Expression of normal and truncated forms of human endoglin

Abstract

Endoglin is a transmembrane glycoprotein 633 residues in length expressed at the surface of endothelial cells as a disulphide-linked homodimer; the specific cysteine residues involved in endoglin dimerization are unknown. Mutations in the coding region of the endoglin gene are responsible for hereditary haemorrhagic telangiectasia type 1 (HHT1), a dominantly inherited vascular disorder. Many of these mutations, if translated, would lead to truncated forms of the protein. It is therefore of interest to assess the protein expression of different truncated forms of endoglin. Infections in vitro or in vivo with recombinant vaccinia virus, as well as transient transfections with expression vectors, were used to express normal and truncated forms of endoglin. Truncated mutants could be classified into three different groups: (1) those that did not produce stable transcripts; (2) those that produced stable transcripts but did not secrete the protein; and (3) those that secreted a soluble dimeric protein. This is the first time that a recombinant truncated form of endoglin has been found to be expressed in a soluble form. Because a chimaeric construct encoding the N-terminal sequence of platelet/endothelial cell adhesion molecule (PECAM-1) antigen fused to residues Ile281-Ala658 of endoglin also yielded a dimeric surface protein, these results suggest that cysteine residues contained within the fragment Cys330-Cys412 are involved in disulphide bond formation. Infection with vaccinia recombinants encoding an HHT1 mutation did not affect the expression of the normal endoglin, and did not reveal an association of the recombinant soluble form with the transmembrane endoglin, supporting a haploinsufficiency model for HHT1.

Investigation of Endoglin Wild-Type and Missense Mutant Protein Heterodimerisation Using Fluorescence Microscopy Based IF, BiFC and FRET Analyses

“…For this purpose, a TGF-β1 regulated signal assay was conducted in HMEC-1 endothelial cells and a rat endothelial cell line (REC), using the (CAGA) 12 -luciferase promoter reporter activated by the ALK5-Smad3 pathway. The capability of dimerisation between human and mouse endoglin had previously been demonstrated [41] and we therefore assumed that endoglin from another rodent species would also interact with human endoglin. We were curious to see whether a possible interaction between human endoglin mutants and endoglin wt from a different species might also interfere with signalling.…”

“…Wild-type and mutant endoglin (G52V, W149C, A160N, L306P, G413V) constructs in the pCMV5 vector and the endoglin-Δcyto (ΔC) construct have been described previously [41]. Further mutations and variations, G191D, S480C and R571H, were generated by site-directed mutagenesis using the pCMV5-endoglin wild-type construct as a template.…”

“…Additional analyses by co-immuno precipitations demonstrated that the mutant proteins also dimerise with wild-type endoglin [40], therefore suggesting that they are capable of interfering with endoglin function. Furthermore, expression of a series of different, engineered, truncated extracellular forms of endoglin, similar to a number of mutant proteins, was found for self-dimerisation, and was found to be extracellularly secreted, but not or only weakly capable of dimerising with wild-type endoglin [33], [40], [41]. In summary, when comparing the different patient sample analyses and recombinant endoglin mutant protein expression studies, the evidence for the proteins of certain mutations becoming expressed and being stable, and the question of whether these are able to dimerise with wild-type endoglin, remains contradictory.…”

Structural and Functional Insights into Endoglin Ligand Recognition and Binding

“…However, the involvement of additional Cys residues in the dimerization process has been suggested [35]. Electrophoretical analysis under reducing and non-reducing conditions of Endo EC and the OD constructs showed bands corresponding to dimeric and monomeric species for the full length endoglin ectodomain and for Endo 362 .…”

Use of Ribosome-Inactivating Proteins from Sambucus for the Construction of Immunotoxins and Conjugates for Cancer Therapy

“…The immunotoxin was completely ineffective on parental L929 cells. 44G4 monoclonal antibody, even linked to nigrin b, does not recognize the murine CD105 present in both the parental and the transfected L929 cells [117]. A strong cytotoxic effect was observed on the viability of HUVEC (human umbilical vascular endothelial cells), which express high levels of endoglin during their proliferation phase in culture [112].…”