Oxidation of membrane proteins and functional activity of band 3 in human red cell senescence

Arch. Gerontol. Geriatr. suppl. 3 (1992) 101-110 9 1992 ElsevierScience Publishers B.V. All rights reserved. 0167-4932/921505.00

101

OXIDATION OF MEMBRANE PROTEINS AND FUNCTIONAL A C T I V I T Y OF BAND 3 IN HUMAN RED CELL SENESCENCE

M.A. CASTELLANA, G. P I C C I N I N I , Go MINETTI, C. SEPPI, C. BALDUINI and A. BROVELLI Dipartimento di Biochimica, Italy

Universit~ degli Studi, Via Bassi, 21, 1-27100 Pavia,

SUMMARY The state of oxidation of membrane proteins was analyzed in red cell subpopulations of different age by q u a n t i f y i n g the oxidation of methionine to its sulfoxide and by determining the amount of thiol groups in ghost membrane preparations and the r e a c t i v i t y of thiols of individual membrane proteins in intact cells. The results obtained show that oxidation of methionine occurs early d u r i n g red cell life in the circulation, and can be detected in middle-aged and senescent cells. Thiol content of ghost membranes is kept constant in all the red cell subpopulations analyzed, but reactivity of thiol groups to the thiol reagent N-(7-dimethyl-amino-4-methyl-coumarinyl) maleimide (DACM) in intact cells decreased 30 ~ in ~ - s p e c t r i n , band 3 ( B 3 ) , 4.1 and 4.2 proteins, probably as a consequence of conformational changes of these molecules. Since the role played by band 3 in the exposure of senescence antigen has been described by many authors, the functional a c t i v i t y of the anion t r a n s p o r t e r has been analyzed by measuring the 4 - 4 " - d i i s o t h i o c y a n o - s t i l b e n e - 2 - 2 - ' - d i s u l f o n a t e (DIDS) binding capacity in d i f f e r e n t red cell subpopulations. The results obtained are in agreement with the possibility that during senescence band 3 undergoes conformational changes involving the anion channel subsite being more exposed to the extracellular space and responsible for binding of DIDS. Keywords:

red cell senescence, membrane proteins, oxidation, band 3, DIDS

INTRODUCTION Since its f i r s t demonstration by Kay (1975), the binding of autologous serum antibodies

to high density

human red cells has been documented

ferent authors (Lutz et a l . , 1984; Galili et a l . ,

1986; Sorette et a l . ,

by d i f -

1991). The

presence of antibodies on the surface of the red cell subpopulation enriched in senescent cells led to develop an autoimmune hypothesis for the removal of senescent cells from the circulation.

IgGs eluted

from

the red cells of highest

density have been shown to recognize isolated band 3 protein Lutz

et a l . ,

1984;

1985; Sorette et a l . ,

Sorette et a l . ,

1991),

~-galactosyl

(B3)

residues

1991), and other epitopes not yet defined

(Kay, 1984;

(Galili et a l . , (Sorette et a l . ,

1991). Although the role played by autologous IgGs in the removal of senescent red cells has not been completely elucidated,

their

presence on the senescent

cell surface has raised questions about the processes leading to the exposure of the senescence antigens. Among these processes oxidation seems to play an important

role,

as suggested by studies carried out on "in v i v o " and "in v i t r o "

models of red cell senescence (Johnson et a l . , al.,

1987; Beppu et a l . ,

1980; Kay et a l . ,

1990). Characterization

1986; Arese et

of density-separated

red cells

102 has provided evidence that the cells of highest density, the cell fraction rich in senescent

cells,

have suffered

1983; Imanishi et al.,

cumulative

1985; Snyder et al.,

oxidation

damage

(Brovelli

et al.,

1985; Jain, 1988); nevertheless the

state of oxidation of membrane proteins in senescent cells has not been investigated in d e t a i l . The state of oxidation of membrane proteins in circulating red cells of different ages was studied in our laboratory by analyzing the oxidation level of methionine (Met) residues and of thiol groups in ghost membranes and the reactivity of membrane protein thiol groups in intact cells. Furthermore, since the involvement of B3 in the expression of membrane properties characteristic

of

senescent red cells has been accepted by many authors (see for details: Clark, 1988), the functional activity of B3 as anion transporter was investigated in red cell subpopulations of different ages. evidences with

Data in the literature

respect to the functional

report conflicting

activity of B3 in senescent e r y t h r o -

cytes. Measuring anion fluxes, increased (Zanner and Galey, 1985) or decreased (Bartosz et al., 1987) anion transport activity, with an increase of the K m and a decrease in the Vma x of sulfate self-exchange (Kay et al.,

1986) have been

reported in senescent red cells. In order

to analyze B3 a c t i v i t y

in

red cell

subpopulations

of different

ages, we have characterized the stoichiometry of binding of the 4-4"-diisothiocyano-stilbene-2-2-'-disulfonate

(DIDS)

to B3 (Ho and Guidotti,

1975).

