Common structural polymorphisms in human erythrocyte spectrin

Common structural polymorphisms in human

erythrocyte spectrin.

W J Knowles, … , S L Marchesi, V T Marchesi

J Clin Invest.

1984;

73(4)

:973-979.

https://doi.org/10.1172/JCI111322

.

Restricted tryptic digestion of erythrocyte spectrin at 4 degrees C followed by

two-dimensional (isoelectric-focusing/sodium dodecyl sulfate) polyacrylamide electrophoresis

yields highly reproducible maps of approximately 50 peptides with molecular weights

between 80,000 and 12,000. Based on molecular weight and isoelectric point (pI), each

unique alpha- and beta-subunit domain can be identified and compared with spectrin

peptides from other individuals. The alpha-subunit of spectrin from 60 Caucasian donors

contains a 46,000-mol-wt tryptic domain, called alpha II-T46, Type 1; more extensive tryptic

digestion of this domain generates peptides with molecular weights of 35,000, 30,000,

25,000, and 16,000. Spectrin from 29 of 37 black donors representing 14 kindreds shows

variation in the molecular weight and/or pI of peptides from the alpha II domain. In the most

common form, Type 2, alpha II tryptic peptides are increased in molecular weight by 4,000,

and the pI becomes more basic. Other alpha II variants are characterized by either the 4,000

increase in molecular weight (Type 3) or by the basic shift in pI (Type 4). When limit peptide

maps of intermediate-sized tryptic and CNBr peptides from the alpha II-domain Types 1 and

2 are compared, a consistent alteration in the chromatographic mobility of one limit peptide

is observed. Polymorphism in the alpha II subunit of spectrin did not itself produce anemia,

nor did it appear to alter […]

Research Article

Common Structural Polymorphisms

in Human

Erythrocyte

Spectrin

W. J. Knowles, M. L. Bologna, J. A.Chasis, S. L. Marchesi, and V. T. Marchesi

Departmentsof Pathology and Internal Medicine, Yale University, New Haven, Connecticut 06510

Abstrtract.

Restricted

tryptic

digestion

of

eryth-rocyte spectrin at

40C

followed by two-dimensional

(iso-electric-focusing/sodium dodecyl

sulfate)

polyacrylamide

electrophoresis yields highly

reproducible maps

of

ap-proximately

50

peptides

with molecular weights between

80,000

and

12,000.

Based

on molecular

weight

and

iso-electric point

(p1),

each

unique

a-

and

#-subunit

domain

can

be

identified

and

compared with spectrin

peptides

from other individuals. The a-subunit

of

spectrin

from

60

Caucasian

donors

contains

a

46,000-mol-wt tryptic

domain,

called

aII-T46, Type 1;

more extensive

tryptic

digestion ofthis domain generates peptides

with molecular

weights of 35,000, 30,000, 25,000, and 16,000. Spectrin

from 29 of 37 black donors representing

14

kindreds

shows

variation

in the molecular

weight and/or

pI of

peptides from the

all

domain.

In

the

mostcommon

form,

Type 2,

all tryptic peptides

are

increased in molecular

weight by 4,000, and the pI becomes

more

basic. Other

all

variants

are

characterized by

either

the

4,000

increase

in molecular

weight (Type 3)

or

by the basic shift in pI

(Type 4). When

limit

peptide

maps of

intermediate-sized

tryptic

and CNBr

peptides

from the all-domain Types

1

and 2 are compared, a

consistent alteration

in the

chro-matographic mobility

of

one

limit

peptide

is observed.

Polymorphism

in the all subunit

of

spectrin

did

not

itself produce anemia,

nor

did

it

appear

to

alter the

expression of

an

underlying hereditary spherocytosis

or

elliptocytosis.

In

six

family studies,

the all

46,000-mol-wt

variations observed

were

consistent

with

Men-delian

inheritance.

