S158 peptide bond for both wild-type and H157Y human TREM2 and for the wild-type murine orthologue. Crucially, we also show that the Alzheimer's disease-associated H157Y TREM2 variant was shed more rapidly than wild type from HEK293 cells, possibly by a novel, batimastat- and ADAM10-siRNA-independent, sheddase activity. These insights offer new therapeutic targets for modulating the innate immune response in Alzheimer's and other neurological diseases. Synopsis Sequence variation in the microglial receptor protein TREM2 is linked to risk for Alzheimer's disease. The disease-linked H157Y variant of TREM2 is found to affect the sheddase site and accelerates proteolytic loss of TREM2 from the cell surface. TREM2 was shed rapidly from primary macrophages and microglia under basal conditions. The sheddase site was identified using peptidomimetic inhibitors and mass spectrometry. The Alzheimer's disease-linked H157Y TREM2 sheddase-cleavage-site variant was shed more rapidly than wild type. For both wild type and variant TREM2 the major sheddase was ADAM10, however an additional proteolytic activity might be recruited by the H157Y variant. The protection of TREM2 from proteolysis might represent a novel therapeutic approach. Introduction Nasu–Hakola disease is a rare but fatal brain and bone disorder (Hakola, 1972; Hakola & Iivanainen, 1973), caused by homozygous inheritance of null or hypomorphic variants of the TREM2 gene (Klünemann et al, 2005). A subset of heterozygous TREM2 variants such as R47H, R62H and H157Y are associated with increased risk of Alzheimer's disease (AD) (Guerreiro et al, 2013; Jonsson & Stefansson, 2013; Jonsson et al, 2013; Slattery et al, 2014; Finelli et al, 2015; Ghani et al, 2016; Jiang et al, 2016). While the prevalence of AD-linked TREM2 variants is low, indeed the most common (R47H) affects 0.3–0.6% of the population (Guerreiro et al, 2013; Jonsson et al, 2013), the relative risk for individual carriers is high, twofold to 11-fold above the general population (Finelli et al, 2015). TREM2 is a single-pass, type I transmembrane protein that includes an extracellular immunoglobulin domain with two N-linked glycans (Park et al, 2015). It is expressed on dendritic cells, macrophages, microglia and osteoclasts and is thought to act as a scavenger receptor (Colonna, 2003). TREM2 is prominent in microglia adjacent to pathological material such as amyloid deposits and cellular debris and may have a role in directing microglial migration (Jay et al, 2015; Kawabori et al, 2015) and phagocytic activation (Keren-Shaul et al, 2017). Studies in cell cultures have shown that at least Nasu–Hakola-linked TREM2 variants are less available on the surface of phagocytic cells because they are incorrectly glycosylated (Kleinberger et al, 2014; Park et al, 2015) and inefficiently trafficked through the synthetic pathway. Most individuals with AD carry wild-type TREM2 and the normal proteolytic processing of the protein is thought to be performed by sequential α- then γ-secretase activity (Wunderlich et al, 2013). It is notable that levels of the shed TREM2 N-terminal fragment (NTF) are raised in the CSF of patients with sporadic AD as compared to healthy controls (Heslegrave et al, 2016; Piccio et al, 2016; Suárez-Calvet et al, 2016b). In this study, we have investigated the properties of the protease (the sheddase) that results in shedding of TREM2 from the surface of primary human and murine myeloid cells. We find that the sheddase activity is sensitive to metalloprotease inhibitors and cleaves at the H157-S158 peptide bond, in both primary human macrophages and mouse microglia. We investigate the effect of the disease-associated H157Y substitution and show that it may recruit a novel sheddase, possibly explaining its pathogenic mechanism. Results TREM2 is expressed on the surface of human cells and the N-terminal fragment is shed into the conditioned medium Non-permeabilised primary human macrophages showed high levels of TREM2 expression on their cell surfaces when probed with the AF1828 polyclonal antibody (Fig 1A, 1, red) but not with