This

fluorochrome is a transport site inhibitor of B3, reducing partially the affinity m

of CI

for the transport site (Falke and Chan, 1986) through its covalent bind-

ing to lysine (Lys) 539 and to a Lys located in the region 814-829 (Jennings et al.,

1986). DIDS site is located in an anion channel subsite exposed to the ex-

tracellular side of the plasma membrane (Jay and Cantley, 1986), and data from Kay et al. (1990) indicate that this subsite contributes to the structure of the epitope recognized by autologous anti-B3 IgGs in senescent red cells. MATERIAL AND METHODS Isolation of red cells from whole blood and separation of erythrocytes of different cell ages. Fresh human blood was collected in 3.8 ~ ( w / v ) rate as anticoagulant.

sodium cit-

Erythrocytes were separated from plasma by centrifuga-

tion at room temperature at 1,700 g for 5 min. The cells were suspended in 1 volume of 154 mM NaCI, 4.5 mM KCI, 5 mM phosphate, pH 7.4 (PBS) containing 0.2 mM phenylmethylsulfonyl

fluoride

(PMSF), passed through a mixture of ~-

and microcrystalline cellulose as described by Beutler et al.

(1976)

and then

washed three times in PBS containing 0.2 mM PMSF. Erythrocytes of different cell density were prepared from human blood as previously described

(Seppi et a l . ,

1991), by a modified version of Murphy's

103 method

(1973).

The sedimented cells were divided

into 8 fractions:

the 2nd,

4th and 8th fractions from the top were collected and the state of oxidation of their membrane proteins and the functional activity of B3 were analyzed. The creatine content of fractionated

red cells ( G r i f f i t h s ,

1964; Fehr and

Knob, 1979) served as a cell-age parameter. Preparation

of ghost membranes.

volumes of 5 mM Tris (containing

Washed erythrocytes

(hydroxymethyl)

were

methylamine ( T r i s ) / H C I

0.2 mM PMSF and 1 mM ethylenediaminetetraacetic

salt (EDTA) as proteinase inhibitors)

lysed

buffer acid,

to prepare ghost membranes

in 10 pH 7.4

disodium

in parallel:

(a) by the method of Marchesi and Palade (1967) and (b) by the same method, but omitting the washing with 50 mM Tris/HCI buffer pH 7.4 containing 0.5 M NaCI. Evaluation of the state of oxidation of membrane proteins was performed on ghost membrane

preparations

by

assaying

content as reported by Seppi et al.

their

methionine

sulfoxide

(MetSO)

(1991) and their thiol content after treat-

ment with 1 ~ ( w / v ) sodium dodecyl sulfate (SDS), using DACM as thiol reagent (Yamamoto et al.,

1977; Seppi et al.,

1991).

Using DACM, the reactivity "in

situ" of the membrane protein thiols was also evaluated as reported by Seppi et al. (1991). Treatment of different red cell subpopulations with DIDS. Washed and packed erythrocytes were suspended in I volume of 0.4 mM DIDS in Krebs-Ringet phosphate buffer incubated at 37~

(KRp)

(Ho and Guidotti,

1975).

Cell suspensions were

for 20 rain by gentle agitation in the dark. Erythrocytes were

washed five times in KRp containing I ~ ( w l v )

bovine serum albumin and once

with PBS. Washed erythrocytes were lysed to prepare ghost membranes by the method

(b).

Functional

activity

of

B3 has been

amount of DIDS bound to B3 in young,

analyzed

by

measuring

the

middle-aged and senescent cells (see

below). SDS-polyacrylamide-gel

electrophoresis

(SDS-PAGE)

of

d e n a t u r e d ghost

membranes was carried out according to Laemmli (1970), in the conditions described

by Seppi et al.

(1991).

Electrophoretic

separation

was performed

on

polyacrylamide gel slabs (14 x 13.5 cm, 1.5 mm thickness) with run times of 4-5 hr at 40 mA. The protein content of each sample was 60 ~g. After electrophoresiso the amount of DACM bound to each protein band was determined as reported elsewhere (Seppi et al.,

1991). Fluorescence intensity emitted by DIDS

bound to B3 was quantified at X > 400 nm (Xex = 366 nm), by scanning gels with a Camag TLC II densitometer.

