recombinant human fam20c (63-c)  (GenScript corporation)

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Ser-Phosphorylation of PCSK9 by Fam20C-Kinase Enhances its Ability to Degrade the LDLR

doi: 10.1161/ATVBAHA.119.313247

Figure Lengend Snippet: Comparative mRNA distribution of mouse Fam20A, Pcsk9 and Fam20C. Sections of whole body P10 WT mouse were subjected to IHyH using mFam20A, mPsck9, and mFam20C cRNA probes. (A) Representative sagittal cryostat sections of P10 mouse with thionin staining. (B-D) X-ray film autoradiography showing: (B) Fam20A mRNA distribution pattern restricted to few tissues including the molar, incisor, cerebellum, liver, kidney papilla and bone; (C) Pcsk9 mRNA distribution pattern restricted to the olfactory tract, molar, cerebellum, liver, stomach wall and small intestine wall; (D) Fam20C mRNA widespread distribution pattern with high levels in bone, molars, incisive, brain with thalamic structures, hippocampus and cerebellum, liver, adrenal gland, kidney cortex and papilla. Abbreviations: Ad – adrenal gland; B – bone; BM – bone marrow; Br – brain; Cb – cerebellum; H – heart; Hi – hippocampus; KCx – kidney cortex; Inc – incisive; Li – liver; LI – large intestine; Lu – lung; Mo – molar; OT – olfactory tract; P – papilla; SI – small intestine; SpC – spinal cord; St – stomach; Te – testis; Th – thymus; Tha – thalamus; UB – urinary bladder; V - vertebrae. (E-H) Adult mouse liver IHyH. (E) High resolution hepatocyte staining, dry film lower resolution IHyH Fam20A (F), mPcsk9 (G) and mFam20C (H). (I) QPCR analysis of mRNA distribution in adult liver mouse extracts and in indicated cell line models. QPCR was achieved using specific Fam20A and Fam20C primers. Data shown represent means ± SEM of at least three independent experiments.

Article Snippet: Recombinant human Fam20C (63-C) was purified from insect cells as described in Xiao et. al. 39 β-casein and PCSK9 peptides were synthesized by GenScript.

Techniques: Staining, Autoradiography

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Ser-Phosphorylation of PCSK9 by Fam20C-Kinase Enhances its Ability to Degrade the LDLR

doi: 10.1161/ATVBAHA.119.313247

Figure Lengend Snippet: Fam20C/Fam20A enhance PCSK9 secretion and activity on the LDLR. (A) HepG2-Fam20C−/− cells were transfected with PCSK9, Fam20A, Fam20C-WT or Fam20C (D478A), a catalytically inactive form. After 18h of serum starvation the media were collected, and cells lysed and subjected to Western blot (WB) analysis with antibodies against the indicated proteins. Representative blots and quantitative densitometry analyses of PCSK9 and Fam20C media are shown. Data represent means of at least three independent experiments. **p<0.01 vs. lane 2, $p<0.05 and $$p<0.01 vs. lane 3, #p<0.05 and ##p<0.01 vs. lane 4, obtained from one-way ANOVA coupled to Tukey's multiple comparisons tests. (bottom panel) Densitometry analysis of total LDLR and the corresponding statistical analysis. Data represent means ± SEM of at least three independent experiments. *p<0.05, **p<0.01, ***p<0.001, two-way ANOVA, Tukey's multiple comparisons tests. iLDLR- ~110 kDa immature form of the LDLR that lacks O-glycosylation. (B) Media of HepG2-Fam20C−/− cells, expressing the indicated constructs were subjected to dephosphorylation by λ-phosphatase then analysed for PCSK9 by WB using anti-V5 antibody. (C) HepG2-Fam20C−/− cells were incubated for 18h with conditioned media (CM) from HEK293 cells expressing Fam20A along with PCSK9 and Fam20C-WT (media swap). The collected cell lysates and CM were analysed as in (A). Representative blots and corresponding densitometry analysis are shown. Data shown represent means of at least three independent experiments. **p<0.01, ***p<0.001 vs. PCSK9-/Fam20C-, ##p<0.01 vs. PCSK9+/Fam20C-, one-way ANOVA coupled to Tukey's multiple comparisons tests. (D) Confocal IF microscopy of cell surface LDLR in HepG2-PCSK9−/− cells incubated 18h with control or PCSK9 CM produced as in (C). np = non-phosphorylated, p = phosphorylated. Representative images are from at least three independent experiments. Scale bar, 15 μm.

Article Snippet: Recombinant human Fam20C (63-C) was purified from insect cells as described in Xiao et. al. 39 β-casein and PCSK9 peptides were synthesized by GenScript.

