human lrp6 ecd fc  (R&D Systems)

Journal: mBio

Article Title: LRP6 Is a Functional Receptor for Attenuated Canine Distemper Virus

doi: 10.1128/mbio.03114-22

Figure Lengend Snippet: Genome-wide CRISPR/Cas9 Knock Out screen identifies potential host factors that are essential for CDV-OP infectivity. (A) Schematic outline of the canine genome-wide CRISPR/Cas9 KO screen. OP neon , mNeonGreen expressing CDV-OP strain. (B) Infection of canine mammary carcinoma P114 cells with CDV-OP (left panel) or CDV wild-type (right panel) expressing mNeonGreen (OP neon and wt neon , respectively). (C) Assessment of cN4-expression in P114 cells by immunofluorescence (IF) analyses, using an anti-N4 antibody. (D) Investigation of cN4 (with GAPDH as a control) mRNA expression via reverse transcription-PCR. (E) Enrichment of cells acquiring CDV-OP-resistance through successive rounds of infection of P114 cells transduced with the canine genome-wide CRISPR library. (F) Cumulative distribution and area under the curve (AUC) analysis of the library representation. The representation was evaluated for the pDNA of the Beauty library (blue graph) as well as for the genomic DNA that was extracted from the D0 population (experiment 1 [E1], red/orange and experiment 2 [E2], green). An ideally distributed library is shown in black. The percentages indicate each library’s representation at 90% cumulative reads, and the AUC values are indicated in the legend. (G) Boxplot representing the log fold change (LFC) (D15, compared to the plasmid DNA of the library [pDNA], obtained for the control population) of genes known to be essential or nonessential. (H) Volcano plot representing the depleted (log fold change [LFC] < 0) and enriched (LFC > 0) genes after four rounds of infection with OP neon , compared to the control population. The LFC and P values were calculated from two independent replicates via a MAGeCK analysis. Each dot represents one gene for which at least two gRNAs (out of four) were used for the analysis. Selected hits are color-coded. (I) Schematic illustration of the LRP6 receptor, consisting of four β-propeller domains linked by epidermal growth factor (EGF)-like motives, low-density lipoprotein receptor (LDLR) class A repeats, a transmembrane domain, and a cytoplasmic tail.

Article Snippet: The gene encoding a soluble form of the canine LRP6 protein (UniProt: A0A8C0NS55_CANLF : aa 20 to 1,246) fused to a human Fc domain (solLRP6-Fc) was synthesized at Twist Bioscience.

Techniques: Genome Wide, CRISPR, Knock-Out, Infection, Expressing, Immunofluorescence, Control, Reverse Transcription, Transduction, Plasmid Preparation

Journal: mBio

Article Title: LRP6 Is a Functional Receptor for Attenuated Canine Distemper Virus

