Expression analysis of four endoglin missense mutations suggests that haploinsufficiency is the...

© 1999 Oxford University Press Human Molecular Genetics, 1999, Vol. 8, No. 122171–2181

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Expression analysis of four endoglin missensemutations suggests that haploinsufficiency is thepredominant mechanism for hereditary hemorrhagictelangiectasia type 1Nadia Pece-Barbara, Urszula Cymerman, Sonia Vera, Douglas A. Marchuk 1 andMichelle Letarte +

Cancer and Blood Research Programme, Hospital for Sick Children and Department of Immunology, University ofToronto, 555 University Avenue, Toronto M5G 1X8, Canada and 1Department of Genetics, Duke University MedicalCenter, Durham, NC, USA

Received June 3, 1999; Revised and Accepted August 16, 1999

ENDOGLIN codes for a homodimeric membraneglycoprotein that interacts with receptors formembers of the TGF- ββββ superfamily and is the genemutated in the autosomal dominant vasculardisorder hereditary hemorrhagic telangiectasia type1 (HHT1). We recently demonstrated that functionalendoglin was expressed at half levels on humanumbilical vein endothelial cells (HUVECs) andperipheral blood activated monocytes from HHT1patients. Two types of mutant protein werepreviously analyzed, the product of an exon 3 skipwhich was expressed as a transient intracellularspecies and prematurely truncated proteins thatwere undetectable in patient samples. Here we reportthe analysis of four proteins resulting from pointmutations, with missense codons G52V and C53R inexon 2, W149C in exon 4 and L221P in exon 5. Meta-bolic labeling of activated monocytes fromconfirmed, clinically affected patients revealedreduced expression of fully processed normal endo-glin in all cases. Pulse–chase analysis with HUVECsfrom a newborn with the C53R substitution indicatedthat mutant endoglin remained intracellular as aprecursor form and did not impair processing of thenormal protein. Biotinylation of cell surface proteins,metabolic labeling and pulse–chase analysisrevealed that none of the engineered missensemutants was significantly expressed at the surface ofCOS-1 transfectants. Thus, these four HHT1 missensemutations lead to transient intracellular species whichcannot interfere with normal endoglin function. Thesedata suggest that haploinsufficiency, leading toreduced levels of one of the major surface glyco-proteins of vascular endothelium, is the predominantmechanism underlying the HHT1 phenotype.

INTRODUCTION

Hereditary hemorrhagic telangiectasia (HHT) is inherited asautosomal dominant trait at a frequency of 1 in 10 000 aexhibits age-related penetrance with variable expressiv(1,2). The most common clinical manifestations involve thdevelopment of vascular abnormalities seen as telangiectaon skin and lesions in nasal mucosa that bleed readily. Slesions are characterized by direct arteriovenous connectithat may develop in several organs and can lead to laarteriovenous malformations (AVM) in brain, lung and liverThese may cause serious complications such as stroke, babscess or hemorrhage (reviewed in ref. 3).

Genetic linkage studies have revealed that HHT is a hetegeneous disorder. The first locus [hereditary hemorrhatelangiectasia type 1 (HHT1)] was mapped to chromosom9q33–34 (4,5), whereENDOGLINwas defined as the affectedgene (6). We have recently defined endoglin (CD105) asaccessory membrane glycoprotein that interacts with signling receptor complexes for several members of the traforming growth factor-β (TGF-β) superfamily (7). It isexpressed at high levels on vascular endothelial cells (however, other sites of expression include activated moncytes, where it is observed at lower levels (9). A second loc[hereditary hemorrhagic telangiectasia type 2 (HHT2mapping to chromosome 12q was shown to beALK1 (activinreceptor-like kinase 1) (10–12). It is also expressedendothelial cells and is a type I receptor of the TGF-β super-family, but its true physiological ligand has not been identifie(13). TGF-β and other members of its superfamily signathrough related serine/threonine kinase receptors, types I an(14,15). Generally, the type II receptor binds ligand, recruitstype I receptor and activates it by transphosphorylation, ththe type I receptor is the primary transducer of signals to dowstream components known as Smads (16–18). These findthat endoglin and ALK1 are mutated in HHT suggest that boproteins are involved in a common pathway controllinvascular development and/or homeostasis. A third variantHHT was reported in a single large family with serious live

+To whom correspondence should be addressed. Tel: +1 416 813 6258; Fax: +1 416 813 6255; Email: [email protected]

2172 Human Molecular Genetics, 1999, Vol. 8, No. 12

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complications (19). Identification of other loci might revealone or more components of a common signalling pathwayinvolving both endoglin and ALK1.

To date, 29 different mutations have been reported in HHT1patients (6,20–24) and 18 distinct mutations have beendescribed for ALK1 (12,25,26). HHT1 families appear to havea higher prevalence of pulmonary AVM while HHT2 familiesin general have a milder phenotype and a later onset of disease(6,23,27,28). Furthermore, clinical heterogeneity withinfamilies is high, suggesting that additional factors are contrib-uting to severity of disease.

Although a dominant-negative model of endoglin dysfunc-tion was initially proposed for HHT1, we recently demon-strated that a mutant protein missing 47 amino acidscorresponding to an exon 3 skip, but with an intact trans-membrane region, was transiently expressed intracellularlyboth in monocytes from an HHT1 patient and in humanumbilical vein endothelial cells (HUVECs) from the child ofthis patient (22). As the cell surface expressed protein was stillable to associate with the TGF-β receptor complex, this indi-cated that it is the reduction in the level of surface endoglin,rather than interference by a mutant protein, that is involved inthe generation of HHT1. The 29 mutations reported to dateinclude various deletions, an insertion and point mutationsleading either to premature stop codons or to frameshifts thatresult in predicted truncated proteins (6,21,23,24). Metaboliclabeling of cells expressing some of these mutations showedthat the corresponding proteins were not expressed at the cellsurface nor were they secreted (22). In addition, three nullmutations have been described where mRNA transcripts wereundetectable (23,24). These data suggest that endoglinhaploinsufficiency is the molecular basis for this geneticdisorder.

