ANNALS O F CLINICA L A N D LABORATORY SC IE N C E, Vol. 10, N o. 3 C opyright © 1980, Institute for C linical S cien ce, Inc.

Structure of Erythrocyte Membrane and Its Transport Functions

SAMIR K. BALLAS M.D. AND STEVEN H. KRASNOW

Cardeza Foundation fo r Hematologic Research, Department o f Medicine, Thomas Jefferson University,

Philadelphia, PA 19107

ABSTRACT

T he red cell m em brane contains approxim ately equal am ounts o f lipids and proteins. M em brane lip ids are e ith e r phospholip ids or neutra l lip ids, m ostly u nesterified cholesterol. M em brane phospholip ids are asym m etri­cally arranged into a lip id b ilayer tw o m olecules thick. C holine phospolip ids are m ore abundan t in the extracellular surface w hereas am ino phospholip ids are m ore concentrated on the in n e r leafle t of the bilayer. C holesterol is in tercala ted b etw een the phospho lip id m olecules. T he relative am ounts of cholesterol and phospholip ids are responsib le for the fluid p roperties o f the e ry th ro c y te m e m b ra n e . A lte ra tio n s in th e m e m b ra n e c h o le s te ro l- phospho lip id ratio resu lt in m orphologically abnorm al erythrocytes w ith decreased life span.

M em brane proteins are also asym m etrically o rien ted w ith in the lip id b ilayer and can be d iv ided into th ree functional sets: structural, catalytic and recep to r proteins. Sprectrin and actin are the two main structural p roteins that together form a subm em branous cytoskeletal m eshw ork that is resp o n ­sible for the viscoelastic p roperties o f the erythrocyte m em brane. Band 3, or the an ion channel, is a major transm em branous protein involved in the transport o f w ater and anions and is a carrier o f the blood-group-I antigen. G lycophorin A, a sialic-acid-rich glycoprotein, is the major contact or re cep ­tor m em brane polypeptide th a t also spans the lip id bilayer. T he MN blood group determ inan ts and possibly o ther biologic receptor sites have been localized on the extracellu lar portion o f glycophorin A. At least 35 to 40 enzym es are confined to the m em brane and, undoubtedly , play a vital role in the m ain tenance of norm al structure and function of the erythrocyte.

In troduction

T he red cell m em brane constitutes only about one percen t o f the dry w eight o f the erythrocyte. N evertheless, it is an im por­tan t organelle tha t serves as a boundary w ith a surface area o f about 140 jum 2 thus d e term in in g the biconcave shape o f the

0091-7370/80/0500-0209 $01.80

erythrocyte. It provides the red cell w ith a deform able and resilien t surface that ena­bles it to traverse capillary and splenic channels sm aller than half its d iam eter. Because o f its u n iq u e transport functions and its selective perm eability to cations, erythrocyte m em brane regulates the con­tents o f the red cell and m aintains an ionic

3.Institute for Clinical Science, Inc.

2 1 0 BALLAS AND KRASNOW

R A P I DF ig u r e 1. Schem atic rep re sen ta tio n of the

phospholipid bilayer of the red cell m em brane. Choline phospholipids (dotted circles) are abundant in the external leaflet whereas amino phospholipids (open circles) are concentrated in the inner half of the bilayer. Arrow in the horizontal plane represents the rapid in terchange and m ovem ent o f phos­pholipid molecules in that direction. Arrow in the vertical plane indicates the slow interchange be­tween the two leaflets of the bilayer (“flip-flop”).

gradien t b e tw een the in tracellu lar and ex­tracellu lar environm ents. F inally , it reg u ­lates the in teraction o f the erythrocyre w ith neighboring cells and w ith the sur­rounding m edium .

Biochem ically, the red cell m em brane is com posed o f proteins,, lip ids and car­bohydrates. A pproxim ately 48 p ercen t of the dry w eigh t of the red cell m em brane is p ro te in , w h ile 44 p e rc e n t o f th e d ry w eigh t is lip id and only 8 p e rcen t o f the mass is m ade up o f carbohydra te .19 T he carbohydrate m oiety is associated e ith er w ith m em brane lip id s (g lycolip ids) or w ith m em brane pro teins (glycoproteins). A lth o u g h d e ta i le d k n o w le d g e o f th e structure-function relationsh ips and o f the protein-pro tein , lip id -lip id and p ro te in ­lip id interactions o f the erythrocyte m em ­brane is lacking at the p re sen t tim e, the last few years have w itnessed m uch prog­ress in this area o f m em brane research. It is the aim o f this p ap er to h igh ligh t the recen t advances in our understand ing of the structure-function re la tionsh ip of the red cell m em brane.

R ed C ell M em brane L ip ids

T he m ost satisfactory cu rren t m odel of m em brane structure is the fluid m osaic m odel proposed by S inger and N icolson

in 1972.46 This m odel (figure 1) postulates the p resence o f a lip id b ilayer of phos­pholip ids arranged into a shee t two m ole­cules th ick and 45°A w ide. T he p hos­pho lip id m olecules are o rien ted in such a w ay th a t th e h y d ro p h o b ic n o n p o la r groups o f the two layers are d irec ted to­w ard one another, form ing lip id -lip id in ­teractions. The hydrophilic polar groups are d irec ted outw ard on both the extracel­lular and in tracellu lar surfaces. C holes­terol is in te rca la ted b e tw een the p hos­ph o lip id m o lecu les. T h e p h o sp h o lip id b ilayer forms a liquid-crystalline m atrix or core o f the red cell m em brane. This lip id m atrix h as th e fo llo w in g im p o rta n t properties:

A s y m m e t r y

A recen t m od ification o f th e b ilay er h y p o th e s is is th e f in d in g th a t p h o s ­pholip ids are asym m etrically organized in the two m em brane m onolayers o f intact red ce lls .43 C holine phospholip ids (phos­phatidyl choline and sphingom yelin) are prim arily co n cen tra ted in th e ex ternal leaflet o f the b ilayer w hereas am ino phos­p h o lip id s (p h o sp h a tid y l e th an o lam in e and phosphatidyl serine) are abundan t in the cytoplasm ic h a lf of the bilayer.