doi: 10.1128/mbio.03114-22

Figure Lengend Snippet: Ablation of LRP6 expression decreases CDV-OP infection in various canine cell lines. (A) Tracking of indels by decomposition (TIDE) analyses. Frequency of frameshift short insertions/deletions of LRP6 KO cells (green) using gRNA 1 or 2 (L1 and L2, respectively) or a nontargeting (NT) gRNA. In-frame mutations are color-coded in blue, and the wt genotype is color-coded in red. (B) Fluorescent syncytium formation of LRP6-expressing or ablated (LRP6 KO ) cells infected with OP neon . L1, LRP6 KO gRNA1; L2, LRP6 KO gRNA2; NT, nontargeting gRNA control. Pictures of stitched 96-wells were taken 3 days postinfection (dpi) (2 dpi for Vero cells). (C) Quantitative assessment of fluorescence emission from OP neon -infected cells, normalized to the NT. The means and standard deviations (SD) of data from three independent experiments are shown. Statistical significance was determined using a one-way ANOVA followed by Dunnett’s multiple-comparison test, using GraphPad Prism v.9 to analyze the differences between the values obtained from the NT and those from the indicated KO cells (*, P < 0.05; ****, P < 0.0001). (D) The viral titers of OP neon in LRP6-expressing and LRP6 KO cells are displayed in Log 10 TCID 50 /mL. The means and standard deviations (SD) of data from six independent experiments are shown. Statistical significance was determined using a one-way ANOVA followed by Dunnett’s multiple-comparison test, using GraphPad Prism v.9 to analyze the differences between the values obtained from the NT and those from the indicated KO cells (**, P < 0.01; ****, P < 0.0001). (E) Assessment of LRP6 expression in the indicated cells via Western blotting analyses, using an anti-LRP6 antibody. As a loading control, actin was detected using a monoclonal anti-actin antibody. (F) Band intensities were recorded with the Bio-1D software. The means and standard deviations (SD) of data from three independent experiments, normalized to the NT, are shown. Statistical significance was determined using a one-way ANOVA followed by Dunnett’s multiple-comparison test using GraphPad Prism v.9 to analyze differences between the NT and the LRP6 KO values (***, P < 0.001; ****, P < 0.0001).

Article Snippet: The gene encoding a soluble form of the canine LRP6 protein (UniProt: A0A8C0NS55_CANLF : aa 20 to 1,246) fused to a human Fc domain (solLRP6-Fc) was synthesized at Twist Bioscience.

Techniques: Expressing, Infection, Control, Fluorescence, Comparison, Western Blot, Software

Journal: mBio

Article Title: LRP6 Is a Functional Receptor for Attenuated Canine Distemper Virus

doi: 10.1128/mbio.03114-22

Figure Lengend Snippet: A point mutation in the CDV-OP receptor-binding protein (A393T) prevents infectivity in Vero and P114 cells. (A) Assessment of LRP6-HA protein expression via Western blotting analyses, using an anti-HA antibody in P114-LRP6 KO cells (P114-L2) reconstituted with cLRP6-HA (P114-L2/L-HA) or not (P114-L2/empty). (B) IF staining of indicated cells, using a monoclonal anti-HA MAb. (C) Quantitative assessment of fluorescence emission from OP neon -infected cells, using an MOI of 1. Images were taken at 3 dpi. The means and standard deviations (SD) of data from three independent experiments are shown. Statistical significance was determined using an unpaired t test, using GraphPad Prism v.9 to analyze the differences between the P114-L2/L-HA and P114-L2/empty values (****, P < 0.0001). (D) The viral titers of OP neon in P114-L2/L-HA and P114-L2/empty cells are displayed in Log 10 TCID 50 /mL. The means and standard deviations (SD) of data from six independent experiments are shown. Statistical significance was determined using an unpaired t test, using GraphPad Prism v.9 to analyze the differences between the values obtained from the indicated cells (**, P < 0.01). (E) Cell-cell fusion induced by cells transiently expressing the indicated H-proteins together with FOP and GFP. Schematic representation of the various H-proteins with the amino acid numberings of the exchanged H-fragments are shown. OP-derived fragments are colored-coded in white, and wt-derived fragments are highlighted in gray. Images were taken 2 days posttransfection (dpt). (F) Cell-cell fusion induced by cells transiently expressing the indicated H-protein variants harboring single (or double) point mutation(s) together with FOP and GFP. Images were taken at 2 dpt. (G) Infection of various cell lines with OP neon carrying a single point mutation (A393T) in the H-protein (OP neon HOP A393T ). Images were taken at 2 or 3 dpi for the Vero or other cell lines, respectively. (H) Quantitative assessment of the fluorescence emission from the OP neon HOP A393T -infected cells. The means and standard deviations (SD) of data from three independent experiments are shown. Statistical significance was determined using a one-way ANOVA followed by Dunnett’s multiple-comparison test, using GraphPad Prism v.9 to analyze the differences between the values obtained from the Vero-cSLAM cells and those from other cell types (****, P < 0.0001).

