Mass spectrometry-based determination of APLP2 γ-cleavage site.

<p>(<b>A</b>) Scheme of experimental procedure: the HA-APLP2CTF-FLAG construct was stably expressed in HEK293 cells. The construct consists of an N-terminal HA-tag (HA), a linker region consisting of five glycines (5G), the APLP2CTF sequence (APLP2) and an C-terminal FLAG-tag (FLAG). Upon γ-secretase cleavage, peptides harboring the γ-secretase cleavage site at their C-terminus were liberated. Peptides were immunoprecipitated using HA-affinity agarose and subsequently analyzed in a MALDI-TOF mass spectrometer. (<b>B</b>) Western Blot to confirm the γ-secretase sensitivity of the construct. Cells stably expressing HA-APLP2CTF-FLAG were treated with (M) or without (C, control) the γ-secretase inhibitor Merck A. In the presence of Merck A the CTFs accumulated in the lysate. The same was observed for cells grown in ‘light’ (L) or ‘heavy’ (H) medium. CTFs were detected with anti-FLAG antibody. Calnexin levels were analyzed as a loading control. (<b>C</b>+<b>D</b>) Mass spectrometry-based identification of the γ-secretase cleavage sites. Under control conditions, seven peaks were detected with a maximal total ion count of 497. Upon Merck A treatment, the intensity (ion count) for the peaks was reduced significantly. Peaks are labeled with identifiers #1–#7 linking them to the respective peptides sequences in the table (D). All peptides were detected with a mass error of less than 0.3 Da. (<b>E</b>) Determination of the labeling efficiency for the SILAC experiment. To determine the labeling efficiency, proteins from the cell lysates of the ‘heavy’ labeled cells from (F) were separated on an SDS-PAGE gel. One band was cut out and tryptic in gel digestion was performed. Peptides resulting from this digestion were analyzed in an LTQ Orbitrap Velos mass spectrometer. Proteins were identified by database search, and the labeling efficiency (Label. Eff.) was determined from the ‘heavy’ to ‘light’ ratios (Heavy/Light) of the quantifiable peptides. Further, coverage of the proteins in % (Coverage), number of unique peptides identified (Peptides), molecular weight of respective protein (MW), SEQUEST score (Score) and number of unique peptides used for quantification (H/L Count) are given. (<b>F</b>) Analysis of γ-secretase-dependent CTF cleavage by quantitative mass spectrometry. Upper panel: Cells without Merck A (Control) grown either in ‘light’ or in ‘heavy’ medium. Supernatants were combined, peptides immunoprecipitated and analyzed in the MALDI-TOF mass spectrometer. Peaks resulting from the ‘light’ and the ‘heavy’ labeled peptides are clearly separated (+10 Da as expected for one heavy labeled arginine). Intensities of corresponding ‘heavy’ and ‘light’ peak clusters are not identical, as the ‘heavy’ clusters are either shifted (+10 Da) or supershifted (+16 Da) due to additional incorporation of a ‘heavy’ proline resulting from arginine- to-proline conversion. Lower panel: upon treatment of the ‘light’ labeled cells with Merck A and the ‘heavy’ labeled cells with DMSO as a solvent control, the ratio between the intensity of the ‘light’ labeled peptides and the ‘heavy’ labeled peptides is significantly reduced if compared to the control experiment. This demonstrates that the identified peptide peaks are generated by γ-secretase. (<b>G</b>) Comparison of the position of the ε- and γ-secretase cleavage sites of APP and APLP2 in an alignment of the juxtamembrane and the transmembrane region of these two proteins. The dark boxes mark the predicted transmembrane domains (TMD), while the light box indicates amino acids which could potentially still be part of the transmembrane domain, as they do not harbor charges.</p

Analysis of APLP2 shedding using protease inhibitors.

