Báo cáo khoa học: Pleiotrophin inhibits HIV infection by binding the cell surface-expressed nucleolin docx

Our results demonstrate for the first time that PTN inhibits HIV infection and suggest that the cell surface-expressed nucleolin is a low affinity receptor for PTN binding to cells and it

surface-expressed nucleolin

Elias A Said1, Jose´ Courty2, Josette Svab1, Jean Delbe´2, Bernard Krust1and Ara G Hovanessian1

1 UPR 2228 CNRS, UFR Biome´dicale des Saints-Pe`res, Paris, France

2 Laboratoire de Recherche sur la Croissance Cellulaire, la Re´paration et la Re´ge´ne´ration Tissulaires (CRRET), FRE CNRS 2412,

Universite´ Paris Val de Marne, Cre´teil, France

The human immunodeficiency virus (HIV) infects

tar-get cells by the capacity of its envelope glycoproteins

gp120-gp41 to attach cells and induce the fusion of

virus to cell membranes, a process which leads to virus

entry The receptor complex essential for HIV entry

consists of the CD4 molecule and at least one of the

members of the chemokine receptor family: CCR5 or

CXCR4 [1,2] Contrary to the virus entry process, the

attachment of HIV particles to cells can occur even

independently of CD4 We have previously

demonstra-ted that HIV attachment is inhibidemonstra-ted by the

pseudo-peptide HB-19 that binds specifically the C-terminal tail

of nucleolin, a cell-surface-expressed protein identified

to be implicated in HIV attachment [3–5] Conse-quently, we have suggested that HIV attachment is achieved by the coordination of at least two events implicating on the one hand heparan sulfate proteo-glycans [6,7] and on the other hand the cell surface-expressed nucleolin [4] In the search for natural ligands of nucleolin that exhibit a potential inhibitory activity against HIV infection, other than midkine [8] and lactoferrin [9], here we show that pleiotrophin

Keywords

binding; HIV; pleiotrophin; receptor; surface

nucleolin

Correspondence

E A Said, UPR 2228 CNRS, UFR

Biome´dicale des Saints-Pe`res; 45 rue des

Saint-Pe`res, 75270 Paris Cedex 06, France

Fax: +33 142862042

Tel: +33 142864136

E-mail: elias.said@umontreal.ca

(Received 11 May 2005, revised 30 June

2005, accepted 18 July 2005)

doi:10.1111/j.1742-4658.2005.04870.x

The growth factor pleiotrophin (PTN) has been reported to bind heparan sulfate and nucleolin, two components of the cell surface implicated in the attachment of HIV-1 particles to cells Here we show that PTN inhibits HIV-1 infection by its capacity to inhibit HIV-1 particle attachment to the surface of permissive cells The b-sheet domains of PTN appear to be implicated in this inhibitory effect on the HIV infection, in particular the domain containing amino acids 60–110 PTN binding to the cell surface is mediated by high and low affinity binding sites Other inhibitors of HIV attachment known to bind specifically surface expressed nucleolin, such as the pseudopeptide HB-19 and the cytokine midkine prevent the binding of PTN to its low affinity-binding site Confocal immunofluorescence laser microscopy revealed that the cross-linking of surface-bound PTN with a specific antibody results in the clustering of cell surface-expressed nucleolin and the colocalization of both PTN and nucleolin signals Following its binding to surface-nucleolin, PTN is internalized by a temperature sensitive mechanism, a process which is inhibited by HB-19 and is independent of heparan and chondroitin sulfate proteoglycans Nevertheless, proteoglycans might play a role in the concentration of PTN on the cell surface for a more efficient interaction with nucleolin Our results demonstrate for the first time that PTN inhibits HIV infection and suggest that the cell surface-expressed nucleolin is a low affinity receptor for PTN binding to cells and

it is also implicated in PTN entry into cells by an active process

Abbreviations

ALK, anaplastic lymphoma kinase; AZT, azidothymidine; CHO, Chinese hamster ovary; HARP, heparin affin regulatory peptide; HB-GAM, heparin-binding growth-associated molecule; HBNF, heparin-binding neurite-promoting factor; MK, midkine; PTN, pleiotrophin; RPTP, receptor-type tyrosine phosphatase.

