The LDL receptor protein that derived from each variant was examined by immunostaining of Western blot with LDL receptor polyclonal antibody (Figure 6)

Download (0)





Expression of LDL receptor cDNA mutants

The effects of seven point changes (D69N, R94H, E207K, C308Y, I402T, A410T, and A696G), two gross deletions (Del e3-5 and Del e6-8) on LDL receptor function were investigated by transient expression in COS-7 cells. In recombinant pcDNA3 plasmids, the entire coding region of LDL receptor is under the control of CMV promoter. Upon transfection into COS-7 cells, the recombinant plasmid reproduces via SV40 origin and transiently expresses high level of LDL receptor protein. Approximately equal levels of LDL receptor mRNA derived from each variant was expressed as quantitated by RT-PCR (Figure 5). The LDL receptor protein that derived from each variant was examined by immunostaining of Western blot with LDL receptor polyclonal antibody (Figure 6). No nonspecific polypeptide was detected in pcDNA3 vector-transfected cells (lane 1). 160 kDa mature and 120 kDa precursor proteins as well as an intermediate form were detected in wild type receptor cDNA-transfected cells (lane 2). A novel intermediate protein that is probably a degradation product and an apparent reduction in mature receptor protein were seen in D69N transfected cells (lane 3). Although the same molecular weight receptor proteins were seen with R94H, E207K, C308Y, I402T and A410T mutations, the amounts of mature protein detected were reduced (lanes 4-8). For A696G, both the processing and the level of the mature protein were close to those of wild type (lane 9).

Conversely, no receptor protein for Del e3-5 and a defect receptor protein for Del e6-8 were detected (lanes 10-11).

LDL receptor function

The amounts and activity of the LDL receptor variants were examined by flow cytometer analysis using Antibody at 4°C and DiI-LDL at 37°C. Analyses of antibody-labeled cells and activity measurements revealed that A696G encodes receptors in amounts and activity comparable with the wild type receptor (98% and 93%, respectively). Del e3-5 encodes receptors that cannot be detected and have no detectable residual activity. All other mutations impair the function, encoding



receptors in reduced amounts (13%~64%) and displaying approximately 12%~64%

residual activity (Table 3).

Fluorescence microscopy of cells expressing the wildtype or the mutant LDL receptor was performed. As shown in Figure 7A, the wild-type and A696G receptor proteins were located in a ring-shaped structure representing the cell surface, whereas most of the mutant receptors accumulated intracellularly. Colocalization studies using a lysosome staining dye (Figure 7B) further established that A410T and Del e6-8 mutant proteins are retained in the endosomal/lysosomal regions, whereas D69N, R94H, E207K, C308Y, and I402T produce proteins that seem to be localized in the endoplasmic reticulum (ER) (Figure 7C).




相關主題 :