D y n a m i c i t y

T h e in d iv id u a l m e m b ra n e p h o s ­pho lip id m olecules are in a dynam ic state w ith in the in tact m em brane .13 T hey move at significant rates along the lateral p lane of the lip id bilayer. It is estim ated that one p h o s p h o lip id m o lecu le show s la te ra l m ovem ent each second in a b ilay er th a t is one /u,m w ide. Exchange of phospholip id m olecules b e tw een the in n er and outer m onolayers (“ flip-flop” phenom enon) is very slow, if it occurs at all.

F l u i d i t y

This refers to the degree of resistance or m icroviscosity a certa in partic le w ould encounter if it w ere able to float freely

STRUCTURE OF ERYTHROCYTE MEMBRANE 2 1 1

w ith in the in terio r o fth e lip id bilayer. It is m ainly d e te rm in ed by the am ount and types of lip ids and fatty acids w hich m ake up the b ilayer and the tem perature o f the system . F atty acids w ith shorter chains, for exam ple, are m ore fluid than longer chain fatty acids. T he g reater the degree o f unsatu ra tion o f fatty acids, th e g reater the flu id ity o f the b ilayer at low er tem ­p e ra tu re s . A n o p tim a l c h o le s te ro l / phospho lip id ratio is req u ired to m aintain norm al fluidity o f the m em brane. M em ­brane lip id flu id ity should not be con­fused w ith the viscoelastic properties of the red cell surface. V isocoelasticity re ­su lts from th e c h a ra c te r is t ic s o f th e spectrin -actin cy toskele ton o f the erythro­cyte w hich w ill be described . L ipids con­trib u te the property o f viscosity b u t not elasticity to the m em brane.

Phospholip ids, neu tra l lip id s (mostly cholesterol) an d glycosphingolip ids are the th ree m ajor constituents o f th e ery th ­rocyte lip id b ilayer. P hospho lip ids ac­count for 54 p ercen t o f the dry w eight and 69 percen t for the m olar concentration of re d ce ll m em b ran e l ip id s .12 T h e four major classes o f m em brane phospholip ids arranged in order o f decreasing concen­tra tio n s a re : p h o s p h a tid y l c h o lin e(lec ith in ), p h o sp h a tid y l e th an o la m in e (c ep h a lin ), sp h in g o m y e lin an d p h o s ­phatidyl serine. Trace am ounts o f o ther phospholip ids, such as lysophospholipids and plasm alogens, are also found. About 50 p ercen t of the fatty acids in red cell m em brane phospho lip ids are saturated and 50 p ercen t are unsaturated . C holes­terol (free and unesterified ) accounts for 29 percen t of the dry w eight and for 43 p e rc e n t o f th e m olar co n cen tra tio n o f m em brane lip id s .12 T here is ev idence that th e convex p o rtio n o f th e m em b ran e m ight be richer in cholesterol than the concave one, suggesting that these m ole­cu les o f ch o le s te ro l co u ld serve as a w edge b en d in g the m em brane into the biconcave shape .35 Polarization and con­densation of cholesterol a t the convex tips o fth e erythrocyte have b een described in

h ered ita ry e llip to cy to sis .40 G lycosph in ­golipids account for 2 percen t of the dry w eigh t and for 3 percen t o f the m olar con­centration o f m em brane lipids. Some red cell m em brane glycolipids have an tigenic activity corresponding to the ABH and P blood g roups .32

Recticulocytes b u t n o t m ature red cells can synthesize phospholip ids and cho les­terol de novo .6 As the re ticu locy te m a­tures, it loses about one-th ird of its m em ­brane surface area, m ostly ow ing to lip id loss. P lasm a free fatty acids, p lasm a phos­pho lip ids and free cholesterol b o u n d to se ru m lip o p ro te in can ex ch an g e w ith th e i r r e s p e c t iv e m e m b ra n e c o u n te r ­parts .12,26,41 Exchange data ind icate that 60 p e rc e n t o f m em b ran e p h o sp a tid y l c h o lin e an d 30 p e rc e n t o f m em b ran e sp h in g o m y elin are ex ch an g eab le w ith th e ir p lasm a coun terparts .41

T here is increasing ev idence tha t the surface area o f th e erythrocyte an d its shape d ep e n d on the am ount o f m em ­brane cholestero l and on the m em brane cho lestero l/phospho lip id m olar ra tio .10,11 In norm al red cells, this ratio is a trifle less than one ranging from 0.83 to 0.95. In table I are sum m arized the repo rted dis­eases characterized by abnorm al com posi­tio n o f re d c e ll m em b ran e l ip id s . In o b s tru c tiv e ja u n d ic e , h e p a titis or c ir­rhosis, red cells have an increased con ten t o f bo th ch o leste ro l and phospho lip ids. T h e c h o le s te ro l in c re a s e is p ro p o r ­tionately m ore than that of phospholip ids, r e s u lt in g in an e le v a te d c h o le s te ro l/ phospho lip id m olar ratio and abundance of target cells. E rythrocytes from patien ts w ith severe hepatocellu lar d isease have m arked ly in c reased m em brane ch o les­terol w ith norm al or slightly increased p h o s p h o l ip id s , le a d in g to s p u r c e ll anem ia w hich is a poor prognostic sign. In abe ta lip o p ro te in em ia (acanthocytosis), the cholesterol conten t of red ce ll m em ­b ra n e is no rm al or s lig h tly in c re a se d w hereas the total phospholip id am ount is decreased , m ostly owing to m arked red u c­tion in lec ith in , w ith a resu ltan t increased

212 B A LL A S A N D K RA SN O W

TABLE I

Lipid Composition of the Red Cell Membrane in Different Disorders

Disorder Membrane Lipid Composition Hematologic Abnormality

I. Severe hepato­cellular disease

Cholesterol in markedly increased but phospholipids are mildly increased. C/Pl* and C/L+ are markedly increased.