(PTN) that binds surface nucleolin inhibits HIV

attachment to cells by its capacity to bind surface

nucleolin as a low affinity receptor

PTN is an 18-kDa protein which was first identified

as a heparin-binding protein that progresses mitogenic

activity in rat and mouse fibroblasts [10] The first

purification was from bovine uterus and neonatal rat,

brain, bone and kidney PTN is rich in basic amino

acids especially lysine in both N- and C-terminal tails

It was also named as

heparin-binding-growth-associ-ated molecule (HB-GAM) [11], heparin-binding

neur-ite-promoting factor (HBNF) [12] or heparin affin

regulatory peptide (HARP) [13]

Biological functions of PTN are mitogenic,

angio-genic and oncoangio-genic activities, cell motility,

differenti-ation, and synaptic plasticity [14] Elevated serum PTN

levels have been detected in patients with testicular,

pancreatic, colon, breast and other cancers [15–17]

Consequently, the circulating levels of PTN have been

proposed to serve as a tumor marker Interestingly,

PTN is expressed in fracture healing [18] and its gene

expression is also upregulated in newly forming blood

vessels, in OX42-positive macrophages, and in

inva-sion-independent pathways of blood-borne metastasis

[14,19,20] PTN is also expressed in adults with

inflam-matory diseases, and proinflammatory cytokines

enhance its expression [21,22] Finally, PTN inhibits

infectivity of human herpes simplex viruses type 1 and

2 and human cytomegalovirus [23]

Several cell surface components have been reported

as potential receptors for PTN, such as the heparan

sulfate proteoglycans of N-syndecan [24] and the

chon-droitin sulfate proteoglycan of receptor-type tyrosine

phosphatase b⁄ f (RPTP b ⁄ f) [25,26] In addition,

ana-plastic lymphoma kinase (ALK) has been reported to

be a receptor that transduces PTN-mediated signals

and the PTN-ALK axis can play a significant role

during development and disease processes [27] PTN

binds the extracellular domain of ALK with a Kd of

32 ± 9 pm [27]

PTN shows a striking structural homology with

another heparin binding growth-associated factor

called midkine, with whom it shares 45% sequence

identity [14,28–31] Therefore, like PTN, the binding of

midkine to heparan sulfate and chondroitin sulfate

proteoglycans could be clearly demonstrated using

purified and soluble components [32,33] Midkine

binds also ALK with a high affinity and this binding is

inhibited by PTN [34] We have previously

demonstra-ted that midkine is a cytokine that binds the cell

sur-face expressed nucleolin as a low affinity receptor

Synthetic and recombinant preparations of midkine

inhibited in a dose-dependent manner infection of cells

by various HIV-1 isolates; this inhibition is due to the capacity of midkine to bind cells specifically and to prevent the attachment of HIV particles to cells [5,35] Nucleolin is a component of the cell surface which could act as a receptor for various ligands Indeed, on the cell surface nucleolin interacts with several mole-cules such as lipoproteins, J factor [50], and the alpha-1 chain of laminin [51] Cell surface-expressed nucleolin could also act as a receptor of viruses such as Coxsackie B [52] and Human Parainfluenza Virus type

3 [53] Indeed, while nucleolin does not have a hydro-phobic domain [54], it is expressed on the cell surface, and it represents 20% of the cytoplasmic portion of nucleolin [55] Our previous results showed that cyto-plasmic nucleolin is found in small vesicles that appear

to translocate nucleolin to the cell surface Transloca-tion of nucleolin is markedly reduced at low tempera-ture or in serum-free medium, whereas conventional inhibitors of intracellular glycoprotein transport have

no effect Thus, translocation of nucleolin is the conse-quence of an active transport by a pathway which is independent of the endoplasmic reticulum⁄ Golgi com-plex [55]

Here, we show that PTN inhibits HIV infection by binding the cell-surface expressed nucleolin leading to the inhibition of HIV-attachment to the cell surface PTN binding to cells involves high and low affinity-binding sites even in cells that are deficient for the expression of both heparan and chondroitin sulfate proteoglycans The synthetic ligand of nucleolin, the HB-19 pseudopeptide, prevents the binding of PTN

to the low affinity receptor, thus suggesting that such

a receptor is the cell surface-expressed nucleolin Accordingly, by confocal immunofluorescence laser microscopy, we show that cell-surface bound PTN colocalizes with that of surface-expressed nucleolin

Results

Inhibition of HIV-infection by PTN

We investigated the inhibitory effect of PTN on HIV infection by using the experimental model of HeLa P4

or HeLa P4C5 cells HIV entry and replication in these cells result in Tat-mediated transactivation of HIV LTR, leading to the expression of the LacZ gene Consequently, the b-galactosidase activity could

be measured in cell extracts to monitor HIV entry The b-galactosidase expression in noninfected cells is considered as the background value in this experi-ment As we had shown previously, midkine inhibited HeLa P4 cells infection by HIV-1 LAI isolate with more than 90% inhibition at 1 lm of midkine [8,35]

