Báo cáo khoa học: Pigment epithelium-derived factor binds to cell-surface F1-ATP synthase pdf

Structured digital abstract l MINT-7711286 : angiostatin uniprotkb: P00747 physically interacts MI:0915 with F-ATPase alpha subunit uniprotkb: P07251 , F-ATPase beta subunit uniprotkb

F1-ATP synthase

Luigi Notari1, Naokatu Arakaki1,2, David Mueller3, Scott Meier3, Juan Amaral1and S P Becerra1

1 Section of Protein Structure and Function, Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, NIH, Bethesda, MD, USA

2 The University of Tokushima Graduate School, Japan

3 Department of Biochemistry and Molecular Biology, Rosalind Franklin University of Medicine and Science, The Chicago Medical School, IL, USA

Keywords

endothelial cells; F 1 -ATPase; F 1 F o -ATP

synthase; PEDF; surface plasmon

resonance

Correspondence

S P Becerra, NIH-NEI, Building 6, Room

134, 6 Center Drive, Bethesda, MD

20892-0608, USA

Fax: +1 301 451 5420

Tel: +1 301 496 6514

E-mail: becerrap@nei.nih.gov

Website: http://www.nei.nih.gov/intramural/

protein_struct_func

(Received 5 October 2009, revised 25

January 2010, accepted 3 March 2010)

doi:10.1111/j.1742-4658.2010.07641.x

Pigment epithelium-derived factor (PEDF), a potent blocker of angiogene-sis in vivo, and of endothelial cell migration and tubule formation, binds with high affinity to an as yet unknown protein on the surfaces of endothe-lial cells Given that protein fingerprinting suggested a match of a

 60 kDa PEDF-binding protein in bovine retina with Bos taurus F1-ATP synthase b-subunit, and that F1Fo-ATP synthase components have been identified recently as cell-surface receptors, we examined the direct binding

of PEDF to F1 Size-exclusion ultrafiltration assays showed that recombi-nant human PEDF formed a complex with recombirecombi-nant yeast F1 Real-time binding as determined by surface plasmon resonance demonstrated that yeast F1 interacted specifically and reversibly with human PEDF Kinetic evaluations revealed high binding affinity for PEDF, in agreement with PEDF affinities for endothelial cell surfaces PEDF blocked interac-tions between F1and angiostatin, another antiangiogenic factor, suggesting overlapping PEDF-binding and angiostatin-binding sites on F1 Surfaces of endothelial cells exhibited affinity for PEDF-binding proteins of 60 kDa Antibodies to F1b-subunit specifically captured PEDF-binding components

in endothelial plasma membranes The extracellular ATP synthesis activity

of endothelial cells was examined in the presence of PEDF PEDF signifi-cantly reduced the amount of extracellular ATP produced by endothelial cells, in agreement with direct interactions between cell-surface ATP synthase and PEDF In addition to demonstrating that PEDF binds to cell-surface F1, these results show that PEDF is a ligand for endothelial cell-surface F1Fo-ATP synthase They suggest that PEDF-mediated inhibi-tion of ATP synthase may form part of the biochemical mechanisms by which PEDF exerts its antiangiogenic activity

Structured digital abstract

l MINT-7711286 : angiostatin (uniprotkb: P00747 ) physically interacts ( MI:0915 ) with F-ATPase alpha subunit (uniprotkb: P07251 ), F-ATPase beta subunit (uniprotkb: P00830 ), F-ATPase gamma subunit (uniprotkb: P38077 ), F-ATPase delta subunit (uniprotkb: Q12165 ) and F-ATPase epsilon subunit (uniprotkb: P21306 ) by competition binding ( MI:0405 )

l MINT-7711113 : angiostatin (uniprotkb: P00747 ) physically interacts ( MI:0915 ) with F-ATPase epsilon subunit (uniprotkb: P21306 ), F-ATPase delta subunit (uniprotkb: Q12165 ), F-ATPase

Abbreviations

BREC, bovine retina endothelial cell; COX-I, cytochrome c oxidase; EBM2, Endothelial Cell Basal Medium-2; F1-ATPase, the F1 portion of ATPase; F1Fo-ATP synthase, ATP synthase; hF1, human F1-ATPase; HMVEC, human microvascular endothelial cell immortalized with telomerase; HUVEC, human umbilical vascular endothelial cell; K1–3, kringles 1–3; K1–5, kringles 1–5; PEDF, pigment epithelium-derived factor; PEDF-R, pigment epithelium-derived factor receptor; SPR, surface plasmon resonance.

