Journal: Journal of Biological Chemistry

Article Title: Comparison of angiopoietin-like protein 3 and 4 reveals structural and mechanistic similarities

doi: 10.1016/j.jbc.2021.100312

Figure Lengend Snippet: Figure 2. ANGPTL3 forms noninterconvertible hexamers and trimers and ANGPTL4 forms a trimer. SEC purified (A) ANGPTL3 peak 1 and (B) ANGPTL3 peak 2 were separately concentrated and reinjected for analysis with SEC–multiangle light scattering. Peak 1 had a molecular weight (MW) of 151 kDa, indicating a hexamer of ANGPTL3 (monomer 26.8 kDa). Peak 2 had a MW of 76 kDa, indicating a trimer of ANGPTL3. The ANGPTL3 trimers and hexamers did not interconvert when reinjected on SEC. Owing to the elution being close together, a small amount of trimer and hexamer can be seen after the peaks were separated and reinjected. C, SEC purified ANGPTL4 was concentrated and injected for SEC–multiangle light scattering analysis. ANGPTL4 resolved as a single peak with a MW of 46 kDa, which indicates a trimer of ANGPTL4 (monomer 15.4 kDa). The UV trace representing protein absorbance at 280 nm is in black and MW is represented in gray. SEC, size-exclusion chromatography.

Article Snippet: ANGPTL4 was probed with a polyclonal rabbit anti-ANGPTL4 antibody (BioVendor) using a 1:5000 dilution and detected with a horseradish peroxidase–conjugated donkey anti-rabbit antibody (Southern Biotech) using a 1:5000 dilution.

Techniques: Multi-Angle Light Scattering, Molecular Weight, Injection, Size-exclusion Chromatography

Journal: Journal of Biological Chemistry

Article Title: Comparison of angiopoietin-like protein 3 and 4 reveals structural and mechanistic similarities

doi: 10.1016/j.jbc.2021.100312

Figure Lengend Snippet: Figure 3. ANGPTL3 and ANGPTL4 have elongated structures. Size-exclusion chromatography–small-angle X-ray scattering scattering data as a log-linear plot with inset showing the real-space distance distribution and the filtered average ab initio models determined using DAMMIN for (A) ANGPTL3 hexamer (SASDJK8), (B) ANGPTL3 trimer (SASDJL8), and (C) ANGPTL4 (SASDJM8). Data deposited in the Small Angle Scattering Biological Data Bank (SASBDB) (61).

Article Snippet: ANGPTL4 was probed with a polyclonal rabbit anti-ANGPTL4 antibody (BioVendor) using a 1:5000 dilution and detected with a horseradish peroxidase–conjugated donkey anti-rabbit antibody (Southern Biotech) using a 1:5000 dilution.

Techniques: Size-exclusion Chromatography

Journal: Journal of Biological Chemistry

Article Title: Comparison of angiopoietin-like protein 3 and 4 reveals structural and mechanistic similarities

doi: 10.1016/j.jbc.2021.100312

Figure Lengend Snippet: Figure 5. Heparin effects LPL inhibition of ANGPTL3 and ANGPTL4. The activity of LPL was determined in the presence of ANGPTL3 using both (A) DGGR and (B) VLDL as substrates. ANGPTL3 experiences a significant loss of inhibition when incubated with 12-mer heparin, but not 6-mer heparin for both substrates. LPL activity was also tested with ANGPTL4 using (C) DGGR and (D) VLDL substrates. ANGPTL4 is an effective inhibitor with addition of either heparin variant. E, biotinylated 6-mer and 12-mer were used to pull down human LPL, ANGPTL3, and ANGPTL4 and assessed via Western blot. Both LPL and ANGPTL4 bound to the 6-mer and 12-mer, whereas ANGPTL3 only showed binding to the 12-mer. Full blots are shown in Fig. S5. DGGR assays were performed with 2.5 nM LPL, 6 μM ANGPTL, 10 μg/ml heparin, and 10 μM DGGR. VLDL assays were performed with 10 nM LPL, 10 nM ANGPTL, 2 nM heparin, and 200 μg/ml VLDL triglycerides. Data were normalized to the activity of LPL without inhibitor present. Dots represent biological replicates; significance was determined by two-tailed Student’s t test. DGGR, 1,2-Di-O-lauryl-rac-glycero-3-glutaric acid 60-methylresorufin ester; LPL, lipoprotein lipase; VLDL, very- low-density lipoprotein.

Article Snippet: ANGPTL4 was probed with a polyclonal rabbit anti-ANGPTL4 antibody (BioVendor) using a 1:5000 dilution and detected with a horseradish peroxidase–conjugated donkey anti-rabbit antibody (Southern Biotech) using a 1:5000 dilution.

Techniques: Inhibition, Activity Assay, Incubation, Variant Assay, Western Blot, Binding Assay, Two Tailed Test