Proc. Nati. Acad. Sci. USAVol. 83, pp. 9512-9516, December 1986Cell Biology

Molecular cloning of protein 4.1, a major structural element of thehuman erythrocyte membrane skeleton

(skeletal proteins/synapsin I)

JOHN CONBOY, YUET W. KAN, STEPHEN B. SHOHET, AND NARLA MOHANDASDepartments of Laboratory Medicine and Medicine, The Cancer Research Institute, and the Howard Hughes Medical Institute, University of California, SanFrancisco, CA 94143

Contributed by Yuet W. Kan, August 29, 1986

ABSTRACT Protein 4.1 is an important structural proteinthat is expressed in erythroid and in a variety of non-erythroidtissues. In mammalian erythrocytes, it plays a key role inregulating membrane physical properties of mechanical sta-bility and deformability by stabilizing spectrin-actin interac-tion. We report here the molecular cloning and characteriza-tion of human erythrocyte protein 4.1 cDNA and the completeamino acid sequence of the protein derived from the nucleotidesequence. Probes prepared from the cloned erythrocyte protein4.1 cDNA hybridized with distinct mRNA species from a widevariety of non-erythroid tissues, including brain, liver, placen-ta, pancreas, and intestine, implying substantial homologybetween erythroid and non-erythroid protein 4.1. The avail-ability of cloned erythrocyte protein 4.1 cDNA should facilitatethe study of the functional characteristics of this protein inerythroid as well as non-erythroid cells.

Protein 4.1 is an important structural component of theerythrocyte membrane (1-3). In mammalian erythrocytes, itstabilizes the spectrin-actin network and helps bind themembrane skeleton to the lipid bilayer through its interactionwith transmembrane proteins band 3 (the anion channel) andglycophorins (membrane sialoglycoproteins carrying bloodgroup specifications) (4-9). Through these interactions, pro-tein 4.1 appears to play a key role in regulating erythrocytemembrane physical properties of deformability and mechan-ical stability (10-12). Confirmation of this important func-tional role was obtained in studies showing that deficiency ofthis protein resulted in marked mechanical instability of themembrane (11) and that normal stability could be restored tounstable protein 4. 1-deficient membranes by reincorporationof purified protein 4.1 into those membranes (13). Recently,protein 4.1-like proteins have been identified in a number ofnon-erythroid tissues and cells such as brain, fibroblasts,platelets, endothelial cells, granulocytes, and lens (14-20).Brain protein 4.1, also known as synapsin I, is the bestcharacterized of these non-erythroid forms. It has beenshown to bind brain spectrin (20) and to interact withmicrotubules (21). Its localization in synaptic vesicles indi-cates a possible role in secretion and/or neurotransmission.

In this paper, we report the molecular cloning and char-acterization of human erythrocyte protein 4.1 cDNA and thecomplete amino acid sequence ofthe protein derived from thenucleotide sequence. We also show that the erythrocyteprotein 4.1 cDNA hybridizes to mRNA species from brainand liver, two tissues in which protein 4.1 has been previ-ously documented, suggesting similarities between erythroidand non-erythroid protein 4.1. In addition, we found thatmRNA species from pancreas, placenta, and intestine (tis-sues in which protein 4.1 has not previously been docu-mented), also hybridized to erythrocyte protein 4.1 cDNA,

suggesting that protein 4.1 may be a membrane component ofa much larger number of non-erythroid tissues than previ-ously thought. The availability of cloned cDNA for erythroidprotein 4.1 should facilitate study of this interesting mem-brane structural protein in erythroid as well as non-erythroidtissues.

MATERIALS AND METHODSReagents. [35S]Methionine and the rabbit reticulocyte ly-

sate in vitro translation system was obtained from NewEngland Nuclear, and [32P]dCTP was from Amersham.Reverse transcriptase was supplied by Molecular GeneticsResources (Tampa, FL), terminal nucleotidyltransferase byPharmacia, DNA polymerase I by Boehringer Mannheim,RNase by Bethesda Research Laboratories, and restrictionendonucleases by Bethesda Research Laboratories and NewEngland Biolabs. Oligo(dT)-cellulose type III, oligo(dT)12_18,and oligo(dC)1218 were obtained from Collaborative Re-search (Waltham, MA). The plasmid vector pSP64 wasobtained from Promega Biotec (Madison, WI).RNA Isolation. Human reticulocyte RNA from whole blood

of sickle cell anemia patients undergoing exchange transfu-sions was prepared as described (22). RNA from liver,placenta, intestine, and pancreas was isolated by theguanidinium thiocyanate method (23). RNA from culturedhuman brain tumor cell lines 188 and 126 (24) was preparedas described (25). Poly(A)+ RNA was prepared by chroma-tography using oligo(dT)-cellulose (26).In Vitro Translation and Immunoprecipitation. Total retic-