Article Snippet: The gene encoding a soluble form of the canine LRP6 protein (UniProt: A0A8C0NS55_CANLF : aa 20 to 1,246) fused to a human Fc domain (solLRP6-Fc) was synthesized at Twist Bioscience.

Techniques: Mutagenesis, Binding Assay, Infection, Expressing, Western Blot, Staining, Fluorescence, Derivative Assay, Comparison

Journal: mBio

Article Title: LRP6 Is a Functional Receptor for Attenuated Canine Distemper Virus

doi: 10.1128/mbio.03114-22

Figure Lengend Snippet: LRP6 acts as a functional receptor for CDV-OP. (A) Assessment of the binding activities of various solH-proteins at the cell surfaces of the indicated cells via immunofluorescence (IF) analyses, using an anti-TST monoclonal antibody. (B) Qualitative assessment of cell-cell fusion upon mixing two cell populations expressing the indicated H/F combinations, the human or canine LRP6 proteins (or empty vector control), the split nanoluciferase (nLuc) reporter proteins, and either GFP or RFP (see Materials and Methods for more details). Images of fluorescence emission were taken 24 h after cell mixing. (C) Quantitative assessment of the cell-to-cell fusion of mixed cell populations. Luminescence triggered (upon the addition of the substrate) by the reconstituted nLuc reporter protein, which was observed using a multiplate reader (Cytation 5 device, BioTek). The means and standard deviations (SD) of data from three independent experiments are shown. Statistical significance was determined using a one-way ANOVA followed by Dunnett’s multiple-comparison test, using GraphPad Prism v.9 to analyze the differences between the values obtained from the hLRP6- or cLRP6-transfected cells and those from the empty vector-transfected control cells. (***, P < 0.001; ns, nonsignificant). (D) Assessment of the binding activity of various TST-tagged, soluble H-protein constructs by mixing them with either an Fc-carrying soluble cLRP6 variant (solLRP6-Fc), an Fc-carrying soluble cSLAM protein (solSLAM.V-Fc), or without any proteins (for control experiments) via coimmunoprecipitation assays and Western blot analyses. Whereas protein G beads were used for the immunoprecipitation step, immunoprecipitated (IP) and coimmunoprecipitated (coIP) proteins were investigated via Western blotting analyses, using an anti-human-Fc or a mouse anti-TST antibody. (E) An ELISA of soluble proteins was used for the coIP experiments. (F) Infection assays in the indicated J3T cells were performed, using VSVΔG pseudotyped with standard CDV-OP glycoproteins (left) or a receptor-binding protein that was defective in LRP6-binding (HOP A393T ; right). The assay was performed in the presence of an F-protein inhibitor (3G), an anti-VSV-G antibody, or DMSO. Since the VSVΔG genome also encodes the firefly luciferase reporter protein, the viral infectivity was assessed indirectly via the recording of the firefly luciferase activity in infected cells at 24 h postinfection. The means and standard deviations (SD) of data from three independent experiments are shown. Statistical significance was determined using a one-way ANOVA followed by Dunnett’s multiple-comparison test, using GraphPad Prism v.9 to analyze the differences between the values obtained from DMSO-treated J3T-NT cells and those from the indicated J3T cells that were treated with inhibitors or DMSO (****, P < 0.0001; ns, nonsignificant).

Article Snippet: The gene encoding a soluble form of the canine LRP6 protein (UniProt: A0A8C0NS55_CANLF : aa 20 to 1,246) fused to a human Fc domain (solLRP6-Fc) was synthesized at Twist Bioscience.

