Figure 8. LBR sterol reductase activity restores glycan processing in LBR-deficient cells.

(A) Immunofluorescence microscopy of ACG1A_1 fibroblasts stained with LBR-specific antibodies; ACG1A_1 carries the homozygous LBR-null mutation. Cells were either not transfected (parental) or transiently transfected with vectors overexpressing the indicated LBR variants; the Greenberg dysplasia–associated p.N547D and p.R583Q missense mutations specifically abrogate C14 sterol reductase activity but do not alter the chromatin/lamin-binding properties of the respective LBR protein. WT, wild type. Scale bar: 10 μm. (B) ACG1A_1 cells were either electroporated without plasmid (mock), with empty vector (vector), or with expression constructs encoding the indicated LBR proteins. Whole-cell protein lysates of nontransfected wild-type controls (CTL) and ACG1A_1 transfectants were obtained 96 hours after electroporation and analyzed by Western blotting with LBR- and LAMP2-specific antibodies. GAPDH immunostaining of the stripped membrane indicates total protein loading. Fast-migrating nonglycosylated LAMP2 is highlighted by a pink asterisk at approximately 30 kDa; intermediate glycosylation products are marked by a red asterisk at approximately 65 kDa. In ACG1A_1 cells overexpressing wild-type LBR, nonglycosylated LAMP2 and intermediate glycosylation products are not detectable and mature LAMP2 has a similar molecular weight to that in nontransfected wild-type fibroblasts with an additional intermediate band at approximately 85 kDa (blue asterisk), likely corresponding to a more highly modified species than that seen upon LBR deficiency, although not fully mature. Note that a higher protein load of nontransfected wild-type fibroblasts was used to visualize the normal electrophoretic mobility pattern of LAMP2 species.