Introduction

It isgenerally agreed that spectrin is essential in determining

thepropertiesof the red cellmembrane, includingitsshapeand

Received for publication 7 July 1983 and in revised form 4 November 1983.

deformability. Structuralanalysis ofthespectrinmolecule has been difficult because of the large size of its two nonidentical

subunits, a(240,000mol wt) and

_#

(225,000mol wt). In such

alarge and complex molecule, small changes inconformation or physicochemical behaviorwould not necessarilycause

sig-nificant alterations in the properties of the native molecule. Our laboratory has recently developed a technique for analyzing

spectrinstructure; thistechnique useslimited tryptic digestion

of spectrinat0C, followedbytwo-dimensional electrophoresis

(isoelectric-focusing, sodium dodecyl sulfate-polyacrylamide gel

electrophoresis

[IEF/SDS-PAGE]).'

This procedure results in remarkably reproducible maps of approximately 50

interme-diate-sizedpeptides whose molecular weights range from 80,000

to 12,000. These peptides may be enzymatically digested or

chemicallycleaved and the resultant limit peptide analyzed by high-resolution mapping techniques. With these procedures, it is possible to assign the origins of all peptides to thea- or

lB-subunit,todetermine relationships between peptides at various stages ofdigestion,and to arrange the unique peptides linearly,

producingadomain structure for each spectrin subunit (1). These techniques have been applied to the study of red cell membranes of patients with hereditary spherocytosis (HS) and hereditary elliptocytosis (HE) and their variants. We and others (2, 3) have recently demonstrated that patients with severe

poi-kilocytichemolyticanemia(hereditarypyropoikilocytosis, HPP) have abnormalities in the80,000-mol-wtdomain of the a-chain.

Inthis study, we describe the occurrence of polymorphisms in the all domain that involve changes in both the molecular weight and the isoelectric point (pI). Thesepolymorphismshave beenfound only in the blackpopulation,arerelatively common, and appearnot to becorrelated with any clinicalabnormality. The first family in which we demonstrated an all poly-morphism was a black family withclassical HS. Heterozygous and homozygous forms of the alI variant were detected and they occurred inpredictableMendelianfashion,butsegregated

independently ofspherocytosis. We subsequently studied 37

blackindividuals from 14kindredsand we discovered a poly-morphicall domain in 29 of theseindividuals, the variations beinginmolecularweightorpIorboth,seeninsingleordouble

1. Abbreviations used inthispaper:DFP, diisopropyl fluorophosphate; HE, hereditary elliptocytosis; HPP, hereditary pyropoikilocytosis; HS, hereditary spherocytosis; IEF/SDS-PAGE, isoelectric-focusing,

SDS-polyacrylamide gel electrophoresis.

973 Spectrin:Common StructuralPolymorphisms J.Clin.Invest.

©TheAmericanSociety for ClinicalInvestigation, Inc. 0021-9738/84/04/0973/07 $ 1.00

13 IV

D4 IV

sCjY

-80

i(~

13 11

-,

3

_o

I

-( 111

-

(§~3I1

1

APO

heterozygote form.Wemadenoattempttodetermine theactual

frequency of these variantsin the blackpopulation.

Methods

Spectrin preparation

Blood was drawn into acid citrate dextrose anticoagulantand storedat 4VC until processed,no morethan 24 h.Erythrocytemembraneswere prepared and spectrinwasextractedaspreviouslydescribed (4). Tryptic digestion of spectrin

200-,ugaliquots of spectrin in 200MAlweredialyzed against 20mM Tris-HCI, pH 8.0, 1 mM2-mercaptoethanol for 20hat4VC.Trypsin

(N-tosyl-L-phenylalaninechloromethyl ketone-treated, 206

Am/mg;

Wor-thington Biochemical Corp., Freehold NJ) was addedatan enzyme/

substrate ratio of 1:20 (wt/wt). The reaction was carried out at4VCfor

lT 46 l

T35 T30

T235

m

m

2I T41 -i

T$ T3

T6OS 1i 33 _1,

Figure 2. Diagrammaticrepresentation of thea-and,8-chainsof

spectrindeducedfromchemicaland enzymaticcleavageand

map-pingofoverlap peptides (8-10). Numbersindicatemolecular weight inthousands.