In this model, PTN inhibited the entry of the X4

HIV-1 LAI isolate in a dose-dependent manner with

IC50 and IC90 values of 60 and 250 nm, respectively

(Fig 1A, HIV-1 LAI) PTN also inhibited infection of

HeLa P4C5 cells by the R5 HIV-1 Ba-L isolate in a

dose-dependent manner with IC50 and IC90 values of

60 nm and 500 nm, respectively (Fig 1A, HIV-1

Ba-L) A similar inhibition profile was obtained with

the infection of MT4 cells (data not shown) HeLa P4

cells preincubated with PTN at 20
C for 45 min and

washed with medium to remove unbound PTN,

resis-ted HIV-1 LAI infection However, incubation of

HIV-1 LAI with PTN and centrifugation at 100 000 g

to pellet the virus gave an HIV pellet that was still

infectious (data not shown) These data indicate that

the inhibitory effect of PTN is mediated through its

action on target cells rather through a direct effect on

virus particles

The effect of PTN on the HIV attachment was

moni-tored by measuring the concentration of the HIV major

core protein p24 in the lysate of HeLa P4 cells that were incubated with HIV-1 LAI at room tempera-ture in the presence of different concentrations of PTN PTN inhibited HIV-attachment in a dose-dependent manner with more 50% and 90% inhibition

at 50 and 250 nm, respectively (Fig 1B) These results demonstrate that the inhibition of HIV infection by PTN is due to its inhibitory effect on the attachment of HIV particles

The inhibiting action of PTN on the HIV-1

infection is mediated through the b-sheet

domains of PTN PTN consists of two b-sheet domains located between N- and C-terminal tails rich in lysine residues [44] In order to locate the domain of PTN responsible of the inhibitory effect on HIV infection, we tested the capa-city of deletion constructs corresponding to various domains of PTN to inhibit infection of HeLa P4 cells

Control

A

B

Control

No HIV 25 50 100 200 250

AZT

Control AZT 30 60 125 250 500

MK 1 µM

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125

250

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0.5 1 1.5

β-Galactosidase Activity (OD) 2 2.5

β-Galactosidase Activity (OD) 2

PTN

[nM]

PTN

[nM]

PTN [nM]

Fig 1 Inhibition of HIV infection by PTN (A) HeLa P4 cells were treated (30 min,

37
C) with midkine (MK) (1 l M ) or PTN (60,

125, 250, 500, 1000 n M ) HeLa P4C5 cells were treated (30 min, 37
C) with PTN (30,

60, 125, 250, 500 n M ) HeLa P4 and HeLa P4C5 cells were then infected with the

HIV-1 LAI or HIV-HIV-1 Ba-L isolate, respectively At

48 h postinfection, the b-galactosidase activ-ity was measured in cell extracts directly to monitor HIV entry (the abscissa; OD, optical density) The histogram AZT represents the background b-galactosidase activity when HIV retrotranscription is inhibited (B) HeLa P4 cells were incubated (45 min, 20
C) with PTN (25, 50, 100, 200, 250 n M ) and the HIV-1 LAI isolate HIV attachment was monitored by measuring the concentration

of the HIV major core protein p24 in cells extracts The histogram No HIV represents the background of p24 concentration in the absence of virus attachment The mean ± SD of triplicate samples is shown.

by HIV-1 LAI The peptide PTN Nt-tail corresponds

to the N-terminal tail of PTN (residues 1–8), the

pep-tide PTN Ct-tail corresponds to the C-terminal tail of

PTN (residues 110–136), PTN (residues 9–110)

corres-ponds to the b-sheet domains, PTN (residues 1–110)

corresponds to the N-terminal tail and the two

b-sheet domains, PTN (residues 9–136) corresponds

to the C-terminal tail and the two b-sheet domains,

PTN-Nf corresponds to the b-sheets on the

N-ter-minal side (residues 9–59), and PTN-Cf corresponds

to the b-sheets on the C-terminal side (residues

60–110)