The observations that every HHT1 family so far has adistinct mutation and that mutations of all types are distributedthroughout the gene are consistent with a haploinsufficiencymodel (29). However, missense mutations often result in dominant-negative mechanisms, constitutive protein activation or newprotein functions (30). Here we study fourENDOGLINmissense mutations to determine whether a mechanism otherthan halploinsufficiency could be associated with HHT1. Toquantitate endoglin expression in adult HHT1 patient sampleswe have further characterized the use of activated monocytesand report that activation of monocytes for at least 21 h inculture is needed to reach steady-state levels of endoglin. Weused these conditions for analyzing missense mutations fromfour HHT1 families. Two missense mutations, G155T andT157C in exon 2, resulting in G52V and C53R codons, and onein exon 4, G447C, creating a W149C codon, were previouslycharacterized as disease associated (24). We report a novelmissense mutation in exon 5, a T662C substitution, leading toL221P conversion. These missense mutations result inmisfolded proteins that are retained intracellularly andexpressed minimally at the surface. Thus, only the normalENDOGLIN allele gets expressed at the cell surface in theseHHT1 patients, correlating with previous data and demonstratingthat haploinsufficiency is the predominant mechanism for thisgenetic disorder.

RESULTS

Endoglin expression is induced on activated monocyteswithin 5 h in culture

Adherence of monocytes induces their differentiation to tmacrophage lineage, which is accompanied by the inductionexpression of several genes. It was shown that adherencperipheral blood monocytes to autologous plasma-coaplastic dishes enhanced their expression of endoglin, whwas then stable for up to 7 days (9). By single color flocytometry we also found that 94% of activated monocytexpressed endoglin (CD105) with a mean relative fluoresceintensity (RFI) of 77 after attachment to plastic and incubatioin culture for 21 h (Fig. 1A). In contrast to published resul(9), there was no further induction with time in culture; in facexpression was slightly reduced when tested at 45 and 11Endoglin expression correlated with that of the monocyte acvation marker CD11b, present on 91% of cells at a mean Rof 102 after 21 h in culture, but also decreased with time. Walso observed that monocyte monolayers were often unstawith longer times in culture and in particular with an autologous plasma coating. For these reasons, we routinely tesendoglin expression on activated monocytes after adherencuncoated plastic for a period not exceeding 1 day in culture

Measuring endoglin levels on peripheral blood monocyt(PBMCs) by selecting the monocyte population using twcolor flow cytometry would facilitate routine testing and overcome the need for activation. Since endoglin expressionnot been reported on PBMCs, we determined the basal levefreshly isolated monocytes and on cells obtained after 1 a21 h adherence. Forward and side scatter analysis of PBMCtime 0 shows four populations of cells: based on size and grularity, the upper right quadrant contains 17% of the total cerepresenting monocytes and contaminating granulocytes,lower right quadrant (61% of total cells) is mostly lymphocyteand the lower left (17%) contains mostly contaminating platlets and red blood cells (Fig. 1B). Analysis of these populatiofor the myelomonocytic marker CD14 (with the FL2 detectousing rhodamine) revealed that the upper right quadrant w>95% positive, confirming that these cells are of the myelolineage. The upper left quadrant (5% of total cells) was alCD14+, while the lower right quadrant contained 22% CD14+

cells. Thus, by far the cleanest monocyte population was inupper right quadrant and a tight region R1 was defined whicontained 99% CD14+ cells with a mean RFI of 1860. A tighterpopulation of CD14+ cells was defined with a second gate (R2and then analyzed for CD105 expression (with the FLdetector, using FITC). Only 22% of the CD14+ cells expressedCD105 at time 0, with a mean RFI of 91.

Attachment of PBMCs to plastic for 1 h, followed bywashing and release of the adherent layer, yielded an enricmonocyte fraction (49% of total cells; upper right quadranAll cells in the monocyte gate, defined by R1, were CD14+

with a mean RFI of 2020. There was no increase in CD1expression on these cells at this time. When PBMCs wplated for 1 h, washed, incubated in culture for a further 20and then released, samples then consisted of 70% monoc(upper right quadrant). There was no significant changeCD14 expression on this activated monocyte population (Rhowever, 99% of these cells were now CD105+ with a mean

Human Molecular Genetics, 1999, Vol. 8, No. 122173

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RFI of 200 (R2). At all time points, the lower right quadrantwas at most 22% CD14+ and <20% CD105+, confirming thatendoglin is not present on lymphocytes (data not shown).Together these data show that the monocyte population isenriched by adherence to plastic and that this attachment isrequired to induce endoglin expression, whereby completeinduction can be observed after 21 h in culture. Thus, it wouldbe feasible to use one or two color flow cytometry to quantifyendoglin levels on activated monocytes, but not on PBMCs asthey do not express sufficient levels.

We previously used metabolic labeling followed by immunoprecipitation analysis as this permits the detection of potenmutant proteins, as well as enabling the quantification of texpression of fully glycosylated endoglin. We thus show thkinetics of induction of newly synthesized endoglin bmetabolic labeling (Fig. 1C). Monocytes were isolated froPBMCs by attachment to plastic for 1 h, washed in methioninfree medium and metabolically labeled for 4 h. The earlieactivation time (time in culture) that was tested by metabolabeling was 5 h, since we established previously that 4

