Báo cáo Y học: Complexation of ytterbium to human transferrin and its uptake by K562 cells pot

Flow cytometric studies on the uptake of fluorescein isothiocyanate labeled Yb3+-bound transferrin species by K562 cells showed that they bind to the cell receptors.. The Yb3+ -trans-ferr

Complexation of ytterbium to human transferrin and its uptake

by K562 cells

Xiu-lian Du1, Tian-lan Zhang1, Lan Yuan1, Yong-yuan Zhao1, Rong-chang Li1, Kui Wang1, Siu Cheong Yan2,

Li Zhang2, Hongzhe Sun2and Zhong-ming Qian3

1

Department of Chemical Biology, School of Pharmaceutical Sciences, Peking University, Beijing, China;2Department of Chemistry and Open Laboratory of Chemical Biology, University of Hong Kong, Hong Kong;3Department of Applied Biology and Chemical Technology, Hong Kong Polytechnic University, Kowloon, Hong Kong

There is an increasing interest in the use of lanthanides in

medicine However, the mechanism of their accumulation in

cells is not well understood Lanthanide cations are similar to

ferric ions with regard to transferrin binding, suggesting

transferrin-receptor mediated transport is possible; however,

this has not yet been confirmed In order to clarify this

mechanism, we investigated the binding of Yb3+to

apo-transferrin by UV-Vis spectroscopy and stopped-flow

spec-trophotometry, and found that Yb3+ binds to

apotransferrin at the specific iron sites in the presence of

bicarbonate The apparent binding constants of these sites

showed that the affinity of Yb3+ is lower than that of

Fe3+and binding of Yb3+in the N-lobe is kinetically

fav-ored while the C-lobe is thermodynamically favfav-ored The

first Yb3+bound to the C-lobe quantitatively with a Yb/

apotransferrin molar ratio of < 1, whereas the binding to the

other site is weaker and approaches completeness by a higher

molar ratio only As demonstrated by1H NMR spectra,

Yb3+binding disturbed the conformation of apotransferrin

in a manner similar to Fe3+ Flow cytometric studies on the uptake of fluorescein isothiocyanate labeled Yb3+-bound transferrin species by K562 cells showed that they bind to the cell receptors Laser scanning confocal microscopic studies with fluorescein isothiocyanate labeled Yb3+-bound trans-ferrin and propidium iodide labeled DNA and RNA in cells indicated that the Yb3+entered the cells The Yb3+ -trans-ferrin complex inhibited the uptake of the fluorescein labeled ferric-saturated transferrin (Fe2-transferrin) complex into K562 cells The results demonstrate that the complex of

Yb3+-transferrin complex was recognized by the transferrin receptor and that the transferrin-receptor-mediated mech-anism is a possible pathway for Yb3+accumulation in cells Keywords: K562 cells; recognition; transferrin; ytterbium

Lanthanides have been suggested for the treatment of a

series of diseases and for diagnosis by magnetic resonance

imaging [1,2] Recent studies also show that they could act

as scavengers of free radicals [3] and therefore protect cells

and tissues from oxidative stress-induced injury Some

lanthanides nuclides were also suggested for palliative

therapy.169Yb (c-emission, t½
32 days) was reported to

provide comparable tumor control and has been considered

as a potential replacement for125I and103Pd in permanent

implants [4,5] Evidently, the intracellular accumulation is

very important in these cases, but its mechanism still remains unclear

It was suggested that the particulate- and protein-bound

Ln enters the cells by endocytosis [6]; the anionic low-molecular-mass complexes, via anion channels [7], whereas free Ln3+ is transported by ionophores [8], Na+/Ca2+ exchange [9] and self-facilitated diffusion [10] It is known that Ln3+is mainly bound to proteins in the extracellular media (e.g plasma) Various studies have demonstrated that considerable amounts of Ln are bound to the iron transport protein transferrin (Tf) in the blood [11–14] As metal ions

of therapeutic and diagnostic interest also bind to Tf at the specific iron sites [15], Tf has been thought of as a
delivery vehicle for metal ions into cells [16–19] Tf takes up Fe3+at

pH 7.4 and transports it into cells via receptor-mediated endocytosis In this transport system, ferric ion binds to apotransferrin (apo-Tf) first to form an iron-loaded Tf, and subsequently the iron-loaded Tf binds to the specific Tf receptor (TfR) The Tf-TfR complex is internalized and iron dissociates from Tf upon acidification of cytoplasm (pH