Whereas lysine-rich peptides corresponding to the N

and C-terminal tails of PTN have no effect on HIV-1

infection, peptides containing the b-sheet domains

[PTN (1–110) and PTN (9–136)], or peptides

contain-ing the b-sheets alone [PTN (9–110)] inhibit HIV

infec-tion by a dose-dependent manner, at an IC50value of

200 and 250 nm for PTN (1–110), and PTN (9–136),

respectively (Fig 2) The most potent inhibitory effect

is observed with the peptide PTN (9–110) that inhibits

HIV-1 LAI infection with an IC50 value of 30 nm

Finally, PTN-Nf does not have an effect on HIV

infec-tion, whereas PTN-Cf inhibits the infection with an

IC50 of 200 nm (Fig 2) These results suggest that the

domains containing the b-sheets are the regions

responsible for the inhibitory effect of PTN on HIV

infection The presence of N or C-terminal tails with

the two b-sheet domains (at residues 9–110) decreased

the inhibitory effect of the two b-sheet domains

with-out the respective tail (Fig 2, compare the results

obtained with PTN 1–110 and PTN 9–136 with PTN

9–110) The presence of either one of the tails alone

might affect the proper folding of such truncated PTN

constructs and consequently affect the inhibitory effect

on HIV infection

Inhibition of HIV particles attachment by PTN

requires a cell surface component other than

heparan and chondroitin sulfate proteoglycans

Described as a HB-GAM, PTN interacts with

glyco-aminoglycans such as heparan sulfate proteoglycans

[25,26], which are also implicated in HIV attachment

to the cell surface [7] In order to investigate whether

the inhibitory effect of PTN on HIV attachment is

due to its interaction with heparan or chondroitin

sul-fate proteoglycans, we used Chinese hamster ovary

(CHO) wild-type cells (CHO K1) and mutant cells

lines that are deficient in the expression of heparan

sulfate (CHO 677), or both heparan⁄ chondroitin

sul-fate proteoglycans (CHO 618) [38,39] Despite lacking

proteoglycan expression, these mutant cell lines

express similar levels of the cell-surface nucleolin [8]

In these HIV attachment experiments, culture super-natants were removed from CHO K1, 677 and 618 cells pretreated with PTN or the nucleolin-binding HB-19 pseudopeptide, before adding the virus preparation on cells The fact that CHO K1 cells do not express the HIV receptor CD4 or the coreceptors CCR5 and CXCR4, demonstrates that HIV attachment should mainly be mediated via the heparan⁄ chondroitin sul-fate proteoglycans and cell-surface expressed nucleolin

Control PTN 200 nM

PTN (1-110)

PTN (9-136)

PTN (9-110)

PTN Nt-tail

PTN Ct-tail

PTN-Nf

PTN-Cf

100 nM

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500 nM

100 nM

200 nM

500 nM

% of HIV infection

Fig 2 The inhibitory of various PTN domains on HIV infection HeLa P4 cells were preincubated or not (30 min, 37
C) with

200 n M of PTN, PTN (1–110), PTN (9–136), PTN (9–110), PTN Nt-tail (1–9), PTN Ct-tail (110–136), PTN-Nf, or PTN-Cf at 100, 200 and

500 n M concentrations Cells were then infected with HIV-1 LAI (90 min, 37
C) The b-galactosidase activity was measured at 48 h postinfection The percentage HIV infection (abscissa) gives the proportion of b-galactosidase activity compared to infected cells without PTN (histogram Control).

[4] HB-19 was included in these binding experiments

in order to estimate the contribution of nucleolin in

the HIV attachment process Accordingly, HB-19

markedly inhibited HIV attachment at a concentration

of 1 lm in all CHO wild-type and mutant cell lines,

thus indicating the capacity of surface expressed

nucleolin to serve as a receptor for HIV binding

inde-pendent of heparan and chondroitin sulfate

proteogly-cans (Fig 3) Interestingly, PTN at a concentration of

500 nm inhibited HIV attachment by about 70% to

the surface of all CHO cell lines used in this assay

(Fig 3) In similar HIV attachment assays, when PTN

was not removed before addition of HIV, then more

than 90% inhibition of HIV attachment was observed

(data not shown) These results do not rule out a

potential role of heparan⁄ chondroitin sulfate

proteo-glycans in the inhibitory activity of PTN, but suggest

the implication of other cell surface component(s) It

should be noted that HIV attachment in control CHO

cell lines is decreased by 50 and 80% in CHO 677

and CHO 618 cells, respectively (Fig 3) This decrease

is probably due to the lack of heparan sulfate and

heparan⁄ chondroitin sulfate proteoglycan expression,

and illustrates the implication of such proteoglycans

in the HIV attachment process The capacity of

HB-19 to inhibit HIV attachment in the CHO

wild-type cells is in accord with our previous results using

various CD4 positive and HIV permissive cell lines;

such results confirm once again that HIV attachment

is coordinated by both proteoglycans and nucleolin

[4]

Role of heparan and chondroitin sulfate proteo-glycans in PTN binding on the cell surface

To evaluate the potential implication of the heparan and chondroitin sulfate proteoglycans in PTN binding,

we used the three CHO cell lines previously described [8] These binding experiments were carried out at

20
C to prevent PTN entry into cells (see Experimen-tal procedures) We first investigated the specific and nonspecific binding of 125I-labeled PTN to the wild-type CHO K1 cells, which express heparan and chon-droitin sulfate proteoglycans by washing cells at 300 and 150 mm NaCl, respectively In cells washed with

