effects of proteases on membrane stability of red …

原著 (Original Contribution) : 膜 (MEMBRANE), 20 (4), 287-295 (1995)

EFFECTS OF PROTEASES ON MEMBRANE

STABILITY OF RED BLOOD CELLS

Kaoru Nagao*•”, Yuichi Takakuwa*, Sumie Manno*

and Hidehiro Suzuki

Departments of *Biochemistry and #Anesthesiology , Tokyo Women's Medical College, 8-1 Kawada-cho, Shinjyuku-ku, Tokyo, 162, Japan

Skeletal proteins of red blood cells appear to be importantly involved in regulating the membrane

mechanical properties that arise with membrane deformability and stability. However, the effects of

limited digestion of skeletal proteins by proteases remain to be clearly determined. To clarify whether

proteases such as trypsin inhibitor-sensitive protease(s) and ƒÊ-calpain, a Ca2+-dependent neutral pro-

tease affect membrane mechanical properties through proteolysis of membrane proteins, assessment

was made of membrane stability of resealed ghosts treated with exogenous ƒÊ-calpain in the presence of

the Ca2+ and/or the trypsin inhibitor . Membrane proteins were analyzed by SDS-polyacrylamide gel

electrophoresis and immunobotting using antibodies against red cell membrane proteins. Untreated

ghosts showed decreased membrane stability and degradation of ankyrin . Inhibition of membrane-

associated protease activity by the trypsin inhibitor restored normal membrane stability and prevented

ankyrin degradation. When membranes previously treated with 4 U/ml of ii-calpain in the presence of

10ƒÊM Ca2+ at 0•Ž were resealed in the presence of trypsin inhibitor, membrane stability decreased and

only ankyrin was degradated, producing a polypeptide of 195-kD . With increase in calpain treatment

time, ankyrin decreased with increase in the 195-kD polypeptide . Correlation between membrane sta-

bility and calpain effects was demonstrated by increase in this polypeptide and decrease in membrane

stability. Elevation of cytosolic Ca2+ to more than 1 ƒÊM would thus appear to cause decrease in mem-

brane stability through Ca2+-activated limited digestion of ankyrin by ƒÊ -calpain .

Key words : red blood cell, protease, membrane stability, calpain, ankyrin

Introduction

Extensive deformation is required for red

cells to pass through small capillaries , and the membrane must be sufficiently strong

to withstand this distortion and resist shear-

induced membrane fragmentation. Such

mechanical stability of erythrocyte mem-

branes is maintained and regulated primar-

ily by the membrane skeleton consisting of

a network of structural proteins 1) . All ma-

jor erythrocyte membrane proteins are af-

fected by one or several types of post-trans-

lational modification 2). Post-translational

modification, such as proteolysis, may be

detrimental to protein functions including

protein-protein interactions on the mem-

brane skeleton. In red blood cells, several

types of proteases are present in membranes

and cytosol 3•`7) . The activity of membrane-

288 Nagao et al. : EFFECTS OF PROTEASES ON MEMBRANE STABILITY OF RED BLOOD CELLS

associated proteases was apparent from the

degradation of membrane proteins by en-

dogenous proteases during incubation of

isolated membranes. One such protease is

serine protease which is inhibited by soy-

bean trypsin inhibitor. In contrast to mem-

brane-associated proteases, most cytosolic

proteases are removed by extensive mem-

brane washing with a hypotonic buffer dur-

ing ghost preparation. Thus, to assess the

effects of cytosolic proteases on membrane

stability, each must be incorporated exoge-

nously into a ghost prior to resealing.

Calpain (EC 3.4.22.17) belongs to a family

of Ca2+-dependent neutral cysteine proteas-

es present in many different cells8- Most

mammalian cells contain two calpains : cal-

pain I or g-calpain, activated by micromolar

Ca2+ and calpain II or m-calpain, activated by

Ca2+ at millimolar concentration. Human

erythrocytes contain only ƒÊ-calpain 12, 13)

Most erythrocyte calpain is present in cy-

tosol as an inactive 80-kD proenzyme. In the

presence of micromolar Ca2f, procalpain

binds to membranes to be converted to the

active 75-kD form by autoproteolysis 14•`19)

Increased erythrocyte intracellular Ca2+

leads to calpain-induced degradation of var-

ious membrane proteins, most notably an-

kyrin and protein 4.120). Up to now, the ef-

fects of such degradation on membrane me-

chanical properties remain unclear.