Figure 1. Kinetics of appearance of endoglin on monocytes. (A) Normal PBMCs from 60 ml of blood were equally divided for three analyses. Monocytes were sepafrom lymphocytes by adherence to plastic, then further activated by incubation in culture. Total time in culture is indicated. Single color flow cytometry analysis of endoglin(CD105, solid black) and the monocyte activation marker (CD11b, grey line) was performed as described in Materials and Methods. A gate (<2% positive)was defined ateach time point with IgG control (black line) and the percentages of CD105+ and CD11b+ cells within this gate are shown. The mean RFI values with increasing timeculture were 77, 41 and 44 for CD105, and 102, 61 and 69 for CD11b. Note the log scale used when expressing fluorescence intensity. (B) Normal PBMCs from 60 ml ofblood, equally divided for three time points, were analyzed by two color flow cytometry. Freshly isolated cells (time 0) and monocytes isolated by adherence to plastic for1 h or further activated for 20 h were stained with MY4–RD1 (anti-CD14) followed by IgG1 or mAb P4A4 (anti-CD105) then FITC-conjugated F(ab′)2 goat anti-mouseIgG. Forward and side scatter analysis and quadrant statistics are shown (left). The upper right quadrant representing monocytes were gated (R1) andanalyzed with the FL2detector (right). A gate was defined with IgG–RD1 as a negative control (<2% +cells). Over 99% cells were CD14+ (solid black) within the R1 gate, with mean RFI valueof 1860, 2020 and 1740 at 0, 1 and 21 h, respectively. A second gate was set on all CD14+ cells (R2) and analyzed with the FL1 detector. A gate was established withIgG control (black line; <2% positive); the percentage of CD105+ cells (solid black) within this gate is shown and the mean RFI values with increasing time were 9and 200. (C) Normal PBMCs from 20 ml of blood were used for each time point. Monocytes were enriched by adherence to plastic for 1 h and further activated16 h in culture. The total time in culture indicated includes a 4 h period of metabolic labeling with [35S]methionine. Cells were solubilized in Triton X-100 and lysates weimmunoprecipitated with mAb P3D1 and fractionated by 4–12% gradient SDS–PAGE under non-reducing (lanes 1 and 2) or reducing (lanes 3 and 4) conditions. D(180 kDa) and monomeric (90 kDa) fully glycosylated endoglin (E) and partially glycosylated precursor (P, 160 and 80 kDa) are indicated. The E band wasquantitated byPhosphorImager and the average pixel values were 13 130 and 18 670 for 5 and 21 h, respectively.


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labeling is needed to reach steady-state levels. Both fullyglycosylated endoglin (E) and the partially glycosylatedprecursor (P) were seen on activated monocytes after 5 h inculture (Fig. 1C). These species were identified by previoussurface labeling and metabolic studies (22). When comparedwith 21 h in culture (including 4 h labeling), where maximumexpression is reached, the expression of glycosylated endoglin(E) was 70% compared with the maximum. Interestingly,

expression of the precursor (P) was higher at 5 h, suggesthat new endoglin synthesis had been recently initiated. Tdifference was more prominent under reducing conditionsuggesting that these species (E and P) are better resounder these conditions. We did not detect a further increaslater time points (data not shown). However, as the monoclayer often becomes more fragile and cells lift off with longetime in culture, reproducible results are more readily obtainedculturing the monocytes for 16–24 h followed by labeling for 4

Endoglin expression is reduced in HHT1 patients fromfour families with missense mutations

Families 5, 85 and 89 were previously characterized andmissense codons, W149C, C53R and G52V, in these famireported (24). The individuals from these families used in th

Figure 2. Sequence of four missense mutations. Exons 2, 4 and 5 were amplifiedby PCR from genomic DNA isolated from normal peripheral blood lymphocytes,lymphocytes of clinically affected HHT1 patients H295, H278, H150 and H277and HUVECs from H319. Purified products were sequenced using a cyclesequencing protocol and resolved using a MicroGeneBlaster Sequencer (VisibleGenetics). The profiles revealing mixed sequence in patients H295, H278, H150and H319 resulting in the indicated substitutions confirm that these patients carrythe previously described missense mutations (K = G or T; Y = T or C; S = G or C).The sequence of exon 5 from patient H277 shows the novel T→C substitution (S)creating the L→P missense codon at position 221.

Figure 3.Schematic diagram of missense mutations in endoglin cDNA and prot(A) The intron–exon boundaries are indicated on the cDNA sequence, as isinitiation codon ATG corresponding to base pair 1 and to M1 of the leader peptThe positions of the four missense mutations relevant to this study are depicThe polypeptide structure of endoglin is illustrated with the approximate positioof cysteine residues (black circles) and the potential N-linked glycosylation s(tridents). The N-terminal residue of the mature protein is E26. mAbs recognizepitopes located in three distinct areas of the extracellular domain of endoglindepicted: P3D1 binding to the N-terminal region (E26–G230); P4A4 and 44binding to the Y277–G331 region; and RMAC8 binding to the C-terminal regi(G331–G586). (B) PCR strategy and primers used to generate constructs withcorresponding missense mutations. The expression construct pcEXV-EndoLused as a template; the positions of primers are shown relative to the cDNA inand the size of each product is indicated above. For the first PCR, two fragmwere made for each mutation that were complementary to the forward (MX5)reverse (MX3) mutagenic primers. For the second PCR, complementary fragmwere amplified in four separate reactions with outer primers AX5 and AX3generate four mutagenized products that were all 827 bp in length. Thfragments were digested withSacII andSbfI as indicated and the 740 bp fragmenwas subcloned back into the expression construct.


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study, H150, H278 and H295, are clinically affected and muta-tions were confirmed by sequencing of appropriate exons(Fig. 2). The HUVEC sample H319, belonging to family 85,was also confirmed as carrying the mutation leading to themissense codon C53R. We identified a novel missense muta-tion in patient H277 that was found by sequencing exon 5(Fig. 2); this point mutation causes conversion of L221 to P.

To determine whether missense mutations inENDOGLINcan also lead to haploinsufficiency, we quantified endoglinexpression in activated monocytes from the four clinicallyaffected adults described above. To maximize detection of amutated protein, expression of endoglin was analyzed withmonoclonal antibodies (mAbs) which recognize epitopes

mapping to three distinct areas of the extracellular doma(31), as shown in Figure 3A. In all samples, fully glycosylateendoglin expression was reduced relative to controls (Fig. 4HUVECs from patient H319 were also reduced compared wnormal HUVECs (Fig. 4B). Quantitation of these gels is showin Table 1 and indicates that endoglin expression is ~50%less, with the exception of patient H278, which had 70relative to normal with the P3D1 antibody. Howeveexpression levels in HUVEC sample H319, from the samfamily and confirmed as having the same mutation (C53Rwere comparable to those seen with the other missemutations.