5.5) The molecular recognition between Tf and TfR is believed to be critical for the iron transport The recognition depends on the conformation of the protein, which is regulated by the metal ion, from the
lobe-open state in the apo-form to
lobe-closed state in the holo-form as revealed from X-ray crystal structures of Tf and the recombinant N-lobe of Tf [20–22]

Although Yb3+has been reported to bind to apo-Tf, it

is not clear whether Yb3+can enter the cells by the same way

Correspondence to K Wang, Department of Chemical Biology,

School of Pharmaceutical Sciences, Peking University, Beijing,

100083, China Fax: + 86 10 6201 5584, Tel.: + 86 10 6209 1539,

E-mail: wangkui@bjmu.edu.cn or H.Z Sun, Department of

Chemistry and Open Laboratory of Chemical Biology,

University of Hong Kong, Pokfulam Road, Hong Kong.

Fax: + 852 2857 1586, Tel.: + 852 2859 8974,

E-mail: hsun@hkucc.hku.hk

Abbreviations: apo-Tf, apotransferrin; Fe 2 -Tf, ferric-saturated

trans-ferrin or holotranstrans-ferrin; FITC, fluorescein isothiocyanate;

FITC-Fe 2 -Tf, FITC labeled Fe 2 Tf; hTf, human serum transferrin; LSCM,

laser scanning confocal microscopy; Tf, transferrin; TfR, transferrin

receptor; ICP-AES, inductively coupled plasma atomic emission

spectrometry.

(Received 9 July 2002, revised 15 October 2002,

accepted 21 October 2002)

In the present work, we report a detailed study of uptake of

Yb3+by human Tf (hTf) by UV-vis and NMR spectroscopy,

and inductively coupled plasma atomic emission

spectro-metry (ICP-AES) The binding of Yb-bound Tf to human

erythroleukemia K562 cells was determined by flow

cyto-metry and the cross-membrane transport was studied by

confocal laser scanning microscopy (CLSM)

E X P E R I M E N T A L P R O C E D U R E S

Materials

Human Tf (catalog no T0519), Hepes, Yb2O3, Lu2O3,

fluorescein isothiocyanate (FITC) and propidium iodide

were purchased from Sigma A Sephadex G-25 fine column

was purchased from Pharmacia K562 cells were obtained

from the Laboratory of Immunology, Peking University

RPMI 1640, penicillin, streptomycin and fetal bovine serum

were all purchased from Gibco All the chemicals used were

of analytical grade All containers were rinsed with

hydro-chloric acid to diminish the influence of metal ions

Ultrapure water was used to prepare solutions

Ytterbium chloride stock solution was prepared by

dissolving ytterbium oxide in a minimum of concentrated

HCl and adjusting pH to
4 with NaOH The solution was

then diluted to the concentration needed prior to use

Ytterbium citrate was prepared by addition of 1 mol equiv

of citrate to YbCl3solution and followed by pH adjustment

to 4–6 Human erythroleukemia K562 cells were cultured in

RPMI 1640 medium supplemented with 10% inactivated

fetal bovine serum and 100 UÆmL)1penicilin and

strepto-mycin at 310 K in a 5% CO2atmosphere All experiments

were performed with a cell density of
5 · 105cells per mL

Preparation of FeC-Tf and ferric-saturated Tf (Fe2-Tf)

Monoferric- (selective loading of Fe3+in the C-lobe of Tf)

and diferric-Tf were prepared as described previously [23]

The apo-Tf was purified by sequential dialysis against 0.1M

Hepes (pH 7.4) with 0.1M NaClO4 for 24 h and then

against 0.1MHepes buffer pH 7.4 containing 0.1MNaCl

[24] The addition of 1 or 2 mol equiv of

Fe(nitrilotriace-tate)2 to apo-TF and incubated the solution at 310 K for

30 min The solution was then passed through Sephadex

G-25 column (1· 10 cm) to remove the low molecular mass

ligands (e.g nitrilotriacetate), and FeC-Tf or Fe2-Tf fraction

was collected Protein concentrations were determined

spectrophotometrically on the basis of e280 93 000,

103 000 and 113 000M )1Æcm)1 for apo-Tf, FeC-Tf and

Fe2-Tf, respectively [23] The iron saturation of FeC-Tf and

Fe2-Tf was also estimated from the ratio of A280nm/A465nm

[23]