control goat serum (Fig 1A, 2). Likewise, stable-expressing hTREM2-HEK293 cells showed TREM2 on their surface (Fig 1A, 3, pink), which was not seen in the untransfected parental cells (Fig 1A, 4). In both cell types, Western blots of the cell lysates and conditioned media showed a range of TREM2 isoforms (Fig 1B). The lysates (L) contained a predominant 35 kDa band with a less intense smear up to 50 kDa. By contrast, this higher molecular weight smear is the major TREM2 species in the culture supernatants (S) from both HEK293 + hTREM2 and human primary macrophages. Figure 1. TREM2 expression, glycosylation and proteolysis Surface TREM2 was detected on non-permeabilised primary human macrophages labelled with anti-TREM2 polyclonal antiserum (1, red), but not a control antiserum (2) and by live cell immunostaining of HEK293 stably transfected with wild-type hTREM2 (3, pink; nuclei stained with Hoechst) but not on parental HEK293 (4). Surface immunolocalisation was also observed. Scale bars = 20 μm. Western blots of lysates (L) and supernatants (S) for hTREM2 from parental HEK293 vs. HEK293+hTREM2 cells showed distinct isoforms of TREM2. The cell lysate (HEK293+hTREM2, L) yielded an immature glycoform at 35 kDa with a less intense smear up to 50 kDa. This smear was the predominant species in the supernatant (HEK293+hTREM2, S). Similar distributions of TREM2 were seen in primary human macrophages (Macrophage, L and S). Subcellular fractionation of macrophages over a time course revealed the fate of surface-biotinylated TREM2 (membrane-associated in blue circles), indicating that most protein was shed into the supernatant (red squares), with a half time of < 1 h, and that little was found in cytosol or nuclear cellular fractions (green triangles & purple inverted triangles). Data plotted as mean ± SEM; n = 4 replicates. Source data are available online for this figure. Source Data for Figure 1 [emmm201707673-sup-0002-SDataFig1.pdf] Download figure Download PowerPoint We investigated the kinetics of TREM2 metabolism in primary human macrophages by biotin pulse labelling of surface-exposed protein and then following the fate of biotin-conjugated TREM2 in various subcellular fractions over time (Fig 1C, raw data Fig EV1). It is notable that the surface-exposed TREM2 has a short half-life of < 1 h and that the bulk of the lost protein appears in the supernatant. Very little surface-expressed TREM2 was seen to internalise into cytoplasmic or nuclear compartments under basal conditions. Click here to expand this figure. Figure EV1. Surface biotinylation and fate of TREM2Surface-expressed TREM2 on human macrophages was biotinylated at t = 0 and fractionated into four pools: supernatant, membrane-associated, cytosolic and nuclear. Similar fractionation was undertaken following incubation for 0.5, 1.0 and 4.5 h. TREM2-associated biotin was purified by immunoprecipitation and quantified by MSD for biotinylated TREM2. The raw and processed data are presented here. The left block of data shows biotin TREM2 measurements for four timepoints for four biological replicates for each of the four subcellular pools. The right block of data represents the normalised TREM2 quantification, with the membrane fraction at t = 0 defined as 100%. Download figure Download PowerPoint Proteolytic release of a 17 kDa TREM2 N-terminal fragment into the supernatants of primary mouse microglia, primary human macrophages and HEK293 cells Soluble TREM2 NTF was detected by Western blotting the conditioned medium from primary murine microglia and primary human macrophages; a similar result was also seen for HEK293 cells (Fig 2A). The high molecular mass smear of immunoreactivity (> 35 kDa) was likely due to extensive and variable glycosylation of the NTF because only a single band of 17 kDa was observed following deglycosylation. In primary human macrophages, and in wild-type hTREM2-expressing HEK293 cells, the ADAM10-specific metalloprotease inhibitor GI254023X reduced shedding. The potencies of GI254023X and a broad-spectrum metalloprotease inhibitor (GM6001) were quantified by a Meso