FUNCTIONAL EFFECT OF NOVEL AMINO ACID VARIANTS OF APOLIPOPROTEIN B IN FAMILIAL HYPOBETALIPOPROTEINEMIA

Abstract

Introduction. Familial Hypobetalipoproteinemia (FHBL) is a codominant disorder characterized by reduced plasma levels of LDL-C and apolipoprotein (apo) B. In 50% of cases FHBL is due to mutations in APOB gene resulting in truncated apoBs of various size. Some mutations in APOB gene resulting in non-conservative amino acid substitutions were reported to cause FHBL. In vitro, these mutations induce the retention of the mutant apoB in the endoplasmic reticulum (ER) and impair the secretion of apoB-containing lipoproteins. In two FHBL subjects we identified two novel amino acid variants (Thr26_27delinsAsn and Tyr102Cys) located in the N-terminal 1000 amino acids of mature apoB. Methods. To investigate the functional effect of these mutations we constructed plasmids containing human apoB-48 cDNAs harbouring the mutations. McA-RH7777 rat hepatoma cells were transiently and stably transfected with wild type or mutant human apoB-48. The secretion efficiency of human apoB-48 was determined by immunoblotting. To evaluate whether the mutant apoB- 48 was able to form apoB-containing lipoproteins, the incubation media were ultracentrifuged to separate the lipoprotein classes. Immunocytochemistry was used to assess the intracellular localization of the mutant proteins. Results. The mutation Thr26_27delinsAsn strongly reduces the secretion of apoB-48 from the transfected cells. The mutant apoB- 48 appears to be retained in ER as demonstrated by the confocal images showing co-localization of the mutant apoB with the ER marker. In stably transfected cells the defect of mutant apoB-48 secretion was confirmed by the absence of apoB-48 containing lipoproteins in the medium. These observations suggest that Thr26_27delinsAsn alters the structure of the beta-barrel of N-terminal domain of apoB (the first 267 amino acids of mature protein) preventing the secretion of apoB-containing lipoproteins. By contrast the mutation Tyr102Cys had no effect on apoB-48 secretion. Conclusions. This finding supports the notion that Thr26_27 delinsAsn is the cause of FHBL.