Spur cell anemia

II. LACT§ deficiencyA. Hereditary Cholesterol is increased and phospholipids are

normal. C/Pl is increased and C/L is normal.Abundant target cells Mild hemolysis

B. Acquired:obstructive Cholesterol and phospholipids are increased Abundant target cellsj aundice to different extent. C/Pl is increased but

C/L is decreased.III. Abeta lipoproteinemia

A. Hereditary Cholesterol is normal. Lecithin is decreased and sphingomyelin is increased. C/Pl is increased and C/L is markedly increased.

Acanthocytosis

B. Acquired:anorexia C/Pl is increased Acanthocytosisnervosa

*Cholesterol/phospholipid ratio §Lecithin cholesterol acyl transferase

tCholesterol/lecithin ratio

c h o le s te ro l /p h o s p h o l ip id ratio. M em ­brane sphingom yelin , however, is signifi­cantly increased in abeta lipoproteinemia. Patients w ith lecith in-cholesterol-acyl- transferase (LCAT) deficiency have mild

hemolytic anemia, abundant target cells an d c h o le s te ro l r ic h e ry th ro cy tes . A n­orexia nervosa is the acquired counter­part o f abeta lipoproteinemia with similar red cell m em brane lipid composition.2

( S P E C T R I N ) 2

S YNDEI NS

ANI ON CHANNEL 34. 14 . 2

4 . 5

ACTI N 5

G 3 P D 6

7

2 4 0 , 0 0 02. 1 ANKYRI N

G P - A D I ME R S

G P - A

G P - CG P - B

1 6 , 0 0 0 DALTONS

F i g u r e 2. E le c ­trophoretic separation of the p ro te in s o f hum an erythrocyte mem brane in SDS-polyacrylamide gels (5.6 percent) stained with Coomassie brilliant blue. Numbers near the top and bottom right side of the gel indicate the molecular weights of spectrin band 1 and g lob in m onom er respectively as indicated by appropriate markers. T he p o sitions w here glycophorins (GP-A, GP-B, GP-C) migrate in PAS stained gels are indicated by arrows.

STRUCTURE OF ERYTHROCYTE MEMBRANE 2 1 3

R ed C ell M em brane Proteins

Red cell m em brane proteins are usually separated and classified by polyacryla­m id e gel e le c t ro p h o re s i s 49 in so d iu m dodecyl sulfate (PAGE-SDS), as shown in figure 2. T here are at least seven major po lypep tide bands d e te rm in ed by this techn ique and, in addition, there is an in­te r m e d ia te n u m b e r o f m in o r p ro te in species. T he normal m em brane pro te in pattern depends on the m ethod o f p re p ­aration and the degree o f w ashing the m em branes. Thoroughly w ashed m em ­branes, for example, lack visible b an d 8 and globin , w h e rea s partia lly w a sh e d m em branes contain these bands and have relatively higher quantities of bands 4.3 and 7 (figure 3). Small amounts of spectrin may also be lost during in vitro lysis and th e w a sh p r o c e d u r e .23 In s ic k le ce ll anemia, it is difficult to p repare m em ­branes free of globin.25 Incubation o f nor­mal e ry th ro c y te s w ith su l fh y d ry l i n ­h ib i to r s , su ch as p a r a c h lo ro m e r c u r i - benzoate (PMB), results in m em branes w ith in separab le b an d 8 and g lob in .55 T h ese observations ind icate that some m em brane proteins may have a cytoplas­mic counterpart and vice versa.

T he fluid-mosaic m odel postulates that m em b ran e proteins are g lobular to ac­count for the high conten t o f alpha helix and are variably and asymmetrically e m ­b ed d e d within the lipid bilayer. M em ­brane proteins, therefore, can be clas­sified as integral or peripheral, according to w he the r or not they interact with the hydrocarbon core of the lipid bilayer, as outlined in table II. Marchesi has recently p roposed31 the classification of the major polypeptide chains of the hum an red cell m em brane into functional sets as ou tl ined in table III and to be p resen ted in the following section.

S t r u c t u r a l o r S u p p o r t i n g P r o t e i n s

At least five or six major po lypeptides of t h e h u m a n e r y th r o c y te s u p p o r t a n d

A B C D E F

F i g u r e 3. E ffec t o f w ash ing on th e SDS- polyacrylamide (5.6 percent) gel electrophoretic pat­tern of red cell membrane proteins. Washed erythro­cytes were lysed in 5mM phosphate buffer pH 8.0 and membranes were separated and washed in the same buffer. M embrane protein pattern was deter­m ined after wash one through six (gels A-F respec­tively). Bands 4.3 and 7 decrease w ith each wash, whereas band 8 and globin disappear after the sixth wash. Gels were stained with Commassie brilliant blue.

stabilize the m em brane. Bands 1,2 (spec­trin) and 5 (actin) join together to form a subm em branous cytoskeleton of the red cell. Bands 2.1 (ankyrin), 2.2 to 2.6 (syn- deins) and 4.1 to 4.2 have b ee n im plica ted in linking spectrin to the m em brane. T he viscoelastic spectrin-actin microfibrillar meshwork influences cell shape and pro­vides anchoring sites at the cytoplasmic m em brane surface for transm em braneous proteins, thus limiting their lateral surface m o b il i ty . T h is m o d if ie s th e S in g e r - Nicholson m em brane model by postu lat­ing a fixed protein matrix instead o f a