The Yb-Tf solution was prepared by mixing YbCl3and

apo-Tf solutions in a molar ratio of 2.5 : 1 and the free Yb

was removed by ultrafiltration As indicated in the Results

section, with this molar ratio apo-Tf was not fully saturated

by Yb3+however, the major Yb-bound Tf species is mainly

Yb2-Tf Fluorescence labeled Yb-Tf and Fe2-Tf were

prepared by incubating Fe2-Tf and Yb2-Tf solution with

FITC in 0.5MNa2CO3-NaHCO3buffer (pH 9.0) at 277 K

for 4–5 h [25] The excess FITC was removed by gel filtration

through a Sephadex G-25 column (1.0· 10 cm) The green

solution of FITC-labeled protein fraction was collected The

ratio of apo-Tf vs FITC and the concentration of FITC-Tf were determined according to the literature [26]

Electronic absorption spectroscopy The apo-Tf solutions were prepared by diluting aliquots of a stock solution to
1 · 10)5M )1with 100 mMHepes buffer (pH 7.4) Immediately before Yb3+was added, an aliquot

of a concentrated solution of NaHCO3was added to give a

5 mM bicarbonate solution For titration experiments, aliquots of the stock solution of Yb3+were added to the apo-Tf cuvette and the spectra were recorded at intervals of

at least 30 min at room temperature

Stopped-flow spectrophotometry The kinetic process of Yb3+binding to apo-Tf and FeC-Tf was monitored on a SF18MV stopped-flow spectropho-tometer (Applied Photophysics) The changes in the absorption at 242 nm were followed upon mixing equal volume of the YbCl3and protein solutions (8.3 and 7.9 lM for apo-Tf and FeC-Tf, respectively) The driving syringes were immersed in a water bath at 298 ± 0.5 K Four-hundred data points were collected over various times (2, 5 and 10 s) for each trace and each curve was obtained by averaging 5–10 traces The dead-time of the instrument under the experimental condition was less than 5 ms

Inductively coupled plasma atomic emission spectrometry

These experiments were carried out on a Perkin-Elmer Plasmaquant 110 Emission Spectrometer using standard methods [27] Yb-loaded proteins were prepared by addition

of appropriate molar equivalent of metal ions to apo-Tf in 0.1MHepes buffer containing 0.1MNaCl, at pH 7.4, and left to incubate at room temperature for about 1 h Then the samples were purified by using Centricon 30 (Amicon) ultrafilters, washed four times with 0.1M Hepes buffer, followed by ultrafiltration after each washing The final protein solutions were diluted with ultrapure water con-taining 1% nitric acid The Yb content measured directly without digestion of the samples using ICP-AES at 369.3 nm

NMR spectroscopy

1H NMR spectra were recorded at 500 MHz on a Bruker DRX500 spectrometer Spectra were acquired using 0.6 mL

of solution in a 5-mm tube at 298 K,
1000 transients, 6-ls pulses, recycle time 1.8 s and 16 K data points A solution of 0.8 mMapo-Tf in 0.1MKCl, 10 mMNaHCO3was used for the1H NMR studies The pH values of NMR solutions were kept at 7.40 ± 0.05 and was checked before and after NMR measurements with a Corning 440 pH-meter, equipped with an Aldrich microcombination electrode and calibrated with standard buffers at pH 4.0 and 7.0, respectively Flow cytometry analysis

The competitive effect of Yb-Tf species on binding of Fe2-Tf

to K562 cells was studied by flow cytometry The solutions with a constant concentration of FITC-labeled Fe-Tf and