To determine whether proteases such as

trypsin inhibitor sensitive protease(s) and ,ƒÊ-

c alp ain have effect on membrane stability,

we measured membrane stability of ghosts

treated with soybean-trypsin inhibitor and

exogeneous ,ƒÊ-calpain in the presence of Ca2+.

Inhibition of membrane-associated trypsin

inhibitor-sensitive protease activity by it's

inhibitor restored normal membrane stabil-

ity and prevented ankyrin degradation. ƒÊ-

Calpain treatment was shown to bring about

decrease in membrane stability with the de-

gradation of ankyrin and the production of

a 195-kD polypeptide.

1. Methods

1.1 Materials

μ-Ca1Dain and calpain inhibitor I were ob-

tamed from Suntory Co. Soybean trypsin in-

hibitor was purchased from Sigma Chemi-

cal Co. All were dialyzed against a hypoton-

ic buffer consisting of 5 mM Tris and 5 mM

KC1 (pH 7.4). Affinity-purified rabbit poly-

clonal antibodies against ankyrin and pro-

tein 4.1 were prepared by the usual meth-

ods21) .

1.2 Preparation of resealed ghosts

After obtaining informed consent, human

venous blood was drawn from healthy vol-

unteers (<25 years old) for use in subsequ-

ent experiments. White membranes were

prepared by lysing and washing human red

blood cells with the ice-cold hypotonic buf-

fer. For restoration of isotonicity, a small

volume of KC1, MgCl2 and dithiothreitol

(DTT) mixture was added to the membrane

suspension, to final concentrations of 150

mM KC1, 1 mM MgCl2 and 1 mM DTT. The

ghost suspension was incubated at 37•Ž for

40 min for ghosts resealing.

1.3 ƒÊ-Calpain treatment of membranes

For this purpose, white membranes were

incubated with 10 ƒÊM CaCl2 and 4 U/ml of

μ-calpain at O℃ for as much as 12 min. To

terminate calpain-catalyzed proteolysis, cal-

pain inhibitor I and EGTA were added to

final concentrations of 0.6 mg/m/ and 1 mM,

膜 (MEMBRNE), Vol. 20 No. 4 (1995) 289

respectively. The membranes were resealed

as described above.

1.4 Measurement of membrane stability

To measure membrane stability, resealed

ghosts were suspended in dextran (40,000 mol wt, 35%wt/vol) and examined by the

ektacytometer as described previously 22) . Briefly, suspended ghosts were subjected to

constant shear stress of 750 dyn/cm2 and

changes in laser diffraction patterns were

examined based on signal designated as the

deformability index (DI). Ghosts fragment

and resultant loss of membrane surface de-

creased DI with time. The rate at which DI

decreases is a measure of the rate of mem-

brane fragmentation, and hence, provides a

quantitative measure of membrane stabili-

ty. The time required for DI to reach half

its maximal value is designated T50 and was

used to evaluate changes in membrane sta-

bility.

1.5 Analysis of membrane proteins

Membrane proteins were analysed by so-

dium dodesylsulphate-polyacrylamide gel

electrophoresis (SDS-PAGE) with a 7.5%

polyacrylamide gel according to Laemmlie

et al.2, 3) and stained with Coomassie Bril-

liant Blue (CBB). Immunoblot analysis was

conducted with affinity-purified polyclonal

antibodies against ankyrin and protein 4 .1. Membrane proteins separated on SDS-PA

GE polyacrylamide gels were also blotted

on PVDF membranes using semi-dry trans-

blots (Nihon Eido Co)24) . Briefly, electro-

transfer of the proteins was carried out in 25

mM Tris containing 20% methanol and 40

Figure 1. SDS-PAGE analysis of ghosts treated with g-calpain in the presence and absence of tryp-

sin inhibitor

Membrane proteins of ghosts were analysed by SDS-PAGE in a 7.5% Laemmlie polyacry-

lamide gel and stained with CBB (lane 1) . Immunoblot analysis was performed with rabbit

antibodies against human ankyrin (lanes 2•`6) and protein 4 .1 (lanes 7•`9).

