Expression analysis of endoglin missense and truncation mutations: insights into protein structure and disease...

Hereditary hemorrhagic telangiectasia (HHT) is an inherited autosomal dominant vascular dysplasia caused by mutations in either endoglin (HHT1) or activin-like kinase receptor-1 (ALK-1) (HHT2). The majority of the mutations in endoglin cause frameshifts and premature stop codons. Although initial reports suggested a dominant-negative model for HHT1, more recent reports have suggested that mutations in endoglin lead to haploinsufficiency. In this study, we investigated six different missense mutations and two truncation mutations in the endoglin gene to examine whether mechanisms other than haploinsufficiency might be involved in HHT1. Expression of the missense mutants alone revealed that they are misfolded and that most show no cell surface expression. When co-expressed with wild-type endoglin, the missense mutants are able to dimerize with the normal endoglin protein and are trafficked to the cell surface. We also show that although one truncation mutation acts through haploinsufficiency, the other acts in a dominant-negative way. This implies that either dominant-negative protein interactions or haploinsufficiency can cause HHT1. The biochemical analyses for the different mutations suggest that the endoglin N-terminus is important for correct protein folding and that cysteine residues in the first 350 amino acids are involved in intramolecular disulfide bonds, whereas cysteines located closer to the C-terminus of the extracellular domain are responsible for inter-molecular disulfide bond dimerization.