Crosslinking of membrane proteins during erythrocyte ageing

Inr. J. Biochem. Vol. 18. No. 4. pp. 377-382. 1986 Printed in Great Bnlain. All rights reserved

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0020-71 IX’86 $3.00 + 0.00 1986 Pergamon Press Ltd

CROSSLINKING OF MEMBRANE PROTEINS DURING ERYTHROCYTE AGEING MARIA GACZY~~SKA and GRZEGORZBARTOSZ Laboratory

of Biophysics

of Development

and Aging, Department 90-237 Zbdi, Poland

(Received 9 Augusf

of Biophysics,

University

of JGdi,

1985)

Abstract-l. Membrane proteins of bovine erythrocytes were crosslinked with cupric di( I ,10-phenanthroline) and analysed by one-dimensional and two-dimensional SDS-polyacrylamide gel electrophoresis. 2. An increase in crosslinking of the Band 3 protein and of spectrin was found with increasing erythrocyte age suggesting an increased aggregation of main membrane proteins in aged erythrocytes.

INTRODUCTION Though the detailed sequence of events in the process of erythrocyte aging is unknown, changes in the structure and mutual arrangement of membrane proteins seem essential for functional alterations of the red cell membrane and for enabling specified recognition of senescent erythrocytes. Various changes of red cell membrane proteins have been reported to occur during the cell aging including an appearance or increase in the content of Band 4.1 protein (Kadlubowski and Harris, 1974; Pfeffer and Swislocki, 1982), increase in polymerization of membrane proteins due to reaction of lipid peroxidation products (Jain and Hochstein, 1980), irreversible spectrin-haemoglobin crosslinking (Snyder et al., 1983) or conformational changes detected with a spin label (Bartosz, 1981) as well as progressive proteolysis (Kay, 1984). Alterations in the structural state of the Band 3 protein may be especially significant for erythrocyte aging since (i) this main integral protein of the red cell membrane performs various important functions such as anion transport (Cabantchik and Rothstein, 1974), binding of haemoglobin (Salhany and Shaklai, 1979) and glycolytic enzymes (Strapazon and Steck, 1977) and anchoring red cell cytoskeleton in the membrane (Bennett and Stenbuck, 1979), and (ii) there is evidence that the “senescent cell antigen” appearing on the surface of old erythrocytes is derived from the Band 3 protein (Kay et al., 1983). As the aggregation state may be an important variable determining protein function, this study was aimed at determination if the aggregation state of main erythrocyte membrane proteins changes in the course of the cell aging. The results obtained indicate an increase in aggregation of the Band 3 protein, and possibly also of spectrin, during the in uiz*o aging of the erythrocyte. MATERIALSAND

METHODS

bottom (oldest) 20% of cells were withdrawn from the stratified cell column and used for comparative studies. Red cell ghosts were prepared according to Dodge et al. (1963). Crosslinking of membrane proteins In most experiments, membrane proteins were crosslinked with cupric di( I, IO-phenanthroline) as follows: membrane aliquots containing 2 mg protein per ml in 5 mM sodium phosphate, pH 7.4, were added with 0.1 mM CuSO,‘5H,0/0.5 mM o-phenanthroline (POCh, Gliwice, Poland) or 0.2 mM CuSO,.SH,O/l mM o-phenanthroline (final concentrations) and incubated for I5 min at room temperature. The reaction was stopped by addition of sodium ethylenediaminetetraacetate up to a final concentration of 20 mM. Electrophoresis The crosslinked samples were submitted to onedimensional SDS-polyacrylamide gel electrophoresis in 7.5% slab gels in the system of Laemmli (1970) or to two-dimensional gel electrophoresis by a modified method of Reithmeier and Rao (1979) in which the first step was accomplished in 7.5% cyllindrical gels under non-reducing conditions while the second step in 10% slab gels under reducing conditions in the system of Laemmli (1970). In all cases the resolving gels were overlaid with 3% stacking gels and the samples were prepared for electrophoresis without a reducing agent. The slab gels were stained with Coomassie Brilliant Blue R-250 by the method of Fairbanks et nl. (1971). Protein bands were quantitated by elution of gel slices with 0.1 M NaOH/0.2% SDS for 48 hr at 37°C and absorbance of the eluatei was measured at 600 nm. Some of the two-dimensional gels were stained by the silver method of Marshall (1984) for qualitative analysis of aggregates. Molecular weights of a part of the aggregates were approximated by comparison of mobilities of the new bands which appeared after crosslinking with those of Band I (240,000). Band 2 (220,000) and Band 2.1 (200,000). The bands were designated according to Fairbanks et al. (1971).