<p>(<b>A</b>) Human neuroblastoma SH-SY5Y cells were transiently transfected with an siRNA pool against APLP2 or with a control pool. Endogenous APLP2 was detected with the N-terminally binding 2D11 antibody. Bands were absent upon knock-down of APLP2 (APLP2KD), demonstrating the specificity of the antibody. Soluble APLP2 (sAPLP2) was detected in the conditioned medium, cellular full-length APLP2 (cell. APLP2), cellular full-length APP (cell. APP) and calnexin as a loading control in the cell lysate. Three forms of APLP2 were detected: CS-GAG modified (*) and two non-CS-GAG modified species (** and ***) with molecular weights of around 115 and 100 kDa respectively. (<b>B</b>) Deglycosylation of APLP2 in conditioned medium and cell lysate of SH-SY5Y cells using endoglycosidase H (H) and N-glycosidase F (F). Deglycosylated forms of APLP2 are indicated (-- and ---). As a control, deglycosylation was performed for BACE1 in the cell lysate of BACE1 overexpressing HEK293 cells. Mature (#), immature (##) and deglycosylated (###) BACE1 is detectable. (<b>C</b>) Representative blots of treatment of SH-SY5Y cells with C3 (1 µM), TAPI-1 (50 µM) or C3+TAPI-1. Upon C3 treatment no significant reduction of sAPLP2 levels was observed. Upon TAPI-1 and C3+TAPI-1 treatment sAPLP2 levels were strongly reduced while no changes in APLP2 levels in the cell lysate were observed. As a control, soluble APP (sAPP) levels were clearly reduced upon TAPI-1 and C3+TAPI-1 treatment. (<b>D</b>) Quantification of experiments in C (mean +/− SEM). C3 treatment did not lead to a significant reduction in sAPLP2 levels while TAPI-1 as well as C3+TAPI-1 treatment led to a significant reduction in sAPLP2 levels (p<0.001 for all three species, n = 6).</p

Transient and stable knock-down of ADAM10 suppresses APLP2 shedding.

<p>(<b>A</b>) SH-SY5Y cells were transfected with siRNA pools against the proteases ADAM10 (A10KD) or ADAM17 (A17KD) or with control siRNA (Con). Both proteases were detected in membrane preparations. The mature active form is indicated with ##, the immature form with #. Actin and full-length APLP2 levels (cell. APLP2) were detected in the cell lysate. Conditioned media were analyzed for total secreted APLP2 (sAPLP2). All three species of sAPLP2 (* CS-GAG modified, ** 115 kDa, *** 100 kDa) were strongly decreased upon knock-down of ADAM10, but not of ADAM17. (<b>B</b>) Quantification of experiments in A (mean +/− SEM). ADAM10 knock-down significantly reduced sAPLP2 (p<0.001 for all three species, n = 6), while ADAM17 knock-down did not lead to any significant changes in sAPLP2 levels. (<b>C</b>) Knock-down of ADAM10 and ADAM17 in HEK293 cells, carried out as in A. (<b>D</b>) Quantification of experiments in C (mean +/− SEM). ADAM10 knock-down significantly reduced sAPLP2 (p<0.001 for CS-GAG modified and 100 kDa APLP2, p = 0.001 for 115 kDa APLP2, n = 6). (<b>E</b>) SH-SY5Y cells with stable shRNA-mediated knock-down of ADAM10 were used. shRNAs sh7 and sh9 are targeting two different regions of ADAM10. As control, a stable SH-SY5Y cell line expressing a non-targeting shRNA was used (Con). sAPLP2 levels were clearly reduced upon ADAM10 knock-down. (<b>F</b>) Quantification of experiments in E (mean +/− SEM). Both shRNAs significantly reduced sAPLP2 (p<0.001 for all three species, n = 6). (<b>G</b>) SH-SY5Y cells were transiently transfected with a siRNA pool against ADAM10 (A10KD) or control siRNA (Con). Cells were treated with DAPT (1 µM). Additionally C3 (1 µM) or DMSO (as a solvent control) was applied. sAPLP2 levels were clearly reduced upon ADAM10 knock-down while cellular APLP2 levels (cell. APLP2) remained unchanged. Two forms of APLP2 C-terminal fragments (CTFs) were detected at around 10 kDa in the cell lysate (-,--). ADAM10 knock-down led to the elimination of the lower molecular weight APLP2 CTF (--) while C3-treatment abolished the higher molecular weight species (-).</p

APLP2 processing in primary cortical neurons.

<p>(<b>A</b>) E16 primary cortical neurons were treated with C3 (1 µM), TAPI-1 (50 µM) or DMSO as a solvent control. sAPLP2 (* CS-GAG-modified, ** 115 kDa, *** 100 kDa) was detected in the conditioned medium of these cells. Full-length cellular APLP2 (cell. APLP2) and actin were detected in the cell lysate. A clear reduction of sAPLP2 levels was observable upon C3 as well as TAPI-1 treatment. (<b>B</b>) Quantification of experiments in A (mean +/− SEM). C3 and TAPI-1 significantly reduced sAPLP2 (C3: p<0.001 for both detected species, TAPI-1: p<0.001 for CS-GAG modified APLP2, p = 0.004 for mature APLP2; n = 6). (<b>C</b>) E16 primary cortical neurons with lentiviral knock-down of ADAM10 were analyzed. shRNAs (sh-I and sh-II) targeting different regions of ADAM10 and a non-targeting shRNA control (Con) were used. A clear reduction of ADAM10 (# immature, ## mature) levels was observed in the cell lysate and of sAPLP2 in the conditioned medium. (<b>D</b>) Quantification of experiments in C (mean +/− SEM). Both shRNAs significantly reduced sAPLP2 (p<0.01 for both detected species for both shRNAs, n = 6).</p

Mass spectrometry-based determination of APLP2 α- and β-shedding sites.