ulocyte RNA was translated in a rabbit reticulocyte lysateusing [35S]methionine to radiolabel newly synthesized pro-teins. 35S-labeled protein 4.1 was immunoprecipitated asfollows: 50 ,ul of translation mixture was diluted 1:5 in 150mM NaCl/1 mM EDTA/0.5% Triton X-100/2% (wt/vol)unlabeled methionine, and 15 ,ul of polyclonal rabbit anti-human erythrocyte protein 4.1 antiserum was added. Themixture was incubated for 60 min at room temperature,followed by a 2-hr incubation at 4°C. Immune complexeswere precipitated and prepared for NaDodSO4/PAGE (27)exactly as described (28) except that protein A-Sepharosewas used in place of Staphylococcus aureus cells.

Construction and Screening of Agtll Library. Ten micro-grams of reticulocyte poly(A)+ mRNA was used to constructa cDNA library in the expression vector Xgtll (29). First-strand cDNA synthesis was primed with oligo(dT)12_18 andcatalyzed by reverse transcriptase. The first strand was tailedwith dATP using terminal deoxynucleotidyl-transferase, andsecond-strand synthesis was primed with oligo(dC)1218 usingDNA polymerase I and RNase H (30). The cDNA wasmethylated with EcoRI methylase, ligated to phosphorylatedEcoRI linkers, digested with EcoRI restriction endonuclease,and separated from free linkers by chromatography on

Abbreviation: kb, kilobase(s).

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Sepharose 2B and by agarose gel electrophoresis. cDNA.700 base pairs long was ligated to EcoRI-digested alkaline-phosphatase-treated Xgtll vector DNA, packaged in vitro(31), and infected into Escherichia coli Y1088 (29). Theresulting library contained =2 x 106 independent recombi-nants.Immunological screening of the cDNA library was per-

formed using affinity-purified polyclonal rabbit anti-protein4.1 IgG. Positive clones were identified by immunoper-oxidase staining using reagents and protocols obtained fromClontech (Palo Alto, CA).The library was also screened usingcDNA inserts obtained

from immunologic screening. These inserts were isolatedeither directly from A phage DNA (26) or from pSP64 plasmidDNA subclones and labeled with 32P by nick-translation (32).DNA Sequence Analysis. cDNA inserts from positive clones

were subcloned into M13mplO and M13mpll (33), andsingle-stranded DNA was prepared and sequenced by thedideoxynucleotide chain-termination method (34). Oligonu-cleotide primers were prepared by using a DNA synthesizer(Applied Biosystems, Foster City, CA) and purified byelectrophoresis in 20% polyacrylamide gels, followed bychromatography on Sep Pak columns (Waters Associates).

Protein 4.1 Secondary Structure Predictions. Secondarystructure of protein 4.1 was deduced from the primary aminoacid sequence derived from the nucleotide sequence usingcomputer algorithms developed by Stroud (35).RNA Blot Analysis of mRNA. RNA blot analysis was

performed as described (36) by electrophoresis of glyoxal-denatured poly(A)+ RNA on 1% agarose gels, followed bytransfer to nitrocellulose filters and hybridization to nick-translated cDNA probes.

RESULTSDetection of Protein 4.1 mRNA in Reticulocytes. Protein 4.1

mRNA was detected in human reticulocyte RNA by virtue ofits ability to direct synthesis ofintact protein 4.1 polypeptidesin an in vitro translation system derived from rabbitreticulocytes (Fig. 1). The [35S]methionine-labeled proteinssynthesized from total human reticulocyte mRNA weresubjected to NaDodSO4/PAGE and visualized by autoradi-ography (lane 1). To determine whether protein 4.1 was oneof the components synthesized, the translated mixture wasimmunoprecipitated with monospecific anti-protein 4.1 anti-

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a

1 2

FIG. 1. Detection of protein4.1 mRNA in human reticulo-cytes by in vitro translation andimmunoprecipitation. Shown isan autoradiograph of a NaDod-S04/PAGE gel (6-15%). Lanes:1, total [35S]methionine-labeledproteins synthesized (only pro-teins >20 kDa are shown); 2,proteins immunoprecipitated byusing nonimmune rabbit serum;3, proteins immunoprecipitatedby using rabbit anti-human pro-

3 tein 4.1 antibody.

body. A single protein having the same electrophoreticmobility as unlabeled pure protein 4.1 was detected in theimmunoprecipitate (lane 3). This polypeptide was not pre-cipitated by nonimmune rabbit serum (lane 2). Densitometricscanning of these autoradiographs indicated that protein 4.1mRNA constituted -0.3% of the non-globin high molecularweight mRNA.

