Functional characterization of Furin-mediated lipoprotein lipase cleavage

Figures

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  Fig. 1.. Forms of endogenous LPL present in tissue. We analyzed endogenous LPL in different tissues from C57BL/6 mice by western blotting. LPL present in conditioned medium of cultured cells was used as control for the position of full-length (FL) LPL and N-terminal (NT) LPL fragments (arrows). Some tissues have unknown bands present in some tissues, which might indicate cross-reaction or bands or LPL cleavage products of unknown origins (indicated by asterisks). LPL expression was highest in heart and adipose tissue, consistent with published RNA expression data.

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  Fig. 2.. Study overview. Two weeks before virus injection (−2) LPL knockout was induced by treatment with tamoxifen for 5 days in 8-week-old whole-body-inducible LPL knockout (iLpl−/−) mice. At week 0, plasma triglyceride levels were checked to confirm the loss of LPL, and mice were treated with LPLWT, LPLR297N or luciferase (negative control) via adeno-associated virus serotype 9 (AAV9)-mediated gene therapy. Blood was collected every two weeks up to week 18. Mice were sacrificed at 10 months, and their blood and tissue collected. (B) Three-dimensional structure of LPL. Indicated is the position of the R297N substitution as deposited at the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB), PDBID: 8ERL.

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  Fig. 3.. Furin-mediated cleavage of LPL in mouse blood and tissues. (A) LPLWT undergoes furin-mediated cleavage in conditioned medium from cultured cells, resulting in both full-length (FL) and N-terminal (NT) cleavage products, whereas LPLR297N does not. Similarly, both FL LPL and NT LPL are present in blood plasma obtained from a C57BL/6 mouse. Both types of LPL were present in the plasma of whole-body inducible LPL knockout (iLpl−/−) mice treated with LPLWT, but only FL LPL was present in mice treated with LPLR297N. (B) In heart tissue, only FL LPL was observed in C57BL/6 mice, mice treated with LPLWT and iLpl−/− mice treated with LPLR297N. (C) In adipose tissue, FL LPL and NT products were observed in C57BL/6 mice and in iLpl−/− mice treated with LPLWT. Only FL LPL was observed in iLpl−/− mice treated with LPLR297N. α-LPL, anti-LPL antibody; α-GAPDH, anti-GAPDH antibody; α-Vinculin, anti-vinculin antibody.

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  Fig. 4.. Plasma triglyceride levels in mice that had been treated with LPLWT or LPLR297N. (A,B) Plasma triglyceride levels in mice treated with LPLWT and LPLR297N were tested every two weeks (week 0−18). Initial levels, at week −2, were low. Mice were then treated with tamoxifen to induce loss of LPL, and triglyceride levels were high when mice were tested at week 0. Mice were treated with LPLWT or LPLR297N at week 0 and plasma triglyceride levels had decreased by week 2. Each data point represents the mean of at least five male and five female mice tested for each condition. Data are the mean±s.e.m. (C,D) To quantify the triglyceride levels over the course of the experiment, the area under the curve (AUC) from week 2 to week 18 was calculated for each mouse. Plots show that average triglyceride levels are lower for LPLR297N than LPLWT in both male and female mice (P=0.03 and P=0.052, respectively, determined by Wilcoxon rank-sum test).

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  Fig. 5.. Lipoprotein profiles for LPLWT vs LPLR297N mice. At 10 months of age iLpl−/− mice treated with LPLWT, LPLR297N or luciferase as a negative control were killed, and blood plasma samples collected and separated on a 24 ml Superose 6 column. (A) Lipoprotein profile of blood from a mouse treated with LPLWT. The area under the curve for the three main peaks (from left to right) was 1596, 1709 and 4611 milli-absorbance units (mAu)×ml. (B) Lipoprotein profile of blood from a mouse treated with LPLR297N. The area under the curve for the three main peaks (from left to right) was 1632, 1950 and 3783 mAu×ml, respectively. (C) As a negative control, plasma from iLpl−/− mice treated with luciferase showed very high levels of triglyceride rich lipoproteins. The area under the curve for the three main peaks (from left to right) was 7184, 1874 and 4707 mAu×ml, respectively. All profiles are representative of two independent trials.

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  Fig. 6.. Liver histology of LPLWT vs LPLR297N mice. (A) Microscopy images showing representative liver sections from C57BL/6 mice, and iLpl−/− mice treated with LPLWT, LPLR297N or luciferase. Samples were stained for lipid droplets using BODIPY(green) and a sodium potassium ATPase antibody was used to mark plasma membrane (red). Imaged sections were centered around the portal vein to select for regions with consistent exposure to plasma triglycerides. Boxed areas are shown magnified (LPLR297N, 4.1×; LPLWT, 5.4×; C57BL/6, 5.7×; Luciferase, 4.4×) bottom left of each image. (B) Violin plots showing the total lipid droplet (LD) area per cell [µm2], calculated using custom Matlab scripts at a log scale [y′=log(y+1)] with medians and IQRs (interquartile ranges). P values shown above brackets were calculated using Bonferroni-adjusted Dunn post-hoc tests.