Pathological vessel growth in the posterior segment of

the eye can perturb the structure and morphology of

the retina, and lead to visual loss If this angiogenesis

is prevented, retinal degeneration is dramatically

restricted Therefore, endogenous angiogenic inhibitors

are likely to play an important role in ocular

neovas-cularization development Pigment epithelium-derived

factor (PEDF) is a potent antiangiogenic,

neurotroph-ic and antitumorigenneurotroph-ic factor [1–5] It is an

extracellu-lar protein present in the interphotoreceptor matrix

and vitreous [6,7], believed to be responsible for the

avascularity of these compartments under

physiologi-cal conditions Moreover, the concentrations of PEDF

in the eye are inversely correlated with ocular

angio-genic development, and overexpression of PEDF or

local PEDF protein delivery prevents ocular

neovascu-larization and tumorigenesis, and delays retinal cell

death in vivo [4,8–18] PEDF induces endothelial cell

apoptosis, inhibits the proliferation and migration of

endothelial cells, and blocks the formation of

endothe-lial capillary-like networks and vessel sprouting

ex vivo from chick aortic rings [1,18,19] However,

little is known about the molecular mechanisms by

which PEDF functions to regulate endothelial cell

behavior

PEDF is a member of the serpin superfamily by

structural homology, but does not have inhibitory

activity against serine proteases [20] Its biological

activities are associated with receptor interactions at

cell-surface interfaces and changes in protein

expres-sion There is evidence for high-affinity PEDF-binding

sites and proteins in retinoblastoma cells, normal

ret-ina cells, cerebellar granule cell neurons and motor

neurons, as well as in endothelial human umbilical vein

endothelial cells (HUVECs) [21–24] We have recently

identified an  85 kDa PEDF-binding protein in the

retina that is a phospholipase-linked membrane

pro-tein, termed PEDF receptor (PEDF-R) [25] PEDF has high affinity for this protein, and stimulates its phos-pholipase A enzymatic activity

It is unclear whether the only receptor for PEDF is PEDF-R Studies on PEDF binding partners have also revealed a PEDF-binding protein of  60 kDa in membrane extracts from bovine retinal tissues and retinoblastoma Y-79 tumor cells [21,22] of as yet unknown identity Preliminary investigations by peptide fingerprinting suggested a match of the bovine retinal protein to Bos taurus F1-ATP synthase b-subunit (Table S1) Until recently, F1Fo-ATP synthase expres-sion was assumed to be strictly confined to mitochon-dria, where it generates most of the cellular ATP Current evidence for extramitochondrial expression of its components is derived from immunofluorescence, biochemistry and proteomics studies [26,27] F1Fo-ATP synthase components have been identified as cell-sur-face receptors for apparently unrelated ligands during studies performed on angiogenesis, lipoprotein metabo-lism, innate immunity, hypertension, or regulation of food intake One of these ligands is angiostatin, which also inhibits ocular angiogenesis [28] and is antitumori-genic [29], like PEDF It has been reported that angiostatin binds and inhibits the F1 catalytic domain

of F1Fo-ATP synthase on HUVEC surfaces, leading to inhibition of migration and proliferation of endothelial cells [30–32] HUVECs possess high ATP synthesis activity on the cell surface [33] Extracellular ATP gen-eration by HUVECs can be detected within 5 s after addition of ADP and inorganic phosphate, and is inhibited by mitochondrial F1Fo-ATP synthase inhibi-tors (e.g efrapeptins, resveratrol, and piceatannol) targeting F1[33] Furthermore, these F1-targeting ATP synthase inhibitors can block tube formation and pro-liferation of HUVECs without affecting intracellular ATP levels [33,34] These observations agree with the

gamma subunit (uniprotkb: P38077 ), F-ATPase beta subunit(uniprotkb: P00830 ) and F-ATPase alpha subunit (uniprotkb: P07251 ) by surface plasmon resonance ( MI:0107 )

l MINT-7711060 : F-ATPase gamma subunit (uniprotkb: P38077 ), F-ATPase beta subunit (uni-protkb: P00830 ), F-ATPase alpha subunit (uniprotkb: P07251 ) and PEDF (uniprotkb: P36955 ) physically interact ( MI:0915 ) by molecular sieving ( MI:0071 )

l MINT-7711313 : F-ATPase epsilon subunit (uniprotkb: P21306 ), F-ATPase delta subunit (uni-protkb: Q12165 ), PEDF (uniprotkb: P36955 ), F-ATPase alpha subunit (uniprotkb: P07251 ), F-ATPase beta subunit (uniprotkb: P00830 ) and F-ATPase gamma subunit(uniprotkb: P38077 ) physically interact ( MI:0915 ) by molecular sieving ( MI:0071 )

l MINT-7711083 : PEDF (uniprotkb: P36955 ) physically interacts ( MI:0915 ) with F-ATPase epsilon subunit (uniprotkb: P21306 ), F-ATPase delta subunit (uniprotkb: Q12165 ), F-ATPase gamma subunit (uniprotkb: P38077 ), F-ATPase beta subunit (uniprotkb: P00830 ) and F-ATPase alpha subunit (uniprotkb: P07251 ) by surface plasmon resonance ( MI:0107 )

idea that the mechanisms of blocking angiogenesis

might involve binding and inhibition of the endothelial

cell-surface F1Fo-ATP synthase

In the current study, we examined the potential

interactions between PEDF and ATP synthase We

used highly purified recombinant yeast F1-ATPase and

recombinant human PEDF in size exclusion

ultrafiltra-tion assays and surface plasmon resonance (SPR)

spec-troscopy We also assessed the binding of PEDF to

endothelial cell-surface ATP synthase, and examined

the effect of PEDF on the extracellular ATP synthesis

activity of human microvascular endothelial cells

(HMVECs) and bovine retinal endothelial cells

(BRECs) Our results provide evidence for high-affinity

interactions between PEDF and F1, as well as for

PEDF-mediated inhibition of extracellular ATP

syn-thesis activity in endothelial cells We discuss how

these interactions provide insights into the mechanisms

of action for angiogenesis inhibition

Results

Direct binding of PEDF and F1-ATPase

To investigate the potential interactions between

PEDF and ATP synthase, mixtures of highly purified

recombinant yeast F1-ATPase ( 360 kDa) and human

PEDF (50 kDa) were first assayed by complex

forma-tion Solutions containing F1-ATPase (14.4 lg) and PEDF (2 or 20 lg) were mixed and incubated at room temperature for 1 h before the mixtures were subjected