Isolation of Protein 4.1 cDNA Clones. A human reticulocytecDNA library containing size-selected inserts .700 basepairs long was constructed in the expression vector Xgtll andscreened with affinity-purified anti-protein 4.1 IgG to detectclones producing protein 4.1 antigen(s). Immunoreactiveclones were detected at a frequency of 1 in 50,000. Fiveclones identified in this manner had inserts of 0.9-1.2kilobases (kb), and these inserts cross-hybridized with oneanother on Southern blots (data not shown). Radiolabeledprobes prepared from clone XHE 4.1-6 were used to isolatethe longer cDNA clone XHE 4.1-8 (Fig. 2). DNA sequenceanalysis of these cDNAs provided incontrovertible evidencefor their identity, as the amino acid sequence predicted bynucleotides 1303-1350 was identical to a peptide of authenticprotein 4.1 sequenced earlier (unpublished results) over astretch of 16 consecutive residues determined for authenticprotein 4.1 (Fig. 3, boxed region).

Characterization of Erythrocyte Protein 4.1 cDNA. Therestriction map of human erythrocyte protein 4.1 cDNAconstructed by analysis of the Xgtll cDNA clones is illus-trated in Fig. 2. Nucleotide sequence of protein 4.1 cDNAand predicted amino acid sequence of the protein are shownin Fig. 3. A single long open reading frame at positions799-2562 in the cDNA encodes a 588-amino acid protein 4.1molecule, which has a calculated molecular mass of 66,303Da. Amino acid residues 1-42 of the predicted sequence areidentical to the amino terminus of erythrocyte protein 4.1determined by direct protein sequencing (T. L. Leto, D. W.Speicher, and V. T. Marchesi, personal communication),confirming the assignment of the initiation codon to the AUGsignal at positions 799-801. In addition, amino acids 405-471of the predicted sequence are identical to another peptide ofprotein 4.1 (the "10 K" domain) (37, 38).The unusually long 5' untranslated region contains four

potential AUG translation initiation sites at nucleotides261-263, 279-281, 461-463, and 611-613. These upstreamsites are unlikely to function as translation signals for thefollowing reasons: all four lie in a different reading frame fromthe functional signal at positions 799-801, all four arefollowed almost immediately by in-phase terminationcodons, and only one contains the required purine nucleotideat position -3 of the consensus sequence surrounding func-tional AUG translation initiation sites (39). In contrast, theAUG at positions 799-801 does conform to the consensussequence and is followed by a long open reading frame.To verify that the long 5' untranslated region represents

true protein 4.1 cDNA sequences and not a cloning artifact,two fragments of cDNA extending from nucleotides 1-254and 254-766 were subcloned separately and radiolabeled foruse in RNA blotting experiments. Both 5' probes hybridizedto a 5.6 kb mRNA, indistinguishable from that recognized bycoding-region probes (data not shown; see Fig. 5 for RNAblot analyses).Primary and Secondary Structure of Protein 4.1. The amino

acid sequence derived from protein 4.1 cDNA was used topredict the major secondary structure features of the proteinas well as to characterize the biochemical properties of thevarious functional domains of the protein (Fig. 4). Thesedomains, defined originally by limited chymotryptic diges-tion (37), serve distinct functional roles critical to the integ-rity of the membrane skeleton. The amino-terminal 30-kDa"membrane-binding" domain, which provides one typeofmembrane skeleton-lipid bilayer linkage by interacting

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Proc. Nadl. Acad. Sci. USA 83 (1986)

3' FIG. 2. Restriction map con-structed by analysis of the iso-lated Xgtll cDNA clones for

AHE 4.1-A protein 4.1. Two of the five* clones (*) isolated by immuno-

logical screening and one clone-1* isolated by screening with radio-

labeled XHE4.1-6 are shown.Box represents the codingdomain.

with transmembrane proteins band 3 and glycophorin (7, 8),is very hydrophobic (36% aromatic plus isoleucine, leucine,methionine, and valine residues). Among these hydrophobicresidues are 33 potential chymotryptic cleavage sites (indi-cated by vertical lines within the boxed portion of Fig. 4) thatare not digested unless high enzyme/substrate ratios are used(37). In addition to its overall hydrophobicity, this domaincontains all seven cysteine residues present in the molecule.Two potential cAMP-dependent phosphorylation sites atSerI85 and Ser237, and three potential N-linked glycosylationsites at Thr5l, Thr74, and Thr'5' are also present in thisdomain. Secondary structure prediction indicates that thisdomain contains a substantial p-sheet structure, in addition toa number of amphipathic a-helical structures.The 16-kDa domain, which resides between the 30-kDa

