Hereditary hemorrhagic telangiectasia type 1 HHT1 is associated with mutations in the ENDOGLIN gene which normally codes for a polypeptide of amino acids expressed at the cell surface as a dimeric glycoprotein.
To maximize 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.
All nine novel mutations reported failed to be expressed other than intracellularly. Several constructs of endoglin Mature no 755 expressed in COS-1 cells; only the full-length protein was processed to the cell surface.
Our studies show that all HHT1 mutants Mature no 755, although expressed to various degrees in COS-1 cells, are either undetectable, present at low levels as transient intracellular forms, or expressed as partially glycosylated precursors in endogenous cells. These mutants do not form heterodimers with normal endoglin and do not interfere with its normal trafficking to the cell surface, further supporting the haploinsufficiency model.
Endoglin is a homodimeric membrane glycoprotein with a molecular mass of kDa which is expressed predominantly on endothelial cells 1.
The polypeptide has an extracellular domain of amino acids from N-terminal E26 to G, M1 being the initiation codona single transmembrane region L—W and a cytoplasmic tail of 47 amino acids terminating in Ala 23. The cytoplasmic tail Mature no 755 rich in serine and threonine residues, three of which are constitutively phosphorylated 245.
There are four potential N -linked glycosylation sites and a potential region of O -linked glycosylation 6. While endoglin "Mature no 755" a glycoprotein primarily expressed on endothelial cells, betaglycan is a membrane proteoglycan expressed in a variety of cells but generally Mature no 755 low levels on endothelial cells Betaglycan can also be enzymatically cleaved from the cell surface and has been detected in Mature no 755, extracellular matrices and cell culture fluids 7 Although low levels of endoglin have been observed in serum, using an ELISA assay 15 — 17no secreted form, or enzymatically cleaved product, has been observed in the media of endothelial cells in culture.
Serum endoglin is likely contained in membrane particles shed from the vascular endothelium. To date, 41 mutations associated with HHT1 have been reported. These are found in the first 12 exons of the gene and include substitutions, deletions and insertions 1820 — A detailed phenotypic analysis of eight families with characterized HHT1 mutations showed that the severity of manifestations was independent on the type and position of the mutation Analysis of endoglin expression in multiple families has provided evidence for haploinsufficiency as the predominant model for HHT1.
Null alleles have been observed 232426 and most mutant proteins are only expressed as transient intracellular species, if expressed at all 222628 We also reported that four missense endoglin mutations were expressed as intracellular precursors and were never processed into mature surface glycoproteins A recent paper, looking exclusively at expression in COS-1 cells, has suggested the presence of dominant negative mutants of endoglin that could be detected at the cell surface as heterodimers or, in one case, secreted In the current study, we performed pulse-chase studies with several large truncation mutants in endothelial cells from HHT1 newborns to optimize detection of potential mutants in endogenous cells.
We also analyzed several novel missense mutations. None of the mutant proteins could be detected at the cell surface or secreted. The expression of different endoglin fragments in COS-1 cells was also studied, particularly to identify potential soluble forms of the protein. The only secreted recombinant fragment generated corresponded to the complete extracellular domain of endoglin.
The data presented in this paper and the extensive protein expression studies performed in about patients do not provide any evidence for dominant negative mutations associated with HHT1. The rate of processing and turnover of normal endoglin in endothelial cells was analyzed prior to undertaking the analysis of truncation mutations associated with HHT1.
HUVEC were metabolically labeled with a 20 min pulse of [ 35 S]methionine, followed by chase periods ranging from 0 to 24 h Mature no 755 immunoprecipitated with monoclonal antibody mAb P4A4, specific for endoglin. Figure 1 A shows a representative experiment. Endoglin is synthesized as a precursor P of 80 kDa, first detectable at time zero and maximum at 30—60 min of chase.
This precursor is processed into the mature glycoprotein Efirst seen after 30 min and maximally expressed at the cell surface after 3 h of chase. Analysis under reducing and non-reducing conditions revealed that partial dimerization of the precursor can be seen as early as time zero.
After 3 h of chase, no residual monomer is seen and the mature glycoprotein is completely dimerized Fig. The relative amount of mature endoglin was estimated at each time relative to the maximum level observed in each of several experiments Fig.
The expression of mature endoglin peaked after 2—3 h and began to decline thereafter. The half-life of "Mature no 755" at the cell surface was estimated at 17 h, indicating that it is a relatively stable glycoprotein. Metabolic labeling of HUVEC and peripheral blood-activated monocytes, using a standard steady state labeling period of 3. However, in the vast majority of cases, no mutant protein could be visualized.
To optimize the detection of potential transient protein species, some of the larger truncation mutations were analyzed by pulse-chase studies in HUVEC.
These cells can readily be expanded in vitro in primary cultures and express much Mature no 755 levels of endoglin than activated monocytes. All mutations described in this paper are shown diagrammatically in Figure 2. When the data is expressed as percentage of the maximum level Mature no 755 surface endoglin E present in the control, it can be seen that the rate of processing of the normal allele is similar in both HHT1 HUVEC, although at lower levels than the control Fig.
A polyclonal antibody to endoglin also immunoprecipitated this mutant, but less effectively than the monoclonal antibodies data not shown.
The mutant is seen as a monomer, estimated at 72 kDa.