2 1 4 BALLAS AND KRASNOW

TABLE I I

Structural Classification of Erythrocyte Membrane Proteins

I. Integral (or intrinsic) proteinsA. Anion transport protein (band 3)B. Glycophorins: A, B, CC. Na--K ATPaseD. Blood group antigens: Rh lipoproteins

II. Peripheral (or extrinsic) proteinsA. Proteins on the cytoplasmic surface of

the membrane1. Spectrin (bands 1 & 2)2. Spectrin binding proteins

a. Ankyrin (band 2.1)b . Synde ins (bands 2.1-2.6)c. Bands 4.1-4.2

3. Actin (band 5)4. Glyceraldehyde-3-phosphate

dehyndrogenase (band 6)5. Band 7

B. Proteins on the extracellular surface ofthe membrane1. Acetylcholine esterase2. Blood group antigens

flu id -m o sa ic in w h ich p ro te in s m ove freely.

Spectrin consists of a com plex of two polypeptides (bands 1 and 2) w hich have m olecular w eights o f about 240,000 and220,000 d a lto n s , re s p e c t iv e ly . T h e se po lypep tides are p re sen t in substan tial am ounts com prising 20 to 25 p ercen t of the total m em brane p ro te in .24 Spectrin is w ater soluble, and its two subunits have

TABLE I I I

Functional Classification of Erythrocyte Membrane Proteins

I. Structural or supporting proteinsA. Spectrin (bands 1 & 2)B. Ankyrin (band 2.1)C. Syndeins (bands 2.1-2.6)D. Bands 4.1-4.2E. Actin (band 5)

II. Catalytic proteinsA. Anion transport protein (band 3)B. Na-K ATPaseC. Glucose transport (band 4.5)D. Other enzymes

III. Contact or receptor proteinsA. Glycophorins: A, B, CB. Blood group antigens

the capacity to form dim ers or tetram ers d ep en d in g upon the conditions of isola­tion and purification .38'39 C urrently , it is b e liev ed that the structural com ponents of th e c y to s k e le to n o f th e re d c e ll a re heterodim ers o f double stranded spectrin w hich form tetram ers by head-to-head as­sociations. T hese tetram ers m ay be con­n ec te d in to m icro fib rilla r n e tw o rk b y oligom eric com plexes o f ac tin .30-38 Each of the two bands of spectrin contains m ulti­p le isoelectric, an tigenic and N -term inal com ponents. It is n o t know n at the p re sen t tim e if th is phenom enon is due to pro­te o ly s is or to th e p re s e n c e o f n o n - detergen t-d issociab le su b u n its .15

U nlike m yosin, spectrin d im ers are less a lp h a h e l ic a l a n d m o re g lo b u la r in shape .24 T here is ev idence that bands 1 and 2 o f spec trin are two d istinctly differ­e n t p o ly p e p tid e c h a in s .21 B and 2, th e sm aller of the tw o com ponents o f spectrin , can be p h o sphory la ted by endogenous p ro te in k in ases w ith ATP^y-32P u n d e r physiologic cond itions3 and it b inds to the in n er surface o f th e ery th rocy te m em ­b ra n e .31 B ands 2.1 an d 4.1 to 4 .2 are th o u g h t to lin k sp ec tr in to transm em - braneous in tegral proteins. Bands 4.1 to4.2 may link spectrin to Band 3 .31 Band 2.1 has b e e n re n a m e d an k y rin (from th e Greek, ankya:anchor) because o f its an­choring function .6

M ore recently , the proteins in the re­gion b e tw een bands 2 and 3 (i.e. bands 2.1 to 2 .6) have b een referred to as syndeins (from the G reek, syndeo: to b ind together) and are thought to b in d spectrin and con­n ec t it to th e ery throcyte m em b ran e .53 M a rin e tt i a n d C ra in h av e re c e n tly suggested that spectrin may in teract w ith phospho lip id clusters on the inner surface of the lip id b ilayer via C a++ bridges that are p resu m ed to occur betw een the car­boxyl groups o f phosphatidyl serine and the carboxyl groups o f spec trin .33

A bnorm alities o f spec trin have b een im plied in a nu m b er of disorders (table

STRUCTURE OF ERYTHROCYTE MEMBRANE 215

IV). S p ectrin d efic ien cy has b e e n d e ­scribed in hered itary spherocytosis o f the m o u se .18 A bnorm al phosp h o ry la tio n of b and 2 occurs in m uscular d ystoph ies .42 T h e m e m b ra n e c y to sk e le to n m ay b e unstab le and friable in hum an hereditary spherocytosis .30 H ered itary elliptocytosis and pyropoikilocytosis are characterized by therm al in s tab ility of sp ec tr in .37 In sickle cell anem ia, th e organization of spectrin may b e a ltered ow ing to the pro­gressive ly in creasin g calc ium and d e ­creasing adenosine triphosphate (ATP) contents o f irreversib ly sickled ce lls .36 In m egaloblastic anem ia, the conform ation o f m em brane proteins, includ ing spectrin may be abnorm al w ith unfo lded po lypep­tide cha ins .4

C a t a l y t i c P r o t e i n s

The A nion Transport C hannel (Band 3). T he band 3 po lypep tide appears as a diffuse band on sodium dodecyl sulfate po lyacrylam ide gels (figure 2) and m i­grates w ith an apparen t m olecular w eight of about 93,000 daltons. It is the p redom i­nant intrinsic m em brane p ro te in com pris­ing approxim ately 25 p ercen t o f the total m em brane protein o f the hum an erythro­cy te .49 I t is not c lear w he ther th is diffuse protein band contains one or several dif­feren t po lypep tide chains. It may exist in a dim eric form w hen it is situated in the in tact m em brane, since it can be cross linked by bifunctional reagents, oxidized u n d e r a p p ro p r ia te c o n d itio n s to a d isu lfide-linked d im er and isolated as a d im e r in th e p re s e n c e o f n o n io n ic d e te rg en ts .54