300 mm NaCl,125I-labeled PTN specific binding occurs

in a dose-dependent manner and reaches saturation at

3 lm of 125I-labeled PTN (Fig 4A), whereas total binding does not reach a saturation limit Because of the considerable amount of nonspecific binding, cells were routinely washed at 300 mm NaCl in all the fol-lowing experiments It is important to note that PTN specific binding resists drastic wash conditions such as normal or acidic culture medium (pH¼ 4) containing (or not) 2 m NaCl (not shown) Interestingly, the

125I-labeled PTN binding profile (binding curve and saturation point) to heparan sulfate-deficient CHO 677 cells (not shown) and to both heparan and chondroitin sulfate proteoglycan-deficient CHO 618 cells was similar to that observed for the wild-type CHO K1 cells (Fig 4B) However, the levels of125I-labeled PTN binding (amount of 125I-labeled PTN bound to cells)

to the CHO 618 and 677 cells was lower than that to the wild-type CHO K1 cells These results indicate that under our experimental conditions, heparan and chon-droitin sulfate proteoglycans may play a role in the overall binding of PTN to cells, even if the specific binding reaches saturation independently of their pres-ence As high affinity binding sites for PTN have been reported in the literature [27], we investigated the pres-ence of such sites on CHO cells Indeed, results show that in CHO K1 cell lines the specific binding of the

125I-labeled PTN reaches saturation at 2 nm (Fig 4C)

A similar saturation curve was obtained in CHO 618 and HeLa cells (not shown) Taken together, these results suggest the presence of low affinity and high affinity binding sites of PTN on the cell surface Scatchard analysis of the 125I-labeled PTN binding using high and low concentrations confirmed the pres-ence of low affinity and high affinity binding sites The estimated Kdvalue for PTN binding to the low affinity binding site on CHO K1 and 618 cells was 1.3· 10)6m (3.6· 107sites per cell) and 1.4· 10)6m(1.9· 107sites per cell), respectively (Table 1) The estimated Kdvalue for the binding of PTN to the high affinity binding site

0

Control

HB-19 1 µM

PTN 0.5 µM

Control

HB-19 1 µM

PTN 0.5 µM

Control

HB-19 1 µM

PTN 0.5 µM

CHO K1

CHO 677

CHO 618

p24 [pg/ml]

Fig 3 Attachment of HIV particles to CD4 – CHO cell lines,

expres-sing or not expresexpres-sing heparan ⁄ chondroitin sulfate proteoglycans is

inhibited by PTN and the nucleolin-binding HB-19 pseudopeptide.

CHO K1 cells (wild type), CHO 677 cells (deficient in heparan

sul-fate proteoglycan) or CHO 618 cells (deficient heparan ⁄ chondroitin

sulfate proteoglycans) were treated (30 min, 20
C) with HB-19

(1 l M ) or PTN (500 n M ) Both reagents were then removed from

the culture before incubation of cells with the HIV-1 LAI isolate

(45 min, 20
C) HIV attachment was monitored by measuring the

concentration of the HIV major core protein p24 in the cells lysate.

The mean ± SD of triplicate samples is shown.

on both CHO K1 and CHO 618 cells was 4.9· 10)11 m (2.5· 105 sites per cell) which is somewhat in accord with the value reported previously [27] The binding of PTN to the high affinity receptor reaches saturation at

2 nm, a concentration that has no effect on HIV particle attachment to cells; the order of PTN concentrations required to inhibit HIV attachment to cells corresponds

to the concentrations of PTN that are required for the interaction and the saturation of the low affinity binding site Consequently, the binding of PTN to the low affin-ity-binding site should be responsible for the inhibitory effect of PTN on HIV infection

PTN binding to the low affinity receptor is blocked by nucleolin-binding HB-19 pseudopeptide

The different CHO cell lines were employed to investi-gate competition experiments for the low and high affinity binding sites Typical results are shown with the CHO K1 cells (Fig 5) The specificity of PTN binding to both binding sites was confirmed by the fact that unlabeled PTN completely inhibited 125I-labeled PTN binding to the low and high affinity binding sites (Fig 5) As expected, the nucleolin binding pseudopep-tide HB-19 prevented 125I-labeled PTN binding to the low affinity but not to the high affinity site (Fig 5) A similar profile of inhibition was observed with MK for the binding of PTN to its low affinity binding site (Fig 5), whereas our previous results showed that PTN inhibited just 50% of125I-labeled MK binding to the low affinity receptor [8], this might be due to the fact that MK interacts with such a binding site with

an affinity higher than that of PTN Interestingly, it was shown that MK binding to its high affinity bind-ing site, which was defined to be ALK also, is com-peted by PTN [34] Our results suggest that PTN and HB-19 share a common receptor, to which both MK and PTN bind with a low affinity Consequently, nucleolin should be the low affinity-binding site of PTN These observations along with the role of surface