various concentrations of unlabeled Yb-Tf (2.5 : 1) were

each incubated with 1 mL of K562 cell suspension

(106cellsÆmL)1) in 10 mMHepes buffer (pH 7.4),

contain-ing 0.15 M NaCl at 310 K for 30 min After chilling

(0C, 30 min) to terminate the reaction, the incubated

mixtures were centrifuged at 500 g for 5 min and then the

pellets were washed twice with ice-cold Hank’s balanced salt

solution to remove the extracellular FITC-labeled Fe2-Tf

The fluorescence intensity of each single cell was recorded

on a FASCAN flow cytometer (Becton-Dickinsin), with

argon laser set at kex488 nm and kem530 ± 26 nm The

results were expressed as the mean channel fluorescence

intensity (FF), which was obtained by integrating the

intensity per cell from 1· 104cells for each sample

Laser scanning confocal microscopy

Entry of FITC-Yb-Tf into K562 cells was investigated by

LCSM A suspension (1 mL) of 106 K562 cells was

incubated with 1 lM FITC-Yb-Tf solution for 30 min at

310 K The unbound FITC-Yb-Tf was removed by

centrifugation with ice-cold 10 mM Hepes buffer at

pH 7.4, containing 0.15MNaCl The cells were visualized

under LCSM (TCS NT, Leica) Fluorescence images were

collected at 512· 512 pixels resolution using confocal

microscopy with an oil immersion objective (apo-planar

40· 1.25) in a Leica inverted microscopy, with kex

488 nm for FITC-Yb-Tf or Fe2-Tf, and kex 568 nm for

propidium iodide The emission fluorescence at kem 530/

30 nm was collected with Band Pass (BP) filter and Long

Pass (LP) 590 nm filter

R E S U L T S

Binding of Yb3+to apo-hTf

The thermodynamics of binding of Yb3+ to apoTf

Binding of Yb3+to apo-Tf was investigated by addition

of aliquots of Yb3+to a solution of apo-TF in 100 mM

Hepes buffer in the presence of 5 mMNaHCO3 As shown

in Fig 1, upon addition of Yb3+to apo-Tf, two absorbance bands appeared at 242 and 292 nm, the characteristic of metal binding to phenolate groups of tyrosine residues at the specific iron bind-sites of apo-Tf The De242 increases linearly with the increase of Yb/apoTf ratio (r) up to a value of r¼ 1 The results indicated that Yb3+bound to tyrosine residues of the Fe3+-binding sites and induced deprotonation From the initial slope of the curve, the molecular absorption coefficient (e) of Tf with one Yb3+ saturated was obtained as 26 500 ± 500M )1Æcm)1 Above

r¼ 1, the absorption increases further but less profoundly, indicative of the occupation of a second site with a lower affinity Under similar conditions, reactions of apo-Tf with

Yb3+-citrate gave rise to the same two bands at 242 and

292 nm, respectively, and the same molecular absorption coefficient We also noticed that the higher the bicarbonate concentration, the higher degree of saturation of trnasferrin

by Yb3+(data not shown)

The two dissociation constants (Kd1 and Kd2) were determined based on the data in Fig 1 According to Bjerrum equation [28], we have,

¼ K1½Yb

3þfreeþ 2K1K2½Yb3þ2free

1þ K1½Yb3þfreeþ K1K2½Yb3þ2free ð1Þ where n¼½Yb3þbind

½Tftotal , K1 (¼ 1/Kd1) and K2 (¼ 1/Kd2) are the stepwise association constants

At low [Yb3+]total, all of the added Yb3+ bind to apo) Tf, [Yb3+]bind¼ [Yb3±Tf ] and therefore,

¼½Yb

3þ Tf

½Tftotal ¼ / ¼

A Amin

Amax Amin

ð2Þ

½Yb3þfree¼ ½Yb3þtotal ½Yb3þbind

¼ ½Yb3þtotal ½Yb3 Tf

¼ ½Yb3þtotal /½Tftotal ð3Þ

By fitting the / 3+]freecurve to Eqn (1) with those data shown in the insert of Fig 1, we get the two dissociation constants Kd1¼ 4.17 ± 0.4 lM and Kd2¼ 18.5 ± 7.0 lM

ICP-AES was also used to study the binding ratio of Yb3+

to apo-Tf After addition of 2.0 and 2.5 mol equiv of Yb3+

to apo-Tf, the final ratios of Yb3+to Tf, after removal of low molecular mass components via ultrafiltration, were
1.70 : 1 and 1.98 : 1, respectively This suggested that both the N- and C-lobe of apo-Tf could be occupied by Yb3+ The kinetics of binding of Yb3+to apo-Tf The kinetics of

Yb3+binding to apo-Tf was studied with the stopped-flow technique The absorbance at 242 nm increased upon mixing Yb3+with apo-Tf and the rise was more rapid with increasing the molar ratio of Yb3+ to apo-Tf (data not shown) The apparent rate constants were obtained from fitting the kinetic curves The quality of the fits was assessed according to the residuals and the normalized variance (Fig 2) The data can be fitted to the bi-exponential function (Eqn 4):

fðxÞ ¼ A1expðR1xÞ þ A2expðR2xÞ þ c ð4Þ Where R1and R2are the rate constants of the two kinetic phases, and A and A are the corresponding amplitudes

Fig 1 UV difference spectra of apo-Tf (11.3 l M apo-Tf in 5 m M

NaHCO 3 and 10 m M Hepes buffer at pH 7.4 and 310 K) after addition

of various amounts of molar ratios of Yb3+ (as YbCl 3 ) The two

absorption bands at 242 and 295 nm are indicative of Yb 3+ binding to

the specific iron sites of Tf Molar ratio from bottom to top: 0.25, 0.50,

0.75, 1.0, 1.25, 1.5, 2.0, 3.0, 3.5 and 4.0 Inset: titration curve (242 nm)

for Yb 3+ binding to apo-Tf.

