Endoplasmic reticulum quality control is involved in the mechanism of endoglin-mediated hereditary haemorrhagic telangiectasia

Abstract

Hereditary haemorrhagic telangiectasia (HHT) is an autosomal dominant genetic condition affecting the vascular system and is characterised by epistaxis, arteriovenous malformations and mucocutaneous and gastrointestinal telangiectases. This disorder affects approximately 1 in 8,000 people worldwide. Significant morbidity is associated with this condition in affected individuals, and anaemia can be a consequence of repeated haemorrhages from telangiectasia in the gut and nose. In the majority of the cases reported, the condition is caused by mutations in either ACVRL1 or endoglin genes, which encode components of the TGF-beta signalling pathway. Numerous missense mutations in endoglin have been reported as causative defects for HHT but the exact underlying cellular mechanisms caused by these mutations have not been fully established despite data supporting a role for the endoplasmic reticulum (ER) quality control machinery. For this reason, we examined the subcellular trafficking of twenty-five endoglin disease-causing missense mutations. The mutant proteins were expressed in HeLa and HEK293 cell lines, and their subcellular localizations were established by confocal fluorescence microscopy alongside the analysis of their N-glycosylation profiles. ER quality control was found to be responsible in eight (L32R, V49F, C53R, V125D, A160D, P165L, I271N and A308D) out of eleven mutants located on the orphan extracellular domain in addition to two (C363Y and C382W) out of thirteen mutants in the Zona Pellucida (ZP) domain. In addition, a single intracellular domain missense mutant was examined and found to traffic predominantly to the plasma membrane. These findings support the notion of the involvement of the ER's quality control in the mechanism of a significant number, but not all, missense endoglin mutants found in HHT type 1 patients. Other mechanisms including loss of interactions with signalling partners as well as adverse effects on functional residues are likely to be the cause of the mutant proteins' loss of function.

Figure 1. The three-dimensional structure of endoglin monomer showing the locations of the twenty five missense mutants studied in this aricle.

Endoglin consists of a small c-terminal intracellular domain and three extracellular domains that include the ZP-C (green), ZP-N (Orange) and orphan (yellow) domains. The endoglin model structure (Llorca et al. 2007) file was provided by Dr Carmelo Bernabeu and then was manipulated using RasMol 2.7 (www.RasMol.org). The ball and stick represntation of the ER-retained (back) and the predominantly plasma membrane (purple) mutants are indicated on the strucure. It is clear that the majority of the mutants affecting the orphan domain resulted in the retention of the protein in the ER whereas those affecting the ZP domains retained their plasma membrane localization.

Figure 2. Comparison of the subcellular localization of wild type Endoglin with two (L32R and C53R) orphan domain HHT1-causing mutants.

HeLa cells were transiently transfected with the C-terminally HA-tagged Endoglin pCMV5 plasmids (panels A-C, G-I and M-O) or co-transfected with the same plasmids and the EGFP-tagged H-Ras plasmid (Panels D–F, J–L and P–R) and processed for fluorescence confocal microscopy as described in the methods. The HA tagged proteins were detected with Anti-HA monoclonal antibodies (red panels A, D, G, J. M and P) and the ER marker calnexin was detected with anti-calnexin polyclonal antibodies (panels B, H and N). GFP-H-Ras staining is shown in panels E, K and Q. The wild type predominantly showed plasma membrane localization as evidenced by its co-localization with Ras (D–F). On the other hand the two mutants (L32R and C53R) showed ER localization (G–R).

Figure 3. The subcellular localization of three orphan domain HHT1-causing mutants (V125D, P165L and I271N).

HeLa cells were transiently transfected with the C-terminally HA-tagged Endoglin pCMV5 plasmids (panels A–C, G–I and M–O) or co-transfected with the same plasmids and the EGFP-tagged H-Ras plasmid (Panels D–F, J–L and P–R) and processed for fluorescence confocal microscopy as described in the methods. In all three cases, the mutants co-localized with the ER marker (A–C for V125D; G–I for P165L and panles M–O for I271N) but not to the plasma membrane (D–F, J–L and P–R for V125D, P165L and I271N, respectively).

Figure 4. Endoglycosidase H (EndoH) analysis of the expressed missense mutants.

The pCMV5 HA-tagged endoglin plasmids were transfected into HEK293 cells and were allowed to express the proteins as described in the methods. This was followed by lysis of the cells and the immuoprecipitation of the proteins with anti-HA monoclonal antibodies. Each immunoprecipitate was divided into two sample with one of them was treated (+) with EndoH or left untreated (−). Both samples were then electrophorese and western blotted using anti-HA monoclonal antibodies as described in the methods section. The majority of the WT protein did not change mobility upon EndoH treatment indicating its maturation and acquisition of complex N-glycan moieties in the post ER compartments. The missense mutants L32R, V49F, C53R, V125D, A160D, P165L, I271N, A308D, C363Y and C382W showed a single band indicating an exclusively ER premature forms. The other mutants either showed two bands or a simple high molecular weight band indicating that at least a significant proportion of the expressed protein matured in post ER compartments. The EndoH treatment data are in agreement with the confocal microscopy data.

Figure 5. The majority of the missense mutants in the ZP and intraceullar domains traffic normally to the plasma membrane.

HeLa cells were transiently co-transfected with the C-terminally HA-tagged Endoglin pCMV5 plasmids and the EGFP-tagged H-Ras plasmid and 24 hours later the cells were processed for fluorescence confocal microscopy as described in the methods. The following missense mutanst were shown to traffic predominantly to the plama membrane as eviednced by their co-localization with GFP-H-Ras: C382G ( data not shoiwn due to space limitations), F403S (panles A–C), S407N (not shown), C412S (not shown), G413V (not shown), N423S (panels D–F), R437W (not shown), A452G (not shown), Q476H (not shown), V504M (panels J–L), R571H (panels M–O ) and P615L (panels P–R).