Figure

1.

_{Electrophoresis}

ofspectrin peptide gen-erated

by

limited

_{tryptic digestion}

at_{4VC. (A)}75

-52

fig

Iyophilizedtryptic digestin 2%SDS,2% 1--46 mercaptoethanol electrophoresedona10-15%

ac-rylamide gradient according

toLaemmli

(5). (B)

-35

200 Mig

tryptic digest

electrophoresedintwo

di--30

mensions (IEF/SDS) according to O'Farrell (6). ThepHgradient (horizontal) extends from 7.2to

4.5

(left

to

_right);

theacrylamide gradient

(verti-cal)is from 10to 15%. Uniquedomains in the

a-and E-chainsarecircled. Lower molecular

_weight

tryptic

digestion

productsof theaII-T46domain -17 areconnected by lines from the parent domain.

Numbers indicate molecularweightin thousands.

90 minandterminated

by

adding diisopropyl fluorophosphateto afinal concentration of 1 mM andcoolingto-80'C. The_{tryptic digests}were

lyophilized

andstoredat-20'C for furtheruse.

-80K

-~~~

-- rn

-

--USUP3U-46 K

_ =

~~=Z n"

woo*.

...0

..

ow*

,

_1/

2, 2,

i, 1,

112

2

1

2

Figure3.Type2variation inthe aII-T46domain of spectrin. Inter-mediatetrypticpeptides of spectrin (75,ug)from members ofa

fam-ilywith all Type 2 polymorphismareelectrophoresedon a10-15%

acrylamide gradient bySDS-PAGE. 1/1:spectrinhomozygous foraII

Type 1.2/2:spectrin homozygous foraIIType2. 1/2:spectrin

dou-bly heterozygous forall Types 1and 2.39 K, 39,000-mol-wt pep-tide;46K,46,000-mol-wt peptide;80K, 80,000-mol-wtpeptide.

A

-B

Em

I

U

UP

NI

A

I

B

I

T7 28Tl

P

SI

_

Polyacrylamide

gel

electrophoresis

One-dimensional electrophoresis. 75 sgofspectrin tryptic peptides pre-paredasdescribed above wassolubilizedin 2% SDS, 2%

2-mercapto-ethanol andelectrophoresed accordingto Laemmli (5) on acrylamide

gradients,asspecified in Figurelegends.

Two-dimensional_{electrophoresis (IEF/SDS-PAGE). 200 ug of}

ly-ophilized spectrin tryptic digests wassolubilizedin 75

_Al

9 M urea, 2% Triton X-100, 5%2-mercaptoethanol,and 2.4%ampholines,and focused on3 X 120 mmpolyacrylamide gelsaccordingto O'Farrell (6). The pH gradient extended from 7.2 to 4.5 and was formed by mixing equal volumes ofampholytes(LKBInstruments, Inc.,RockvilleMD) at pH 4-6, pH 5-7, and pH 3.5-10 (2.4% finalampholyte concentration). Afterelectrophoresis_{for 5,700 V/h, the gels were removed and}

equil-ibrated for 10 min in 10% glycerol, 3% SDS, 1 mM EDTA, and 2%

2-mercaptoethanol beforeelectrophoresisin the seconddimension ona 10-15%acrylamidegradient using Laemmli buffers (5).Molecularweights weredetermined byreferenceto known protein standards.

CNBr

cleavage

Lyophilized spectrin (1 mg) was dissolved in 70% formic acid. A 500-fold molar excess of CNBr (Pierce Chemical Co.,Rockford, IL) with

respecttomethionine_{was added and the reaction was allowed to proceed}

overnight at 37°C. The reaction was terminated by drying with N2,

followed by repeated resuspension in water andlyophilization.The sam-ples were then subjected totwo-dimensional electrophoresisaccording toO'Farrell (6) asdescribedabove.

1/1

---.

.

"W

_0I

--wr.

El

_

.