to size-exclusion ultrafiltration through membranes with 100 kDa exclusion limits (C-100) Figure 1 shows PEDF immunostaining and Ponceau Red staining of bands for F1 subunits and PEDF after SDS⁄ PAGE One-tenth of the reaction mixture was removed before size-exclusion ultrafiltration, and analyzed in separate lanes as control of starting material (Fig 1, lanes 10 and 11) PEDF immunostaining was proportional to the PEDF amount added to the reactions Ponceau Red bands for a-subunits and b-subunits were detected

in both reaction mixtures In lane 10 of Fig 1, the rel-ative intensities of Ponceau Red bands suggested a lower ratio of PEDF to a⁄ b-subunit (about or less than 1 : 10) than in lane 11, in which the band intensi-ties for each a⁄ b-subunit and PEDF appeared at an approximately 1 : 1 molar ratio for these components After ultrafiltration, only the reactions with equimolar amounts of PEDF and a⁄ b-subunits showed detectable levels of PEDF (Fig 1, lane 2), indicating the forma-tion of PEDF complexes with F1-ATPase Omitting

F1-ATPase (Fig 1, lanes 3 and 4) or replacing it with BSA (66 kDa) (lane 9) did not result in PEDF com-plexes A 1 mm Mg2+⁄ ATP combination is known to increase the stability of the F1-ATPase multimeric protein, as detected by an increase in Ponceau Red

PEDF

=α, β

-γ

-PEDF (50 kDa)

BSA- Ova-

CA- BSA- Ova-

CA-PEDF (µg) 2 20 2 20 2 20 0 0 20 2 20

F1-ATPase (14.4 µg) BSA (14.4 µg) ATP/MgCl2(1 mM)

+ + – – + + + + – + + – – – – – – – – + – – – – – – + + – + – – –

1 2 3 4 5 6 7 8 9 10 11

C-100 1/10 rxn

No C-100

Fig 1 Assays for complex formation between soluble recombinant human PEDF and recombinant yeast F1-ATPase Proteins were incu-bated for 1 h at room temperature, and the mixtures were then subjected to size exclusion ultrafiltration, using membranes with size exclu-sion limits of 100 kDa (lanes 1–9, indicated by C-100) The amounts of each component in each reaction mixture are indicated at the top The total protein complexes retained by the membrane for each reaction were applied to lanes 1–9 of a 10–20% polyacrylamide gel, and resolved by SDS ⁄ PAGE One-tenth of the reactions corresponding to lanes 1 and 2 before being subjected to ultrafiltration were applied to lanes 10 and 11 (indicated by 1 ⁄ 10 rxn, No C-100) of the same gel Proteins were transferred from the gel to a western blot, stained with Ponceau Red (bottom blot), and then immunostained with antibodies against PEDF (top blot) The migration positions of PEDF, F 1 a-subunit,

F1 b-subunit and F1 c-subunit are indicated to the right, and those of protein standards to the left (BSA,  66 kDa; Ova, ovalbumin,

 48 kDa; CA, carbonic anhydrase,  31 kDa).

staining of the F1 subunits (see bottom of Fig 1 and

compare lanes 1, 2 and 7 with lanes 5, 6 and 8)

Bind-ing reactions in the presence of 1 mm Mg2+⁄ ATP

resulted in a proportional increase in the amount of

PEDF–F1 complexes (compare lanes 2–6 in Fig 1)

Formation of fluorescein-conjugated PEDF complexes

with F1-ATPase was also observed (Fig S3) These

observations revealed that PEDF bound specifically to

F1complexes

To determine the biophysical binding parameters for

the PEDF–F1-ATPase interactions, real-time SPR

spectroscopy was performed Sensorgrams with PEDF

immobilized on the surface of a CM5 sensor chip

revealed binding response units for the yeast F1

-AT-Pase that were above those of reference cells (without

PEDF) (Fig 2A) They indicated specific, reversible

and concentration–response binding of F1 to PEDF

(Fig 2B) The kinetic parameters for the SPR

interac-tions between F1-ATPase and PEDF were consistent

with 1 : 1 Langmuir binding, implying one-site binding

between F1 and PEDF They revealed high binding

affinities (KD= 1.51 nm) with high association rates

and low dissociation rates between PEDF and

F1-ATPase in vitro (Fig 2B) Similarly, the SPR

inter-actions between F1 and angiostatin kringles 1–5 (K1–

5) were assessed (Fig 2C) Table 1 summarizes the

results obtained with several batches of F1-ATPase

proteins The yeast F1-ATPase had higher affinity for

PEDF surface sensor chips than for angiostatin K1–5

surface sensor chips (> 10-fold) Altogether, these

results implied that soluble and immobilized PEDF

can interact with F1

Competition between PEDF and angiostatin for

F1-ATPase binding

Angiostatin binds the a⁄ b-subunits of F1-ATPase [31]