RESULTS

General

Erythroc.vte membranes

characteristics

of the crosslinking

Cupric di( 1, lo-phenanthroline) (CUP) catalyses the oxidation of protein sulfhydryl groups by molecular oxygen which leads to formation of covalent crosslinks between polypeptide chains whose sulfhydryl groups are in close proximity. The disulfide bridges

Bovine blood, obtained in a local abbatoir, was anticoagulated with sodium citrate and processed immediately. Erythrocytes were separated according to age by the method of Murphy (1973). Top (youngest) 20%. middle 20% and 377

378

MARIA GACZY~KA and GRZEGORZ BARTOSZ

I.

Table without

Relative

protein

a reducing

bar&aggregates

agent

ratio5

and crosslinked

(containing

DTT

with

or ME)

in membrane

CUP

assumed

(in

%).

samples

Respective

as 100%

(mean

+ SD.

Crosslinked Samples Ratm Bd 3;Ag

I

Bd 3.Ag

2

2. Relative

membranes

reducing

aeenl

0.1 mM10.5

3

ratios

h3?

IO

Bd

I + 2:Ag

2

60 i

II

ratms

of various

age fractions

“young”

x

cells assumed

and

“old”

~.~

One-dimensional

Bd3/Agl

L~~_~~~

S?_t ~~..~_

respective DitTerence

mM

CUP

0.2 mM/I

19

68k

Bd 3/Ag

1-3

Bd 3/Ag

2-3

Bd

I + Z/Agl-I

Bd I + 2/Ag2-I

mM

CUP Old 7s +9

IO

80?

I3

72*

IO

113+

IO

93 t

5

I8

Two-dimensional

electrophoresis mM

76i + 2

f SD,

89i

Old

+ 2

significant

(mean

Medium

Old

0.1 mMiO.5 Ratio

not

electrophoresis

64 k 9

71+21

+ 2iAg2

(in%;

as 100%).

membranes

-

Bdl

for CUP-crosslinked

to paired “1” test) at P = 0.05 n= ? nr A\

Medium

+ 2iAgl

4I

ii

II + I 0 . I

0.1 mM/0.5

Bdl

! I

17i2

~~

Bd3/Ag2

I mM Ii

lo+2

(according

Rat10

WmM

72 * 6

of youngest

for

CUP

I

value for the fraction between

mM

I3 z 2

691

band/aggregate

II

68 i: I9 -

wnple\

sample\

I ? 2:Ag

protein

prepared

II = 3)

Bd

of erythrocytes

statistically

for control

wthout

formed may be easily disrupted by treatment with reducing agents like 2-mercaptoethanol (ME) or dithiothreitol (DTT) thus enabling identification of aggregate-forming polypeptides by two-dimensional electrophoresis containing a reducing agent in the second dimension gel (Peters and Richards, 1977). We observed a rapid aggregation of bovine erythrocyte membrane proteins, especially of bands 1, 2, 2.1, 3, 4.2 and 5 by CUP (Fig. 1). The reaction was rapid and practically the same pattern of aggregation was seen after exposure to CUP in the range of several seconds-up to 10 min. No significant differences were observed in the crosslinking pattern between incubation at 0°C and room temperature at the both CUP concentrations employed. CUP concentrations higher than 0.5 mM/2.5 mM resulted in an almost complete crosslinking of all membrane proteins, with an appearance of intensely staining bands at the borderline of the stacking and resolving gels. The crosslinking caused by disulfide bridges formation occurred also to some extent by simply omitting a reducing agent during sample preparation for electrophoresis (Fig. 1). The extent of crosslinking can be quantitated as a ratio of a protein band content to that of an aggregate content. Four such ratios were analysed: Band 3 to aggregates in the stacking gel (Ag 1), Band 3 to aggregates in the resolving gel above spectrin (Ag 2), Bands 1 + 2 to Ag 1 and Bands 1 + 2 to Ag 2, respectively. Values of these ratios found for erythrocyte membranes prepared for electrophoresis without a reducing agent and crosslinked with CUP are given in Table 1.