2

1 _

2/2

-*&

a-A

_w-e

2

_{_}

0

ob

_~~

: _4w 4 _

kW212

Figure

4.Two-dimensional IEF/

SDS-PAGE

showing

homozy-gousand

_heterozygous

_Type

2

polymorphism

in the aII do-main. The parent domains

(all-T46 and

_aII-T50,

_{Type 2),}

are

circled and connectedbylinesto

thesubdomains

_aII-T35

and

aII-T39

(Type

2), respectively. Gel conditionsare asinFig. I

B;

alltypesare

_designated

asin

Fig.

3.

2SI-peptide mapping

Two-dimensional

peptide

mapswere_prepared

_by

_{the method of Elder}

etal.

_{(7) using}

CoomassieBlue-stained

_proteins

cutfrom

_acrylamide

gels

and labeled with

_{Na'25I (New England Nuclear, Boston, MA)}

_as

previously

described

_{(8, 9). lodinated gel}

sliceswere

_digested

with 50 Mgof

_{a-chymotrypsin (Worthington}

_{BiochemicalCorp.)in 1 ml of 50} mM ammonium

bicarbonate,

pH 8.0,for 24 hat37°C. The

_lyophilized

sampleswere

_subjected

totwo-dimensional

_{peptide mapping;}

details of thisprocedurehave been described earlier

_{(8, 9).}

Results

Two-dimensional

_{electrophoresis}

_of

intermediate-sized

_tryptic

peptides

of

spectrin.

We haveisolated

_spectrin

fromthe red cells of 97individuals and from 10 cordblood

_samples,

cleaved it

by

limited

_tryptic

_digestion

at

_0°C,

and

_analyzed

the resultant

intermediate-sized

_peptides

_by

one-and

two-dimensional

elec-trophoresis.

The

peptide

patterns obtained are

highly

repro-ducible,

and blood

_given

_by

a

single

donoron

multiple

occasions has

_always

_produced

thesame

_{spectrin peptide}

_{patterns, whether}

normal orabnormal. A

_typical

_spectrin

_digest

canbeseen on

one-dimensional SDS-PAGEin

_Fig.

1A andon_{two-dimensional}

IEF/SDS-PAGE

in

_Fig.

I B. Itisapparentfrom

_Fig.

1 that the

complexity

ofthe

spectrin

digest

requires

two-dimensional

elec-trophoresis

to resolve all

spectrin

domains and

_subdomains,

975

Spectrin:

Common Structural

Polymorphisms

1/2

0

i_

2

_{_}

_{_50K}

M

_

346K

-39K

I

M

~3

"

_5K

and it is this techniquethat we have used toanalyzethe structure

ofthespectrin molecule. Inearlier work inourlaboratorythat

combined two-dimensional electrophoresis and peptide

map-ping, we identified the origin andrelationships ofthe majority oftryptic peptides separated on two-dimensional gels (8-10). The unique a- and (3-chain domains are identified in Fig. 1 B andindiagrammatic form inFig. 2.

In analyzing spectrin from normal volunteers and from

families with HS, HE, and HPP,we have detecteda polymor-phism within the alldomain.The common alltrypticdomain

ofspectrin from 60 Caucasian donorshas amolecular weight of46,000 (aII-T46) and is called Type 1(Fig. 1 B). This peptide has two prs,possiblyowingtovariabledeamidation,whichare

identical by peptidemapping(datanotshown). Trypticdigestion

at 00C produces a subfragment ofall that has a molecular

weight of 35,000; peptides with molecular weights of 30,000, 25,000, and 16,000canbeproducedbymoreextensive tryptic digestion. Peptide maps (Fig. 5) have confirmed that the lower

molecularweight peptidesarefragmentsof the 46,000-mol-wt

domain,and amonoclonalantibodyhas been made whichreacts

with a determinant common to all but the 16,000-mol-wt frag-ment(10).