To determine whether PEDF and angiostatin share a

binding site(s) on F1-ATPase, the SPR interactions

between angiostatin and F1-ATPase were subjected to

competition by PEDF Injections of yeast F1-ATPase

mixed with increasing concentrations of PEDF

decreased the SPR response to angiostatin surface

sen-sor chips in a dose–response fashion (Fig 3A) and

with an estimated half-maximum inhibition, IC50, of

 12 nm PEDF Control injections of yeast F1-ATPase

mixed with PEDF onto PEDF surfaces also decreased

the SPR response of F1-ATPase (Fig 3B; estimated

IC50of 17 nm PEDF), and PEDF by itself was

defi-cient in binding to either surface (data not shown)

Competition between fluorescein-conjugated PEDF

and angiostatin or unmodified PEDF for F1-ATPase

binding was also observed by size-exclusion

ultrafiltra-A

B

C

Fig 2 Real-time SPR binding analyses of F1-ATPase and PEDF inter-actions (A) SPR spectroscopy of recombinant yeast F 1 -ATPase with recombinant human PEDF immobilized on a CM5 sensor chip Sen-sorgrams of SPR responses (relative units, RU) of 200 n M F1-ATPase solutions injected onto surfaces with PEDF or without PEDF (refer-ence surface) are shown (B, C) Sensorgrams were recorded with PEDF (B) or human angiostatin K1–5 (C) immobilized on CM5 sensor chips, and injections of F 1 -ATPase solutions [100, 50, 20, 10, 5, 1 and

0 n M F 1 -ATPase in (B); 500, 300, 200, 100, 50, 20 and 0 n M F 1 -ATPase

in (C)], using a BIAcore 3000 biosensor and BIAEVALUATION software The SPR responses for the blank surface and for the 0 n M F 1 -ATPase were subtracted from the ones obtained at the various concentrations during the evaluation with BIAEVALUATION software (y-axis), and are shown as a function of time (s, x-axis) The kinetic and thermody-namic values were k a (1 ⁄ M · s) = 6.89 · 10 3

; k d (s)1) = 1.04 · 10)5 and KD= 1.51 n M for PEDF in (B), and ka (1 ⁄ M · s) = 962; k d (s)1) = 1.88 · 10)4and KD= 195 n M for angiostatin in (C).

tion (Fig S4) These results indicated that PEDF

effi-ciently blocked the F1-ATPase interactions with

an-giostatin by competing for the anan-giostatin-binding

site(s)

Binding of PEDF to endothelial cell-surface ATP

synthase

As illustrated in Fig 4A,B, PEDF bound to BRECs

with high affinity (KD= 3.04–4.97 nm) and with

39 000–78 000 sites per cell (two different batches of

cells) Competition of radioligand PEDF binding with unlabeled PEDF showed an EC50(4.1–4.6 nm) similar

to the KD The physicochemical parameters of these interactions are in agreement with previously reported ones for the binding of PEDF to HUVECs (KD= 5.2 ± 2.3 nm; Bmax= 42 000–54 000 sites per cell; EC50= 5.1 nm [24]), and the affinity for purified PEDF and yeast F1-ATPase subunits (see above) These results demonstrated that the binding of PEDF

to the surface of endothelial cells was specific, was con-centration-dependent, was saturable, and had high

Table 1 Summary of SPR kinetic parameters for the interactions between yeast F 1 -ATPase and human PEDF or human angiostatin K1–5.

ND, not determined.

SPR

Surface

F 1 -ATPase

(batch No.) Fit method

k a (1 ⁄ M · s ± SE a )

k d (1 ⁄ s ± SE a )

K A (1 ⁄ M ± AVEDEV b )

K D (n M ± AVEDEV b ) PEDF 1 1:1 (Langmuir) binding 8.8 · 10 3 ± 127 5.5 · 10)5± 9.4 · 10)7 1.6 · 10 8 ± 2.7 · 10 6 6.30 ± 0.11

with drifting baseline

6.9 · 10 3 ± 76 1.0 · 10)5± 3.0 · 10)7 6.6 · 10 8 ± 1.9 · 10 7 1.51 ± 0.04 PEDF 1 1:1 (Langmuir) binding 3.6 · 10 4 ± 323 1.7 · 10)4± 7.8 · 10)7 2.1 · 10 8 ± 1.9 · 10 6 4.82 ± 0.04

± 1030 1.5 · 10)4± 8.4 · 10)7 5.6 · 10 8

± 6.8 · 10 6

1.79 ± 0.02

with drifting baseline

8.6 · 10 4 ± 883 7.2 · 10)4± 4.8 · 10)6 1.2 · 10 8 ± 1.2 · 10 6 8.39 ± 0.09

with mass transfer

4.7 · 10 5

± 9200 1.4 · 10)3± 2.5 · 10)5 3.2 · 10 8

± 6.4 · 10 6

3.08 ± 0.06 Angiostatin 2 1:1 (Langmuir) binding 1.3 · 10 3 ± 29 2.0 · 10)4± 2.2 · 10)6 6.5 · 10 6 ± 1.5 · 10 5 154 ± 3.5 Angiostatin 2 1:1 (Langmuir) binding 0.5 · 10 3

± ND 6.9 · 10)5± ND 7.3 · 10 6

Angiostatin c 3 1:1 (Langmuir) binding 0.96 · 10 3 ± 4.5 1.9 · 10)4± 2.9 · 10)6 5.1 · 10 6 ± 7.9 · 10 4 195 ± 3.0

a SE values were obtained from the files of the SPR kinetic analyses using the BIAEVALUATION software program b AVEDEV values were calculated from ka± SE and kd± SE values using EXCEL ‘s Statistical functions c An additional SPR bioevaluation estimated the kdvalue to be 2.10E-05 1 ⁄ s and the K D value to be 230 n M for the interactions between F 1 -ATPase (batch No 3) and angiostatin surfaces (P Schuck, per-sonal communication).