Band 3 is a g lycoprotein th a t contains 5 to 8 percen t carbohydrate on a w eight basis. T he m ajor com ponents of the car­bohydra te m oiety are m annose, N-ace- tylglucosam ine and glactose in the ap­proxim ate ratios of 1:2:2 .31,48 T he 93,000 dalton po lypep tide spans the m em brane asym m etrically and can be proteolytically

TABLE IV

Spectrin Abnormalities

I. Spectrin deficiency A. Hereditary spherocytosis of the

mouse

II. Abnormal phosphorylation of band 2 A. Muscular dystrophies

III. Abnormal stability of spectrinA. Diminished stability: hereditary

spherocytosisB. Thermal instability of spectrin

1. Hereditary elliptocytosis2. Congenital pyropoikilocytosis

IV. Altered organization of spectrin A. Sickle cell anemia

V. Abnormal conformation A. Megaloblastic anemia

d isse c te d in to th re e large frag m en ts. T hese consist o f ou ter surface, transm em ­brane and cytoplasm ic surface dom ains w ith a p p a re n t m o le c u la r w e ig h ts o f38,000,17,000 and 40,000 daltons, respec­tively .48 T he integral ou ter surface frag­m en t is a g lycopeptide w hich intercalates into the b ilayer w here it is anchored by h y d ro p h o b ic a sso c ia tio n s . T h e tra n s ­m e m b ra n e 17 ,000 d a lto n s s e g m e n t trav erses th e h y d ro p h o b ic core o f the m em brane and can only be lib era ted from the b ilay er w ith detergen ts . T he cyto­plasm ic fragm ent carries the am ino te r­m inus of band 3, in contrast to o ther in te ­gral m em b ran e p ro te in s w hose am ino term inus is at the ou ter surface o f the m e m b ra n e . A ld o la se an d g ly ce r- a ld e h y d e -3 -p h o sp h a te d e h y d ro g e n a se b in d rev ersib ly , in v itro , to th is cyto­plasm ic dom ain of band 3. R ecently, it has b e e n sh o w n th a t th e cy to p lasm ic com ponent of band 3 can be cross-linked to spectrin ban d 1 at physiologic pH and isotonic cond itions .29 This supports the concep t that b an d 3 is attached to the subm em branous cytoskeletal netw ork of the red cell m em brane. O ther p ro teins that have b een show n to b in d to the cyto­plasm ic portions of band 3 include hem o­

216 BALLAS AND KRASNOW

globin and bands 4.1 to 4 .2 .48 Band 3 has recently b een assigned as the I antigen carrier.9

Functionally , band 3 is involved in the tran sfe r o f an io n s an d p o ss ib ly w a te r across the lipid bilayer.8,48 There are two lines of ev idence linking band 3 to anion transport. F irst, co v a len t in h ib ito rs of anion exchange, notably stilbene deriva­tives such as 4, 4’-diisothiocyano-2, 2’- s t i lb e n e d isu lfo n a te (3H -D ID S ) b in d ra ther selectively to band 3. Second, par­tially purified ban d 3 (Triton X-100 ex­tracts o f red ce ll ghosts) increases the a n io n tra n s p o r t a c tiv ity o f s y n th e tic lecith in vesicles by th ree to tenfold. Re­cent studies have show n that covalent in ­hibitors o f anion flux p referren tially b ind to the 17,000 dalton m em brane-spanning dom ain o f th e m o lecu le , m ak in g th is fragm ent a notable candidate for future studies on th e structural basis for anion transport. T he actual m echanism o f anion transport by band 3 is not known at the presen t tim e. K inetic data suggest a dou­b le d isp lacem ent or “ ping-pong” m echa­nism. It is proposed th a t single anions are a lte rn a te ly tran sp o rte d from one com ­partm ent to the o ther in a reciprocating cycle that may be conform ational in na­tu re .48

N a +-K+ ATPase. T he N a+-K+ ATPase com plex responsib le for the active trans­port of N a+ and K+ across cell m em branes is an in tegral p ro te in that spans the red cell m em brane and contains both a large po lypep tide of approxim ately 95,000 dal- tons and a sm aller glycoprotein o f about45,000 daltons .20,48 T he Na+-K+ ATPase po lypeptide has b een identified in the band 3 region of sodium dodecyl sulfate polyacrylam ide gels as the acyl-32P cova­len t in term ediate . T he polypeptide, how ­ever, contributes m uch less than one per­cen t of the po lypeptides in the band 3 area. T he stoichiom etry of ligand b in d in g by this enzym e is such that for each pair of large p o ly p ep tid e m olecules, one ATP m olecule can be b o u n d or one o f the

po lypep tide chains m ay be phosphoryl- a ted by ATP.

Glucose Transport. In itia l reports have im plicated the b and 3 po lypep tide as the p ro te in involved in facilitating glucose tra n s p o r t ac ro ss th e re d c e ll m e m ­b ran e .28,31 M ore recen t studies, how ever, have suggested that glucose transport m ay be m ed ia ted by an o th e r g ly co p ro te in , tentatively iden tified as band 4.5 w ith ap­p aren t m olecular w eight of 55,000 dal- tons .47 This polypeptide contains only 24 percen t acidic and basic am ino acids, 7 percen t glucosam ine, 5 p e rcen t neutra l sugars (mostly galactose) and 5 p ercen t sialic acid.