Table

(unfractlonated) ratios

CUP

t

I

(‘UP

Decrease of the protein band/aggregates ratio calues with increasing erythrocyte age was observed for all the ratios analysed and all the conditions employed (Fig. 1, Table 2). In addition to oncwith samples crosslinked dimensional gels, 0.1 mM/0.5 mM CUP were analysed by twodimensional gel electrophoresis. In the latter case. aggregates originated only from Band 3 or spectrin and migrating in the region corresponding to the stacking gel in one-dimensional electrophoresis were designated Ag l-3 and Ag l-l + 2, respectively. The Ag 2-l + 2 region corresponded to the Ag 2 region in one-dimensional gels while the aggregates designated Ag 2-3 corresponded to the main aggregate of the Band 3 protein (Fig. 2). Not all the changes observed were statistically significant; those bearing statistical significance are reported in Table 2. Qualitatiae

characteristics

of’ the crosslinking

Clear-cut cell-age dependent changes in the aggregate pattern were seen in the stacking gel after one-dimensional electrophoresis. While for control samples, prepared with DTT or ME, this region contained no band of protein aggregates, such aggregates appeared in the stacking gel for samples prepared without a reducing agents or crosslinked with CUP in a number generally increasing with cell age (Fig. 1). Electropherograms of samples prepared without reducing agent showed one aggregate band (at the top of the stacking gel) for membranes of young erythrocytes, while, moreover, a second band appeared below in the stacking gel for membranes of medium and old erythrocytes. Electropherograms of samples crosslinked with 0.1 mM/O.S mM CUP. showed one and two aggregate bands for membranes of young and old cells, respectively, and changeable amount (1 or 2) for membranes of middle-age erythrocytes. After two-dimensional electrophoresis, aggregates in the region corresponding to the stacking gel were. in general, hard to identify. However, two bands originated from Band 3 and one originated from spectrin were observed for samples treated with 0.2 mM/l mM CUP. This gel region contained also a big aggregate of Band 2.1. Composition of the main aggregates determined by two-dimensional gel electrophoresis is shown in Table 3. Samples prepared without a reducing agent but not treated with CUP yielded only small, hardly identified aggregates. Molecular weight of the main Cup-induced aggregates are given in Table 4. The main crosslinking product of Band 3 was a big aggregate of approxi-

.

Fig. 1. One-dimensional electrophoresis of crosslinkcd erythrocyte membranes from cells of various age fractions: Lane 1 (from the left)+ontrol sample (membranes of medium fraction, prepared with DTT). Lanes 2 A-membranes of young, medium and old erythrocyte fractions, respectively. crossllnked with 0.1 mMi0.5 mM CUP. Band 3 and gel regions delined as “Ag I” (stacking gel) and “Ag 2” (resolving gel above spectrin) are marked. The gel was stained with Coomassie.

37’)

Fig. 2. Two-dimensional electrophoresis of erythrocyte membranes crosslinked with 0.05 mMi0.25 mM CUP: membranes of young (A), medium (B) and old (C) erythrocytes. The 1st dimension (horizontal) non-reducing conditions, the 2nd dimension (vertical)-reducing conditions. Spectrin. Band 3 and crosslinking products of Band 3 are marked. Silver staining of the gels.

380

381

Membrane proteins and erythrocyte ageing 3. Composition

Table

of

membrane

the main aggregate of bovine erythrocyte moteins induced bv CUP Crosslmking

Location of aggregate Stacking

gel

conditmns

(mM CUP)

0. I xi.5

0 WO.25 Bd 1.2.syndeins.

Rd 1.2. syndeins

Bd 3. 4.

Bd 3

I, 4.2.

0.2:t Bd l-3

4.5. 5 Top

of resolving

Bd I to 3,4.2. 5

gel Resolving

gel

Bd I. 2. 3

Rd 1to 3.4.1.

Bd

4.2. 5

4.1.4.2.

Bd

I, 2,3.

4.5

I to 3, 5

Bd I. 2. 3,

45 Table 4. Apparent molecular werghts of the main producrs of Cup-induced crosslinking. Products of crosslinking of the Band 3 protein

in italics

Crosslinking

Erythrocyte

conditions

(mM

CUP)

age fraction

0.1,0

5

0.2,

Medium

230,000

280.000

300.000

Old

‘?3o,oou

280,QoQ

300.000

G?: 000 300.000 23mi3l3 300.~~

I

280.000 280.000

mate molecular weight of 230,000. It was well visible in two-dimensional gels (Fig. 2) but masked by spectrin in most one-dimensional gels (Fig. 1). Pronounced age-dependent changes were visible on with electropherograms of samples treated 0.05/0.25 CUP: a progressive decrease in lowermolecular weight aggregates (230,00~250,000) accompanied by an increase in higher-molecular weight aggregates (over 260,000) in membranes of aged erythrocytes, both for Band 3 and spectrin crosslinking products (Fig. 2). DlSCUSSlON

