Analysis of several endoglin mutants reveals no endogenous mature or secreted protein capable of interfering with normal endoglin function

Hereditary hemorrhagic telangiectasia type 1 (HHT1) is associated with muta tions in the ENDOGLIN gene which normally codes for a polypeptide of 653 am ino acids expressed at the cell surface as a dimeric glycoprotein. To maxim ize the detection of potential mutant proteins, we analyzed by pulse-chase experiments the expression of large truncation mutants in endothelial cells from newborns with HHT1. A mutant truncated at residue 490 (Delta 490) and the Delta 517 mutant, previously suggested to act as dominant negative, we re undetectable. Proteins Delta 471 and Delta 571 were barely detectable as transient monomers of 62 and 72 kDa. A de novo 13 bp deletion in exon 11 e ncoded a monomeric protein of 70 kDa (Delta 557), present at low levels in activated monocytes, Six novel missense mutants and Delta S411 were express ed only as the 80 kDa intracellular precursor of surface endoglin, suggesti ng impaired processing. All nine novel mutations reported failed to be expr essed other than intracellularly. Several constructs of endoglin were expre ssed in COS-1 cells; only the full-length protein was processed to the cell surface. Recombinant Delta 586, corresponding to the complete extracellula r domain, was secreted as monomeric and dimeric glycosylated species. Our s tudies show that all HHT1 mutants analyzed, although expressed to various d egrees in COS-1 cells, are either undetectable, present at low levels as tr ansient intracellular forms, or expressed as partially glycosylated precurs ors in endogenous cells. These mutants do not form heterodimers with normal endoglin and do not interfere with its normal trafficking to the cell surf ace, further supporting the haploinsufficiency model.