Lane 1 : unsealed membrane ; Lane 2 : unsealed membrane treated with calpain inhibitor I

followed by digestion with 4 U/ml calpain at 0•Ž for 12 min ; Lane 3 : unsealed membrane

digested with ƒÊ-calpain at 0•Ž for 12 min ; Lanes 4 and 7 : resealed ghosts ; Lanes 5 and 8 :

ghosts resealed in the presence of trypsin inhibitor ; Lanes 6 and 9 : resealed ghost treated

with ƒÊ-calpain in the presence of the trypsin inhibitor.


mM E-amino-n-Caproic acid for 1 h at 250

mA. The membranes were washed once in

0.2% Tween 20 and blocked with skim-milk

solution (Block-Ace, Yukijirushi Co) for 1

h, followed by incubation for 1 h at room

temperature with anti-ankyrin and anti pro-

tein 4.1 in skim-milk solution. Antigen-an-

tibody complexes were developed using the

peroxidase conjugate substrate kit of Dupon/ NEN. Briefly, the immunoblots were incu-

bated at room temperature for 1 h with an-

ti-rabbit IgG conjugated peroxidase and de-

veloped using the substrate solution of a

chemiluminescence reagent kit. The mem-

branes were exposed to Kodak X-ray film.

To determine the amounts of proteins, chem-

iluminescence profiles were analyzed with a

densitometer (ATTO AE6,900M). Protein

concentration was determined according to

the method of Lowry et al.2 5)

2. Results

2.1 Immunoblot analysis of red cell mem-

brane proteins digested with proteases.

Red cell membrane proteins digested with

membrane-associated protease(s) and ii -

calpain were analyzed by SDS-PAGE and

immunoblotting using antibodies against

ankyrin and protein 4.1 (Fig. 1). Immuno-

blot analysis of calpain-untreated mem-

branes showed most ankyrin to appear as

the 210-kD polypeptide with a minor band of

200-kD (Fig. 1, lane 2). Ankyrin on unsealed

membranes apparently underwent digestion

by 4 U/m/ of i-calpain at 0•Ž for 12 min to

produce a 195-kD band (Fig. 1, lane 3). A

200-kD band diminished by g-calpain. When

the calpain-untreated membranes were in-

cubated for ghost resealing at 37•Ž for 40

min, ankyrin was digested with consequent

production of the 195-kD polypeptide as well

as several small fragments (Fig. 1, lane 4).

Presence of soybean trypsin inhibitor dur-

ing incubation at 37•Ž for 40 min prevented

ankyrin degradation (Fig. 1, lane 5). How-

ever, calpain-induced degradation of anky-

rin was unaffected by the trypsin inhibitor

or incubation of membranes at 37•Ž for 40

min (compare Fig. 1, lanes 3 and 6). Im-

munoblot analysis indicated protein 4.1 not

to be affected by the trypsin inhibitor or

calpain (Fig. 1, lanes7-9). All major prote-

ins except ankyrin were apparently not affect-

ed on the SDS-PAGE gel (data not shown).

2. 2 Effects of 1.1 -calpain on membrane

stability.

Ghost resealing in the presence of the tryp-

sin inhibitor prevented decrease in mem-

brane stability. Calpain treatment decreas-

ed membrane stability. Typical changes in

Figure 2. Membrane stability of ghosts Under high constant shear stress of 750 dyn/ cm2, untreated ghosts (line b) began to frag-ment at 10 sec and the T50 was 18 sec. Ghosts resealed in the presence of trypsin inhibitor

(line a) began to fragment at 15 sec and T50 was 30 sec. Ghosts treated with ,u-calpain in the presence ofthe trypsine inhibitor (line c) fragmented in a manner similar to that noted for untreated ghosts.

膜(MEMBRNE),Vol. 20 No. 4 (1995) 291

A B

C

DI are shown in Fig. 2. When resealed ghosts

were subjected to an applied shear stress of

750 dyn/cm2, membrane fragmentation oc-

curred for a certain period of time. For com-

parative assessment of membrane stability, T50 was determined. For calpain-untreated

ghosts resealed in the presence and absence of the trypsin inhibitor, T 50 was 30 and 18

sec, respectively. For calpain-treated ghosts

resealed in the presence of the trypsin in-

hibitor, T 50 was 16 sec. When membranes

previously treated with calpain together with calpain inhibitor I were resealed in the pres-

ence of the trypsin inhibitor, the ghosts frag-

mented in a manner similar to that of the

calpain-untreated ghosts resealed in the

presence of this inhibitor (data not shown).