Scale Discovery (MSD) assay for shed TREM2 and were shown to be similar in macrophages (Fig 2B), inhibiting shedding by ~50% at 20 μM. Under the same conditions, the broad-spectrum serine protease inhibitor PMSF did not inhibit TREM2 shedding at concentrations exceeding 100 μM (Fig 2B). GI254023X and GM6001 also inhibited shedding in HEK293 cells; additionally, the MMP2/9 inhibitor SB-3CT failed to inhibit shedding (Fig 2C). Figure 2. Shedding of glycosylated TREM2 NTF is sensitive to inhibitors of ADAM10 and matrix metalloproteinases Western blotting of the conditioned media from primary murine microglia (left panel), transfected HEK293 cells (middle panel) and primary human macrophages (right panel) revealed the presence of shed wild-type TREM2. This soluble TREM2 appeared as a > 35 kDa smear of various glycoforms and upon deglycosylation was reduced to a single band of 17 kDa (arrowhead). The ADAM10 inhibitor, GI254023X, blocked shedding in transfected HEK293 cell and macrophage cultures. The concentration of shed TREM2 in the supernatant of macrophage cultures was reduced to ˜50% by 20 μM of both GI254023X and the broad-spectrum metalloprotease inhibitor GM6001, as measured by an MSD assay. By contrast, the broad-spectrum serine protease inhibitor PMSF did not reduce shedding. Experiments were repeated three times and for two donors of the macrophage progenitors. Data plotted as mean ± SEM. In HEK293 cells, GI254023X and GM6001 had comparable potencies; however, the MMP2/9 inhibitor SB-3CT did not block shedding. Data plotted as mean ± SEM; n = 3 replicates. Source data are available online for this figure. Source Data for Figure 2 [emmm201707673-sup-0003-SDataFig2.pdf] Download figure Download PowerPoint Peptidomimetic protease inhibitors point to residues 158–160 as the site of sheddase cleavage We synthesised a tiled library of reverse-sequence d-amino acid (retro-inverso) polypeptides that replicated both the biophysical characteristics and the specific side-chain interactions of peptides constituting the peri-membrane region of TREM2 (Li et al, 2010) (Fig 3A). Unlike natural l-amino acid polypeptides, these peptidomimetics are resistant to proteolysis (Taylor et al, 2010) and at millimolar concentrations predictably act as competitive protease inhibitors. The levels of TREM2 NTF in the conditioned media of macrophage cultures treated with the peptidomimetics were compared with the levels in the media of untreated cultures. We found that all retro-inverso peptides that included residues 158–160 (C'-fedahvehsis-N', C'-hvehsisrsll-N', C'-hvehsis-N' & C'-sisrsll-N') inhibited shedding, whereas other members of the library (C'-sllegei-N', C'-geipfpp-N' & C'-fpptsil-N'), analogous to nearby peri-membrane regions of TREM2, did not (Fig 3B and C). Figure 3. Peptidomimetic protease inhibitors point to residues 158–160 as the site of sheddase cleavage An overlapping library of retro-inverso peptides were designed to mimic the extracellular peri-membranous domain of TREM2 in the region of the sheddase site (TM: transmembrane). Blue boxes represent retro-inverso peptides that reduce TREM2 shedding; red boxes those that do not. The black box indicates the three residues that are common to all the inhibitory retro-inverso peptides. The peptidomimetics were incubated with primary human macrophages, and the resulting levels of shed TREM2 NTF were quantified by MSD ELISA. Blue = inhibitory; red = non-inhibitory. Values plotted: mean ± SEM; each experiment was repeated for 3–5 independent human donors. Peptides including amino acids 158–160 (blue) inhibited TREM2 shedding more than retro-inverso peptides that did not (red). Values plotted: mean ± SEM; two-tailed Student's t-test, ***P = 0.003; each experiment was repeated for 3–5 independent human donors. Forward and reverse TREM2 peptidomimetics containing residues 158–160 suppressed TREM2 shedding equally. Values plotted: mean ± SEM; n = 3 replicates; two-tailed Student's t-test; ns = not significant. Download figure Download PowerPoint To understand whether the protease inhibition was strictly