O ther C ata ly tic Proteins. B esides the catalytic proteins m entioned previously, at least 35 to 40 enzym es w hose activity is confined to the m em brane have b een d e­scribed .44 T he classes o f these enzym es are sum m arized in table V. T he p ictu re is further com plicated by the fact th a t about 20 m ore enzym es, w hose activity is found bo th in the cytosol and the m em brane com partm ents o f th e ery throcyte, have also b een d esc rib ed .44 M ost of the en ­zymes of carbohydrate m etabolism and the enzym e acetylcholine esterase are ex­ternally o rien ted on the surface o f the red ce ll. T h e en zy m es C a++-A T Pase, like N a+-K+ ATPase, are a m inor in tegral pro­tein that span the red cell m em brane. T he structure-function relationships o f these enzym es aw ait fu ther elucidation .

C o n t a c t o r R e c e p t o r P r o t e i n s

Sialogly coproteins (G lycophorins). R e­ceptor or contact proteins are those com ­ponents on the surface of the red cell that m ediate its in teraction w ith the surround­in g e n v iro n m e n t. T h e y h av e u n iq u e m olecular features that confer biologic specificity and conform ation. B iochem i­cally they are e ith e r glycolipids or glyco­proteins in nature. R eceptor proteins b in d tigh tly to surface m em branes, usually by d irec t insertion of hydrophobic pep tide

STRUCTURE OF ERYTHROCYTE MEMBRANE 217

portions into the lip id bilayer. T he sialo- glycoprotein, g lycophorin A, is a sialic acid-rich glycoprotein and, to date, is the b es t stud ied contact pro tein and is the p ro ­totype o f recep to r g lycopolypeptides o f th e ery throcyte .17,45

W hen red cell m em branes are analyzed by the standard sodium dodecyl sulfate p o ly acry am id e gel e le c tro p h o re s is , a t least th ree bands can b e dem onstra ted by periodic acid Schiff (PAS) stain ing (figure 2). T he dim eric form o f glycophorin A (previously labelled PAS 1) m igrates w ith an apparent m olecular w eigh t o f 83,000 daltons. G lycophorin A m onom er (PAS 2) has a m olecular w eight o f approxim ately45.000 and glycophorin B (PAS 3) o f about25 .000 d a lto n s .14 F u r th e rm o re , w h e n sialoglycoprotein fractions ob ta ined from hum an re d b lood ce ll m em b ran es are a n a ly z e d b y o th e r so d iu m d o d ec - lysulfate-gel techn iques, d ifferen t ban d ­ing patterns are obtained, indicating that sialoglycopeptides may have m ore than one electrophoretic form. To com plicate this p icture further, a th ird , apparently distinct, sialoglycoprotein o f th e hum an erythrocyte m em brane, g lycophorin C, has recently b een d esc rib ed .16

T h e m ajor s ia lo g ly c o p ro te in o f th e hum an red cell, glycophorin A, is a trans- m em branous pro tein that spans the lip id b ilay er7 and am ounts to 1.6 p e rcen t o f the total m em brane pro tein as de term ined by radioim m unoassay .16 Its p o lypep tide por­tion com prises about 40 p e rcen t o f its total dry w eight and is m ade up o f 131 am ino acids that can be g rouped into th ree d is­tin c t dom ains .47,51 T h e am ino-term inal segm ent o f the m olecule (amino acid resi­due one through 70) is located extracellu- lary, contains a very h igh concentration of th reonine and serine residues and is heav­ily g ly co sy la ted , h av in g 16 o lig o sac ­charide chains per p ep tid e ch a in .51 R e­cently, it has b ee n show n th a t the m ultip le am ino acids a t positions 1 and 5 are re­sp o n s ib le fo r th e M N b lo o d g ro u p specificities .52 O ther b lood group deter-

TABLE V

Erythrocyte Membrane Enzymes

I. Enzymes confined to the membraneA. Enzymes of carbohydrate metabolism (14)*B. Protein kinases (2)C. Proteinases or proteases (3)D. Enzymes of nucleotide metabolism

excluding ATPases (4)E. ATPases (6)F. Phosphatases (3)G. Other miscellaneous enzymes (3)

II. Enzymes present both in the membrane and thecytosol

A. Enzymes of glucose metabolism (9)B. Enzymes of glutathione metabolism (2)C. Enzymes of nucleotide metabolism (6)D. Phosphatases (2)

♦Number in brackets is the number of enzymes described in each category.

m inants, and possibly o ther biologic re ­ceptor sites for lectins, may be localized on the glycosylated en d of glycophorin A. T he central dom ain o f the pro tein has a s e q u e n c e o f 23 s u c c e s s iv e n o n p o la r am ino ac ids (re s id u es 71 th ro u g h 90) w hich span the lip id b ilayer and w hich may be involved in p ro te in -p ro te in in ­teractions in the m em brane. T h e carboxyl term inal dom ain o f glycophorin A (resi­d u es 91 th ro u g h 131) con tains a large num ber o f charged am ino acids and a sub­stantial nu m b er o f p roline residues that may play a major role in determ in ing its confirmation.

Blood G roup A ntigens. O f all the blood group an tigens, only the MN d e te rm i­nants are defin ite ly localized to glyco­phorin A as m entioned previously. En (a-) hum an erythrocytes have m odified forms of the glycophorin A m olecule or may lack it a ltogether.50 T he I an tigen9 and some Duffy blood groups22 may be localized in the extracellular dom ain of b an d 3. E ry th ­rocytes th a t lack the Duffy-blood-group antigens are resistan t to invasion by Plas­m odium vivax.34 T he ABH and P antigens are both g lycosphingolipids in nature b u t their re lation to the lip id b ilayer and other surface proteins is no t w ell estab lished . T he Rh determ inants are lipopro teins and

218 BALLAS AND KRASNOW

th e ir absence in the Rh nu ll syndrom e renders the erythrocytes stom atocytic in shape w ith decreased life span and in­creased potassium transport across the m em brane .27 T he M cLeod phenotype, a K ell n u ll v a rian t, is a c co m p an ied by a b u n d a n t acan thocy tes, re ticu locy tosis a n d a c o m p e n sa te d h em o ly tic s ta te .1 Localization of the blood group antigens on the red cell surface and its im plication in the structure-function relationsh ips of the m em brane is a fascinating field that aw aits fu rther exploration.