The presence of 10 ,a1VI Ca2+ alone or the cal-

pain inhibitor alone failed to alter stability

(data not shown). It follows from these re-sults that the trypsin inhibitor restores mem-

brane stability and that calpain activation

Figure 3. Degradation of ankyrin and pro-

tein 4.1 by g-calpain treatment

Unsealed membranes were treated

with g-calpain for 0 to 12 min, fol-

lowed by incubation at 37•Ž for 4C

min in the presence of trypsin in-

hibitor. Immunoblot analysis of

these resealed ghosts were carried

out with antibodies against (A)

ankyrin and (B) protein 4.1. (C)

Density of the 195-kD polypeptide

(●)andprotein4.1(○)wereplot-

tedagainstcalpaintreatmenttime.


lessens membrane stability. The addition of

μ-calpain to the outside of ghosts after re-

sealing did not alter membrane stability

(data not shown). The effects of g-calpain on membrane stability would thus appear

due to its effects on the cytoplasmic side of

the membrane.

2.3 Time dependent degradation of an-

kyrin by p-calpain treatment

To monitor the time-dependent degrada-

tion of ghost membrane proteins by g-cal-

pain treatment, immunoblotting using anti-

bodies against ankyrin and protein 4.1 was

performed (Fig. 3). Ankyrin on ghost mem-branes was clearly digested by 4 U/ml of

μ-calpain at 0℃ with time, with consequent

increaseintheamountofthe195-kDpoly-

peptide(Fig.3A).Thephotographicdensity

of the 195-kD band was plotted against ,a-

calpain incubation time (Fig. 3C). Analysis

with immunoblotting showed protein 4.1 not

to be affected by g-calpain treatment (Fig.

3BC). g-Calpain treatment under the pre-

sent conditions thus brings about the degra-

dation of only ankyrin.

2. 4 Correlation between membrane sta-

bility and formation of the 195-kD polypep-

tide.

Membrane stability of ghosts decreased

with increase in g-calpain incubation time

(data not shown). In Fig. 4, T 50 is plotted

against the photographic density of the 195-

kD band obtained from Fig. 3C. This plot

clearly shows decrease in membrane stabili-

ty can be seen to be correlated with increase

in the 195-kD polypeptide and hence, with

decrease in ankyrin.

3. Discussion

The present study demonstrates for the

first time that g-calpain catalyzes the limit-

ed digestion of ankyrin on erythrocyte mem-

branes, with consequent decrease in mem-

brane stability.

For measurement of membrane stability,

resealed ghosts were prepared by membrane

incubation at 37•Ž for 40 min. Under these

conditions, ankyrin on the membranes was

clearly degradated by membrane-associa-

ted protease(s) to produce several peptides

with the molecular weight of 195-kD or less.

The digestion of ankyrin was prevented by

the presence of the soybean trypsin inhibitor,

indicating trypsin-inhibitor sensitive pro-

tease(s) to be present on erythrocyte mem-

branes. From these results, it follows that

the trypsin inhibitor may be used in all cas-

es for assessing g-calpain effects on the de-

Figure 4. Correlation between membrane

stability and amounts of 195-kD

polypeptide. T 50 is plotted against the photo-

graphic density of 195-kD polypep-tide. T50 for ghosts resealed in the

presence of the trypsin inhibitor

was normalized to 100%. Increases

in the amounts of this polypeptide

decreased T50.

膜 (MEMBRNE), Vol. 20 No. 4 (1995) 293

gradation of membrane proteins and mem-

brane stability.

When membranes treated with 4 U/m/ ex-

ogenous IL -calpain in the presence of 10 gM

Ca2+ at 0•Ž for 12 min were incubated in the

resealing buffer containing the trypsin in-

hibitor, only ankyrin was degradated, pro-

ducing the 195 kDa polypeptide. This find-

ing is basically consistent with the previous

report showing digestion of purified ankyrin

by ,u-calpain to produce many fragments in-

cluding this polypeptide26' . It is important

to note that ankyrin on membranes produces

the 195-kD polypeptide without further de-

gradation by ,u -calpain. This polypeptide

differs from the 195-kD product of ankyrin

digestion by trypsin inhibitor-sensitive ser-

ine protease(s), since g-calpain is a cysteine

protease which removes the last 196 amino

acids from the C-terminus of ankyrin to pro-

duce the 195-kD polypeptide24) . This poly-

peptide was previously shown to bind to an-

kyrin-depleted inside-out vesicles with eight-

fold reduction in affinity but with binding

capacity twice that of undigested ankyrin 2 4) .