membrane-binding domain and the 10-kDa spectrin-bindingdomain is quite hydrophilic (only 14% hydrophobic residues)and has an unusually high proline content (9.3%). a-Helical

structure is the principal feature of the secondary structure ofthis domain.The "10 K" spectrin-binding domain (37), which is respon-

sible for the interaction ofprotein 4.1 with spectrin and actin,is highly charged (46% aspartic acid, glutamic acid, arginine,lysine, and histidine). Secondary structure predictions indi-cate that this region contains a long a-helix as its mostprominent feature. A potential cAMP-dependent phospho-rylation site is located at Ser467.

Finally, the carboxyl-terminal domain (22-24 kDa), asreported earlier (37), is quite acidic. The most remarkablefeature of this domain is its short length: it contains 117 aminoacids with a calculated size of 12,636 Da, far below itsapparent size in NaDodSO4/PAGE of 22-24 kDa. Themolecular basis for this discrepancy is unknown.RNA Blot Analysis of Erythroid and Non-Erythroid Protein

4.1 mRNA. To determine the size of erythroid protein 4.1mRNA and to study the expression of non-erythroid protein4.1 mRNA species, we hybridized an erythroid protein 4.1

TCC CGT GGA GCA GAG

GGC CGA AAA TTC AU

ACA CAM GGT GMA AGC

CAA ATC TCA GOT GTC

CCC AAT TOC AMC TCC

TmT CTO AAG CAT GTG

GTA AAA GAa GMA AMC

Cys Val Val Glu LysTGT GTT GTG GAM AAA

Tzp Lou Asp Ser AlaTOG CTG GAT TCC 0CC

Tyr Lou Cys Lou GlnTAT TTA TGT CTT CAG

Lou His Gly Val AspCTC CAT GGC GTG OAT

Lou Glu Ph. Lou Glu

Lys Asp Lys Lou ArgAAA GAT AAG CTG AGa

Gly Ph. Lys Lou ProGGT TTC AAA CTT CCC

Gly SOr Lys Ph ArgGTM TCC AA Tm CGA

Asp Gly Ala Ala AlaGAT GGT GCA GCA GCT

Val Pro Lys Ala GlnGTC CCT AMA GCA CAG

Glu AMn 11 Tyr IleGAa AAC ATT TAT ATC

Pro Glu Pro Arg ProCCA GAM CCA CGG CCT

Val Thr Ile SOr AspGTC ACC ATC TCA GAT

Ii. S-r Glu Thr ArgATT TCA GAG ACA COT

Thr Lys Val Val ValACC AAM GTG GTC GTC

CTG CCA TAG TCA GAC

CTT TTT CTA TAT TAG

GGG CAA AGT GOC AGG

GCA cca ACA GM TA

TTC TM TGG AGA CAC

cTA GTA AGA AGG CAA

TGa ACC GGA ACT Caa

AAT ATT ATC OCG ASC

AAC CTC TTA AAG GOC

AA GGG TOA GGA AC

TCC TAC ACA TGA AGA

AGA AMT AGA GTC AGA

AAC AGA CCC ATC TOT

GTT AAA AGT CCT GCT

OCT CAA TCA AAA GCA GMA ACA GMA TTA AAA20His Ala Lys Gly Gln Asp Lou Lou Lys AgCAT GCT AMG GGA CAM GAT TTG CTT AAA cTA60Lys Glu Il Lys Lys Gln Vol Arg Gly ValAAA GAA ATA AAA AAG CMG GTT CGT GGT GOC100Lou Arg Gln Asp Ile Val Ala Gly Ar LouCTT CGO CMG GAC ATA GTT GCA GM CGT CTG140Tyr Val SOr Asp Ph. Lys Lou Ala Pro AsnTAT GTT AMT GAT OTT AMA CTG GCC CCG AMT180AMn Ala Lys Lys Lou OSr Mat Tyr Gly ValMTv nCC AM Aa TnG TCT ATO TAT GGA GTT220Il AMn ArM Ph. Pro Tip Pro Lys Val LouATT AAC CGC TTC CCT TGG CCC AAA GTG CTG260Ser Tyr Arg Ala Ala Lys Lys Lou Trp LysAMT TAC CGA GCA GCT AMG AAA TTA TOG AAA300Tyr SOr Gly Arg Thr Gln Ala Gln Thr ArgTAC AGT GGC COG ACT CMA GCT CAG ACC AGG340Vol Asp Oar Ala Asp Arg Sr Pro Arg ProGOC GAT TCG GCT GAC CGA AGT CCT COG CCC380Lys Glu Thr Val Lys Ala Glu Val Lys LysAM GAM ACA GmT AM GCT GMA GTO AAA AAM420AMg His SOr An Lou Mat Lou Glu Asp LouAU CAT AGC AT TT ATO TTG GM GAT TTM460SOr Glu Trp Asp Lys Ag Lou SOr Thr HisAMT GAM TGG GAT AAA COC TTA TCC ACT CAC500An Ala AMn Ala Val Lys Ser Glu Ile ProAMT GCC AMT OCT GTG AMA AGT GMA ATC CCA540Ile Glu Lys AM Ile Val Ile Thr Gly AspATT GAA AAM AGA AOT GTO AC Aca TA GAT580His Gln Glu Thr Glu Il- Ala Asp Glu INCAC CAG GMG ACC GM6 ATT GCT GAT GAM TGA