Techniques: Functional Assay, Binding Assay, Immunofluorescence, Expressing, Plasmid Preparation, Control, Fluorescence, Comparison, Transfection, Activity Assay, Construct, Variant Assay, Western Blot, Immunoprecipitation, Enzyme-linked Immunosorbent Assay, Infection, Luciferase

Journal: mBio

Article Title: LRP6 Is a Functional Receptor for Attenuated Canine Distemper Virus

doi: 10.1128/mbio.03114-22

Figure Lengend Snippet: Oligonucleotides used for genome editing

Article Snippet: The gene encoding a soluble form of the canine LRP6 protein (UniProt: A0A8C0NS55_CANLF : aa 20 to 1,246) fused to a human Fc domain (solLRP6-Fc) was synthesized at Twist Bioscience.

Techniques: Sequencing

Journal: mBio

Article Title: LRP6 Is a Functional Receptor for Attenuated Canine Distemper Virus

doi: 10.1128/mbio.03114-22

Figure Lengend Snippet: PCR primers used for TIDE analysis

Article Snippet: The gene encoding a soluble form of the canine LRP6 protein (UniProt: A0A8C0NS55_CANLF : aa 20 to 1,246) fused to a human Fc domain (solLRP6-Fc) was synthesized at Twist Bioscience.

Techniques: Sequencing

Journal: Translational Psychiatry

Article Title: A role for APP in Wnt signalling links synapse loss with β-amyloid production

doi: 10.1038/s41398-018-0231-6

Figure Lengend Snippet: a APP overexpression enhanced Wnt-β-catenin signalling as measured by TCF/LEF transcriptional activity using SuperTOPflash luciferase reporter assay in HEK293A cells; activity was measured in relative light units (RLU). The enhancement of Wnt-β-catenin signalling was only detectable in cells also overexpressing Wnt3a (a “canonical Wnt”) and not in the presence of the “non-canonical Wnt”, Wnt5a. b APP overexpression also enhanced Wnt-PCP signalling as measured by AP1 transcriptional activity (APRE-luciferase) in HEK239A cells. The enhancement of Wnt-PCP signalling was only detectable in cells also overexpressing Wnt5a and not in the presence of Wnt3a. c APP co-immunoprecipitated from lysates of primary rat cortical neurons with Wnt-β-catenin co-receptor protein, LRP6 and with Wnt-PCP co-receptor protein, Vangl2. (The full blots are presented in the supplemental data.) d Schematic illustrating the sites of APP-LRP6 interaction determined by peptide array hybridisation. Overlay of APP peptides with recombinant extracellular domain (ECD) of LRP6 demonstrated binding between the LRP6 and APP ECDs. e Schematic illustrating the site of APP-Vangl2 interaction as elucidated by peptide array. Overlay of APP peptides with HEK293A lysates overexpressing Vangl2-HA demonstrated binding of Vangl2 to a single site in the APP-ICD. f Schematic of the precise binding regions of LRP6/Vangl2 binding within the APP sequence. Binding of LRP6 is restricted to the ECD with one region lying close to the site affected by the Swedish APP mutation. In contrast, Vangl2 binding at the APP-ICD occurs at the YENPTY motif, which has been shown to control APP internalisation. g Overexpression of LRP6 enhanced the activity of APP in Wnt-β-catenin signalling as measured by TCF/LEF transcriptional activity using SuperTOPflash luciferase reporter assay in HEK293A cells. h Overexpression of Vangl2 enhanced the activity of APP in Wnt-PCP signalling as measured by APRE luciferase reporter assay in HEK293A cells. Data are plotted as mean+/− standard deviation ( n ≥ 3). Statistical significance was determined by one-way ANOVA and post-hoc Tukey’s test (* p < 0.05; ** p < 0.01); p values are indicated for key comparisons only

Article Snippet: Arrays were incubated with recombinant human LRP6 (Fc-chimera, R&D systems) or total lysate from HEK293A cells overexpressing HA-tagged Vangl2.