In activated monocytes from all patients except H278, tband migrating as the partially glycosylated precursor (P) wstronger than normal (Fig. 4A). An elevated precursor was aseen in the HUVEC sample H319 (Fig. 4B), which has thC53R mutation. This suggested that mutant proteins wmigrating in that position or could be interfering with thsynthesis of normal protein. As it was difficult to obtain >30 mof blood from all adult patients, it was not feasible to usmonocytes for pulse–chase experiments. We thus testedsynthesis and stability of both fully processed endoglin (E) athe partially processed form (P) by pulse–chase experimewith HUVEC H319, as this was the only cord sample availabfrom the families in this study (Fig. 5). In both normal anHUVEC sample H319, the maximum expression of fullglycosylated endoglin is reached after 2 h. From the analysismultiple experiments, maximum expression of normal endoggenerally persists up to 4 h. However, as shown in Figureexpression at 3.5 h was 90% of that observed at 2 h, in bnormal and H319 samples, and probably reflects experimenvariation. Although total endoglin (E) was reduced in thpatient (H319) relative to normal, the rate of appearancefully processed endoglin (E) when expressed relative to ttotal (E + P) was not significantly different between these twsamples. This was observed when mAbs recognizing epitoin all three regions of the extracellular domain were us(Fig. 5). Total expression of fully processed endoglin (H31relative to normal was similarly reduced at each time poinindicating that processing of the normal protein is not affectby the presence of a mutant protein (Fig. 5B). However, tband migrating as the precursor was more stable inpatient’s cells and was still visible after a 3.5 h chase time.contrast, precursor levels were reduced to 10% in normal cells

Figure 4. Analysis of endoglin expression on activated monocytes from fourHHT1 families. (A) PBMCs from 30 ml of blood from clinically diagnosed HHT1patients H295, H278, H150 and H277, with the missense mutations indicated, orfrom normal controls (N) were activated by adherence to plastic and incubation inculture for at least 21 h, which includes labeling with [35S]methionine for 4 h.Metabolically labeled activated monocytes were processed as described in Figure1A except that both mAbs P3D1 and P4A4 were used to immunoprecipitateendoglin. Equivalent c.p.m. (estimated by trichloroacetic acid precipitation ofaliquots of total lysates prior to immunoprecipitation) were loaded for patients andmatched controls in each experiment. (B) H319 and normal HUVECs weresimilarly labeled and analyzed as in (A). Duplicate samples from both H319 andnormal HUVECs were loaded. Total radioactivity in the normal fully glycosylatedendoglin (E; 90 kDa) band for each mAb P3D1 and P4A4 immunoprecipitate werequantitated by PhosphorImager and the pixel values for each patient relative to thematched normal controls are reported in Table 1.

Table 1.Clinical and molecular data for four HHT1 families with missense mutations analyzed

Levels of fully glycosylated endoglin expression were determined by metabolic labeling with [35S]methionine and quantitative immunoprecipitation in blood-derived activated monocytes or HUVECs relative to controls. Total radioactivity in the normal fully glycosylated endoglin (E; 90 kDa) band of each mAbP3D1 and P4A4 immunoprecipitate, run under reducing conditions were quantitated by PhosphorImager and the pixel values for each patient are srelative to normal.

Family no. Clinical manifestations in family members Patient Mutation Missense codon Fully glycosylated (% HHT/normal)

P3D1 P4A4 Mean

89 PAVM, CAVM 295 adult G155→T G52→V 37 57 47

85 PAVM, CAVM 278 adult T157→C C53→R 71 50 61

85 PAVM, CAVM 319 newborn T157→C C53→R 40 35 38

5 PAVM, CAVM 150 adult G447→C W149→C 26 29 28

84 PAVM 277 adult T662→C L221→P 41 29 35


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2 h (Fig. 5C). Similar results were obtained using a polyclonalantibody (pAb) to human endoglin (data not shown). Interest-ingly, mAb RMAC8, recognizing an epitope between G331and G586 of the extracellular domain of endoglin, was slightlymore reactive with the precusor form of HUVEC H319. Thesedata suggest that the intracellular precursor pool is a combinationof mutant and normal at time 0 and 1 h wheareas it is mostlymutant after 2 h of chase. Thus, the mutant precursor ismisfolded and retained intracellularly, where it is eventuallydegraded.

HHT1 missense mutants expressed in COS-1 cells are notfully processed and not detectable at the surface

To establish whether missense proteins were expressed at thecell surface, we generated each mutation by overlap PCR (seeMaterials and Methods; Fig. 3B). COS-1 cells transientlytransfected with each mutant were compared with those trans-fected with normal endoglin by both metabolic labeling andcell surface biotinylation. As shown in Figure 3A, all fourmissense codons fall within the first 200 amino acids, wherethe P3D1 epitope was mapped, whereas the P4A4 epitope fallsoutside this region. Thus, we expected that at least one of thesetwo antibodies would react with each mutant protein. Aftermetabolic labeling for 4 h (Fig. 6A), the mutants G52V andC53R were barely detectable (compare lanes 1 and 2 with lanes7 and 8). The total expression in COS-1 cells of these mutantsrelative to normal endoglin was 3 and 8% with mAbs P3D1

and P4A4, respectively (Fig. 6B). Mutant W149C was ondetectable with mAb P4A4 (lanes 3 and 9) at 16% of norm(Fig. 6B). Mutant L221P exhibited the highest expressiolevel, 24% compared with normal, but mAb P3D1 was moreactive with this mutant than mAb P4A4. The metabollabeling also showed that the mutants co-migrated with tpartially processed precursor (P) of normal endoglin and wethus not fully processed, and would be degraded intracellulaThe highest levels of mutant detected relative to normprecursor (P) (Fig. 6C) were 9 (G52V), 17 (C53R), 45 (W149Cand 110% (L221P). Cell surface biotinylation (Fig. 6A and Drevealed that only proteins with the mutations W149C aL221P were detectable at the surface of COS-1 transfectabut at levels of 3 and 9% of normal endoglin. In transfectiosystems, expression levels are very high, thus a smpercentage of misfolded proteins might escape degradationbe detectable at the cell surface. These data suggest thafour missense mutants are not processed fully and are preas transient species degraded intracellularly.