TTC AGA CTT TM AGMA TMTC TAA ATC ACC

GAT ATC AGA ATT GTT CAM CTT TTC ACT CTA

GMAG GGCGOC GMC CGA GAA CGC GGT CGG

CAT AM CTC AM CCA MA MA ACC TCA MCA

CTT GM CMAA CAA GGA GCG Gm ASC AlA

TAA M;A Am AGG T&A AMO MG TCA Gm MA

GGA TCT TCA TTC ATT AAG CAM TGC AMA AAC

CAG GMA GAM CTC MA GMA CAT CCA GAT TCT

GCT TCC CAA AAM CCA ATC AMA AAA CAC AGG30Val Cys 6lu His Lou Asn Lou Lou Glu GluGTA TGT GAG CAT CTC AAT CTT TTG GMA GMA70Pro Typ Asn Phe Thr Ph. Asn Val Lys PhsCCT TGG AAT TT ACA TTT AAT GTA AAM TT110Pro Cys Ser Ph. Ala Thr Lou Ala Leu LouCCC TOT TCC OTT GCA ACC TTA GCA TTA TTA150Gln Thr Lys Glu Lou Glu Glu Lys Vl MatCAM ACC AAG GA CTT GAA G14 AAG GTC ATG190Asp Lou His Lys Ala Lys Asp Lou Glu GlyGOT CTT CAT AAA GCA AAM GAC TO GM GA230Lys Ile SOr Tyr Lys Arg SOr S-r Ph. PhbAAG ATT TCT TAT AAM CGT AMT AGC TT TTC270Val Cys Val Glu His His Thr Ph. Ph. ArgGTC TGT GTh GM CAT CAC ACG TTT TTC aGA310Gln Ala Ser Ala Lou Ile Asp Arg Pro AlaCAA GCT AGT OCT CTA ATT GAC AGG CCT GCC350Thr Ser Ala Pro Ala Ie Thr Gln Gly GlnACT TCT GCA CCT GCC AT ACT CAG GGT CAM390Glu Asp Glu Pro Pro Glu Gin Ala Glu ProGA GAC GAG CCA CCT GMG CAA GCT GMG CCA430Asp Lys SOr Gln Glu Glu Ile Lys Lys HisGAC AAM aGT CAA GM6 GMG ATC AMA AM CAT470SOr Pro Ph. Arg Thr Lou asn I1. Asn GlyTCA CCC TTC CGA ACT CTT MAC ATC ART G00510Thr Lys Asp Val Pro I1e Vol His Thr GluACC AM GAC GTC CCT ATT GTC CAC ACT GM550Ala Asp Ile Amp His Asp Gln Val Lou ValOCT GAT ATT GAC CAT GOT CAG GTC CTT GTA

GCT CAG GAA CTA ACC TAC CCC AAC TCT GCC

AGA AM TTh ATT TCA GTT TCT ATT GOG AGT

TaG ACT GTT OTA AGA GTT TTG GGG GGT TTT

CCC GGT CCC CGC COC

GGA GGA ATC TTG TCA

AAG CAG AMG ACT TTC

OAT AGA ATT TGG AAC

ACA 0CC TCA CCA TTa

GMA ATT AAG Q&A GG

Nat His Cys LysaaC ATG CAC TGC AAM40Asp Tyr Ph. Gly LouGAC TaT TOT GOT CTa80Tyr Pro Pro Asp ProTAT CCA CCT GAC CCA120Gly SOr Tyr Thr IleGGT TCT TAC ACC ATC160Glu Lou His Lys SOrGAA CTO CAT AAG TCA200Val Asp Ile Ile LouGTA GAT ATC ATC CTA240Ie Lys Ie arg ProATC AAG ATS CGO CCT290Lou Thr SOr Thr AspTTG AMA TCT ACA GAC320Pro His Ph. Glu ArgCCA CAC TTC GMG CGT360Val Ala Glu Gly GlyGTT GCA GA GGT GOC400Glu Pro Thr Glu AlaGMG CCC ACK GMA GCA440His Ala SOr Ile SOrCAT GCC Aac AMC AMT480Gln Ile Pro Thr GlyCAM ATC CCC ACA GA520Thr Lys Thr Ie ThrACC AAG ACC ATC ACT560Gln Ala Ii Lys GluCAA GCC ATC AAG GM;