Techniques: Activity Assay, Transfection, Western Blot, Glycoproteomics, Expressing, Construct, De-Phosphorylation Assay, Incubation, Microscopy, Control, Produced

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Ser-Phosphorylation of PCSK9 by Fam20C-Kinase Enhances its Ability to Degrade the LDLR

doi: 10.1161/ATVBAHA.119.313247

Figure Lengend Snippet: In vitro and cellular validation of Fam20C-targets on PCSK9. (A) Schematic representation of potential Fam20C-Ser substrates (S-X-E motif) on hPCSK9-V5. Pro- Pro domain; Catalytic- Catalytic Domain; H- hinge region; CHRD- C-terminal Cys-His–rich domain. (B) In vitro Fam20C kinase assay measurements with the PCSK9 synthetic peptides as substrates and their specific activity values are indicated (top panel). The peptide sequences are presented and their predicted Ser-phosphosites are in bold and underlined, whereas natural mutations are in bold (bottom panel). The data were obtained from three independent experiments in duplicate. ****p<0.0001, obtained using one-way ANOVA coupled to Tukey's multiple comparisons tests. (C and D) Levels of non-phosphorylated and phosphorylated PCSK9 peptides spanning (C) Ser47 (S47EEDGLAEAPEHGTTATFHR) and (D) Ser688 (HLAQAS688QELQ) as measured by PAC-qMS assay in the culture medium of HepG2-Fam20C−/− cells (C), naive HepG2 cells with PCSK9 overexpression, and naive HepG2 cells with PCSK9 and Fam20C overexpression (D).

Article Snippet: Recombinant human Fam20C (63-C) was purified from insect cells as described in Xiao et. al. 39 β-casein and PCSK9 peptides were synthesized by GenScript.

Techniques: In Vitro, Biomarker Discovery, Kinase Assay, Activity Assay, Over Expression

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Ser-Phosphorylation of PCSK9 by Fam20C-Kinase Enhances its Ability to Degrade the LDLR

doi: 10.1161/ATVBAHA.119.313247

Figure Lengend Snippet: Characterization of Fam20C-mediated Ser-phosphorylation on PCSK9 activity. (A) HepG2-PCSK9−/− cells were transfected with V5-tagged control (empty vector) or PCSK9 constructs (WT, R46L, S668R and E670G) in the presence of Fam20C/Fam20A to maximise phosphorylation. After 18h, the cells were lysed and analysed by WB using antibodies against the indicated proteins. Representative blots (top panel) and densitometry quantification (bottom panel) are shown. Data represent means ± SEM of at least three independent experiments. *p<0.05, ***p<0.001, one-way ANOVA coupled to Tukey's multiple comparisons tests. (B) HepG2-PCSK9−/− cells were incubated for 18h with the CM of HEK293 cells expressing V5-tagged empty vector or PCSK9 constructs (WT, R46L, S668R and E670G) in the presence of Fam20C/Fam20A (media swap). The cells were then lysed and analysed as in (A). Representative blots and densitometry analysis are shown (top and bottom panels, respectively). Data represent means ± SEM of at least three independent experiments. *p<0.05, **p<0.01, one-way ANOVA coupled to Tukey's multiple comparisons tests. (C and D) HepG2-PCSK9−/− cells were incubated for 18h with the CM of HEK293 cells expressing V5-tagged control or PCSK9-WT and mutated constructs (E670G, D374Y, S47A, S666A, S668A, S688A and S666A/S668A) in the absence (−) or presence (+) of Fam20C/Fam20A as indicated. The cells were then lysed and analysed as in (A). Representative blots (top panels in C, and D) and densitometry quantification (D and bottom panel in C) are shown. (C) Data represent means ± SEM of five independent experiments. *p<0.05, **p<0.01, ***p<0.001, obtained by two-way ANOVA coupled to Tukey's multiple comparisons tests. (D) Data represent means ± SEM of three independent experiments. *p<0.05, obtained by one-way ANOVA coupled to Tukey's multiple comparisons tests.

Article Snippet: Recombinant human Fam20C (63-C) was purified from insect cells as described in Xiao et. al. 39 β-casein and PCSK9 peptides were synthesized by GenScript.

Techniques: Phospho-proteomics, Activity Assay, Transfection, Control, Plasmid Preparation, Construct, Incubation, Expressing

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Ser-Phosphorylation of PCSK9 by Fam20C-Kinase Enhances its Ability to Degrade the LDLR