that show the contribution of individual kinetic phases to

the observed change in the absorption The molar

absorp-tivity (e) and the normalized rate constant k can be obtained

from the fitted values of A and R divided by the protein

concentration (e¼ A/[Tf] and k ¼ R/[Tf]) The

depend-ence of e and k on the molar ratio of Yb3+to apo-Tf is

shown in Fig 3, upper and lower, respectively

In order to understand the two kinetic phases, Tf with

iron selectively loaded into the C-lobe (FeC-Tf) was

prepared and the binding kinetics of Yb3+to FeC-Tf was

studied under the same experimental conditions Quite

differently, the kinetic data for the reaction of Yb3+and

FeC-Tf can readily be fitted to a single exponential function

f(x)¼ Aexp(–Rx) + c The molar absorptivity (e) and the

normalized rate constant k can be obtained similarly and

were shown in Fig 2 for comparison It was noticed that e1

and e (13 300 ± 500M )1Æcm)1) were approximately the

same and were only half the value of e2 The slope of the

curve of rate constant k1 vs [Yb3+]/protein (1.268· 105

s)1) was again almost the same as that of k vs [Yb3+]/

protein (slope: 1.216· 105s)1)

Citrate competition

The competition between Tf and citrate for Yb3+binding

was investigated by addition of small aliquots of citrate to

the solution containing apo-Tf and 2.5 mol equivalents of

Yb3+in the presence of 5 mM bicarbonate The protein

bound Yb3+ (converted from DA242) decreased almost

linearly upon addition of 2, 4, 6, 8 and 10 mol equiv of

citrate, and finally reached to < 10% of its original value

The very minor decrease in absorbance upon further

addition of citrate was probably due to other factors

(Fig 4)

Competition with ferric iron When Fe3+was added to a

solution of apo-Tf containing 2.5 mol equiv of Yb3+, a new

broad band centred at 465 nm appeared and increased in

intensity gradually up to 2.0 mol equiv of Fe3+(data not shown) This indicated that 2 mol equiv of iron were sufficient to completely displace Yb3+from Yb-bound Tf species in solution of 2.5 : 1 mol ratio, suggesting that Fe3+ binds to Tf more tightly than Yb3+(data not shown)

The binding mode and the conformational changes induced by Lu3+and Yb3+:1H NMR studies

These experiments were carried out in order to investigate the order of lobe loading and the conformational change induced by Yb3+ Because the signals of Yb3+-Tf systems were broadened, due to paramagnetic effect of Yb3+, a parallel study on Lu3+ and apo-Tf was conducted for comparison that will provide insight into the nature of

Yb3+binding and the conformational changes in detail

A1H NMR spectrum of apo-Tf in the presence of 10 mM bicarbonate was recorded after addition of Yb3+(or Lu3+)

Fig 2 Kinetics of binding of Yb3+to apo-Tf The final concentration of

the protein is 4.15 l M , and the molar ratio of Yb3+to apo-Tf is 1.75.

Also shown are the residuals to the bi- (middle) and mono-exponential

fits (the lower), suggesting that the bi-exponential function is a better fit.

Fig 3 The dependence of e (top) and k (bottom) on the molar ratio of

Yb3+to apo-Tf In the lower panel the data of k 1 , k 2 and k over the range of the ratio [Yb3+]/protein between 2 and 6 are linearly fitted:

k 1 ¼ 1.268x + 2.449, R 2

¼ 0.979; k 2 ¼ 0.704x + 0.110, R 2

¼ 0.952;

k ¼ 1.216x + 0.508, R 2

¼ 0.996.

in steps of 0.5 mol equiv Typical1H NMR spectra of

apo-Tf in aliphatic and aromatic regions are shown in Figs 5 and

6 The resonance appeared in the high-field region

(+0.5 to)1.0 p.p.m., Fig 5A) was attributed to the methyl

groups close to the surface of the aromatic rings of Phe, Trp

and Tyr residues due to the ring current effects [29] Signal b

()0.33 p.p.m) was related to the resonance of the CH3

group of Leu122, which lies directly above the face of

Trp128 [29–31] Signals g and h were assigned to Leu122

and Val246, respectively; other signals were assigned to

methyl groups in the C-lobe [29] Following the addition of

1 mol equiv of Lu3+, signal c disappeared, signal d

increased in intensity, while signal j decreased, and signal i

shifted to the lower frequency The other signals remained

unchanged This suggests that Lu3+binds to the C-lobe

first No changes appeared after further addition of Lu3+,

which is similar to that of Bi3+[32] More changes were

observed in the1H NMR spectrum upon addition of Yb3+

to Tf solution (Fig 6)