sequence specific, we synthesised a D-polypeptide with the reverse sequence of the most effective inhibitor (C'-hvehsisrsll-N'). This reverse retro-inverso peptide was equally effective at preventing TREM2 shedding (Fig 3D), indicating that access to the protease is determined less by the specific amino acid sequence as by general biophysical characteristics such as charge. Mass spectrometry identifies His157-Ser158 as the main sheddase cleavage site in wild-type and disease-linked variants of TREM2 Immunoprecipitation, using a goat polyclonal anti-hTREM2 (AF1828, R&D Systems) from the conditioned medium of primary human macrophages, followed by deglycosylation, yielded two specific bands on a silver-stained protein gel (Fig 4A, single biological replicate run in duplicate lanes). The upper band had a molecular mass of 17 kDa, consistent with the expected size of the NTF (arrowhead 1); running slightly ahead of this was a 15 kDa protein (arrowhead 2). SDS–PAGE purification and trypsin digestion of these bands yielded peptides that were analysed by LC-MS/MS, using the published TREM2 sequence as the search guide (PubMed references: human: NP_061838.1 & mouse: NP_001259007.1). Similar mass spectrometric assays were performed independently and in duplicate for the conditioned media of primary human macrophages, primary murine microglia and HEK293 cells stably expressing hTREM2. Murine TREM2 was immunoprecipitated with a rat anti-mTREM2 monoclonal (MAB1729, R&D Systems). The conditioned media were derived from two donors (macrophages and microglia) or from two independent cultures of HEK293 cells. Those peptides with C-terminal Arg and Lys residues were considered to result from trypsin cleavage while peptides with other C-terminal residues were likely the result of sheddase activity. For experiments that yielded non-trypsin-derived peptide fragments, we plotted their frequency against the peri-membrane sequence of TREM2 (Fig 4B). Figure 4. Mass spectrometry identifies His157-Ser158 as the sheddase site TREM2 was immunoprecipitated and deglycosylated from primary human macrophage conditioned media. Two specific bands were visible on a silver-stained SDS–PAGE gel (arrowheads 1 & 2). These bands were excised, digested with trypsin and analysed by LC-MS/MS. TREM2 in the conditioned media of primary human macrophages (black), primary murine microglia (white) and HEK293 cells stably expressing hTREM2 (grey) was digested with trypsin and the resulting peptides identified by mass spectrometry. The most frequent C-terminal residue not consistent with trypsin digestion was H157 (from two donors/biological replicates of HEK293 cells; each replicate assayed in separate mass spectrometry laboratories; n = total number of peptides identified; where peptide sequences differ between species they are shown as human/mouse). Trypsin digestion (red sites) of band 1 provided almost complete coverage of macrophage TREM2 (bold), lacking only the peptide expected to have R52 at its C-terminus. Non-trypsin cleavage (blue) was observed predominantly at H157. The absence of the peptide with K42 at its C-terminus suggests that N-terminal truncation is responsible for generating band 2. Underlined: predicted secretion signal peptide. Peptides from the supernatants of HEK293 cells transiently expressing wild-type (black) and H157Y (white) human TREM2, and co-expressing human DAP12, were identified by mass spectrometry. The most common C-terminus was residue 157 for both TREM2 isoforms (one biological replicate; n = total number of peptides identified; where WT and variant sequences vary they are shown as WT/variant). Schematic of the TREM2 protein. SP, signal peptide; IG domain, immunoglobulin domain; TM, transmembrane domain; triangles, N-glycosylation sites; arrow, site of proteolytic shedding; all numbers relate to amino acid positions. Source data are available online for this figure. Source Data for Figure 4 [emmm201707673-sup-0004-SDataFig4.pdf] Download figure Download PowerPoint Mass spectrometry yielded almost complete coverage of the extracellular domain of TREM2 (Fig 