doi: 10.1161/ATVBAHA.119.313247

Figure Lengend Snippet: Effects of PCSK9-phosphosphomimetics on LDLR degradation. (A) HepG2-PCSK9−/− cells were incubated for 18h with CM from HEK293 cells expressing V5-tagged control, PCSK9-WT or mutated constructs (E670G, D374Y, S47A, S47D, S666A, S666E, S668A, S668E, S688A and S688D) in the presence of Fam20C/Fam20A. The cells were then lysed and analysed by WB using antibodies against the indicated proteins. Representative blots (top panels) and densitometry quantification (bottom panel) are shown. The data represent means ± SEM of at least three independent experiments. **p<0.01, ***p<0.001 and ****p<0.0001, obtained by one-way ANOVA coupled to Tukey's multiple comparisons tests. (B) Dil-LDL uptake. HepG2-PCSK9−/− cells were incubated for 20h with phosphorylated PCSK9 CM from HEK293 cells expressing V5-tagged control or PCSK9 constructs (WT, E670G, D374Y, S47A, S47D, S666A, S666E, S668A, S668E, S688A, S688D and S668R), in the presence of Fam20C/Fam20A. During the last 3h of incubation, Dil-LDL was added to the CM. The cells were then fixed and the Dil-LDL fluorescence quantified. The data represent means ± SEM of at least three independent experiments in quadruplicates. *p<0.05, **p<0.01 and ***p<0.001 vs. Control, #p<0.05 and ##p<0.01 vs. WT, $p<0.05, one-way ANOVA coupled to Tukey's multiple comparisons tests. (C) Confocal IF of cell surface LDLR in HepG2-PCSK9−/− cells incubated with phosphorylated PCSK9 CM from HEK293 cells expressing V5-tagged control or PCSK9-WT, and mutated constructs (E670G, D374Y, S47A, S47D, S666A, S666E, S668A, S668E, S688A, S688D and S668R), in the presence of Fam20C/Fam20A. After 18h of incubation the cells were fixed and processed for IF. Representative images are shown for each condition. Scale bar, 15 μm. (D) Quantifications were derived from analyses of 8 fields/condition/experiment. Data are averages ± SEM of two independent experiments. ***p<0.001 and ****p<0.0001, obtained by one-way ANOVA coupled to Tukey's multiple comparisons tests.

Article Snippet: Recombinant human Fam20C (63-C) was purified from insect cells as described in Xiao et. al. 39 β-casein and PCSK9 peptides were synthesized by GenScript.

Techniques: Incubation, Expressing, Control, Construct, Fluorescence, Derivative Assay

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Ser-Phosphorylation of PCSK9 by Fam20C-Kinase Enhances its Ability to Degrade the LDLR

doi: 10.1161/ATVBAHA.119.313247

Figure Lengend Snippet: Effect of PCSK9 pro-domain and Furin in PCSK9-mediated LDLR degradation. (A) Effect of PCSK9 Δ33-58 deletion. HEK293 cells were incubated for 18h with CM from HEK293 cells expressing V5-tagged control, PCSK9-WT or mutated constructs, D374Y, E670G, Δ33-58 and Δ33-58-E670G in the presence of Fam20A (non-phosphorylated, left panel) or Fam20C/Fam20A (phosphorylated, right panel). Cell lysates were analysed by WB using the indicated antibodies. Representative blots (top panels) and densitometry quantification (bottom panels) are shown. Data represent means ± SEM of at least three independent experiments. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, obtained by one-way ANOVA coupled to Tukey's multiple comparisons tests. (B-C) Fam20C affects Furin-mediated PCSK9 cleavage. (B) HEK293 cells were transfected with V5-tagged PCSK9-WT or Fam20C/Fam20A. After 18h of serum starvation, PCSK9 media were collected and analyzed by WB. Representative blots of at least three independent experiments are shown. The migration position of the Furin-cleaved PCSK9 (PCSK9-Δ218) as well as the % cleavage by endogenous Furin are emphasized. (C) CM from CHO-FD11 Furin−/− cells transfected with the indicated constructs (i.e., PCSK9-V5, Furin-V5 and Fam20C-WT/Fam20A). Cell lysates and media were analysed by WB and representative blots of at least three independent experiments are shown. (D) HepG2-PCSK9−/− cells were incubated for 18h with the media produced in (C) and LDLR levels analysed by WB. Representative blots (top panels) and densitometry quantification (bottom panels) are shown. Data represent means ± SEM of at least three independent experiments. *p<0.05, ***p<0.001, ****p<0.0001, obtained by two-way ANOVA coupled to Tukey's multiple comparisons tests; ns- non-significant. (E) Relative PCSK9-LDLR binding assay. Microplates pre-coated with recombinant LDLR EGF-AB domain, which contains the PCSK9 binding site, were incubated for 18h with non-phosphorylated and phosphorylated PCSK9 CM obtained from HEK293 cells expressing V5-tagged PCSK9-WT in the absence or presence of Fam20C/Fam20A, respectively. The binding affinity of PCSK9 to LDLR was determined following analyses with 8 different concentrations per sample and the calculated relative EC50 values of WT phosphorylated PCSK9 (WT-P) were normalized to WT non-phosphorylated PCSK9 (WT-NP) as shown in (B). Data are averages ± SEM of two independent experiments. *p<0.05, obtained by unpaired two-tailed student t-test.

Article Snippet: Recombinant human Fam20C (63-C) was purified from insect cells as described in Xiao et. al. 39 β-casein and PCSK9 peptides were synthesized by GenScript.

Techniques: Incubation, Expressing, Control, Construct, Transfection, Migration, Produced, Binding Assay, Recombinant, Two Tailed Test