Since pulse–chase experiments of HUVEC sample H3(C53R) suggested that the precursor was more stable and amulated intracellularly, we studied this mutant by pulse–chaexperiments in COS-1 cells (Fig. 7). At time 0, only precurswas observed for both the C53R mutant and normal endogLevels of expression relative to normal were 40% witRMAC8 and 13% with P4A4 (Fig. 7 compare lanes 2 and 3As the mAb RMAC8 reacts with the C-terminal portion of thextracellular domain, whereas the C53R substitution liwithin the N-terminal portion, it is likely to be more efficient adetecting this mutant. This also indicates that the C53R mutis fully synthesized. A pAb to human endoglin also preciptated the mutant equally well as P4A4 (data not shown). Thit is critical to use mAbs reacting with different antigeniregions of endoglin (Fig. 3A) when analyzing expressionmutant forms. After a 3.5 h chase no fully glycosylated for(E) of the mutant C53R is expressed at the cell surface, whnormal endoglin is almost exclusively found (Fig. 7, compalanes 6 and 8 with lane 7). The C53R mutant is still detectabbut only as a precursor, intracellular form (Fig. 7, P, lane 7The level of this mutant protein was higher when detected wRMAC8 mAb than with P4A4 and similar to levels of mutanW149C shown in Figure 6. Thus, the COS cell data confirthat missense mutants are expressed, but only as intracelforms.

DISCUSSION

ENDOGLIN is the gene mutated in HHT1. It is expressedhigh levels on endothelial cells, but at lower levels on activatmonocytes. We previously used both cell types to analyexpression of normal and mutant endoglin in samples froHHT1 patients (22). Since endothelial cells from adult patienare not readily available, we are limited to the use of peripheblood to analyze endoglin expression in these HHT1 patienBy flow cytometry, we now confirm that maximum endoglinexpression is reached on activated monocytes within 1 dayculture and is not further increased even after 5 days. We ademonstrate by two color flow cytometry that in peripherblood only cells of the myeloid lineage (CD14+) express endo-glin, and at low levels. These endoglin-positive cells represeonly 4% of total PBMCs. Enrichment of the monocyte popul

Figure 5. Pulse–chase analysis of endoglin in HUVECs expressing the C53Rmissense mutation. (A) H319 and normal HUVECs were pulsed with[35S]methionine for 20 min and chased with medium containing Met. At each timepoint, extracts were prepared, immunoprecipitated with mAbs P3D1, P4A4 andRMAC8 to endoglin and fractionated as described in Figure 1A. Total radioactivityin the normal fully glycosylated endoglin (E; 90 kDa) and precursor (P; 80 kDa)was quantitated by PhosphorImager with ImageQuant software. (B) Rate ofappearance of fully glycosylated endoglin (E) for H319 (closed symbols) andnormal HUVECs (open symbols); total pixel values for E relative to the total(E + P) at each time point and for each antibody was plotted versus chase time.Circles, P3D1; squares, P4A4; triangles, RMAC8. (C) Stability of partiallyglycosylated precursor (P) for H319 (closed symbols) and normal HUVECs (opensymbols); total pixel values for P relative to total (E + P) at each time point and foreach antibody was plotted versus chase time. Circles, P3D1; squares, P4A4;triangles, RMAC8.


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tion by attachment to plastic and incubation for 1 day in cultureare required for maximal endoglin expression. After 21 h inculture, 99% of activated monocytes expressed twice as muchendoglin as monocytes at time 0. By metabolic labeling,expression of fully processed endoglin was activated within5 h in culture and was at 70% of the maximum observed after21 h in culture. Thus, in order to measure levels of endoglin onmonocytes, either by flow cytometry or metabolic labeling,activation for 21 h in culture is optimal.

Mutations resulting in reduced protein expression as seen forHHT1 may arise from a number of mechanisms, such as dele-tions and truncations caused by nonsense and frameshiftmutations, as well as some promoter and splice site mutations(30). Missense mutations are more likely to be dominant-

negatives or they might enhance or disrupt protein functionexpressed at normal levels, such mutants could revealfunctional residues. To test this possibility, we analyzemutant proteins corresponding to four missense mutatioFully glycosylated endoglin in activated monocytes from clinically affected patients having missense mutations in exons4 and 5 was reduced to mean values ranging from 28 to 61%normal (Table 1). As multiple patient samples are difficult tobtain, in particular from elderly patients, as is the case wH278 (where a value of 71% was observed with mAb P3D1experimental variation must be considered. In an independstudy, three separate experiments performed with a confirmHHT1 patient gave endoglin levels of 35± 11, 42± 8 and 60±10% of normal. Furthermore, the variation observed doesreflect the mutationper se, as evidenced by the differenceobserved for H319, with a mean level of 38% relativenormal, compared with 61% for H278 carrying the sammissense mutation. Recently, we have obtained data for tpatients from another family carrying a missense mutationcodon 383. Analysis of monocyte cultures from both thepatients revealed 63± 7 and 28± 5% of normal levels. Thus,there is experimental variation in the level estimated forsingle patient as well as for different family members with thsame mutation. The range of all mean values obtainedaffected individuals in all HHT1 families analyzed to date~30–70% (32). There is also variation in the level of endogestimated for normal individuals with mean values ranginfrom 73 to 140% with an average of 100± 35% (33). Alongwith experimental variation, the range of expression couindicate that endoglin gene transcription is not stably inducand might reflect differences in initiation and deactivation otranscription. In a recent publication, where a stochastic moof gene expression was used to estimate product levels frone and two alleles, it was demonstrated that fluctuations frthe expected values of 50 or 100% product were greater whonly one allele is expressed (34). Thus, even in an ideal repsentation of haploinsufficiency, such as a null mutation, texpression level from the normal allele will deviate from ththeoretical 50%.

Serious complications of HHT, such as pulmonary arterivenous malformation (PAVM), were reported in all four famlies, while cerebral arteriovenous malformation (CAVM) wafound in three of them, indicating that missense mutations ccause as severe a phenotype as other types of HHT1 mutaThis is consistent with a previous study where 30 individuafrom eight different HHT families were analyzed with respeto age of presentation and severity of clinical symptoms. Thewas a lack of correlation between genotype and clinical mafestations, supporting a haploinsufficiency model (23). So f>60 families with reduced endoglin expression have been idtified and only four mutant proteins have been detected, alowith the present four detected in this study; in all cases mutproteins were transiently expressed, intracellularly (22; unpulished data).