<p>(<b>A</b>) Scheme of experimental procedure. A TEV-protease cleavage site followed by a FLAG-tag was introduced into APLP2 isoform 763 starting after amino acid M653 (APLP2TF). The TEV-FLAG site is positioned 39 amino acids N-terminally of the assumed start of the transmembrane domain (TMD). Shedding yields sAPLP2TF, which was immunoprecipitated and digested with TEV-protease, leading to a small peptide harboring the N-terminal FLAG-tag as well as the C-terminal cleavage site resulting from the shedding. This peptide was analyzed in a mass spectrometer. (<b>B</b>) HEK293 cells were transiently transfected with the APLP2TF construct and either treated with TAPI-1 (50 µM) or DMSO as a solvent control. Upon TAPI-1 treatment secreted sAPLP2TF levels were clearly decreased in the conditioned medium of the cells. An accumulation of APLP2TF levels was observed upon TAPI-1 treatment in the cell lysate. Actin was analyzed in the cell lysate as a loading control. (<b>C</b>) Peptides produced according to the scheme in A were obtained from HEK293 cells transiently overexpressing APLP2TF and either luciferase as a control, ADAM10 or BACE1. Mass spectrometric analysis of the peptides revealed two peaks for control and ADAM10 overexpression (*,**) while upon BACE1 overexpression only one peak with a lower m/z ratio was observed. Total Ion Counts (TIC) and centroid peak masses of the first isotopic peak are given for each isotopic peak cluster. All peaks are revealed from singly-charged peptides. (<b>D</b>) Determined (Det.) masses of the peaks in C (*,**,***) were compared to calculated (calc.) masses. For each mass, a corresponding peptide could be computed with less than 0.5 Da error. (<b>E</b>) Western Blot analysis of the samples used in C. sAPLP2TF was detected in the conditioned media, full-length APLP2 (APLP2TF), ADAM10 (A10, # immature and ## mature), BACE1 and calnexin as a loading control were detected in the cell lysates. A clear increase in sAPLP2TF levels was observed upon ADAM10 and BACE1 overexpression. sAPLP2TF has a slightly reduced apparent molecular weight upon BACE1 overexpression if compared to control (Con). (<b>F</b>) Summary of known ADAM10 cleavage sites in different substrates aligned with the newly detected ADAM10 cleavage site for APLP2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021337#pone.0021337-Caescu1" target="_blank">[41]</a>. (<b>G</b>) Schematic comparison of the ectodomain shedding sites of APP and APLP2. The APP α-cleavage occurs 12 amino acids N-terminally of the transmembrane domain (TMD), for APLP2 it occurs 22 amino acids N-terminally of the TMD. For both proteins β-cleavage occurs N-terminally of an aspartate.</p

Regulated Intramembrane Proteolysis and Degradation of Murine Epithelial Cell Adhesion Molecule mEpCAM

<div><p>Epithelial cell adhesion molecule EpCAM is a transmembrane glycoprotein, which is highly and frequently expressed in carcinomas and (cancer-)stem cells, and which plays an important role in the regulation of stem cell pluripotency. We show here that murine EpCAM (mEpCAM) is subject to regulated intramembrane proteolysis in various cells including embryonic stem cells and teratocarcinomas. As shown with ectopically expressed EpCAM variants, cleavages occur at α-, β-, γ-, and ε-sites to generate soluble ectodomains, soluble Aβ-like-, and intracellular fragments termed mEpEX, mEp-β, and mEpICD, respectively. Proteolytic sites in the extracellular part of mEpCAM were mapped using mass spectrometry and represent cleavages at the α- and β-sites by metalloproteases and the b-secretase BACE1, respectively. Resulting C-terminal fragments (CTF) are further processed to soluble Aβ-like fragments mEp-β and cytoplasmic mEpICD variants by the g-secretase complex. Noteworthy, cytoplasmic mEpICD fragments were subject to efficient degradation in a proteasome-dependent manner. In addition the γ-secretase complex dependent cleavage of EpCAM CTF liberates different EpICDs with different stabilities towards proteasomal degradation. Generation of CTF and EpICD fragments and the degradation of hEpICD via the proteasome were similarly demonstrated for the human EpCAM ortholog. Additional EpCAM orthologs have been unequivocally identified <i>in silico</i> in 52 species. Sequence comparisons across species disclosed highest homology of BACE1 cleavage sites and in presenilin-dependent γ-cleavage sites, whereas strongest heterogeneity was observed in metalloprotease cleavage sites. In summary, EpCAM is a highly conserved protein present in fishes, amphibians, reptiles, birds, marsupials, and placental mammals, and is subject to shedding, γ-secretase-dependent regulated intramembrane proteolysis, and proteasome-mediated degradation.</p></div

Determination of the sheddase cleavage amino acid sequence in mEpEX.

