Analysis of naturally occurring mutations in the human lipodystrophy protein seipin reveals multiple potential pathogenic mechanisms

Abstract

Aims/hypothesis: In humans, disruption of the gene BSCL2, encoding the protein seipin, causes congenital generalised lipodystrophy (CGL) with severe insulin resistance and dyslipidaemia. While the causative gene has been known for over a decade, the molecular functions of seipin are only now being uncovered. Most pathogenic mutations in BSCL2 represent substantial disruptions including significant deletions and frameshifts. However, several more subtle mutations have been reported that cause premature stop codons or single amino acid substitutions. Here we have examined these mutant forms of seipin to gain insight into how they may cause CGL.

Methods: We generated constructs expressing mutant seipin proteins and determined their expression and localisation. We also assessed their capacity to recruit the key adipogenic phosphatidic acid phosphatase lipin 1, a recently identified molecular role of seipin in developing adipocytes. Finally, we used atomic force microscopy to define the oligomeric structure of seipin and to determine whether this is affected by the mutations.

Results: We show that the R275X mutant of seipin is not expressed in pre-adipocytes. While the other premature stop mutant forms fail to bind lipin 1 appropriately, the point mutants T78A, L91P and A212P all retain this capacity. We demonstrate that wild-type human seipin forms oligomers of 12 subunits in a circular configuration but that the L91P and A212P mutants of seipin do not.

Conclusions/interpretation: Our study represents the most comprehensive analysis so far of mutants of seipin causing lipodystrophy and reveals several different molecular mechanisms by which these mutations may cause disease.

Fig. 1

The R275X mutation in seipin leads to loss of protein expression in differentiating adipocyte precursors. (a) HEK293 cells were transfected with empty vector (−), the long form of wild-type seipin bearing N-terminal triple-FLAG and C-terminal Myc tags (WT) or identically tagged forms with the point mutations E113X, R138X, R275X or Q391X. Lysates were separated by SDS-PAGE and immunoblotted for FLAG (α-FLAG), Myc (α-Myc) and calnexin (α-Calnexin). (b) Confluent C3H10T1/2 cells were transfected with empty vector (−) or with epitope-tagged WT or mutant forms of seipin as in (a). Cells were induced to differentiate for 2 days; lysates were separated by SDS-PAGE and immunoblotted for FLAG, Myc and calnexin. In (a) and (b) blots are representative of at least three independent experiments. (c) Quantitative analysis of WT, E113X, R138X, R275X or Q391X forms of seipin protein in C3H10T1/2 cells. FLAG–seipin bands from replicated blots as shown in (b) were normalised to calnexin expression in the same samples and expressed as means ± SEM, n = 3. *p < 0.05 vs WT. (d) C3H10T1/2 cells were transfected with empty vector (m) or with epitope-tagged WT or mutant forms of seipin as in (a). Cells were induced to differentiate for 2 days, and expression of mRNA encoding transfected human seipin (hBSCL2 mRNA) was determined by real-time PCR. Data are normalised to cyclophilin A and expressed as means ± SEM, n = 4. (e) Subconfluent C3H10T1/2 pre-adipocytes were transfected with epitope-tagged WT or mutant forms of seipin, fixed and immunostained for seipin using anti-FLAG antibody or with anti-calnexin antibody to reveal the ER. Individual images are shown in greyscale, and merged images show overlay of FLAG–seipin (green) and calnexin (red). Scale bars, 10 μm

Fig. 2

The pathogenic point mutations T78A, L91P and A212P do not affect seipin protein expression, but L91P and A212P seipin partially mislocalise to the nuclear envelope. (a) HEK293 cells were transfected with empty vector (−), the long form of wild-type seipin with N-terminal triple-FLAG and C-terminal Myc tags (WT) or with identically tagged T78A, L91P or A212P forms. Lysates were separated by SDS-PAGE and immunoblotted for FLAG (α-FLAG) and calnexin (α-Calnexin). (b) C3H10T1/2 cells were transfected with empty vector (−) or with epitope-tagged WT or mutant forms of seipin as in (a). Lysates were separated by SDS-PAGE and immunoblotted for FLAG and calnexin. In (a) and (b) blots are representative of at least three independent experiments. (c) Quantitative analysis of WT, T78A, L91P or A212P forms of seipin protein in C3H10T1/2 cells. FLAG–seipin bands from replicated blots as shown in (b) were normalised to calnexin expression in the same samples and expressed as means ± SEM, n = 3. *p < 0.05 vs WT. (d) C3H10T1/2 cells were transfected with empty vector (m) or with epitope-tagged WT or mutant forms of seipin as in (a). Cells were induced to differentiate for 2 days and expression of mRNA encoding transfected human seipin (hBSCL2) was determined by real-time PCR. Data are normalised to cyclophilin A and expressed as means ± SEM, n = 4. (e) Subconfluent C3H10T1/2 pre-adipocytes were transfected with epitope-tagged WT or mutant forms of seipin and were fixed and immunostained for seipin using anti-Myc antibody or with anti-calnexin antibody to reveal the ER. Individual images are shown in greyscale and merged images show overlay of Myc–seipin (green) and calnexin (red). Scale bars, 10 μm

Fig. 3

The pathogenic point mutations T78A, L91P and A212P do not affect the capacity of seipin to bind the PA phosphatase lipin 1. (a) HEK293 cells were transfected with empty vector (−), FLAG-tagged long form of wild-type seipin (WT) or with identically tagged T78A, L91P, A212P forms of seipin in the absence or presence of lipin 1β. Cell lysates or anti-FLAG immunoprecipitates were immunoblotted for seipin using anti (α)-FLAG antibodies or lipin 1β using and anti-lipin 1 antibodies, as indicated. Lysates were also probed for calnexin as a loading control. (b) The binding of lipin 1β to WT or mutant forms of seipin was quantified in replicate immunoblots (n = 3), normalised to expression levels and expressed as a fold of that observed with wild-type seipin. Data shown are means ± SEM

Fig. 4

Wild-type human seipin and T78A mutant seipin form high-molecular-mass oligomers, while L91P seipin and A212P seipin are unable to do so. (a–d) tsA 201 cells were transfected with the long form of wild-type seipin with an N-terminal triple-FLAG tag and a C-terminal Myc tag (a) or with the T78A (b), A212P (c) or L91P (d) mutant forms of this protein. Isolated proteins were subjected to AFM imaging. Scale bar, 200 nm; shade-height scale, 0–5 nm. (e–h) Galleries of zoomed images of wild-type seipin (e) and the T78A (f), A212P (g) and L91P (h) mutants. Scale bar, 20 nm; shade-height scale, 0–5 nm. (i–l) Frequency distributions of molecular volumes of wild-type (i), T78A (j), A212P (k) and L91P (l) seipin. The curves indicate the fitted Gaussian functions

Fig. 5

Both wild-type human seipin and T78A mutant seipin form dodecamers. (a) AFM images of wild-type seipin either alone or after incubation with anti-Myc or anti-HA antibodies. (b) AFM images of T78A mutant seipin either alone or after incubation with either anti-Myc or anti-V5 antibodies. Arrows indicate multiply decorated seipin complexes. Scale bar, 100 nm; shade-height scale, 0–5 nm. (c, d) Gallery of zoomed images showing multiply decorated wild-type (c) or T78A mutant seipin (d). Scale bar, 20 nm; shade-height scale, 0–5 nm. (e, f) Frequency distributions of angles between pairs of antibodies bound to either wild-type (e) or T78A mutant seipin (f). The curves indicate the fitted Gaussian functions