CTT CTC CCA TCC AM

TTA TAC CAA GAG ATT

TAA TTG GGT GGT TTO

ACC CAG CCC AGA GAA

AAC AMC AGC TGA AGG

ACG ACT ATT CTC CTC

CAG TCT ?GA T&A AGA

CAM TTA AGA ATT ATT

GAA GGA CTT GAA GAG

Val SOr Lou Lou AspGTT TCT OTG TTG OATAla Ile Trp Asp AsnGCC ATT TGG GAT AAC

Ala Gln Lou Thr GlUGCA CAG TTA ACA GA

Gln SOr Glu Lou GlyCAG TCT GMA CTG GGATyr Arg SOr tNtI1ETAC AGG TCC ATGIMI

Gly Val Cys SOr SOrGGT GTC TGC TCT AGT

Gly Glu Gln Glu GlnGGM GAG CAM GAG CAG

Thr 11 Pro Lys SOrACC ATT CCC AAA AMCThr Ala SOr Lys argACA GCA AGT AAM COG

Val Lou Asp Ala SOrGTC CTa GAT GCC TCT

Trp Lys Lys Lys ArgTOG AAG AAA AAG AGA

Glu Lou Lys LyS AsnGAM CTM AAA AAG AAC

Glu Gly Pro Pro LouGA GGA CCT CCC CTG

Tyr Glu Ala Ala GlnTAT GM OCT GCC CAG

Ala Lys Glu Gln HisGCA AAG GMG CAM CAC

AMA MC CAC GMA AAT

CTT CTA OAT CTC ATT

TMA CCC CTT CM CCT

GAG TTT AGT GAC TGA 120

AGA TMA TTG GTG TGA 240

GTT TCT CAA AAG GCC 360

GAT CAT TTm AAA G¢C 480

TT aGA GTC TTC TTA 600

TOC TCC AMA ATA GAA 72010Asp Thr Val Tyr GluGAC ACA GTT TAT GAA 84050Ala Thr Oar Lys ThrTCA ACC TCT AAG ACA 96090Asp Iie Thr Arg TyrGAC ATA ACA AGA TAT 1080130Asp Tyr Asp Pro GluGAC TAC GAC CCA GAA 1200170Pro Ala Gin Ala AsprLrGCTQWL.O&L1320210Giy Lou Lou Val TyrGGC CTT CTO GTT TAC 1440250Tyr Glu SOr Thr IeTAT GAA AGT ACC ATC 1560290Lys Ph. Lou Ala LouAAA TTT CTT GCG CTA 1680330Ala SOr Arg SOr LouGCG TCC COG AGC CTC 1800370Ala Lys Lys Thr ValOCT AAA A ACA GTG 1920410Glu Arg Lou Asp GlyGAA A" CTA GAT GGT 2040450Ph. Met Glu SOr ValTTC ATG GMG OCT GTA 2160490Val Lys Thr Gln ThrGTG AMG ACA CAA ACT 2280530Thr Val Lys Gly GlyACT GTA AAA GOT G0G 2400570Pro Asp Mat Oar ValcCA GAC ATO TCA GTG 2520

GAT AMA GAA OCT AAC 2640

OAT CCT TT GAA GAG 2760

aG 2867

FIG. 3. Nucleotide sequence of protein 4.1 cDNA and predicted amino acid sequence of the erythrocyte protein 4.1. Boxed area indicatesamino acid sequence determined directly by analysis of a cyanogen bromide-cleaved tryptic peptide of purified protein 4.1.