The typical1H NMR spectrum of Tf in the region of 2.0–

2.2 p.p.m featured three sharp intense signals (Figs 5B and

6B), which are assigned to the N-acetyl moieties of AcNeu

and GlcNac of the di-antennary glycan chains in the C-lobe

[29] Upon the addition of 1 mol equiv of Lu3+, a new signal appeared at 2.097 p.p.m (signal C), while the signal

at 2.088 p.p.m split into two (A and B) The relative intensities of the peaks altered upon further addition of

Lu3+, but no shift occurred Although the signals were broadened and shifted by Yb3+due to its paramagnetic character, the appearance of a new signal at 2.120 p.p.m and the splitting of signals at 2.084 and 2.045 p.p.m were still visible (Fig 5B); Yb3+binding to the C-lobe can thus

be inferred These results suggested that Yb3+bind to the protein and probably altered the conformation of the protein in a manner similar to Fe3+

The signals in the region of 6.2–8.5 p.p.m were usually attributed to His dCH resonance As shown in Fig 5C, when 0.5 mol equiv of Lu3+ was added, signal p (6.360 p.p.m) decreased in intensity, while the new signal

q appeared at 6.420 p.p.m Further addition of Lu3+led to the disappearance of p and further increase of q, whereas a new signal r emerged at 7.75 p.p.m No further changes were observed up to 1.5 mol equivalent Lu3+was added In 2D-TOCSY1H NMR spectra, the signals at 6.34 p.p.m (p) and 7.72 p.p.m (r) were previously demonstrated to be correlated [32] These changes induced by Lu3+indicated the disturbance on the microenvironment around His residues In contrast, the signals of apo-Tf were severely broadened when Yb3+was added (data not shown) Binding of Yb2-Tf on K562 cell membrane

Binding of FITC-Yb2-Tf to K562 cells The binding of Yb-bound Tf species in solution with 2.5 : 1 mol ratio to K562 cellular membrane was quantitatively evaluated by sorting the cells after incubation with FITC-Yb2-Tf by flow cytometric technique based on the fluorescence excited

at 488 nm As shown in Fig 7A, the mean channel fluorescence intensity (FF), which reflects the amount of FITC-Yb-Tf species bound to the cells, was found to increase significantly at low concentration of FITC-Yb-Tf (< 0.1 lM Tf), probably due to specific binding of the protein to Tf receptor (TfR) When the concentration of FITC-Yb-Tf was over 0.1 lM, the increase in intensity became less profound and reached saturation at 0.2 lM

In contrast, the mean channel fluorescence intensity of

Fig 5 1 H NMR Spectra of apo-Tf and its Lu 3+ complexes (A) In the

high-fi eld region ( )1.0 to +0.5 p.p.m), (B) N-acetyl region, and (C) in

the aromatic region.

Fig 6.1H NMR spectra of apo-Tf and its Yb3+complexes in the high-field region ()1.0 to +0.5 p.p.m) (A) and N-acetyl region (B) Fig 4 Competition between apo-Tf and citrate Changes in percent of

Yb bound to Tf (converted from the molar absorption coefficient at

242 nm) with increasing citrate concentration at 298 K and pH 7.4.

FITC-Fe2-Tf increased more evidently and reached

satura-tion at a much lower concentrasatura-tion (0.05 lM) than that of

FITC-Yb-Tf species under the same conditions This result

confirmed the binding of Yb-Tf to K562 cellular membrane

The competitive effect of Yb2-Tf on the binding of Fe2-Tf

to K562 cells K562 cells were incubated with a constant

concentration of FITC-Fe-Tf solution in the presence of

different molar ratios of Yb-Tf (2.5 : 1) at 273 K for

30 min The cells were then sorted according to FITC fluorescence excited at 488 nm (Fig 7) The results showed that upon the addition of Yb2-Tf and increasing the molar ratio of Yb-Tf /FITC-Fe2-Tf, the cell populations shift to lowered fluorescence (Fig 7B), while the mean fluorescence intensity (FF) decreased with increasing concentration of

Yb-Tf (Fig 7C) The FF decreased to half of its original value when 18 mol equiv of Yb-Tf was incubated with cells in the presence of FITC-Fe2-Tf Therefore part of the FITC-Fe2

-Tf was inhibited from binding to -TfR by Yb Tf These results suggested that Yb-bound Tf species compete with

Fe2-Tf for the specific Fe2-Tf binding sites to the receptor on the cell membrane