As proteins with missense codons are nota priori distinctfrom normal ones in patient samples, we expressed aanalyzed these mutants in COS-1 cells. By metabolic labeliwe found mutant proteins expressed in COS-1 transfectantsmigrating with partially processed normal endoglin precursoNone of these mutant proteins was significantly expressedthe cell surface, indicating that they are retained intracellula

Figure 6. Expression of endoglin with missense codons in COS-1 cells. COS-1cells were transiently transfected with pcEXV-EndoL (normal endoglin) andpcEXV constructs containing the generated missense codons in endoglin G52V,C53R, W149C and L221P (as illustrated in Fig. 3). (A) Transfected cells werelabeled with [35S]methionine for 4 h, solubilized in Triton X-100,immunoprecipitated with mAbs P3D1 (lanes 1–6) and P4A4 (lanes 7–12),analyzed and quantitated as in Figure 1A. Lanes 13 (P3D1) and 14 (P4A4)represent images of normal endoglin as seen with the PhosphorImager withImageQuant software and were used to identify both fully glycosylated endoglin(E) and partially glycosylated precursor (P) for quantitation. Cells were also surfacelabeled with biotin and extracts containing equivalent total protein from surfacelabeled transfectants were immunoprecipitated with mAbs P3D1 (lanes 1–6) andP4A4 (lanes 7–12). Eluates were fractionated, gels were transferred to PVDFnylon membranes, probed with streptavidin–horseradish peroxidase and detectedby enhanced chemiluminescence. Multiple exposures were obtained and gels werequantitated using a densitometer and ImageQuant software. (B) The total pixelvalues for all bands in each lane were calculated and background fromcorresponding regions in the empty vector (pcEXV-1) control was subtracted. Thevalues for each missense mutant transfectant were plotted relative to normalendoglin transfectant. (C) As metabolically labeled mutant proteins co-migratedwith the partially glycosylated normal precursor, total pixel values for eachprecursor minus background from empty vector control were plotted as apercentage of normal precursor. (D) The total amount of mutant protein measuredby surface labeling from all bands in each transfectant was expressed relative tothat obtained for normal, after subtracting background (vector alone) from each.Note the difference in scales of the histograms.


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as transient species. This is in agreement with patient sampleswhere elevated precursor levels were noted, suggesting amixture of mutant and normal precursors. Pulse–chase experi-ments with HUVEC H319 demonstrated that precursor accu-mulated in particular after 2 h of chase. Yet, the rate ofprocessing of endoglin produced from the normal allele wasnot affected. In addition, pulse–chase experiments in COS-1cells confirmed that mutant proteins are indeed expressed,albeit intracellulary and not at the cell surface.

Mutations leading to a stable cell surface expressed proteinassociated with HHT1 have yet to be described. Such mutantswould be beneficial in structure–function analyses. However,from the analysis of expression of the missense mutants andconsidering effects of these specific mutations on structure,one can begin to ascertain key residues in protein folding. Theuse of mAbs reactive with epitopes mapping to the differentregions of the extracellular domain of endoglin is critical in theanalysis of mutant proteins (31). Mutations in exons 2 and 4were more disruptive than that in exon 5, as judged from thelevel of precursor protein produced. The mutation created atC53 is located 13 residues downstream from C40; bothcysteines are conserved in endoglin and betaglycan amongstspecies (35,36) and likely form an intracellular disulfide bond.Replacing C53 with R, a positively charged residue, did notdisrupt dimer formation (data not shown), but altered foldingsuch that the P3D1 epitope was completely destroyed, P4A4reactivity was reduced and maximum reactivity was observedwith RMAC8 (Fig. 3). The mutation creating G52V is equallydisruptive and this mutant protein also dimerizes. This regionmust be required for proper folding of the N-terminal domain,early in the synthesis of endoglin. As two or more consecutivedomains are thought to fold co-translationally, disruption ofthis domain likely results in a completely misfolded proteinthat would be degraded in the endoplasmic reticulum (37).Introduction of a C at position W149 could also be disruptingformation of a disulfide bond. The fourth mutation in exon 5,converting L221 to P, likely disrupts folding, as it occurs in apredicted α-helix (38,39). P4A4 reactivity, which mapped

previously to a region downstream of this position (Fig. 3),completely abolished. This indicates structural alteratiobeyond the mutation at L221 and implies that the P4Aepitope, although still detectable in a synthetic denaturprotein (31), requires proper folding for maximum detection

We conclude from our studies that these missense mutatilead to transient intracellular species and are not significanexpressed at the cell surface. To date, all HHT1 patieanalysed at the protein level have revealed reduced levelsfully glycosylated normal endoglin at the cell surfaceEndothelial cells express ~106 endoglin molecules on the cellsurface, where it is known to function and interact with thTGF-β receptor complex (7,40). Cell surface endogliproduced from the normal allele in HUVECs expressing a trasient intracellular mutant with an intact transmembrane regwas previously found to function normally and thus interawith the TGF-β1 receptor complex (22). We demonstrate thin HHT1 patients the missense mutants are also intracellumisfolded proteins that by virtue of their localization woulnot alter the function of the normal allele expressed at the csurface. These data are consistent with haploinsufficienbeing the predominant mechanism associated with this genvascular disorder.

MATERIALS AND METHODS

Cell culture and transfections

All materials were from Gibco BRL, Canadian Life Technologies (Burlington, Ontario, Canada), unless otherwise specifiPBMCs were obtained from whole venous blood as describ(22). Briefly, blood samples were depleted of erythrocytesgravity sedimentation through Dextran T-500 and mononclear cells recovered by Ficoll-Paque density gradient centrigation. Separation of monocytes from lymphocytes wachieved by adherence to plastic for 1 h at 37°C. This adher-ence step initiates the activation of monocytes into a macphage-like phenotype. Lymphocytes were collected and ufor DNA isolation. Monocyte layers were then washed anincubated in culture for periods of time varying from 4 h todays, to determine optimal conditions of endoglin expressio

HUVECs were derived from newborns of HHT1 familieand control babies by previously published procedures (4For assays, equivalent cell densities and passage numbersemployed with patient and matched controls as published (2

COS-1 cells were maintained and transiently transfectwith expression constructs using the DEAE/Dextran/chlorquine method as reported (42,43). Assays were performedays post-transfection.