20000

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CHO K1 cells

CHO 618 cells

CHO K1 cells

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Concentration of PTN [nM]

2.5 2

Fig 4 The binding of 125 I-labeled PTN to CHO cell lines Binding at

high concentrations of PTN is shown in (A) and (B) The nonspecific

(squares) and specific (circles) binding of 125 I-labeled PTN to cells

was investigated using wild-type CHO K1 cells (A) and the

hepa-ran⁄ chondroitin sulfate-deficient CHO 618 cells (B) using different

concentrations of the 125 I-labeled PTN (abscissa) C Binding of

125 I-labeled PTN to CHO K1 cells was carried out as in the sections

A and B but at lower concentrations of the 125 I-labeled PTN The

specific binding was measured after washing cells in 300 m M NaCl

(see Experimental procedures) The ordinate gives the values of

measured c rays in counts per minute (c.p.m.) Each point

repre-sents the mean ± S.D of duplicate samples.

Table 1 High affinity and low affinity Pleiotrophin receptors on CHO wild-type and proteoglycan-free cells Scatchard analysis of the binding data on CHO K1 and CHO 618 cells (heparan ⁄ chondro-itin-proteoglycan-deficient cells) carried out as shown in Fig 3 sug-gested the presence of high and low affinity binding sites for PTN The K d values and the number of sites per cell are as indicated Cell lines Affinity K d ¼ (M) Receptors per cell

nucleolin in HIV attachment to cells point out that the

inhibitory action of PTN on HIV infection could be

the consequence of PTN binding to the cell-surface

expressed nucleolin

Internalization of PTN is independent of heparan

and chondroitin sulfate expression but it is

inhibited by the nucleolin-binding HB-19

pseudopeptide

The use of specific anti-PTN antibodies in confocal

laser immunofluorescence microscopy experiments,

demonstrated internalization of PTN in HeLa cells at

37 but not at 20
C (not shown), thus indicating that PTN entry occurs by an active process In order to investigate the role of surface nucleolin in the PTN internalization process, PTN entry was monitored in the different CHO cell lines Because heparan sulfate proteoglycans are implicated in the internalization of FGF-2 [45], we also monitored entry of PTN in these same cells Our results show that PTN enters efficiently

in the heparan sulfate-deficient CHO 677 and hepa-ran⁄ chondroitin sulfate-deficient CHO 618 cells as in the wild-type CHO K1 cells (Fig 6) In contrast,

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PTN [log (M)]

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b I-PTN/HB-19

c I-PTN/MK

PTN [log (M)]

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% Binding of I-labeled PTN 0

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I-PTN/HB-19 I-PTN

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Fig 5 The effect of the nucleolin binding molecules on the binding of PTN to the low and high affinity-binding site (A) Inhibitory effect of HB-19 on the binding of the

125 I-labeled PTN to the low affinity-binding site CHO K1 cells were incubated with the

125

I-labeled PTN (25 n M ) in the presence of various concentrations of unlabeled PTN (a), HB-19 (b) or midkine (c) The cells were washed in culture medium containing

300 m M NaCl to monitor the specific bind-ing The mean ± SD of duplicate samples is shown (B) HB-19 prevents the binding of the 125 I-labeled PTN to the low but not the high affinity-binding site CHO K1 cells were incubated with 2 n M for the high affinity binding site or 25 n M for the low affinity binding site with the 125 I-labeled PTN in the presence of 100-fold higher concentrations

of PTN, HB-19 or midkine (MK) as it is indi-cated Cells were washed in culture med-ium containing 300 m M NaCl to monitor for specific binding Each histogram represents the mean ± SD of duplicate samples The ordinate gives the percentage 125 I-labeled PTN binding to cells in the presence of the different reagents.

FGF-2 internalization occurs only in the CHO K1

cells thus confirming the requirement of proteoglycans

in its entry process The internalization of125I-labeled

PTN was also monitored in CHO K1, 677 and 618 cell

lines by treating cells with trypsin to eliminate cell

surface bound PTN (not shown) These experiments

demonstrated once again that internalization of PTN

occurs at 37
C and does not require heparan and

chondroitin sulfate proteoglycans

In accord with the inhibition of PTN binding to

cells by the nucleolin-binding HB-19 pseudopeptide

(Fig 5B), PTN entry was inhibited almost completely

by HB-19 (Fig 7) It is of interest to note that a

syn-thetic peptide composed of nine arginine residues [8]

has no apparent effect on the binding and

internalizat-ion of PTN (not shown), thus pointing out that the

inhibitory effect of HB-19 is an specific event on the

binding of PTN to its low affinity binding site and not simply due to the basic nature of HB-19 These obser-vations further confirm that PTN binding and internal-ization in cells is mediated by the cell-surface nucleolin