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60 120 180 240 300 360 420

NH2

480 54 10 588

30K * I 16K J 1OK J 22-24KI 1 I III I1 I I II III I I I l! I I I II |COOH

SHSH SH SH SH SH SHP p

_~~.- - - -- - - = --C 1-{

FIG. 4. Secondary structural features of protein 4.1. Scale indicates amino acid number. Arrows indicate the boundaries of the four domainsof protein 4.1 molecule identified by limited chymotryptic cleavage (ref. 37; T. L. Leto, D. W. Speicher, and V. T. Marchesi, personalcommunication). Potential chymotryptic sites are indicated by vertical lines within the closed box. Potential glycosylation sites (Asn-Xaa-Ser)(40) are indicated by asterisks (*); potential cAMP-dependent phosphorylation sites (basic-basic-Xaa-Ser) (41) are indicated by "P"; "SH"denotes cysteine residues. Secondary structure symbols are as follows: o, a-helix; o, amphipathic a-helix; ., hydrophobic a-helix; -, (-sheet;----, turns and random coils.

cDNA probe to size-fractionated mRNAs from several hu-man tissues by using RNA blot analysis (36). Reticulocyteprotein 4.1 mRNA was -5.6 kb long (Fig. 5, lane 1). DistinctmRNAs that hybridized with erythrocyte protein 4.1 cDNAwere also detected inRNA isolated from two different humanbrain tumor-derived cell lines (lanes 2 and 3), as well as inhuman liver (lane 4), pancreas (lane 5), placenta (lane 6), andintestine (lane 7). A closer examination of the RNA blotsrevealed small differences in the sizes of non-erythroidprotein 4.1 mRNAs. While liver and pancreas protein 4.1mRNAs were the same size as the erythrocyte mRNA, thebrain protein 4.1 mRNA was slightly larger (a differencereproducibly seen on three different RNA blots). Interest-ingly, intestine mRNA consisted of two distinct mRNAspecies. In contrast to these non-erythroid RNAs, HeLa cellRNA gave a very weak signal even when substantial quan-tities of poly(A)+ RNA (-5 ,g) was analyzed in an identicalfashion (data not shown).The probe used in the RNA blot in Fig. 5 was derived from

XHE4.1-A, indicating that erythroid and non-erythroidmRNAs are homologous in the region surrounding thecarboxyl-terminal coding sequence. To determine whetherhomology extends toward the amino-terminal coding se-quences and into the 5' untranslated region, hybridizationswere performed with additional cDNA probes. A probederived from XHE4.1-6, representing the carboxyl-terminaltwo-thirds of protein 4.1, hybridized to all of the non-erythroid mRNAs tested (data not shown). Moreover, asubcloned portion of XHE 4.1-8 representing 5' untranslatedsequence (nucleotides 254-766) hybridized to the brain-

4 8kb--

1 2 3 4 5 6-

FIG. 5. RNA blot analysis of erythroid and non-erythroidmRNAs. Lanes: 1, reticulocyte mRNA (1 ,ug); 2 and 3, mRNA fromhuman brain tumor cell lines 188 (5 ,g) and 126 (5 ,ug); 4, livermRNA(5 ug); 5, pancreas RNA (<0.5 ug); 6, placenta RNA (2 pg); 7,intestinal RNA (2 ,ug). Faint band seen for pancreas is due to thesmall quantity ofRNA available. Probe used for hybridization is theinsert from XHE 4.1-A (see Fig. 2). Standards used for size estimateswere ribosomal 18S RNA (-1.8 kb) and 28S RNA (-4.8 kb).

specific protein 4.1 mRNAs (data not shown). Hybridizationagainst other non-erythroid mRNAs has not been tested.

DISCUSSIONThe results presented here indicate that human reticulocytesin the peripheral circulation retain intact mRNAs encodingerythrocyte membrane skeletal protein 4.1. This RNA wasused to construct a Xgtll expression library, from whichseveral protein 4.1 cDNA clones were isolated. The identityof these clones was verified by the finding that the predictedamino acid sequence contained regions identical to authenticprotein 4.1 sequences determined earlier.

Earlier biochemical studies of erythrocyte protein 4.1 havedefined four domains with distinct physical properties anddifferent binding interactions with membrane components(37, 38, 42). The complete amino acid sequence we havededuced from the nucleotide sequence of its cDNA allows usto begin a more detailed chemical and structural analysis ofeach of these domains. The amino-terminal domain (30 kDa),involved in binding to transmembrane proteins (42) and tophospholipids (43) is hydrophobic and rich in P-sheet struc-ture. It contains all seven cysteine residues present in theprotein, as well as all three potential glycosylation sites. Thehydrophobic nature of this domain, the P-sheet structure,along with the many cysteine residues available for partici-pation in intramolecular disulfide bond formation suggest atightly compacted tertiary structure, which may explain theobserved resistance of this domain to proteolytic digestion(37). No particular binding function has yet been defined forthe second domain (16 kDa), which is quite hydrophilic. Thisdomain may simply act as a linker between the membrane-glycophorin binding domain and the spectrin-actin bindingdomain that follows it. The spectrin-binding domain (10 kDa)is highly charged and the structure of this short domaincontains a relatively long a-helix. This domain also containsa site for phosphorylation by a cAMP-dependent proteinkinase (37, 44), suggesting a potential mechanism for regu-lation of the assembly of the spectrin-actin-protein 4.1complex. Finally, the carboxyl-terminal domain (22-24 kDa)is quite acidic, as reported earlier by Leto and Marchesi (37).In contrast to the other three domains for which the measuredmolecular mass is very close to the molecular mass calculatedfrom the derived amino acid sequence, a large discrepancyexists for the carboxyl-terminal domain. The calculated sizeis 12,636 Da, far below its apparent size in NaDodSO4/PAGEof22-24 kDa. This discrepancy may be due in part to aberrantmigration of this charged domain in NaDodSO4/PAGE gels.One question as yet unresolved by cDNA sequence analysisconcerns the nature of the difference between the closelyrelated forms of protein 4.1 designated as 4.1a and 4.1b (3),which appear to differ by -2 kDa in the carboxyl-terminaldomain (37).