500 400 300 200 100 0

Time (s)

PEDF (n M ) 1 10 20 25 100

Angiostatin surface

PEDF surface

Time (s)

350 300 250 200 150 100 50 0

PEDF (n M ) 1 10 100 300

Fig 3 Ligand competition for F 1 -ATPase binding to angiostatin (A) or PEDF (B) surfaces was performed F 1 -ATPase (100 n M ) was premixed with increasing concentrations of PEDF (as indicated), and injected onto each surface for 300 and 250 s, respectively, at a flow rate of

20 mLÆmin)1 Dissociation was performed with running buffer for 600 and 300 s, respectively SPR response differences with respect to blank surfaces were aligned to 0 in the region preceding the injections (D Resp Diff.), and are shown as a function of time Half-maximal inhibition values determined by nonlinear regression of SPR response differences at saturation and dissociation time points as a function of PEDF concentration were as follows: IC50= 11.8 ± 0.3 n M PEDF for angiostatin surface, and IC50= 17.3 ± 2.1 n M PEDF for PEDF surface.

affinity, and suggested that PEDF interacts with a

pro-tein(s) at the surface of endothelial cells

To determine whether the endothelial PEDF-binding

component was related to cell-surface F1Fo-ATP

syn-thase, we prepared subcellular fractions of plasma

membrane proteins from endothelial cells We

con-firmed that they were depleted of mitochondrial

mem-brane markers and contained plasma membrane

markers (Fig 4C) In western blots of

detergent-solu-ble membrane protein fractions from HMVECs and

BRECs, we detected proteins that were

immunoreac-tive to antibody to the b-subunit of human heart

mitochondrial F1Fo-ATP synthase (anti-hF1), which

comigrated with  60 kDa proteins of yeast and human heart mitochondrial F1-ATPase controls (Fig 4D) The b-subunit-immunoreactive band was also detected in plasma membrane extracts from nor-mal bovine retina and human retinoblastoma Y-79 tumor cells However, PEDF-R was undetectable in endothelial cell membrane extracts

SPR interactions of PEDF and endothelial cell membrane proteins

To investigate whether the endothelial cell-surface

F1Fo-ATP synthase binds to PEDF, real-time SPR

0.00 0.01 0.02 0.03

PEDF (n M )

Specific binding (pmoles per point) 0.00 0.01 0.02 0.03

0.0025 0.0050 0.0075 0.0100

Specific binding

1000 2000 3000 4000 5000 6000

Log [PEDF (n M )]

97.4 66.2 45.0 31.0 21.5

Mr × 10–3

-F1β-subunit

-PEDF-R

BRECsY-79 HMVECsBov ret.HH Mit

COX-I

Na+/K+-ATPase

Lys Mb

2 1

1 Fig 4 PEDF binding to endothelial cell surfaces (A, B) Radioligand-binding assays were performed with 2 n M [ 125 I]PEDF and 0–200 n M

unlabeled ligand on BRECs attached to collagen-coated plates at 4 C for 90 min Cells were washed with binding medium, and bound radio-activity was determined in cell extracts detached with 0.1 M NaOH Binding competition with unlabeled PEDF (A), and saturation isotherm, nonlinear regression of transformed binding in function of PEDF concentration (B) with a Scatchard plot in the inset are shown The satura-tion isotherm was calculated by nonlinear regression of transformed binding data in funcsatura-tion of PEDF concentrasatura-tion The Scatchard plot was calculated by linear regression of the transformed binding data Both were determined using GRAPHPAD software (C) Western blots of HMVEC total lysate (Lys) and plasma membrane (Mb) extracts with antibodies against Na + ⁄ K + -ATPase, a plasma membrane marker, and to COX-1, a mitochondrial membrane marker, are shown Samples were loaded onto the gel as follows: lane 1, total homogenate from HMVECs (52 lg of protein); and lane 2, HMVEC membrane fraction (4 lg of protein) (D) Western blots of BREC, Y-79, HMVEC and bovine retinal (Bov ret.) membrane extracts with antibodies to F1b-subunit Western blots of the same samples of HMVECs and bovine retina with antibodies to PEDF-R are also shown (bottom) Detergent-soluble plasma membrane protein fractions were prepared and loaded onto gels

as follows (protein amounts): lane 1, BRECS (8 lg); lane 2, Y-79 cells (8 lg); lane 3, HMVECs (5 lg), and lane 4, bovine retina (5 lg) Lane 5 contained human heart mitochondria (HH Mit.) (1 lg), a positive control for F1-ATP synthase.