The results obtained indicate that the Cup-induced crosslinking of the Band 3 protein occurs more easily in membranes of older bovine erythrocytes. The crosslinking products appeared progressively in the Ag 1 and Ag 2 regions, depending on the CUP concentration and cell age. The main product of Band 3 crosslinking had an apparent molecular weight of about 230,000. Molecular weight of the monomer of bovine Band 3 protein was estimated to be 112.000 & 3000 (Nakashima and Makino, 1980) so the 230,000 aggregate corresponds to Band 3 dimer. This covalent dimerization of Band 3 by CUP reflects the fact that non-covalent dimer is the main aggregation state of human and bovine Band 3 protein in a native erythrocyte membrane (Makino and Nakashima. 1982; Staros and Kakkad, 1983). Band 3 tetramers were not identified in our experiments, though they might be present in the aggregate band at the very top of the resolving gel. On the other hand, covalent tetramers might be not formed, too, due to steric limitations in mutual accessibility of -SH groups of Band 3 polypeptides. Cell-age dependent changes in the crosslinkability of spectrin were less evident. It might be partly due to the migration of Band 3 crosslinking products very close to the spectrin region in one-dimensional gels, obscuring alterations in the spectrin itself. In fact,

both qualitative and quantitative analysis of twodimensional electropherograms point to changes in spectrin aggregability parallel to those of Band 3, suggesting a cell-age dependent increase in spectrin crosslinkability. too. The observed increase in the crosslinkability of the main proteins of the erythrocyte membrane seems to reflect an increased protein aggregation in the membranes of aging erythrocytes. Alternative expianations of these findings (i.e. increased -SH reactivity or increased lateral mobility of membrane proteins in senescent erythrocytes) do not seem tenable as (i) the content of membrane -SW groups does not change signifi~dntly during aging of bovine erythrocytes (Bartosz, unpublished), (ii) the changes in protein crosslinking were practically the same at O-C (when protein mobility is severely restricted) and at room temperature, and (iii) the order of membrane lipids increases rather than decreases with red cell age thus hampering rather than facilitating protein mobility (Bartosz, 1981). Therefore, although no differences were found in the distribution of intramembrane particles between young and old erythrocytes (Fischbeck et al., 1982), the present results suggest that the main membrane proteins, especially the Band 3 protein, become more aggregated as the erythrocyte ages. This change in protein topography may inffuence protein functions and proteolytic susceptibility. REFERENCES

Bartosz G. (198 1) Aging of the erythrocyte. IV. Spin-label studies of membrane lipids, proteins and permeability. Biochim.

biophys.

Acta 664, 69-13.

Bennett V. and Stenbuck P. J. (1979) The membrane attachment protein for spectrin is associated with band 3 in human erythrocyte membrane. Nurure 280, 468473. Cabantchik Z. I. and Rothstein A. (1974) Membrane proteins related to anion permeability of human red blood cells. I. Localization of disulfonic stilbene binding sites in proteins involved in permeation. J. Membrane Biol. 15, 207-226. Dodge J. T., Mitchell C. and Hanahan D. J. (1963) The preparation and chemical characteristics of hemoglobin free free ghosts of human erythrocytes. Archs Biorhem. Biophys. 100, 119-130. Fairbanks G., Steck T. L. and Wallach D. F. H. fI97L) Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry 10, 2606-2616.

Fischbeck K. H., Bonilla E. and Schotland D. L. (1982) Freeze-fracture characterization of “young” and “old” human erythrocytes. Biochim. biophys. Acta 6885,207-210. Jain S. K. and Hochstein P. (1980) Polymerization of membrane components of aging red blood cells. Biochem. biophys. Rex Commun. 92, 247-254. Kadiubowski M. and Harris J. R. (1974) The appearance of a protein in the human erythrocyte membrane during aging. FEBS Lett. 41, 252-254. Kay M. M. B. (1984) Band 3, the predominant transmembrane polypeptide, undergoes proteolytic degradation as cells age. Monogr. &I. Biol. 17, 245-253. Kay M. M. B., Goodman S., Sorensen K., Whitfield C., Wong P.. Zaki L. and Rudloff V. (1983) The senescent cell antigen is immunologically related to Band 3. Proc. natn. Acad. Sci. U.S.A.

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382

MARIA GACZYQSKA and GRZEGORZ BARTOSZ

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