The all domain ofspectrin from 29 of 37 black donors

studied shows alterationsinmolecularweight and/orpI.These

variations occur in what appear to be heterozygous and ho-mozygous forms. In the most common form ofthe variant, Type 2, theaII-T46 domain and the aII-T35 subdomain increase in apparent molecular weight by 4,000 and become slightly more basic. The all Type2 polymorphism can beseen on a

one-dimensional gel(Fig.3) by the loss ofa46,000-mol-wtband in homozygotes and the presence ofa 39,000-mol-wt band. Two-dimensional maps arerequired, however, for adequate

res-olution andanalysisofindividualpeptidesin thisregion(Fig. 4). Thedifference in pI and molecularweightismost noticeable

in the heterozygous condition 1/2,where both the normal and variant peptides are found(Fig. 4,sections labeled 1/2).

Otherdifferences among the all Types 1/1, 1/2, and 2/2 appear on the one-dimensional gels in the 70,000-90,000 region.

Two-dimensionalgelsfrom normal controls show some degree ofvariabilityin this group ofpeptides, whichareknownfrom

limitpeptide mapping to represent several different domains. Using one monoclonal antibody to one aII-T46 domain (see

reference 10), wedetected no allpeptidesin this groupof pep-tides.

Limit peptide maps of variant aIItryptic domains. Limit chymotryptic maps of the Type 1 and Type2 all domains are shown inFig.5.Maps ofthe 46,000-mol-wt (Type 1) and 50,000-mol-wt (Type 2) peptides (Fig. 5, A and B), and of 35,000-mol-wt (Type 1) and 39,000-35,000-mol-wt (Type 2) peptides (Fig. 5, C and D) showadifferencein a singleiodinatablepeptide, the Type 2 variant having a relative decrease in mobility in the

chromatographic dimension. Sincethese maps show only

io-dinatable peptides, predominantlythose that contain tyrosine, and onthe average only 33% of allpeptides contain tyrosine,

otherpeptides may also be altered but remain undetected by this procedure.

A

!

C

B

f

*

p.'

_;

it

,1

S

:

VW

D

.9

6.P

9

E

'V0

V

>_4 0

:1:-h

4

a: 2

0

cc (.

F

* l

ELECTROPHORESIS

Figure5.Autoradiographs of limit chymotryptic mapsof iodinated intermediate tryptic peptides of the all domain: comparison of maps from all Type 1 and Type 2. A, C,E areall Type 1:46,000, 35,000,

16,000 molwt, respectively. B, D, F areall Type 2: 50,000, 39,000, 20,000 molwt, respectively. The small arrowsineach map denote thepeptide withachangein mobility in the chromatographic dimen-sion.

The subfragments ofthe aII domain withmolecularweights <35,000are notnormally produced bythe mildtryptic digestion procedure used in these experiments. Type 1 aII-T30,aII-T25,

Variant aII domains are also produced by CNBr cleavage. Since the allpeptidesdiscussed above were generated by tryptic digestion, it seemedpossiblethat a lysine or arginine normally present on one end ofthe all domain was either buried or substituted in the Type 2 variant.Trypticdigestion would then havecleaved the next lysine or arginine, possibly producing a slightly larger (50,000-mol-wt) domain. To characterize further the variant all domains, spectrin homozygous for Type 1 or Type 2 was cleaved with CNBr in 70% formic acid. All peptides that resulted from the cleavage wereidenticalwith oneexception:

Type 1 spectrin contained a 23,000-mol-wt peptide that was slightly more acidic than a corresponding 26,000-mol-wt peptide in Type 2spectrin. Limitchymotrypticpeptide maps of these CNBrpeptides showed that both 23,000-mol-wt and 26,000-mol-wtpeptides are derived from the all domain and that they

differin themobilityof onechymotryptic peptidein the same manner as the maps of the 46,000-mol-wt (Type 1) and 50,000-mol-wt(Type 2) tryptic peptides (Fig. 6, A and B). The prelim-inary interpretation of thesefindingsis that the all variants are produced not simply by burial or substitution of a single lysine

or arginine, but that the polypeptide chain in this region is increased in apparent molecular weight byapproximately4,000.