Cross-linking of surface-bound PTN results in the clustering of surface nucleolin

In general, the cross-linking of a ligand leads to the clustering or capping of its surface receptor Previously,

we had reported that antibody-mediated cross-linking

of ligands of nucleolin, such as the pseudopeptide HB-19, HIV virions, midkine and lactoferrin, results in clustering of surface nucleolin and its coaggregation with the specific ligand [5,8,9] Similarly, here we show that cross linking of cell surface bound PTN with

CHO K1 (HS, CS)

A PTN

B FGF-2

Fig 6 Internalization of PTN does not require expression of heparan (HS) ⁄ chondroitin sulfate (CS) proteoglycans CHO wild-type K1, the heparan sulfate-deficient CHO 677, and the heparan ⁄ chondroitin-deficient CHO 618 cells were incubated (60 min, 37
C) in fresh culture medium containing 10% (w ⁄ v) FBS and 200 n M PTN or FGF-2 Cells were then washed and fixed with 3.7% PFA and permeabilized with Tri-ton X-100 The primary antibodies were goat anti-PTN and anti-FGF-2, which was revealed by FITC-conjugated rabbit anti-(goat IgG) Ig For each condition, a scan corresponding to a cross-section towards the middle of the cell monolayer is shown along with the respective phase contrast.

anti-PTN Igs results patching of nucleolin at one pole

of the cell, which coincided with the PTN signal

(Fig 8) This observation is consistent with the surface

nucleolin being a binding site of PTN

Discussion

Here we show for the first time that the growth factor

PTN inhibits HIV-1 infection by its capacity to inhibit

HIV attachment to the cell surface The b-sheet

domains of PTN, especially those located on the

C-ter-minal side, appear to be implicated in this inhibitory

effect PTN binding to the cell surface is mediated by

high and low affinity binding sites, the low affinity

binding sites being nucleolin Following binding, PTN

enters cells by an active process that is independent of

heparan and chondroitin sulfate proteoglycans, but is

inhibited by the nucleolin-binding pseudopeptide

HB-19 Cross-linking of surface-bound PTN with a

specific antibody results in the clustering of cell

surface-expressed nucleolin and the colocalization of both PTN

and nucleolin; thus confirming the interaction of PTN with surface-expressed nucleolin The interac-tion of PTN with surface nucleolin is in accord with a previous report showing that PTN binds nucleolin in solution [46]

PTN inhibits HIV attachment to CD4+ permissive cells as well as to CD4– nonpermissive CHO cell lines that express (or not) heparan⁄ chondroitin-sulfate pro-teoglycans In such CD4+ or CD4– cell lines HIV attachment is also inhibited by the HB-19 The demon-stration that both PTN and HB-19 inhibit HIV attach-ment even in the absence of heparan⁄ chondroitin sulfate proteoglycans (such as in CHO 618 cells), sug-gests that their inhibitory effect on virus attachment should be due to binding to the cell-surface-expressed nucleolin We have previously reported that the initial attachment of HIV particles to cells is coordinated on one hand by heparan⁄ chondroitin sulfate proteoglycans

PTN

PTN + HB-19

Phase contrast

Phase contrast

Fig 7 Internalization of PTN is inhibited by the HB-19

pseudopep-tide HeLa P4 cells in culture medium containing 10% (w ⁄ v) FBS

were incubated (45 min, 37
C) with PTN (200 n M ) in the absence

or presence of HB-19 (2 l M ) Cells were then washed and fixed

with 3.7% PFA and permeabilized with Triton X-100 The primary

antibody was anti-PTN polyclonal antibody, which was revealed

using FITC-conjugated rabbit anti-(goat IgG) Ig A scan

correspond-ing to a cross-section toward the middle of the cell monolayer is

shown.

Nucleolin-TR

Pleiotrophin-FITC

Fig 8 PTN induced clustering of nucleolin in MT4 cells: colocaliza-tion of PTN with nucleolin at the surface of PTN treated cells MT4 cells were incubated with 1 l M of PTN at 20
C for 45 min Cells were then washed before incubation at 20
C for 45 min in the presence of anti-PTN antibody to induce lateral clustering while inhibiting PTN entry At this stage, cells were first partially fixed with 0.25% PFA before the addition of the monoclonal antibody against nucleolin (mAb D3; 20
C, 45 min) After washing, cells were fixed, and the primary anti-PTN Ig was revealed by FITC-con-jugated rabbit anti-(goat IgG) Ig, whereas mAb D3 against nucleolin was revealed by Texas Red dye-conjugated horse anti-(mouse IgG)

Ig A cross-section towards the middle of cells for each staining with the merge of the two colors in yellow are presented.