spectroscopy was performed with detergent-soluble

plasma membrane extracts from HMVECs on a PEDF

surface sensor chip Sensorgrams revealed binding

response units with injections of membrane extracts

that were above those of reference cells (without

PEDF) (Fig 5A), indicating specific binding of a

com-ponent(s) in HMVEC membranes to PEDF Upon

stopping the injection of extracts, the bound

compo-nents remaining on the PEDF sensor chip become

available to be selectively captured with injections of

specific antibodies This was clearly demonstrated by

capturing purified yeast F1-ATPase on PEDF sensor

chips with polyclonal antiserum against yeast

F1-ATPase (Fig 5D) To determine whether the

PEDF-binding component(s) in endothelial membranes

included F1Fo-ATP synthase, solutions of antibodies

to F1-ATPase were subsequently injected onto the sur-face As shown in Fig 5A, injections of anti-hF1 increased the SPR response units above those of HMVEC plasma membrane extracts In contrast,

an F1-unrelated antibody that immunorecognized

Na+⁄ K+-ATPase in HMVEC plasma membrane extracts (Fig 4A) did not increase the SPR response (Fig 5B), and anti-hF1 alone (control injections) did not bind to the PEDF surface (Fig 5C) Figure 5E,F shows that the F1 b-subunit and the previously identi-fied PEDF-R [14] from bovine retina plasma mem-branes bound to PEDF PEDF-R was undetectable in endothelial cell membranes extracts by SPR capture (L N., personal observations), in agreement with

3400

2600 3000

0

Time (s)

B Sto p

A

b-hF1

p

3000

2200 2600

0

Time (s)

B S p

A

b-R A S p

5200

4960

5080

0

Time (s)

NoneStop A

b-hF1

p

0

Time (s)

1900

1660 1780

HM V

Cs

p

Ab-Na + /K+

-ATPase Sto p

A

b-yF1 4600

3480 4040

0

Time (s)

yF1 Sto p

p

HM V

Cs

5200

4960 5080

0

Time (s)

p

A

b-hF1

p

Fig 5 PEDF-binding proteins in cell membranes from HMVECs and bovine retina SPR spectroscopy on PEDF surfaces of detergent-soluble membrane proteins from HMVECs (A, B) and bovine retina (BR) (E, F), and no extracts (C) and control yeast F1-ATPase (yF1) (D) Antibody capture was performed with antibodies against human F 1 -ATPase b-subunit (Ab-hF 1 ), yeast F 1 -ATPase b-subunit (Ab-yF 1 ) and PEDF-R

(Ab-RA) Protein extracts (34 lgÆmL)1) were injected for 300 s at a flow rate of 20 lLÆmin)1, and after 600 s of dissociation, the flow rate was decreased to 5 lLÆmin)1and specific antibodies (5 lgÆmL)1) were injected for 600 s Sensorgrams relative to the reference surface (without PEDF) are shown Dashed lines in the sensorgrams point to time of injection of proteins and antibodies, as well as cessation of injection.

tern blotting results (see above) Altogether, these

results clearly demonstrated that the ATP synthase F1

b-subunit in plasma membrane extracts of endothelial

cells was a PEDF-binding component They suggest

that interactions of extracellular PEDF ligands with

the b-subunit of F1 on endothelial cell surfaces may

regulate ATP metabolism

Effects of PEDF on the extracellular ATP

synthesis activity of endothelial cells

First, we determined the ATP synthesis activity of

HMVECs The cell-surface ATP synthase activity was

measured by extracellular ATP production after

addi-tion of ADP and inorganic phosphate to intact

HMVECs Extracellular ATP production increased

lin-early during the first 60 s of incubation, whereas the

intracellular ATP levels did not change significantly

with incubation time or when inorganic phosphate was

not included in the reactions (Fig 6A) These results

demonstrate extracellular ATP synthase activity in

these cells, as observed before for HUVECs [33]

Then, we examined the extracellular ATP synthesis

activity of endothelial cells in the presence of PEDF

The cell-surface ATP synthase activity was measured

in HMVECs treated with PEDF for the indicated

peri-ods of time Extracellular ATP generation was assayed

within 60 s after addition of ADP and inorganic phos-phate, in the presence or absence of PEDF Treatment for 30 min with 1 nm PEDF decreased extracellular ATP synthesis (Fig 6B) The positive control, picea-tannol, was also a potent inhibitor, requiring £ 5 min

of preincubation time for effective blocking Other

4000 9000

14 000

19 000

24 000

A

B

C

Time (s)

Extracellular ATP

Intracellular ATP

0 5000

10 000

15 000

20 000

1 min 5 min

Additions

Additions

0 5000

10 000

15 000

20 000

25 000

30 000

35 000

30 min

Fig 6 ATP production by HMVECs (A) Extracellular ATP

produc-tion by and intracellular ATP levels of HMVECs ATP synthesis was

initiated by the addition of a solution containing ADP and inorganic

phosphate to a culture of HMVECs At the indicated times,

extra-cellular medium and intraextra-cellular pools were prepared, and the ATP

content in those pools was determined Each point corresponds to

an average of triplicate samples for: extracellular medium (•);

intra-cellular pools (s); and reactions without inorganic phosphate ( ,

extracellular; ·, intracellular) (B) HMVECs were incubated in

EBM2 ⁄ BSA in the presence of PEDF (1 n M ) or piceatannol (20 l M )

for an increasing period of time (top) Extracellular ATP synthesis

activity was determined after incubation for 60 s with ADP and

inorganic phosphate in the presence of the indicated inhibitors

(x-axis) (C) HMVECs were incubated in EBM2⁄ BSA containing

increasing PEDF concentrations, angiostatin K1–5 or piceatannol for

30 min Extracellular ATP synthesis activity was determined as in

(B) Box-and-whisker plot representations of replicates for

extracel-lular ATP synthesis determination are shown Each point

corre-sponds to a measurement from one well, measurements in each

condition were performed in triplicate wells, and measurements in

all conditions were repeated with three batches of cells Values

inside the boxes correspond to the central 50% of measurements,

the internal horizontal bars correspond to median values, and the

vertical lines outside the boxes correspond to variances of

mea-surements Inhibitor concentrations are indicated on the x-axis.