Family studies ofspectrin aII variants. The initial family

in which we detected theallType 2 variation was a black family with classical HS (Fig. 7). A father and three of the five children tested hadclassical HS: the three affected brothers had sphe-rocytes on smear, abnormal osmotic fragility tests, and hema-tocrits of 34-36% with elevated reticulocyte counts. Two of the sons and the father had had their spleens removed. The two

unaffecteddaughters had normalosmoticfragilitytestsand one had ahematocrit of 40% in 1976 at age 23. When studied in 1981, both daughters had mild anemia with hematocrits 36-37%. Red cell indices,peripheralsmears,ferritin,andtransferrin

levels suggested mild iron deficiency.

When we examined the tryptic digest of spectrin from this

familybytwo-dimensional IEF/SDS-PAGE, wenotedaII-T46

Type 2 in either the heterozygous or homozygous form in five

ofsevenfamily members(Figs. 4 and 7). Thedistribution of

A

aII-T46 Type 2 followed classical Mendelian genetics but did notcorrelate with the presence of spherocytosis. The two child-ren who were homozygous for the aII-T46 variant did not have HS.

Wesubsequently demonstrated other structural variations within the all domain of black donors. In the Type 3 variation, only the molecular weight ofthe domain changes (increasing

by 4,000), and the pI remains the same. Type4 consists of a pI change only, thedomainbecomingslightlymore basic. Fig.

8 shows the spectrin maps of donors whose spectrin has both

all Types 1 and 3 (1/3), Types 3 and 4 (3/4), and Types 2 and4 (2/4). The bottom right panelofFig. 8 is a composite

drawingofthe relative positions of peptides in all variants 1 through 4.

Note that the twotypes of all peptides found in one in-dividual are not always present in equalquantities(e.g.,Fig.8, 1/3), which one would expect if the donor wereatrue hetero-zygote. This may be due to different susceptibilities ofeach variant to tryptic digestion or may suggest that there are more than two alleles for eachspectrin subunitin the genome.Athough family studieswereincomplete,the all variants observedwere

consistentwithsimple Mendelian

genetics

insix familiesstudied.

The distribution ofvariant

spectrins

seen in the 37 black individuals (14kindreds) studiedis indicated below:

Type-No. Type-No. Type-No. Type-No.

1/1-8

1/2-17

1/3-1

1/4-3

2/2-3

2/3-3

2/4-1

3/3-0

4/4-0

3/4-1

Thepolymorphic spectrins were discovered duringastudy of

patients with HS, HE, and relateddisorders;threeofthe black

familiesstudiedhaveHS,one hasHE, and three have HE and HPP,representing 27 of the 37 blackindividuals. The presence of theaIIvariant didnotappeartoaffecttheclinical

presentation

of these disorders.

B

4,

i.o

ELECTROPHORESIS o

Figure6.Autoradiographsof limit chymo-trypticmapsof iodinated CNBrcleavage products. (A) aIl Type 1, 23,O00-mol-wt peptide. (B) aII Type 2, 26,000-mol-wt peptide.Arrows denotepeptidewith

changein mobilityinthechromatographic

dimension.

977 Spectrin:Common StructuralPolymorphisms

I

oit

0.

0

I-a

0

C:)

ocCIType 0

Figure

7.

Genealogy

of

family

with

2 ~~~classical HS and allType2

polymor-I/2

phism.

Discussion

The searchforstructural abnormalitiesin alargemoleculesuch asspectrin requiresamethod that notonly surveystheentire molecule but that is sensitive enoughtopick upsmallchanges

in molecular weights or pI within a domain. The analysis of intermediatetryptic peptides of nativespectrin by two-dimen-sionalelectrophoresisdescribed above appearsto be onemethod

suitableforthistask. Furthermore, it iscompatible withsmall volumes of bloodobtainablefrompatients (including children)

with hemolyticanemias.Thepeptidesarewell resolved and the maps arehighly reproducible.

