Journal: EMBO Molecular Medicine
Article Title: TREM2 shedding by cleavage at the H157‐S158 bond is accelerated for the Alzheimer's disease‐associated H157Y variant
Figure Lengend Snippet: 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.
Article Snippet: Concentrated medium from human cells (HEK293, macrophages) or mouse microglia were incubated with goat anti‐human TREM2, or rat anti‐mouse TREM2, respectively (both 10 μg/ml from R & D Systems) and immunoprecipitated using Dynabeads® Protein G (ThermoFisher).
Techniques: Mass Spectrometry, Immunoprecipitation, Staining, SDS Page, Liquid Chromatography with Mass Spectroscopy, Stable Transfection, Expressing, Variant Assay