The transport of FITC-Yb3+-apo-Tf into K562 cells observed by LCSM

LSCM was employed to visualize the entry of FITC labeled Yb-bound Tf species into K562 cells In Fig 8A,B, the green fluorescence resulted from the emission of FITC labeled Yb-Tf when cells were excited at 488 nm As the protein (Tf) was randomly labeled with FITC, its position and intensity represent the location and relative concentra-tion of the labeled Yb-Tf The red fluorescence resulted from the emission of propidium iodide when the same cells were labeled with propidium iodide and excited at 560 nm The appearance of green fluorescence inside K562 cells (Fig 8C) clearly demonstrates that FITC labeled protein not only binds to K562 cellular membrane but also enters the cells

D I S C U S S I O N

There is an increasing interest in the use of lanthanide in medicine and biology [4–7].169Yb has been considered as a potential replacement for125I and103Pd It was also chosen

as a model nuclide for chemically related trivalent metal ions such as 90Y3+and the clinically used 153Sm3+[4,5] However, not enough about transport proteins and the chemical structure in which169Yb is incorporated into cells

is understood; in order to improve their accumulation in tumors, such knowledge is essential Tf is a glycoprotein (80 kDa) present in blood plasma with a concentration of

35 lM It is only 30% saturated with iron in blood plasma and has the capacity for binding to other metal ions of therapeutic and diagnostic interest [16] Therefore, it has been suggested that Tf can act as a nature
carrier for metallodrugs (e.g.67Ga, Ru and Ti anticancer agents) [33] Complexation of metal ions to the phenolic group of the tyrosine residues in the specific iron site perturbs the p–p* transitions of the aromatic group and leads to two absorption bands at
240 and 295 nm in the UV difference spectrum The molar absorption coefficient (26 500M )1Æcm)1) upon binding of Yb3+ to apo-Tf, is similar to other Ln to apo-Tf, for example, 20,400, 21 000 and 22 000M )1Æcm)1for Lu3+, Sm3+and Eu3+, respect-ively [34] Both UV and ICP-AES data suggested that two

Yb3+bind to apo-Tf in the specific iron binding site and two tyrosines are involved in binding of Yb3+in both the N- and C-lobe as the case for Fe3+ The X-ray crystal structure of

Sm2-lactoferrin revealed that Sm3+indeed binds to two tyrosine residues in both lobes of the protein and induces the

Fig 7 Saturation curve of binding of FITC-Fe 2 -Tf (filled square) and

FITC-Yb-Tf (filled red circle) to the K562 cellular membrane (A),

his-tograms of cellular fluorescence excited by 488 nm (B), and the inhibition

of Yb-Tf on FITC-Fe 2 -Tf bound to K562 cells (C) The three curves in

(B) represent the negative populations (curve
a), cell populations after

incubation with 0.48 M FITC-Fe 2 -Tf solution (curve
b) and cell

populations after incubation with solution with Yb 2 Tf: Fe 2 -Tf ¼ 18,

0.48 M FITC-Fe 2 -Tf (curve
c).

same overall structural changes in this protein as Fe3+[35].

However, binding of Yb3+to the strong site (the C-lobe)

was in a molar ratio Yb3+/apo-Tf < 1: 1, while the binding

to the other site is much weaker

To examine the kinetics of the uptake of Yb3+in the

individual lobes, we carried out stopped-flow experiments

and found two kinetic phases with twofold differences in rate

constants The almost identical molar absorptivity of e1and e

and approximately the same slope of the k1and k (Fig 3)

suggests that thenatureofthefirstkineticphaseofthereaction

between Yb3+and apo-Tf is the same as that of the reaction

between Yb3+and FeC-Tf Therefore the rapid kinetic phase

should correspond to the binding of the metal to the N-lobe,

although the binding favors the C-lobe thermodynamically as

judged by1H NMR The more open conformation of the

N-lobe may facilitate Yb3+ion binding [21]

Although1H NMR signals were considerably broadened

upon addition of Yb3+to the protein due to paramagnetic

properties, information can still be obtained by comparison

with its analogue Lu3+ The sharp resonances at 2.0–

2.2 p.p.m are attributable to the N-acetyl moieties of the

glycan chains (NAcGlc and NacNeu residues) in the C-lobe

of Tf As these resonances were perturbed only on addition of

the first mol equiv of Yb3+and Lu3+(Figs 5 and 6), this

suggests preferential binding of Yb3+(and Lu3+) to the

C-lobe of Tf occurs Similar behavior has been observed for

several other metal ions [29,30,32] The changes in shifts

induced by progressive addition of Lu3+(and Yb3+) may be

due to the conformational change induced by metal ions, a

common feature that was observed previously [16,29,30,32]