<p>(<b>A</b>) Schematic representation of mEpCAM-TF containing a TEV protease recognition site and a Flag-Tag in the mEpEX domain 42 amino acids before the predicted transmembrane domain. After cleavage by sheddases, the largest part of mEpEX can be removed through digestion with TEV protease and the resulting small fragment immunoprecipitated using Flag-specific antibodies. (<b>B</b>) Representative mass spectrometry spectrum of HEK293, NIH3T3, and mF9 cells stably expressing mEpCAM-TF and of vector control HEK293 cells as a control. Four major peak species are indicated. (<b>C</b>) Tabular overview of sheddase cleavage sites within mEpEX as determined upon mass spectrometric analysis and alignment to potential molecular weights. Calculated and determined masses are given in Dalton including error and charge of each peptide. (<b>D</b>) Representative mass spectrometry spectrum of HEK293 cells stably expressing mEpCAM-TF after treatment with DMSO, the metalloprotease protease inhibitor TAPI-1, and the phorbol ester PMA. (<b>E</b>) Representative mass spectrometry spectrum of HEK293 cells stably expressing mEpCAM-TF after treatment with DMSO, the BACE1 protease inhibitor C3, and after transient transfection of BACE1 expression plasmid. (<b>F</b>) Sequence alignment of murine and human EpCAM (top), and murine EpCAM and murine Trop-2 (bottom). metalloprotease protease cleavage (a-secretase) and BACE1 cleavage sites (b-secretase) are indicated.</p

Cleavage of murine EpCAM in membrane assays.

<p>HEK293 cells were stably transfected with full-length mEpCAM in fusion with YFP (EpCAM-YFP). (<b>A</b>) Schematic representation of cleavage processes resulting in the generation of soluble EpEX, CTF-YFP, and intracellular EpICD-YFP fragments. (<b>B–C</b>) Membranes of stable transfectants were isolated and either kept at 0°C (0 h) or incubated at 37°C in reaction buffer for the indicated time points. Thereafter, pellets and supernatant were collected upon differential centrifugation. Pellets (<b>B</b>) and supernatants (<b>C</b>) of membrane assays were separated in a 10% SDS-PAGE and probed with mEpICD- and YFP-specific antibodies. (<b>D</b>) Embryonic stem cell line E14TG2a and teratocarcinoma cells mF9 were treated with DMSO (control) or the γ-secretase inhibitor DAPT before being subjected to a membrane assay. The total fraction of the membrane assay was separated in a 10% SDS-PAGE, and probed with a YFP-specific antibody. Treatment with DAPT resulted in the accumulation of CTF-YFP and in the inhibition of mEpICD-YFP formation. Protein bands corresponding to mEpCAM-YFP, CFT-YFP, and mEpICD-YFP are indicated in each immunoblot. Shown are the representative results of three independent experiments.</p

Schematic representation of EpCAM presenilin-dependent regulated intramembrane proteolysis (PS-RIP) and endocytosis.

<p>Murine EpCAM (mEpCAM) is cleaved at the plasma membrane to release soluble EpEX (smEpEX). The resulting C-terminal fragment (mCTF) is a substrate for γ-secretase, which cleaves mCTF to generate soluble, extracellular mEp-β fragments (γ-cleavage) and intracellular mEpICD fragments, which are prone to proteasomal degradation. Additionally, mEpICD can be endocytosed and processed either by BACE1 in acidic intracellular compartments (endosome) and/or by acidic hydrolases in lysosomes.</p

Sequence conservation of cleavage sites in orthologs of EpCAM found in fishes, amphibians, birds, to placental mammals.

<p>Amino acid sequences of 52 orthologs of human EpCAM were aligned using ClustallW and sequence conservation of each amino acid was calculated (maximum score 11. Shown are the mean conservation score throughout all orthologs (mean) and conservation scores of single amino acids ranging positions p⁻³ to p⁺³ around determined cleavage sites of metalloproteases (<b>A</b>), BACE1 (<b>B</b>), γ-cleavage of γ-secretase (<b>C</b>), and ε-cleavage of γ-secretase (<b>D</b>).</p