The same gels were then stained with Coo-

massie blue and scanned at 561 nm. The amount of DIDS bound to B3 was evaluated

by measuring

the

ratio between

fluorescence

intensity

fluorochrome and absorbance due to Coomassie blue staining.

emitted by the

104 Analytical methods.

The assay of MetSO content of membrane proteins was

carried out on ghost membranes prepared with the method ( a ) , with b u f f e r s not containing

PMSF.

Ghosts

(0.2

ml corresponding

hydrolyzed for 5 hr in 4 ml of 14 -% ( w / v ) al.,

to =0.8 mg of protein)

Ba(OH) 2 x 8H20 at 120~

were

(Krehl et

1946), and MetSO was determined by ion exchange chromatography as de-

scribed by Moore and Stein (1951) using an LKB 4101 Aminoacid A n a l y s e r . tein content was determined by the method of Lowry et al. albumin as a standard. with the D r a b k i n ' s

Pro-

(1951) using serum

Hemoglobin content of red cell suspensions was assayed

reagent as described by Beutler (1984). Creatine content of

red cells was assayed on density-separated

red cell subpopulations as reported

by G r i f f i t h s (1964). RESULTS The state of oxidation of membrane proteins in red cells of different ages. We analyzed the state of oxidation

of two s u l f u r - c o n t a i n i n g

amino acids,

cysteine (Cys) and Met,in membrane proteins of e r y t h r o c y t e s of d i f f e r e n t ages. The

results

during

obtained

show that oxidative

processes

the red cell life in the circulation.

appears

to involve d i f f e r e n t l y

affect

membrane

proteins

The expression of these processes

Met and Cys residues of membrane proteins

in

red cell subpopulations of various ages. Oxidation of Met to its sulfoxide (MetSO) is evident in both middle-aged and senescent red cells (Figure I ) , while quantitation gent-treated and

of thiol

content of membrane proteins,

ghost membranes, did not show differences

senescent e r y t h r o c y t e s

used

for

thiol

titration,

(Figure

2).

was applied

performed in young,

on determiddle-aged

When the same thiol-reagent

to

analyze

the

reactivity

of

(DACM) membrane

protein thiol groups in intact cells, and the response of individual proteins was quantified,.~-spectrin, their

reactivity

(Figure 2).

B3, 4.1 and 4.2 proteins showed a decrease of 30 96 of

to DACM

in

In middle-aged

senescent

as compared

red cells a r e a c t i v i t y

to middle-aged

red

cells

similar to that of young cells

was found (not shown). Functional analysis of B3 protein in red cells of different ages. Since B3 protein and some proteins present in the B3 environment are concerned in the decreased thiol r e a c t i v i t y characteristic of senescent cells (Figure 2),

and since the involvement of B3 i n

characteristic 1984;

of senescent

Lutz et a l . ,

1984;

cells

the expression of membrane properties

has been

described

by

many authors

Low, 1989), we have performed a functional

B3 in red cells of different ages, by analyzing

(Kay,

s t u d y of

the stoichiometry of binding of

DIDS to B3 in intact red cells of various ages. A f t e r having verified the specificity of DIDS binding to B3 in our e x p e r imental conditions (Brovelli et a l . , 1991),:the amount of fluorochrome bound to

105 %

200 -

150 -

100

-

-//,

,,

190

//,, 170

,//,

130

110

, 70

, 50

creatine lxg / g Hb

age F i g u r e 1. MetSO c o n t e n t in m e m b r a n e p r o t e i n s of y o u n g , m i d d l e - a g e d and s e n e s c e n t red cells. T h e r e l a t i v e cell a g e is g i v e n as i n t r a c e l l u l a r c r e a t i n e c o n t e n t { G r i f f i t h s , 1964, F e h r a n d K n o b , 1979). MetSO c o n c e n t r a t i o n s w e r e m e a s u r e d as n a n o m o l e s / m g of m e m b r a n e p r o t e i n a n d p l o t t e d t a k i n g t h e y o u n g v a l u e 100 96. % 150

-

100 % and and and d

50

--//, 190

,

,

170

~,

,

130

,

110

//

,

70

,

1 3 4.1 4.2

69 70 69 72

+ + + +

20 19 25 27

,

50

creatine ~tg / g Hb

age F i g u r e 2. Thiol c o n t e n t of m e m b r a n e p r o t e i n s in red ceils a s s a y e d on d e t e r gent-treated ghost membranes prepared from yot~ng, middle-aged and senescent red cells (in 96 of the young f r a c t i o n ) : ( [ ] ), and the r e a c t i v i t y to the thiol reagent DACM ( / ~ ) of membrane protein fractions in middle-aged and senescent e r y t h r o c y t e s (in 96 of the middle-aged f r a c t i o n ) . The relative cell age is given as intracellular creatine content ( G r i f f i t h s , 1964; Fehr and Knob, 1979).