4C). For the 17 kDa band 1 from primary human macrophages, we observed peptides that included the N-terminus through to the most distal non-trypsin cleavage sites. These cleavages, likely due to the sheddase, occurred most commonly at H157, with such peptides accounting for up to 10% of all TREM2 peptides detected. Other nearby non-trypsin sites at positions 158 and 159 each accounted for < 0.1% of the peptides observed. Across all three cell types, H157 was the most common site with other non-trypsin sites each accounting for < 1% of the total peptides observed. The 15 kDa band 2, from primary human macrophages, yielded similar peptide fragments but lacked the most N-terminal sequence, making it likely that a second endoproteolytic event within the first 34 amino acids of TREM2 is a common occurrence. Control experiments in which cell-free, full-length, recombinant TREM2 was digested with trypsin did not yield peptides with H157 at the C-terminus, instead the expected tryptic peptides ending with R161 were observed (data not shown). In the same way, we identified peptides in the conditioned medium of HEK293 cell lines transiently expressing wild-type (Fig 4D, black) and H157Y (white) human TREM2 and co-expressing human DAP12. For both isoforms, residue 157 was the predominant C-terminal residue indicating that the site of proteolysis is largely unaffected by the substitution of a non-polar tyrosine for the polar histidine at the P1 position (see Fig 4E for schematic of cleavage site). The H157Y substitution accelerates TREM2 shedding from HEK293 cells The H157Y variant of TREM2 carries an increased risk of AD (Jiang et al, 2016) although, like the R47H variant, the mechanism is unknown. To test whether H157Y substitution alters the proteolytic metabolism of TREM2, we expressed the wild-type and the variant proteins in HEK293 cells. Levels of cell-associated TREM2 in cell lysates (Fig 5A) and shed TREM2 in conditioned media (Fig 5C) TREM2 were measured by Western blot (full-length TREM2 species quantified in Fig 5B and cleaved fragments in Fig 5D). Both wild-type and H157Y variant TREM2 expressed at similar levels. However, for H157Y TREM2 there was a reduction in mature glycoforms in the cell lysates, whereas the immature glycoforms were equivalent (blot in Fig 5A, quantified in Fig 5B). This altered ratio is unlikely the result of impaired delivery of the variant to the cell surface because levels of both the TREM2 C-terminal fragment (CTF, blot in Fig 5A, quantified in Fig 5D) and NTF (blot in Fig 5C, quantified in Fig 5D) are higher for the H157Y variant as compared to wild type. Rather it appears that mature H157Y TREM2 is shed more rapidly from the cell surface. Figure 5. The disease-linked H157Y variant of TREM2 is shed more rapidly than wild-type TREM2 Western blot for TREM2 in lysates of HEK293 cells transiently expressing either wild-type (WT) or the H157Y variant protein (N = 3): levels of immature TREM2 (major band at 35 kDa) were unchanged by the H157Y substitution; however, total levels of the variant were reduced as compared to WT because of a more marked reduction in the levels of the glycosylated isoform. The proteolytic cleavage of TREM2 generated a truncated C-terminal fragment (CTF) that was more abundant in lysates from cells expressing H157Y TREM2. GAPDH was the loading control; DAP12 was co-expressed with TREM2. Molecular mass markers in kDa. Quantitation of the full-length TREM2 isoforms as shown in panel (A) (data plotted as mean ± SEM; N = 12). Western blot for the shed TREM2 NTF from the conditioned medium of HEK293 cell cultures (N = 3): levels of H157Y TREM2 NTF were higher than WT. A secreted fragment of the amyloid precursor protein (sAPPa) was the loading control. Molecular mass markers in kDa. The proteolytic fragments of TREM2 as shown in panel (A) (CTF, N = 3) and panel (C) (NTF, N = 15) were corrected for the total full-length TREM2 (FL) from each cell lysate: the levels of the shed N-terminal fragment (NTF) of TREM2 were higher in cells expressing the H157Y variant as compared to WT. Data plotted as mean ±