PEDF and the positive controls angiostatin (10 n M ) and piceatannol

(2 l M ) inhibited ATP synthesis.

investigators have demonstrated inhibition of

extracel-lular ATP synthesis by pretreatment of HUVECs for

30 min with much higher doses of angiostatin

krin-gles 1–3 (K1–3) (50 lm [35]) and piceatannol (1–20 lm

[33]; 500 lm [35]) than those used here As shown in

Fig 6C, pretreatment with PEDF for 30 min inhibited

extracellular ATP synthesis activity in a

dose-depen-dent fashion The range of distribution of the

measure-ments reflected the variability of the assay The median

value of the inhibitory activity of PEDF on

extracellu-lar ATP synthesis varied between 27%, 43% and 53%

with 0.1, 1 and 10 nm PEDF, respectively No

signifi-cant statistical difference was observed between PEDF

and angiostatin at 10 nm (P£ 0.096) Moreover,

treat-ment with PEDF or angiostatin for up to 48 h did not

decrease the intracellular levels of ATP; if anything, it

slightly increased them (Fig S1) These results

demon-strated that extracellular PEDF additions inhibited the

extracellular ATP synthesis activity of endothelial cells

Discussion

PEDF, a potent inhibitor of neovascularization, targets

endothelial cells [3] We have shown that PEDF

directly binds and inhibits endothelial cell-surface

F1Fo-ATP synthase These two proteins interact when

they are in solution and when either one is

immobi-lized PEDF can bind recombinant yeast F1 in a

puri-fied version or native mammalian F1 in membrane cell

extracts or in intact cells We observed that PEDF

chemically modified at primary amines (e.g

fluorescein-conjugated PEDF) also binds to F1-ATPase (Figs S3

and S4) The interactions are specific, reversible, and of

high affinity, and take place between PEDF and the F1

b-subunit Furthermore, inhibition of extracellular

ATP synthesis in intact endothelial cells demonstrates

that the PEDF interaction blocks the structural

deter-minant required for the activity of the cell-surface ATP

synthase PEDF shares these properties with

angiosta-tin, and the observed competition for binding to

F1-ATPase between these two factors implies that the

b-subunit of F1-ATPase has an overlapping site(s) for

binding both proteins These conclusions suggest that

interactions between extracellular PEDF ligands and

the F1 b-subunit on endothelial cell-surfaces may

regu-late ATP metabolism They imply that inhibition of

ATP synthase may form part of the biochemical

mechanisms by which PEDF exerts its antiangiogenic

activity

Previous reports have described a PEDF-binding

protein of 60 kDa in plasma membranes from

HUVECs [36], normal bovine retina [22], and human

retinoblastoma Y-79 tumor cells [21], but have not

shown its identity The present results reveal that the

 60 kDa PEDF-binding protein is the b-subunit of

F1Fo-ATP synthase in endothelial cells, as well as in retina and Y-79 cells (Figs 4D and 5E,F; Table S1 [21,22]) Other subunits of the F1Fo-ATP synthase holoenzyme, such as the a-subunits and b-subunits of

F1, and the b-subunits and d-subunits of Fo, have also been identified in plasma membranes of HUVECs, several tumor cells, adipocytes, and myocytes [27] Interestingly, the entire F1Fo-ATP synthase has demonstrable activity in the endothelial cell-surface, with the ability to synthesize ATP and transport pro-tons [27,30] Our data provide further lines of evidence for the extramitochondrial expression of ATP synthase

in the surfaces of endothelial cells The presence of the

F1 b-subunit in retinoblastoma Y-79 cell surfaces

is consistent with previously reported expression of

F1Fo-ATP synthase in tumor cell surfaces [27], and suggests a role for interactions between cell-surface

F1Fo-ATP synthase and PEDF in mediating differenti-ating activity in retinoblastoma cells PEDF affinity column chromatography of plasma membrane extracts revealed different migration patterns of PEDF-binding proteins among bovine retinal cells, Y-79 cells, and BRECs (Fig S2 [21,22]) All gave bands corresponding

to F1-ATPase a⁄ b-subunits of  60 kDa, but only bovine retinal cells and Y-79 cells gave detectable bands for PEDF-R of  85 kDa Peptide fingerprint-ing of the PEDF-bindfingerprint-ing protein of 60 kDa matched it

to the F1-ATPase b-subunit (Table S1) The inability

to detect PEDF-R in endothelial cell membranes sup-ports the idea that endothelial cell surfaces express a different set of PEDF-binding protein(s) than neural retinal cell surfaces, which may distinctly and specifi-cally trigger angiostatic activities upon interacting with PEDF ligand

We compared the interactions of the purified

F1-ATPase and PEDF proteins, and those that occur with cells The KD values of the SPR binding of yeast

F1-ATPase to immobilized human PEDF match those for the interactions between PEDF and the surface of endothelial cells (KD= 3–7.5 nm) (Fig 4A,B [24]), as well as the concentration of PEDF capable of inhibit-ing about 50% of the maximum extracellular ATP syn-thase activity in HMVECs (Fig 6C) The estimated