R eferences1. Al l e n , F. W., J r .: Null types of the human

erythrocyte blood groups. Amer. J. Clin. Path. 66:467-474, 1976.

2. Am r e in , P. C., F r ie d m a n , R. Ko s in s k i , K., and E l l m a n , L.: Hematologic changes in anorexia nervosa. J. Amer. Med. Assoc. 274:2190-2191, 1979.

3. A v r u c h , J. and F a ir b a n k s , G.: Phosphoryla­tion of endogenous substrates by erythrocyte m em brane protein kinases. I. A monovalent ca tio n -s tim u la ted reaction . B iochem istry 13 :5507-5514, 1974.

4. BALLAS, S. K.: U n p u b lish e d o b se rv a tio n s .5. Ba l l a s , S. K. a n d B u r k a , E . R. : P a th w ay s o f de

novo p h o s p h o lip id sy n th e s is in re tic u lo c y te s . B io ch im . B io p h y s . A cta 337 :239-247, 1974.

6. B e n n e t , V. and STENBUCK, P. J.: Identification and partial purification of ankyrin, the high af­finity mem brane substructure of human eryth­rocyte spectrin. J Supramol. Struct. 10:227- 239,1979.

7. Br e t s c h e r , M. S.: Major human erythrocyte glycoprotein spans the cell membrane. Nature 231 :229-232, 1971.

8. Br o w n , A. P., F e i n s t e in , A. B., and Sh a ’a f i , R.I.: Membrane proteins related to w ater trans­port in human erythrocytes. Nature 254:523- 525, 1975.

9. C h i l d s , R. A. F e i z i , T., F a k u d u , M., and HAKOMORI, S .: B lood-group-I activ ity as­sociated w ith band 3, the major intrinsic mem­brane protein o f human erythrocytes Biochem. J. 173:333-336, 1978.

10. C o o p e r , R. A.: A b n o rm a litie s o f c e ll m e m b ra n e f lu id ity in th e p a th o g e n e s is o f d ise a se . N ew E ng . J. M ed . 297:3 7 1 -3 7 7 , 1977.

11. C o o p e r , R. A.: Influence of increased mem­brane cholesterol on membrane fluidity and cell function in human red blood cells. J. Sup­ramol. Struct. 8:413-430, 1978.

12. C o o p e r , R. A.:Lipids of human red cell mem­brane: Natural composition and variability in disease. Semin. Hematol. 7:290-322, 1970.

13. E d id in , M.: Rotational and translational diffu­sion in membranes. Ann. Rev. Biophs. Bioen- gin. 3:179-201, 1974.

14. F a ir b a n k s , B., St e c k , T. L., and W a l l a c h , D.

H. F.: Electrophoretic analysis of the major polypeptides of the human erythrocyte mem­brane. Biochemistry 10:2607-2617, 1971.

15. F u l l e r , G. M ., B o u g h t e r , J. M ., and M o r a z - ZANI, M.: Evidence for m ultiple polypeptide chains in the m em brane p ro te in spec trin . Biochemistry 13 :3036-3041, 1974.

16. FURTHMAYR, H.: Glycophoreins, A B, and C: A family of sialogycoproteins. Isolation and pre­lim inary characterization of trypsin derived peptides. J. Supramol. Struct. 9:79-95, 1978.

17. F u r t h m a y r , H. and M A R C H E S I, V. T.: Sub­units structure of hum an erythrocyte glyco- phorin A. Biochemistry 75:1137-1144, 1976.

18. G r e e n q u is t , A. C., Sh o h e t , S. B ., and B e r n ­s t e in , S. E.: Marked reduction of spectrin in hereditary spherocytosis in the common house mouse. Blood 51:1149-1155, 1978.

19. G uiD O TTI, G.: The composition of biological membranes. Arch. Intern. Med. 229:194-201,1972.

20. H o k in , L. E., D a h l , J. L., D e u p r e e , D ., D ix o n , J. F ., H a c k n e y , J. F ., and P e r d u e , J. F.: Studies on the characterization of the sodium- potassium transport adenosine triphosphatase. J. Cell. Biol. 218:2593-2605, 1973.

21. H su, C. J., Le m a y , A., E s h d a t , U., and Ma r - CHESI, V. T.: Attachment site for human eryth­rocyte spectrin. J. Biol. Chem. 254:2533-2541, 1979.

22. Je n s e n , N. J. and F u r c h t , L. T.: Localization of Duffy antigen on the red cell m em brane components. Transfusion 18 :643, 1978.

23. Ka n s u , E., Kr a s n o w , S. H., and Ba l l a s , S. K.: Immunological determination of spectrin loss during RBC lysis. B lood52 (Suppl. 1):99, 1978.

24. Kir k pa t r ic k , F . H.: Spectrin: C urrent under­standing o f its physical, biochemical and func­tional properties. Life Sciences 19:99, 1978.

25. Kr a s n o w , S. H. and Ba l l a s , S. K.: U npub­lished observations.

26. L a n g e , Y. and D’A l e s s a n d r o , J. S.: The ex­changeability o f human erythrocyte m em brane cholesterol. J. Supramol. Struct. 8 :391-397,1978.