Antibodies

P3D1 and P4A4 mAbs were provided by Dr E.A. Wayne(Seattle, WA). RMAC8 was obtained via the 5th InternationLeukocyte Differentiation Workshop (44). The mAbs wershown to react with epitopes located between the N-termiresidues E26 and G230 for P3D1, between residues Y277G331 for P4A4 and 44G4 (38,45) and between G331 and G5for RMAC8 (31). These epitopes are depicted in Figure 3 aare shown relative to the four missense mutations studiedthis paper. FITC-conjugated F(ab′)2 goat anti-mouse IgG was

Figure 7. Pulse–chase analysis in COS-1 cells expressing recombinant endoglinand the mutant C53R. COS-1 cells were transiently transfected with pcEXV-LEndo (normal endoglin), the pcEXV construct containing the generated C53Rmissense codons in endoglin and a combination of both. Transfected cells werepulsed with [35S]methionine for 20 min and chased with medium containing Metfor 0 and 3.5 h. At each time point, extracts were prepared, immunoprecipitatedwith mAbs P4A4 and RMAC8 and fractionated as described in Figure 1A. Totalradioactivity in the normal fully glycosylated endoglin (E; 90 kDa) and precursor(P; 80 kDa) was quantitated by PhosphorImager with ImageQuant software.


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purchased from Tago (Burlingame, CA). Rhodamine (RD1)-conjugated MY4 (IgG2b) reactive with CD14, and mAb MO-1,reactive with CD11b, were used as markers of the myelomono-cytic lineage and were obtained from Coulter Electronics(Hialeah, FL), as were IgG2b and IgG1 isotype controls.

Patient samples and mutation analysis

Informed consent was obtained from all individuals participatingin the study, including clinically diagnosed HHT parents or theirspouses, in the case of newborn umbilical cord samples. Positivediagnoses were based on established criteria (1,46). All patientsand families were given a number and patients are referred to withthe prefix H (for HHT). Patients H295 and H278 (as well as babyH319) belong to large families whose clinical history and linkageto 9q33 were previously reported to have substitutions G155T andT157C in exon 2 of theENDOGLINgene, leading to the missensecodons G52V and C53R (24). In this paper, these two families aredesignated families 89 and 85, respectively. Patient H150, alsoincluded in this study, belongs to a family designated here family5. This family was previously described as having the missensemutation G447C that creates a W149→C conversion (24). PatientH277, with the novel missense mutation T662C in exon 5 creatingan L→P substitution at codon 221, belongs to family 84.

All mutations in individuals tested in the study wereconfirmed by sequencing as well as in two additional clinicallyaffected members from family 5. Genomic DNA was isolatedfrom peripheral blood lymphocytes using DNAzol reagent(Gibco BRL). Appropriate exons were amplified by PCR andsequenced using a cycle sequencing protocol described (22)and primers reported previously (6). Products were resolved ona MicroGeneBlaster Sequencer (Visible Genetics, Toronto,Ontario, Canada). Positions of these mutations are shown inFigure 3A.

Preparation of expression constructs

Full-length endoglin cDNA in the mammalian expressionconstruct pcEXV-EndoL was provided by Dr C. Bernabeu(Centro de Investigaciones Biológicas, CSIC, Madrid, Spain)(47). Four missense mutations in endoglin cDNA were generatedby an overlap PCR strategy (Fig. 3B). The unique restriction sitesSacII, at position 32 within the insert of pcEXV-EndoL, andSbfI, atposition 772, were used to subclone final mutagenized fragments.Outer primers (forward, AX5, 5′-CTGCAGGGAATTCCGT-GGACAGCAT-3′; reverse, AX3, 5′-AGATCTGCATGTTGT-GGTTGGCGTCGAT-3′) were thus designed to flank these tworestriction sites and encompass all four missense mutations.Complementary mutagenic primers were designed for eachmutation:

G155T (M1X5, 5′-ACTAGCCAGGTTTCGAAGGTCTGCGTGGCTCA-3′, andM1X3, 5′-TGAGCCACGCAGACCTTCGAAACCTGGCTAGT-3′);T157C (M2X5, 5′-ACTAGCCAGGTTTCGAAGGGCCGCGTGGCTCA-3′, andM2X3, 5′-TGAGCCACGCGGCCCTTCGAAACCTGGCTAGT-3′);G447C (M3X5, 5′-AGACCCAGATCCTTGAGTGCGCAGCTGAGAG-3′, andM3X3, 5′-CTCTCAGCTGCGCACTCAAGGATCTGGGTCCTGCCG-3′);T662C (M4X5, 5′-ACAAGGAGGCGCACATCCCGAGGGTCCTGCCG-3′, andM4X3, 5′-CGGCAGGACCCTCGGGATGTGCGCCTCCTTGT-3′).

The mutations generated with each set of primers are in bold.For M1 and M2, a T was introduced instead of a C, shownunderlined. This destroys theBsaI site, but the codon remainsunaffected. For M3 the mutation creates anFspI site and for