Techniques: Over Expression, Activity Assay, Luciferase, Reporter Assay, Immunoprecipitation, Peptide Microarray, Hybridization, Recombinant, Binding Assay, Sequencing, Mutagenesis, Control, Standard Deviation

Journal: The FEBS journal

Article Title: Haemorrhagic snake venom metalloproteases and human ADAMs cleave LRP5/6, which disrupts cell-cell adhesions in vitro and induces haemorrhage in vivo.

doi: 10.1111/febs.14066

Figure Lengend Snippet: Fig. 2. VAP1 cleaves LRP6 at the activation region. (A) HeLa cells were incubated with or without 0.4 lgmL1 VAP1 in serum-free medium for 2 h at 37 °C. The cells (1.5 9 107 cells) were harvested with a lysis buffer (25 mM Tris/HCl, pH 7.4, 300 mM NaCl, 1.5 mM MgCl2, 0.5% Triton X-100, 1 mM PMSF, 4 mM EDTA). Samples were immunoprecipitated with an anti-LRP6 antibody. The precipitates were subjected to SDS/PAGE (10% separating gel) and western blotting by using an anti-LRP6 antibody as a primary antibody. (B) Ectodomain recombinants of human LRP6 and mouse LRP5 at 17 lgmL1 and 50 lgmL1 purified bovine fibrinogen were incubated with 0.03 lgmL1 VAP1 in PBS for 0, 1 and 3 h at 37 °C. Samples (12 lL) were subjected to SDS/PAGE (10% separating gel) and silver staining. (C) Human fibronectin at 15 lgmL1 was incubated with each dose of VAP1 in PBS for 1 h at 37 °C. Samples (12 lL) were subjected to SDS/PAGE (10% separating gel) and silver staining. (D) The VAP1-cleavage point is shown by a black arrow in the schematic representation of LRP6. Fragment sequences of 140-kDa and 60-kDa bands of LRP6 detected by mass spectroscopic analysis are shown by a double underline and dashed underline, respectively. (E) A mouse ectodomain recombinant of LRP6 at 10 lgmL1 was incubated with 0.1 lgmL1 VAP1 in PBS for 1 h at 37 °C. Samples (15 lL) were subjected to SDS/PAGE (10% separating gel) and silver staining.

Article Snippet: Recombinant human LRP6 with an Fc-tag, human ADAM8 and ADAM12, VEcadherin, and mouse LRP6 and LRP5 having a His-tag were purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Activation Assay, Incubation, Lysis, Immunoprecipitation, SDS Page, Western Blot, Silver Staining, Recombinant

Journal: The FEBS journal

Article Title: Haemorrhagic snake venom metalloproteases and human ADAMs cleave LRP5/6, which disrupts cell-cell adhesions in vitro and induces haemorrhage in vivo.

doi: 10.1111/febs.14066

Figure Lengend Snippet: Fig. 3. VAP1 cleaves LRP5 at the same site as that of LRP6. (A,B) A mouse ectodomain recombinant of LRP5 at 26 lgmL1 was incubated with each dose of VAP1 (A) and VAP2 (B) in PBS for 1 h at 37 °C. Samples (15 lL) were subjected to SDS/PAGE (10% separating gel) and silver staining. (C) Mouse LRP5 ectodomain recombinant at 70 lgmL1 was incubated with 7 lgmL1 VAP1 in PBS for 1 h at 37 °C. Samples (60 lL) were subjected to SDS/PAGE (5–20% precast SDS/PAGE gel) and stained with Coomassie Brilliant Blue R250. (D) The VAP1-cleavage point is shown by a black arrow in the schematic representation of LRP5. The fragment sequences of the 140-kDa band of LRP5 detected by mass spectroscopic analysis is shown by a double underline. (E) Amino acid residues from cleavage site position P5 to P50 of LRP6 and LRP5 are aligned. The VAP1-cleavage point is shown by a black arrow.