27. La u f , P. K. and Jo in e r , C. H.: Increased potas­sium transport and ouabain binding in human Rh null red blood cells. Blood 48:457—468,1976.

28. L i n , S . and S p u d i c h , J. A.: B in d in g of cytochalasin B to a red cell mem brane protein. Biochem. Biophys. Res. Commun. 61:1471- 1475, 1974.

29. Liu, S. C. and P A L E K , J.: Cross linkings be­tw een spectrin and band 3 in hum an erythro­cyte membranes. J. Supramol. Struct. 10:97- 108, 1979.

30. Lux, S. E.: Spectrin-actin m em brane skeleton of normal and abnormal red blood cells. Semin. Hematol. 76:21-51, 1979.

31. MARCHESI, V. T .: F u n c tio n a l p ro te in s o f th e h u m a n r e d b lo o d c e l l m e m b r a n e . S e m in . H e m a to l. 2 6 :3 -2 0 , 1979.

32. M a r c u s , D . M ., M a s h a h a r u , N., and Ku n d u ,S. K.: Abnormalities in the glycosphingolipid content of human p l and p erythrocytes. Proc. Nat. Acad. Sci. 73:3263-3267, 1976.

STRUCTURE OF ERYTHROCYTE MEMBRANE 2 1 9

33. M a r in e t t i , G. V. and C r a in , R. C . : Topology of amino-phospholipids in the red cell membrane. J. Supramol. Struct. 8:191-213, 1978.

34. M i l l e r , L . H., M a s o n , S. J., C l y d e , D. G ., and M cGlNNISS, M.: The resistance factor to plas­m odium vivax in Blacks. The Duffy-blood- group genotype FyFy. New Eng. J. M ed. 295:302-304, 1976.

35. M u r p h y , J. R.: Erytrocyte metabolism. VI. Cell shape and the location of cholesterol in the erythrocyte m em brane. J. Lab. C lin . Med. 65:756-774, 1965.

39. RALSTON, G. B.: Physico-chemical characteri­zation of the spectrin tetramer from bovine eryth- sickled red cells. J. Supramol. Struct. 10:79-96,1979.

37. P a l e k , J ., L iu , P . A., L iu , S. C ., P r c h a l , J. T ., a n d C a s t l e b e r r y , R. P. : T h e rm a l in s ta b il i ty o f s p e c t r i n in h e r e d i t a r y p y r o p o ik i lo c y to s i s (H P P ). C lin . R es. 27:303A, 1979.

38. RALSTON, G. B.: Physical chemical studies of spectrin. J. Supramol. Struct. 8:361-373, 1978.

39. Ra l s t o n , G. B.: Physico-chemical characteri­zation of the spectrin tetram er from bovine erythrocyte m em branes. Biochim. Biophys. Acta 455:163-172, 1976.

40. R e b u c k , J. W . and VAN SLYCK, E.J.: An unsus­pected ultrastructural fault in human ellipto­cytes. Amer. J. Clin. Path. 4 9 :1 9 -2 5 , 1968.

41. R e e d , C. F.: Phospholipid exchange betw een plasma and erythrocytes in man and dog. J. Clin. Invest. 47:749-760, 1968.

42. Ro s e s , A. D. and APPEL, S. H.: Erythrocyte spectrin peak II phosphorylation in D uchenne muscular dystrophy. J. Neurol. Sci. 29:185-193,1976.

43. R o t h m a n , J. E. and LENARD, J.: Membrane asymmetry. Science 295:743-753, 1977.

44. Sc h r ie r , S. L.: Human erythrocyte membrane enzymes: Current status and clinical correlates. Blood 50:227-237, 1977.

45. S c h u l t e , T. H. and M ARCHESI, V. T.: Confor­mation of human erythrocyte glycophorin A and its c o n s titu e n t p e p tid e s . B iochem istry 28:275-280, 1979.

46. S i n g e r , S. J. and N lC O LSO N , G. L.: The fluid- mosaic model o f the structure of cell mem­branes. Science 275:720-731, 1972.

47. SOGIN, D. C. and H IN K LE, P. C.: Characteriza­tion of the glucose transporter from hum an erythrocytes. J. Supramol. Struct. 8:447-453,1978.

48. STECK, T . L.: T h e b a n d 3 p ro te in o f th e h u m a n r e d c e l l m e m b ra n e : A re v ie w . J. S u p ra m o l. S truc t. 8:311-324, 1978.

49. STECK, T. L.: The organization of proteins in the human red cell membrane. J. Cell Biol. 62:1-19, 1974.

50. T a n n e r , M. T. A. and An s t e e , D. J.: The mem­brane change in En(a—) human erythrocytes. Biochem. J. 253:271-277, 1976.

51. T o m it a , M . and M ARCHESI, V. T .: Amino acid sequence and oligosaccharide attachm ent sites of human erythrocyte glycophorin. Proc. Nat. Acad. Sci. 72:2964-2968, 1975.

52. W a s n i o w s k a , K., D r z e n i e k , A., and LlSOWSKA, E.: The amino acids of M and N b lood group g ly co p ep tid es are d iffe ren t. Biochem. Biophys. Res. Commun. 76:385-390,1977.

53; Yu, J. and G o o d m a n , S. R.: Syndeins: The spec­trin binding protein(s) of the human erythrocyte membrane. Proc. Nat. Acad. Sci. 76:2340-2344,1979.

54. Yu, J. and St e c k , T. L.: Association of band 3, the predom inant polypeptide of the hum an ery tro cy te m em brane . J. B iol. C hem . 250:9176-9184, 1975.

55. Z a il , S. S. and v a n d e n H o e k , A. K.: Mem­brane proteins of incubated eythrocytes: Effect o f su lphhydryl inhibition . Brit. J. Haem at. 37:353-361, 1977.