106

Ly,539

...I-

DIDS SITES

- - .

8,.829

reprintedfrom: Kay r al., 1990

Young

Middle-aged

creatine

141 + 20 lig/g Hb n=4

80 + 12 lig/g HI) n--4

F.L/O.D.%

100

89 + 14

Middle-aged

Senescent

creatine

102 + 25 ~tg/g Hb n=5

47 + 12 ttg/g Hb n=5

F.I./O.D.%

100

90 ---- 5 9

Figure 3. Binding of DIDS to B3 in y o u n g , middle-aged and senescent cells. Relative cell ages are expressed as creatine content of the analyzed e r y t h r o c y t e subpopulations. DIDS concentrations bound to B3 are compared as fluorescence intensities/optical densities ( F . I . / O . D . ) on a percentage basis as shown. The symbol 9 indicates significant difference at p < 0.01. A scheme of the s t r u c t u r e of B3 integral domain is presented, indicating the localization of 3 Met residues (9 and of DIDS binding sites. B3 was quantified by measuring the ratio between fluorescence intensity emitted by B3 protein and its absorbance due to Coomassie blue staining.

The results

obtained comparing the amount of DIDS bound to B3 in young, middle-aged and senescent e r y t h r o c y t e s are summarized in Figure 3, and they show a decrease in the amount of DIDS bound to B3 in senescent cells. DISCUSSION Many authors chain

have suggested

of events that

in senescent

that oxidation red

is the process initiating

cells modifies

structure,

topology

the and

topography of B3, and finally produces the exposure of the senescence antigen on the

red cell

surface,

Results

reported

in this

paper

show that

affects membrane proteins t h r o u g h o u t the red cell life in the circulation,

oxidation since a

significant oxidation of Met residues to the corresponding sulfoxide is evident in middle-aged and senescent red cells. Thiol state of membrane proteins proved to be constant in all red cell subpopulations

analyzed;

however the r e a c t i v i t y

to

the thiol reagent DACM of B3, a - s p e c t r i n ,

4.1 and 4.2 proteins due to Cys of

107 membrane

proteins

middle-aged

decreased

red ceils.

30 ~ in

intact

It must be noted that

senescent

cells

the decreased

with

respect

reactivity

to

to the

thiol reagent was observed in particular in B3 and in other proteins interacting with

this molecule

in the supramolecular

arrangement

of the membrane

(Low,

1989). On the basis of the hypothesis that the observed response could be related to a modified conformation and function of the anion t r a n s p o r t e r , analyzed the functional a c t i v i t y of B3, by q u a n t i f y i n g

we have

the binding stoichiometry

of B3 with DIDS, a t r a n s p o r t site i n h i b i t o r of this membrane protein.

We found

that the amount of DIDS bound to B3 molecule decreases in senescent red cells. This behavior could depend on oxidation of some Met residues of B3. Although we have no data about the state of oxidation of B3 protein,

we could t r y

to

relate observations about the impairment of B3 to bind DIDS and the state of oxidation of membrane proteins d u r i n g red cell senescence. Human B3 contains 5 Cys and 24 Met residues (Tanner et a l . ,

1988; Lux et a l . ,

1989). Oxidation of

Met, evident in middle-aged and senescent red cells, is known to produce short range conformational aminoacidic

changes

sequence,

in the affected

deduced

by

the

proteins.

sequence

of

By the analysis of B3

the

corresponding

cDNA

(Tanner et a l . , 1988; Lux et a l . , 1989) it can be observed that Met 559 and 578 and Met 833 are near to the Lys residues (539 or 542 and in the region 814829) involved sidues could

in the binding modify

sidues to bind

of DIDS (Figure 3).

B3 conformation

DIDS in senescent

and,

cells.