μ-Calpain can also remove 119 amino acids

from the C-terminus of ankyrin, possibly

producing the 200-kD polypeptide, based on

the results of immunoblot analysis.

It should be pointed out that spectrin, pro-

tein 4.1, and band 3 on membranes may not

necessarily undergo degradation by exoge-

nous ,a-calpain. However, purified erythro-

cyte spectrin a and ,6 chains are readily di-

gested by calpain, as are also purified an-kyrin and protein 4.126' . Band 3 has been

shown to be degradated in g-calpain-treated.

ghosts prepared from red blood cells of el-

derly people (>70 years old) 2 7) . However, in

the present study, fresh human venous blood

from healthy young volunteers was used

(<25 years old).

Membrane stability is essential for the

passage of red blood cells to pass through

small capillaries without fragmentation un-

der shear stress. The membrane skeleton

may be the means for maintaining mem-

brane stability. Most data in this regard in-

dicate lateral interactions such as spectrin

dimer association and spectrin-protein 4.1-

actin association rather than vertical inter-

actions such as band 3-ankyrin-i3-spectrin

association and glycophorin-protein 4.1 as-

sociation. Alteration in the binding of anky-

rin to band 3 may be the cause for changes

in membrane mechanical properties. The

artificial dissociation of band 3 and ankyrin

is induced by certain sulfhydryl reagents2 8) ,

competing antibodies29' and elevation in pH

to near 930). Low et al. have shown eleva-

tion in intracellular pH above 8.5 to gradu-

ally release band 3 from ankyrin and mem-

branes to become unstable 31) . They hypoth-

esize that the ankyrin-band 3 linkage of the

membrane skeleton with the lipid bilayer is

essential for maintaining red blood cell sta-

bility. However, the high pH in their study

may have affected the chemical nature and

interactions of membrane lipids and pro-

teins as well as cell shape32) . In this study,

erythrocyte membranes were treated with it-

calpain under physiological pH and degra-

dation of ankyrin resulted in decreased

membrane stability. Production of the 195-

kD polypeptide may have been the cause for

this decrease by reducing affinity for bind-

ing to membranes2 6) . This would confirm

Low's report that membrane stability de-

creases through alteration of the band 3-an-

kyrin-ƒÀ-spectrin linkage.

That Ca2+ at more than 1 M was found

in this study to induce decrease in mem-


brane stability by Ca2+ activation of ,u-cal-

pain implies that, for red blood cells to main-

tain normal membrane stability, intracellu-

lar Ca2+ must be less than 1ƒÊM. The re-

markably efficient Ca2+-ATPase system is

able to maintain such a concentration in nor-

mal red cells33, 34) . Failure to maintain low

normal intracellular Ca2+ has been docu-

mented in abnormal cells such as sickle and

thalassemic cells as well as senescent

cells35•`40) In these cells, Ca2+-dependent

functions, such as those of Ca2+-dependent

protein kinase C, the Ca2+-calmodulin com-

plex and calpain become active. Protein ki-

nase C phosphorylates protein 4.1, band 4.9

(or dematin) and adducin in the presence of

micromolar Ca2+ 41•`43) However, little is

known about the effects of the phosphoryla-

tion of these proteins on membrane mechan-

ical properties. Membrane stability has been

shown to decrease with increasing Ca2+ con-

centration at more than 1ƒÊM in the pres-

ence of calmodulin 44) . In contrast to revers-

ible Ca2+-dependent functions, Ca2+ higher

than 1ƒÊM is shown by the present study to

also activate ƒÊ-calpain which irreversibly

catalyzes the proteolysis of ankyrin, with

consequent decrease in membrane stability.

This irreversible process may be part of the

mechanism for the limited life span of red

blood cells.

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effects of proteases on membrane stability of red …

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