Article Snippet: Recombinant human LRP6 with an Fc-tag, human ADAM8 and ADAM12, VEcadherin, and mouse LRP6 and LRP5 having a His-tag were purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Recombinant, Incubation, SDS Page, Silver Staining, Staining

Journal: The FEBS journal

Article Title: Haemorrhagic snake venom metalloproteases and human ADAMs cleave LRP5/6, which disrupts cell-cell adhesions in vitro and induces haemorrhage in vivo.

doi: 10.1111/febs.14066

Figure Lengend Snippet: Fig. 4. Substrates and cleavage points of VAP1. (A) Substrates cleaved by VAP1 are indicated. The cleaved fragments were subjected to N-terminal sequence analysis. P5 to P50 amino acid residues of the cleavage points are shown. (B) Docking model of VAP1 and the cleavage site moiety of LRP6. The inset indicates the substrate-binding cleft. VAP1 is shown by a white space-filling model. The cleavage site moiety of LRP6 is shown by a red space- filling model (in the upper figure) and by a ball-and-stick model (in the lower figure). Zn2+ and Glu336A, both of which are involved in catalysis, and Leu363A are shown by grey spheres. S10 and S30

Article Snippet: Recombinant human LRP6 with an Fc-tag, human ADAM8 and ADAM12, VEcadherin, and mouse LRP6 and LRP5 having a His-tag were purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Sequencing, Binding Assay

Journal: The FEBS journal

Article Title: Haemorrhagic snake venom metalloproteases and human ADAMs cleave LRP5/6, which disrupts cell-cell adhesions in vitro and induces haemorrhage in vivo.

doi: 10.1111/febs.14066

Figure Lengend Snippet: Fig. 5. LRP6 cleavage by VAP1 is involved in disruption of cell–cell junctions and haemorrhage. (A,B) A human ectodomain recombinant of LRP6 at 30 lgmL1 was incubated with 30 ngmL1 VAP1 and with 2 mgmL1 LRP-cleavage site antibody or control antibody in PBS for 1 h at 37 °C. Samples (15 lL) were subjected to SDS/PAGE and silver staining (A). Cleavage inhibition is shown by the 140-kDa fragment density score (B). The data (n = 5; error bars correspond to standard errors) were compared by Student’s t test. (C) HUVECs were incubated with 140 ngmL1 VAP1 and with 1.35 mgmL1 LRP6-cleavage site antibody or control antibody in a medium for 1 h at 37 °C. After being fixed, the cells were stained with anti-VE-cadherin antibody. Arrows indicate the remaining membrane VE-cadherin. Scale bar, 50 lm. (D,E) VAP1 with LRP6 cleavage site antiserum or control serum was intradermally injected into mice bisymmetrically. One hour later, haemorrhagic plaques on the inner surface of the skin were observed (D). Scale bar, 10 mm. Densitometry scores of VAP1-induced haemorrhage with the antiserum or control serum in each individual were compared by paired t test (E).

Article Snippet: Recombinant human LRP6 with an Fc-tag, human ADAM8 and ADAM12, VEcadherin, and mouse LRP6 and LRP5 having a His-tag were purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Disruption, Recombinant, Incubation, Control, SDS Page, Silver Staining, Inhibition, Staining, Membrane, Injection

Journal: The FEBS journal

Article Title: Haemorrhagic snake venom metalloproteases and human ADAMs cleave LRP5/6, which disrupts cell-cell adhesions in vitro and induces haemorrhage in vivo.

doi: 10.1111/febs.14066

Figure Lengend Snippet: Fig. 6. VAP1-cleavage site and/or LDLa domains of LRP5/6 are deleted in many venom-resitent animals. Deleted regions of LRP6 (A) and LRP5 (B) in animals are shown in schematic representation. LRP5/6 of animals associated with tolerance to snake bite-induced haemorrhage are indicated by red boxes. LRP5/6 of animals with moderate resistance and animals with unknown sensitivity to snake bite- induced haemorrhage are shown by orange and green boxes, respectively. The presented data are from NCBI RefSeq (Table S1). Except for humans, mice and rats, the data are all predicted sequences from the genome of each animal. Amino acid sequences of the cleavage site and LDLa region are shown in Fig. 7. Regarding the predicted king cobra LRP6 (ETE69657.1), although the genome sequence of the full- length mRNA region of LRP6 contains several gaps, the indicated deleted region does not contain gaps.