M4 the mutation destroys anSauI site. The first PCR consistedof eight reactions using pcEXV-EndoL as template and AXwith each mutagenic reverse primer and AX3 with each mugenic forward primer, as shown in Figure 3B. An aliquot of 2ng of template was amplified in a 100µl volume containingPerkin-Elmer reagents (Perkin-Elmer, Roche MoleculSystems, Branchburg, NJ) in PCR reaction buffer: 2.0 mMgCl2, 200 nM each dNTP, 200 ng of primers, 2 U AmpliTaq(Perkin-Elmer, Roche Molecular Systems) and 1 U VenR(New England Biolabs, Mississauga, Ontario, Canada) DNpolymerases. Cycling conditions were: two cycles of 5 min94°C, 3 min at 58°C, 3 min at 72°C, then 23 cycles of 30 s at94°C, 30 s at 58°C, 60 s at 72°C, and a final extension of 5 minat 72°C. PCR products were fractionated by agarose gel eltrophoresis, excised and purified with Qiaex (Qiagen, Chaworth, CA). The second PCR consisted of four reactions uspurified fragments complementary to the mutagenic primeand AX5 with AX3 outer primers to create the full-lengthproducts containing the mutations. Cycling conditions wethe same. All four products of 827 bp were subjected to restrtion analysis to confirm the presence of mutations, then pufied as above. Mutagenized fragments were extracted wchloroform:isoamyl alcohol (24:1), then precipitated prior tdigestion withSacII andSbfI to produce 740 bp fragments thawere subcloned into pcEXV-EndoL. PCR was performedpicked bacterial colonies, followed by restriction analysiPositive clones were sequenced in both forward and revedirections with primers just upstream (forward, 5′-AAGAACTGCTCCTCAGTGAT-3′) and downstream(reverse, 5′-AAGGAGTATTCTCCAGT-3′) of AX5 and AX3,respectively, by ACGT (Toronto, Canada).

Flow cytometry

Normal PBMCs were obtained and monocytes were enrichby adherence, activated and incubated in culture as descriabove. Adherent cells were released with 10 mM EDTAphosphate-buffered saline (PBS) (Ca2+, Mg2+-free), washedand resuspended in PBS containing 2% fetal bovine serum.samples were incubated with 2.0% autologous plasma at estaining step to prevent Fc receptor-mediated binding. Fsingle color flow cytometry, PBMCs from 60 ml of blood wereequally divided and harvested after 21, 45 and 114 h. Activamonocytes were incubated with saturating amounts of m44G4 or the IgG1 isotype control followed by FITCconjugated F(ab′)2 goat anti-mouse IgG. For two color flowcytometry, PBMCs from 60 ml of blood were also equalldivided and used at time 0 and after 1 and 21 h adherenPBMCs or activated monocytes were then incubated wsaturating amounts of IgG–RD1 and MY4–RD1, followed bextensive washing and incubation with mAb P4A4 or IgGplus FITC-conjugated F(ab′)2 goat anti-mouse IgG. A gate wasset on CD14+ cells and these were analyzed for CD105 expressiPercentage of positive cells and mean fluorescence intenwere determined relative to negative controls. All samplwere analyzed on the FACScan (Becton Dickinson, MountaView, CA) and in each experiment an unstained sample wused for compensation and calibration of the FACScan. FITwas detected with the FL1 detector and RD1 with the FLdetector.


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Metabolic labeling and pulse–chase experiments

Endoglin expression in activated monocytes from 20–30 ml ofblood, equivalent numbers of HUVECs or transfected COS-1cells was quantitated by metabolic labeling according topublished procedures (22). Briefly, cells were incubated with100 µCi/ml [35S]methionine (Trans-label; ICN Pharmaceuti-cals Canada, Montreal, Quebec, Canada) in methionine-freeDulbecco’s modified Eagle’s medium (DMEM) (low glucose;Gibco BRL) for 4 h, solubilized in lysis solution containing 1%Triton X-100, equivalent counts per minute (c.p.m.) valueswere estimated by trichloroacetic acid precipitation and lysatesimmunoprecipitated with saturating amounts of antibodies(P3D1, 4 µg; P4A4, 1.0 µg). Immune complexes werecollected with bovine serum albumin-adsorbed Gamma-bind GSepharose (Pharmacia Biotech Canada, Montreal, Quebec,Canada), washed, eluted, equivalent c.p.m. values were frac-tionated by SDS–PAGE (4–12% gradient gels; Novex Experi-mental Technology, San Diego, CA) and gels were eithersubjected to fluorography or autoradiography with BioMaxMS film and the BioMax TranScreen LE intensifying screensystem (Eastman Kodak, Rochester, NY). Multiple exposuresof each experiment were obtained. Gels were then quantitatedusing a PhosphorImager and ImageQuant Software (MolecularDynamics, Sunnyvale, CA) as described. For pulse–chaseexperiments using HUVECs, equivalent cell numbers werepulse labeled for 20 min with 250µCi/ml [35S]methioninefollowed by two washes, then incubated further in serum-freeDMEM for a chase period of between 0 and 3.5 h. Cells werelysed and analyzed as described above.

Cell surface biotinylation

Equivalent numbers of transiently transfected COS-1 cellswere surface labeled with biotin as reported (22). Cells werelysed and immunoprecipitated as described under Metaboliclabeling, except that lysates were precleared for at least 1 hwith protein A–Sepharose CL-4B (Pharmacia BiotechCanada). Eluates were fractionated on 4–12% SDS–PAGEgels, electrotransferred to PVDF nylon membranes for 1 h at45 V, blocked for 1 h in TBS-T (0.02 M Tris, pH 7.5, 0.137 MNaCl, 0.1% Tween-20) containing 5% dry skimmed milkpowder and probed with streptavidin–horseradish peroxidase(400-fold dilution in TBS-T; Amersham Life Sciences,Oakville, Ontario, Canada) for 20 min. Biotinylated proteinswere detected using enhanced chemiluminescence (AmershamLife Sciences); multiple exposures were obtained. Bands onautoradiographs were quantitated using a densitometer andImageQuant Software (Molecular Dynamics).

ACKNOWLEDGEMENTS

We gratefully acknowledge all the patients who participated inthe study and the support of the HHT Foundation International.We are also grateful to J. MacDonald and Dr A.E. Guttmacherfor providing clinical information on the patients. We thank DrC. Bernabeu (Madrid, Spain) for providing the expressionconstruct END and the pAb to endoglin, Dr E.A. Wayner(Seattle, WA) for mAbs P3D1 and P4A4, L. Gunaratnam forassisting on establishing conditions for the macrophagecultures, K.A. Galley and Dr G. Boileau for advice on site-

directed mutagenesis, and Dr J. Wrana for insightful criticisand helpful discussions. We thank Visible Genetics Inc. ffinancial support and use of the MicroGeneBlaster SequencThis work was supported by grant no. NA3434 from the Heaand Stroke Foundation of Ontario and from a grant from tMedical Research Council of Canada.

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Expression analysis of four endoglin missense mutations suggests that haploinsufficiency is the...

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