Our flow cytometric data (Fig 7) clearly demonstrate

that the Yb-bound Tf species can be recognized by and bind

to TfR on the surface of K562 cells in a manner similar to

Fe2-Tf However, the higher saturation concentration of

Yb2-Tf (0.2 lMvs 0.05 lMfor Fe2-Tf) and its competition

with iron indicated a lower affinity of cell receptors for Yb2

-Tf than for Fe2-Tf

LCSM offers an effective way to investigate the transport

of extracellular molecules across the membranes and to

identify the locations of the molecules within individual cells

using appropriate fluorescent probes [36–38] The green

fluorescence inside cells indicated that FITC-Yb2-Tf entered

cells and may have been located in cytoplasm when

compared with cells labeled with propidium iodide These

results are in good agreement with flow cytometric study

However, they are merely the static pictures, reflecting the

position of the Yb-bound Tf species at that moment

Further investigation is therefore needed to confirm whether

Yb3+can be released from Yb-Tf species Our data on the

stability of the Yb3+–Tf complexes as a function of pH have

shown that Yb3+ was released under acidic conditions

(pH < 6) Intracellular Yb has also been detected after

incubation of Yb-Tf solution (mol ratio 2.5 : 1) with

U87-MG cells by ICP-MS (K Wang, X Du, Y Chang, R Li, J Situ, H Sun & Z Qian, unpublished data) Thus, beside anion channel and other mechanism (e.g citrate), the cellular uptake of Yb3+via Tf receptor-mediated endocy-tosis is highly possible

Different transport or uptake mechanisms seem to exist for the complexes of Yb3+ and uptake of 169Yb-citrate was previously shown to be an active cellular transport process It

is only dependent on the metabolic activity of the cells, however, and is not tumor specific [5] The citrate concentra-tion in blood plasma (
100 lM) is comparable to Tf (35 lM) Our citrate competition data indicated that binding

of Yb3+to Tf is slightly stronger than to citrate (Fig 4) The metal can be essentially removed from the protein in the presence of 10 mol equiv of citrate Therefore, Tf is probably one of the (major) targets in blood plasma in addition to citrate Previous in vivo and in vitro studies using labeled blood serum have suggested that the major target of Yb3+is Tf [12,13] It is known that malignant cells have a higher iron requirement and subsequently express much higher Tf receptors [39] Then the uptake of Yb-bound Tf by the tumor cells might be much higher than the normal cells and the uptake of iron by the tumor cells will be retarded much more than by the normal cells Yb-Tf uptake mechanism via the receptor-mediated endocytosis is possible and this mechan-ism could selectively facilitate the accumulation of Yb3+in tumor cells It is known that67Ga citrate, a commonly used radiopharmaceutical for soft tumors and abscess diagnosis, enters tumor cells via the Tf mediated endocytosis [11,40,41]

C O N C L U S I O N S

The spectroscopic studies have shown that Yb3+binds to the two specific iron-sites of apo-Tf The1H NMR data show that Yb3+preferentially occupies the C-lobe rather than the N-lobe, as has been observed for several other metal ions and that Yb3+ can be replaced by Fe3+ Interestingly, the binding to the N-lobe is kinetically favored The binding of Yb2-Tf to K562 cell membranes (TfR) was demonstrated and was weaker than that of Fe2

-Tf and only part of Fe2-Tf could be displaced The confocal microscopic studies indicated that Yb2-Tf is likely to enter the cells in a way similar to Fe2-Tf, and at the same time almost without affecting the function of Tf It will be interesting for the future to investigate whether Tf can enhance Yb3+accumulation in tumor cells

A C K N O W L E D G E M E N T S

This work was funded by National Natural Science Foundation of China (No 29890280), the University of Hong Kong and the Hong Kong Polytechnic University We thank the Area of Excellence Scheme

of University Grants Committee (Hong Kong) for their support.

Fig 8 Transport of FITC-Yb-Tf into K562 cells visualized by LSCM (A) K562 cells after incubation with FITC-Yb-Tf (B) propidium iodide-stained K562 cells and (C) merging the images The green fluorescence indicated FITC- Yb 2 Tf entered into cells.

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S U P P L E M E N T A R Y M A T E R I A L

The following material is available from http://www.black-wellpublishing.com/products/journals/suppmat/EJB/ EJB3326/EJB3326sm.htm

Fig S1 The effect of pH on the binding of Yb3+to apo-Tf