IC50 values of PEDF for blocking binding of yeast

F1-ATPase to immobilized angiostatin ( 12 nm) or PEDF ( 17 nm) suggest similar affinities for PEDF when in solution and when immobilized on sensor chips This observation implies that only minimal changes in affinity occurred upon PEDF immobiliza-tion In contrast, angiostatin K1–5 at concentrations

£ 270 nm (five-fold the F1-ATPase concentration) could

not compete with immobilized PEDF on sensor chips

(L N unpublished observations), in agreement with a

lower affinity for the yeast F1-ATPase–angiostatin

interactions In spite of the higher affinity of yeast

F1-ATPase for immobilized human PEDF than for

human angiostatin K1–5 as determined by SPR

(Table 1), no significant statistical difference was

observed between PEDF and angiostatin in inhibiting

endothelial extracellular ATP synthase activity

(Fig 5C) A previously reported value of an apparent

dissociation constant [Kd(app)= 14.1 nm] for binding of

human angiostatin K1–3 to purified bovine heart

F1-ATPase immobilized on plastic [30] suggests higher

affinity for these interactions than for binding of yeast

F1-ATPase to angiostatin K1–5 sensor chips (KD=

130–237 nm; Table 1) The affinity of F1-ATPase–

angiostatin interactions is likely to be species-specific,

and the observed affinity of the F1-ATPase-angiostatin

interaction as determined by SPR is lower than that in

mammalian cells In addition, alterations of structural

determinants in angiostatin that are critical for binding

F1-ATPase might also affect the affinity of these

inter-actions For example, immobilization of molecules on

the SPR sensor chips by conjugation of primary

amines (lysines and N-terminal ends) to the CM5

sur-faces may decrease the affinity of the angiostatin

mole-cule for F1-ATPase As mentioned above, PEDF is not

affected by this Moreover, piceatannol, which is

known to target the catalytic F1-ATPase⁄ ATP synthase

domain at the b-subunit [37], does not affect the SPR

interactions of F1-ATPase with PEDF, either when it

is coinjected or when it is included in the SPR running

buffer (L N and S P B., personal observations) This

implies that the structural determinants required for

binding PEDF and piceatannol do not overlap

Our results have biological implications The

interac-tions of extracellular PEDF ligands with cell surface

F1Fo-ATP synthase molecules may regulate the levels

of ATP and ADP, which in turn may affect the

behavior of endothelial cells; for example, PEDF

may interact with the ATP–P2X and ADP–P2Y

recep-tor-mediated signaling pathways by regulating the

availability of the ATP and ADP ligands, similarly to

angiostatin [27] It has been shown that blocking the

ATP synthase by targeting the F1 catalytic domain

with angiostatin or piceatannol can trigger

caspase-mediated endothelial cell apoptosis, and inhibit the

tube formation and proliferation that are necessary for

antiangiogenesis [32–34] Similarly, blocking the ATP

synthase with PEDF may trigger signal transduction

to mediate apoptosis in endothelial and⁄ or tumor cells

In summary, this is the first report

demonstrat-ing that PEDF binds the endothelial cell-surface

F1-ATPase⁄ ATP synthase b-subunit, and inhibits endothelial extracellular ATP synthesis activity The findings imply that F1Fo-ATP synthase may act as a receptor for PEDF on the surfaces of endothelial cells, and that PEDF can inhibit this extramitochondrial ATP synthase, which catalyzes ATP synthesis The interactions between PEDF and ATP synthase might

be a critical biochemical step for the angiostatic effects exerted by PEDF on the neovasculature

Experimental procedures Proteins

PEDF was human recombinant PEDF, as described previ-ously [38] Recombinant yeast F1-ATPase was obtained and highly purified as described previously [39] Human angiost-atin K1–5 was purchased from Calbiochem (La Jolla, CA, USA) Human angiostatin K1–3 was from Sigma (St Louis,

MO, USA) Polyclonal antibodies directed against the b-subunit of the yeast F1-ATPase were made in rabbits using b-subunit purified from recombinant yeast F1ATPase by SDS⁄ PAGE Mouse monoclonal antibody against human

F1Fo-ATP synthase b-subunit (anti-F1Fo-b; Ab-hF1) (cat

no MS503), and human heart mitochondrial extracts (cat

no MS801-50) were from MitoSciences (Eugene, OR, USA)

Cells HMVECs immortalized with telomerase were a generous gift from R Shao, and were cultured as described previ-ously [40] BRECs were from Vec Technologies, Inc (Rens-selaer, NY, USA) These cells were sensitive to the angiostatic effects of PEDF

Size-exclusion ultrafiltration Complex formation was analyzed by size exclusion ultrafil-tration, using Centricon-100 devices with membranes with

100 kDa exclusion limits, as described previously [41] This assay is based on the fact that PEDF of 50 kDa passes through the membranes, but PEDF in complexes of

‡ 100 kDa does not The components retained by the mem-brane after centrifugation and washes of the devices were analyzed by western blotting

SPR spectroscopy The interactions between PEDF and yeast F1-ATPase were analyzed by SPR using a BIAcore 3000 instrument (BIAcore, Uppsala, Sweden) with immobilized PEDF ligands, as described previously [42] PEDF ligand (4 ng) was immobilized on a CM5 sensor chip by