Article Snippet: Recombinant human LRP6 with an Fc-tag, human ADAM8 and ADAM12, VEcadherin, and mouse LRP6 and LRP5 having a His-tag were purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Combined Bisulfite Restriction Analysis Assay, Sequencing

Journal: The FEBS journal

Article Title: Haemorrhagic snake venom metalloproteases and human ADAMs cleave LRP5/6, which disrupts cell-cell adhesions in vitro and induces haemorrhage in vivo.

doi: 10.1111/febs.14066

Figure Lengend Snippet: Fig. 8. Recombinant human ADAM8 and ADAM12 cleave LRP6 at the same site as that of VAP1. (A) Recombinant human LRP6 at 64 lgmL1 was incubated with 20 lgmL1 recombinant human ADAM8 for 3 h at 37 °C. Samples (10 lL) were subjected to SDS/PAGE (12% separating gel) and silver staining. (B) LRP6 at 20 lgmL1 was incubated with 20 lgmL1 recombinant human ADAM12 for 16 h at 37 °C. Samples (15 lL) were subjected to SDS/PAGE (10% separating gel) and silver staining. (C) LRP6 at 20 lgmL1 was incubated with 200 lgmL1 recombinant human ADAM12 for 18 h at 37 °C. Fragments containing C-terminal Fc peptide (20 lL) were purified with protein A–Sepharose. Samples were subjected to SDS/PAGE (15% separating gel) and silver staining. (D) The LRP6 140-kDa fragments treated with ADAM8 (A) and ADAM12 (B) and the LRP6 60-kDa fragment treated with ADAM12 (C) were analysed by mass spectrometry. Detected fragments by mass spectrometry and the proposed cleaved sites are shown.

Article Snippet: Recombinant human LRP6 with an Fc-tag, human ADAM8 and ADAM12, VEcadherin, and mouse LRP6 and LRP5 having a His-tag were purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Recombinant, Incubation, SDS Page, Silver Staining, Mass Spectrometry

Journal: The FEBS journal

Article Title: Haemorrhagic snake venom metalloproteases and human ADAMs cleave LRP5/6, which disrupts cell-cell adhesions in vitro and induces haemorrhage in vivo.

doi: 10.1111/febs.14066

Figure Lengend Snippet: Fig. 9. Hypothetical roles of ADAM and LRP5/6 in haemorrhage and invasion. (1) Cell–cell junctions with cadherin and catenin are normally stable. LRP5/6 may form a complex with cadherin and catenin. (2) Haemorrhagic SVMP, which is an ADAM-type toxin, can cleave LRP5/6 at the activation region. Cleaved LRP5/6 may form a dimer or multimer. (3) Cleaved LRP6 is involved in c-catenin and VE-cadherin relocation and in disruption of cell–cell adhesions. (4) Cleaved LRP6 mediates haemorrhage. Some animals with snake venom tolerance have deleted LRP5/6. (20) Invasion-associated ADAMs are located at the tips of invadopodia in invasive cells such as leukocytes and cancer cells. The ADAMs can cleave the same site of LRP6. (40) Cleaved LRP6 has the potential to cause disruption of cell–cell adhesions and to induce invasion. Summarizing the above, it is thought that ADAMs, as cell barrier openers, cleave novel ADAM receptors, LRP5/6, to induce haemorrhage and potentially invasion.

Article Snippet: Recombinant human LRP6 with an Fc-tag, human ADAM8 and ADAM12, VEcadherin, and mouse LRP6 and LRP5 having a His-tag were purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Activation Assay, Disruption