Innormalspectrin,mostof thelysineandarginine residues

areprotected fromtryptic digestion bythe nativeconformation ofthemolecule. Smallchangesin sequence, abnormal subunit

association, or oxidative damage could change conformation enough to expose or bury an arginine or lysine residue and

thereby result in altered trypticdigestion of native spectrin.For example, inspectrin obtained from patients withHPP, an ab-normalconformationwithin the aI-T80domaincauses a new

trypticcleavagewhichgenerates twopeptidesnotobtained from

normalspectrin (2).

The allvariants illustratedabove demonstrate well the sen-sitivityofthismethod to small structural changes inspectrin.

In contrast to the findings in HPPspectrin, however (2), the allvariants are not the resultofnewtrypticcleavages. Theall

polymorphismsdescribedhereconsist oftwodistinct structural

changes, a molecular weight increase, as in Type 3, andabasic shift in pI, as in Type 4. The Type 2 all variant, which has both the molecular weight and pI change, appears to be the result of two changes within a singledomain.Thelimitpeptide

maps and the cyanogenbromide digests from allType 2spectrin

suggest that thealterations are notthe resultof burialor sub-stitution ofalysineorarginine residue,butaredue,atleast in part, to asequence alteration that increases itsapparent mo-lecular weight by 4,000. This apparent increase in momo-lecular weight may be the result of a 4,000-mol-wt insert (roughly 35 aminoacids) possibly occurringatanexon-intron interfaceor

atan mRNAsplice junction. Alternatively, specific amino acid substitutionshave been showntoincreasethe apparentmolecular

weight of a protein by 2,000 (11). The change in pI may occur

at adifferentsite within thepolypeptide chain,butit ispresent in the firstmajorcleavage product

(all

T35).

*

(9

4 _ ,

0

2/4

...

WCr

__

4-j2

. ,

4 _

2 3

481

4 1

__d

2 3

C21O CubP

4 1 0

Figure 8. Two-dimensionalIEF/SDS-PAGEof

spec-trin fromindividualsdoubly heterozygousfor all

variants 1, 2, 3, and 4. A schematic representation

of therelativepositions of Types 1, 2, 3,and 4

pep-tides is shown in the lowerright panel;solidareas areforreferencetootherspectrin peptidesseen on

two-dimensionalelectrophoresis.

1/3

aII Type 2 spectrin occurred most frequently in theblack

population we studied, the sample size was small, and two kindredsthat accountedfor14ofthe 37 blackindividualsstudied werepredominantly 1/2 variants; therefore, no conclusionscan

be drawn from this study about frequency of spectrin all vari-ation in the black population, nor about the most common type of variation.

Weobserved no change in behavior of the red cell caused byall polymorphism, at least by clinical criteria. This is in

contrast toalterations in the aI-T80 domain that are associated with HPP and HE (2, 3 and Knowles, W. J., J. S. Morrow,

S. L.Marchesi, and V. T. Marchesi, manuscript in preparation). Although manyof our HS, HE, and HPP patients have poly-morphic all domains, we do not yet have enough data to de-termine whether this subtly influences the occurrenceor

expres-sionof thesehereditaryhemolytic anemias.Thequestionarises

astowhetherpolymorphism suchasthat seen in the all domain and a structural changecausinghemolyticanemiamust occur

onthe samepolypeptide chain foramplificationof the potential forhemolysis. Alternatively,doesapolymorphismonone

sub-unit increase the hemolytic potential of a structural changeon

the othersubunit? These questions will be answered by con-tinuingphenotypic andgenotypicanalysisofspectrinand other structuralproteins of the red cell membrane.

Acknowledaments

Theauthorswouldliketoacknowledge Dr.David Speicherfor hishelp in analyzingthepeptidemaps. We alsoacknowledge theassistanceof Drs.JosephBove,David Seligson,and PeterMcPhedran incollecting blood samples and inperformingclinical laboratorytests.

Thiswork wasfundedbyU.S.PublicHealthServicegrantAM27932.

References

1. Marchesi, V. T. 1983. The red cell membrane skeleton: recent

progress. Blood. 61: 1-11.

2.Knowles,W.J.,J.S.Morrow,D. W.Speicher,H.S.Zarkowsky,

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