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Protein 4.1 analogs have been detected by immunologicalmeans in several mammalian non-erythroid tissues, includingbrain (15, 20), platelets (16, 18), endothelial cells (19), lenscells (17), fibroblasts (14), and lymphoid cells (16). Ourfinding that liver, pancreas, placenta, and intestine all containmRNAs that hybridize to erythroid protein 4.1 cDNA (Fig. 5)indicates that the distribution of protein 4.1-like proteins iseven more widespread than previously realized. Our under-standing of the structural and functional relationships amongthe various protein 4.1 species, however, is currently limitedto a few cases in which these proteins have been purified.Analysis of bovine lens protein 4.1 shows that it is similar oridentical to bovine erythrocyte protein 4.1 with respect tosize and two-dimensional peptide maps (17). In the aviansystem, a more complex array of protein 4.1 species, whoseexpression changes during differentiation of erythroid andlens cells, is observed (45). The structural relationship amongthese proteins is unknown. In contrast, brain protein 4.1 (alsoknown as synapsin I) is clearly a distinct protein with a uniquetwo-dimensional map, a different mobility in NaDodSO4/PAGE gels, and a different isoelectric point (20).Our finding that erythrocyte protein 4.1 cDNA hybridizes

to non-erythroid mRNAs that vary slightly in size raises thepossibility that tissue-specific expression of related protein4.1 mRNAs is responsible for production ofrelated structuralproteins in these tissues. This result was most obvious inintestine mRNA, which contained two distinct protein 4.1mRNA species (Fig. 5), but reproducible size differenceswere also present between erythrocyte and brain protein 4.1mRNAs. Since erythroid 4.1 cDNA hybridized to non-erythroid mRNAs, it should be feasible to isolate the corre-sponding cDNAs from non-erythroid cDNA libraries byusing erythroid probes. Sequence analysis of non-erythroidprotein 4.1 cDNA will clarify the relationships among thenon-erythroid protein 4.1 molecules and the genetic mecha-nism(s) responsible for this diversity. At least two differentmechanisms can be invoked to explain the diversity betweenerythroid and non-erythroid forms. First, it may be thatseveral protein 4.1 genes exist in the genome to generatediverse protein forms, such as those expressed in brain anderythrocytes. These genes likely would be clustered in theshort arm of chromosome 1, because this is the only regionthat hybridized to erythrocyte protein 4.1 cDNA probesunder conditions identical to those used in our RNA blots(46). Alternatively, a single protein 4.1 gene might generatemultiple mRNAs utilizing tissue-specific alternate splicingpathways. These mRNAs might then be translated to yieldproteins that share certain exons and contain other uniqueexons. Precedence for this type of mechanism exists, par-ticularly for structural proteins such as mouse myelin basicprotein (47), the rat fibrinogen gene (48), the troponin T gene(49), and the human tropomyosin gene (50). Further charac-terization of the protein 4.1 gene(s) from genomic DNA andfrom non-erythroid cDNA clones will be required to distin-guish between these alternate mechanisms.

We thank Dr. Dan Cashman for preparing synthetic oligonucleo-tides, Dr. Robert Stroud for helping us derive the secondarystructure predictions of protein 4.1, Drs. Dennis Deen and LarryMarton for providing human brain tumor cell lines, Dr. Wing Kam forproviding pancreas and placental RNA, Drs. Joel Anne Chasis andYuichi Takakuwa for helping purify protein 4.1 antibody, Mrs. MaryRossi for excellent technical assistance in deriving the amino acidsequence of protein 4.1, Charlotte Wang and Mary Marx for DNAsequencing, and Mr. James Harris for his expert assistance inpreparing this manuscript. This work was supported by grants fromthe National Institutes of Health (AM-16666 and AM-32094) and bythe Howard Hughes Medical Institute.

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