[6,48,49], and on the other hand by the

surface-expressed nucleolin [4,5] Consequently, HIV

attach-ment could be inhibited either by FGF-2 which uses

heparan-sulfate proteoglycans as low affinity receptors

[45], and by various specific ligands of nucleolin such

as the HB-19 pseudopeptide, midkine, PTN, and

lacto-ferrin [4,5,8,9,35] The capacity of HB-19 to inhibit

HIV attachment to CHO 618 cells that are deficient

in both heparan⁄ chondroitin sulfate proteoglycans

expression (the results herein) provides further evidence

illustrating that surface nucleolin is implicated in the

HIV attachment process

Several groups have previously shown that PTN

interacts with heparan⁄ chondroitin sulfate

proteogly-cans [24–26,47] Accordingly in our experiments,

although PTN binds specifically CHO cells deficient in

the expression of heparan and chondroitin sulfate

pro-teoglycans, the total amount of binding is much lower

compared to wild-type CHO cells expressing both

proteoglycans The latter therefore suggests that

hepa-ran and chondroitin sulfate proteoglycans are also

implicated in the mechanism of PTN binding to cells

This is somewhat analogous to the mechanism of HIV

binding cells in which both heparan and chondroitin

sulfate proteoglycans and nucleolin are implicated

Accordingly, both HIV attachment and PTN binding

to cells is decreased at a similar level of by 50 and

80% in CHO 677 and 618 cells, respectively In

con-trast to cell binding, nucleolin-mediated PTN entry

appears to be independent of heparan and chondroitin

sulfate proteoglycans Thus it is plausible that heparan

and chondroitin sulfate proteoglycans might be

neces-sary for the concentration of PTN on the cell surface

for an efficient interaction with nucleolin

The b-sheets located on the C-terminal side of PTN

(amino acids 60–110) appear to be responsible for its

inhibitory effect on HIV infection Accordingly, the

construct representing the b-sheets located on the

C-terminal side inhibits the HIV infection, whereas its

counterpart on the N-terminal side has no apparent

inhibitory effect Interestingly, the construct that

con-tains both b-sheet domains is more active in the

inhi-bition of HIV infection This latter could be due to

conformational effects on the PTN structure that is

optimal for the interaction with nucleolin Finally, the

presence of the N- and C-terminal tails of PTN along

with the b-sheet domains results in a decrease in the

inhibitory effect of PTN on HIV infection, indeed this

might affect the folding of such PTN constructs

and consequently affect the inhibitory effect on HIV

infection

Little information about the conditions of PTN

expression is available Nevertheless, its expression in

inflammatory deceases was described [56–58] PTN is found at high concentration in the serum of patient suffering from pancreas, colon, testicular and breast cancer [59–61] Also PTN is expressed during fracture healing [62] Whereas, at low concentrations, PTN enhance the proliferation of the PBMCs (Achour et al 2001), PTN has not been detected in resting or activa-ted T lymphocytes [35] Thus, this information does not allow us to form a clear idea about a potential role

of PTN in in vivo HIV infection Thus the conditions

of PTN expression and its role in in vivo HIV infection have to be studied

Taken together, our results demonstrate that PTN uses the cell surface-expressed nucleolin as a low affin-ity cell surface receptor This binding and its internal-ization by an active process might be implicated in the mechanism of action of PTN as a mitogenic and growth regulatory factor Consequently, HB-19 that prevents PTN binding to surface nucleolin provides a potential inhibitor of PTN

Experimental procedures

Materials Recombinant human PTN (rh PTN) and human midkine (rh MK) produced in Escherichia coli were purchased from

R & D systems Basic fibroblast growth factor (FGF-2) produced in E coli was from Sigma (St Louis, MO, USA) PTN was iodinated (2.2· 103

lCiÆlmol)1) using the Bolton-Hunter reagent (PerkinElmer Life Sciences, ON, Canada) by a procedure as recommended by the manufac-turer The HB-19 pseudopeptide was synthesized as described previously [4]

Antibodies Goat anti-(human PTN) Ig, and anti-(human FGF-2) Ig were purchased from R & D systems The monoclonal anti-body (mAb) D3 specific for human nucleolin was provided

by J.S Deng, Veterans Affairs Medical Center, Pittsburgh,

PA, USA [36]

Cell lines and virus preparation The MT4 is a human T lymphocyte cell line that was pro-pagated in RPMI 1640 (BioWhittaker, Verviers, Belgium) Human HeLa-CD4-LTR-LacZ cells expressing or not expressing CCR5 were referred to as HeLa P4-C5 and HeLa P4, respectively These HeLa cells (provided by

P Charneau and O Schwartz, Institut Pasteur, Paris, France) were cultured in Dulbecco’s modified Eagles’s medium (Invitrogen, Carlsbad, CA, USA) supplemented with G418