Oxidation

therefore,

of these Met re-

the capacity

By performing

of Lys re-

competitive

inhibition

assay using synthetic peptides to adsorb IgGs isolated from senescent e r y t h r o cytes Kay et al. (1990) have shown that the red cell senescent antigen contains residues 538-554 and residues 812-827. These regions bear DIDS binding sites, and the results obtained in our functional study of B3 agree with the observations of K a y ' s g r o u p , undergoes during

namely that

the conformational

changes the B3 protein

red cell aging concern the region of the anion channel more

exposed to the extracellular space, where the DIDS-binding site is located. Oxidation

of Met could produce

the DIDS binding

capacity,

conformational

changes affecting,

besides

the r e a c t i v i t y of the Cys 843 toward the thiol-rea-

gent DACM. Investigations are in progress in our laboratory to evaluate the capacity of B3 to bind eosin 5-maleimide and eosin 5-isothiocyanate

in young,

and senescent red cells.

inhibitors

These compounds are specific

middle-aged of the anion

t r a n s p o r t a c t i v i t y of B3, whose binding subsites seem to be located more deeply in the anion channel

(own unpublished

observation).

The results obtained

in

these studies are also in agreement with the possibility that the conformational changes the B3 undergoes during

senescence involve the anion channel region

more exposed to the extracellular space (own unpublished observation).

108 Our results suggest the possibility that oxidation of Met could initiate the chain of events finally leading to the exposure of the senescence antigen. The results obtained by treating e r y t h r o c y t e s with p h e n y l h y d r a z i n e as an oxidant reagent have shown that oxidation affects anion t r a n s p o r t a c t i v i t y of B3 (Petty et a l . ,

1991).

It is, however, worth

reminding that oxidation of other amino

acid residues, essential for t r a n s p o r t a c t i v i t y like a r g i n i n e , Iysine and glutamic acid (Jay and Cantley, 1986), and of h i s t i d i n e , involved in the conformational changes occurring d u r i n g the anion t r a n s p o r t

(Hamasaki et a l . ,

1989), could

play a role in the functional impairment of B3 d u r i n g e r y t h r o c y t e senescence. F u r t h e r investigations about the state of oxidation of B3 would help to understand better the role played by oxidative processes in the exposure of the senescence antigen. ACKNOWLEDGEMENTS Work supported by funds from MURST and from CNR: Target Project Biotechnology, and Target Project Aging to C.B. REFERENCES Arese, P., Bussolino, F . , Flepp, R., Stammler, P., Fasler, S. and Lutz, H.U. (1987): Diamide enhances phagocytosis of human red cell in a complementand anti band 3 antibody-dependent process. Biomed. Biochim. Acta, 46, S84-S87. Bartosz, G., Gaczynska, M., Grzelinska, E., Soszynski, M., Michalak, W. and Gondko, R. (1987): Aged e r y t h r o c y t e s e x h i b i t decreased anion exchange. Mech. Ageing Dev., 39, 245-250. Beppu, M., Mizukami, A . , Nagoya, M. and Kikugawa, K. (1990): Binding of a n t i - b a n d 3 auto-antibody to o x i d a t i v e l y damaged e r y t h r o c y t e s . J. Biol. Chem., 265, 3226-3233. Beutler, E., West, C. ~and Blume, K.G. (1976): The removal of leukocytes and platelets from whole blood. J. Lab. Clin. Med., 88, 328-333. Beutler, E. (1984): Red cell metabolism. In: A Manual of Biochemical Methods. 3rd edition, pp. 8-19. Editor: E. Beutler. Grune and Stratton, New York. Brovelli, A . , Seppi, C . , Pallavicini, G. and Balduini, C. (1983): Membrane processes d u r i n g "in vivo" aging of human e r y t h r o c y t e s . Biomed. Biochim. Acta, 42, S122-S126. B r o v e l l i , A . , Castellana, M . A . , Minetti, G., Piccinini, G., Seppi, C . , De Renzis, M.R. and Balduini, C. (1991): Conformational changes and oxidation of membrane proteins in senescent human e r y t h r o c y t e s . In: Red Blood Cell A g i n g , pp. 59-63. Editors: M. Magnani and A. De Flora. Plenum Press, New York. C l a r k , M.R. (1988): Senescence of red blood cells: progress and problems. Physiol. Rev., 68, 503-554. Falkeo J.J. and Chan, S . I . (1986): Molecular mechanisms of band 3 i n h i b i t o r s . I . T r a n s p o r t site i n h i b i t o r s . Biochemistry, 25, 7888-7894. Fehr, J. and Knob, M. (1979): Comparison of red cell creatine level and r e t i culocyte count in appraising the severity of hemolytic processes. Blood, 53, 966-976. Galili, U . , Macher, B . A . , Buehler, J. and Shohet, S.B. (1985): Human natural anti-alfa-galactosyl IgG. II. The specific recognition of a l f a ( 1 - 3 ) - l i n k e d galactose residues. J. Exp. Med., 162, 573-582.

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