Functional characterization of recombinant human red cell alpha ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Val. 268, No. 20, Issue of July 15, p p . 1478&14793,1993 Printed in U.S.A .

Functional Characterizationof Recombinant Human Red Cell a-Spectrin Polypeptides Containing the TetramerBinding Site* (Received for publication, November 2, 1992, and in revised form, March 18, 1993)

Leszek Kotulal-, Tara M. Desilva, David W. Speicher, and Peter J. Curtis4 From The Wistar Instituteof Anatomy and Biology, Philadelphia, Pennsylvania19104

Spectrin, a heterodimer composed of a and p sub- Both chains consist mostly of a repeat unit of 106 amino units, interacts with itself head-to-head to form tetra- acids, with 21 repeats in cy and 17 in 0. A triple helical model mers in the erythrocyte membrane cytoskeleton. The has been proposed for the repeat unit, where helices 1 and 2 NH2-terminal region of cr-spectrin, encompassing the of oneunitinteract withhelix3 of the following repeat a1 80-kDa domain, was expressed in Escherichia coli. (Speicher and Marchesi, 1984) which has been further refined In addition to the correctly initiated polypeptide, four smaller polypeptides were producedby initiation at (Speicher etal., 1993). The different domainsof spectrin, a1-aV and PI-PIV, have internal codons. Only the full-length polypeptide was able to bind to spectrin dimers, p monomers, and to a been characterized by limited tryptic digestion, which gives recombinant polypeptide containing the COOH termi- rise to several large polypeptides (Speicher et al., 1982). The nus of 8-spectrin. The head-to-head interaction with p- a1 80-kDadomain, derived from the NH2 terminus of cyspectrin was also retained by a recombinant polypep- spectrin, retains the ability to interact with spectrin dimers tide containing the NH2-terminal 158 amino acids of and /3-spectrin (Morrow etal., 1980), whereas QV and PIV are the a subunit. Deletion of the first 2 7 or 49 NH2- important for the nucleationof a / @dimer formation (Speicher terminal amino acids abolished binding of this polypep- et al., 1992). tide to the # monomer. I The phasing used to design these Defects of the cytoskeletal protein network have beenfound recombinant polypeptides was based on a conforma- to be the primary cause of various hereditary hemolytic anetional model recently refined by Speicher etal. (Speicher, D. W., Desilva, T. M., Speicher, K. D., mias, including hereditary spherocytosis (HS),’ elliptocytosis Ursitti, J. A., Hembach, P., and Weglarz, L. (1993) J. (HE), andpyropoikilocytosis (HPP). Themajority of HE and with diminished tetramer formation Biol. Chem. 268, 4227-4235), where the structural H P P cases are associated unit begins and terminates around residue 30 of the and abnormal peptide patternsof limited trypsin proteolysis repeat unit. The binding properties, mobility on gel (Knowles et al., 1983; Lawler et al., 1982) affecting primarily filtration, and circular dichroism data of the recombi- the CUI80-kDa domain (see review by Palek and Lambert, nant polypeptides indicated that most polypeptides 1990). Mutations have also been located in the COOH-terwere able to assume their native conformation. minal portion of the /3 subunit (Eber et al., 1988; Tse et al., 1990; Pothier et al., 1990; Yoon et al., 1991; Tse et al., 1991; Gallagher et al., 1991). Interestingly, some of these /3 mutaThe erythrocytemembraneskeleton, a complex protein tions induce the same aIj74 tryptic peptide asobserved previously in a subset of a-chain mutations. To explain these network underlying the plasma membrane, determines the unique shape and flexibility of red blood cells. A number of results, it has been proposed that helices 1 and 2 of the /3 membrane skeletal proteins have beenidentified including COOH-terminal repeat interact with helix 3 of the Q NHzet 1990). This hypothesis is supported actin, ankyrin, protein 4.1, and spectrin, which is the major terminal repeat (Tse al., proteolysis component of the network (see review by Bennett and Lam- by mapping of the tetramer binding site using bert, 1991). Spectrin consists of two polypeptide chains, the protection experiments (Speicher etal., 1993). a and p subunits, which associate side-to-side in an antiparIn this study, we report on the functional characterization allel orientation to form heterodimers that, in turn, associate of recombinant polypeptides derived from the a1 domain of head-to-head(spectrin self-association) toformtetramers, erythrocyte spectrin. The phasing of the repeat unit in the the basic functionalunitinthemembraneskeleton.The recombinant polypeptides was chosen on the basis of preprimary structure of a and @-spectrin has been deduced from dicted conformational models for a repeat unit (Speicher and amino acid analysisandcDNAsequencing(Speicherand Marchesi, 1984; Speicher et al., 1993). This phasing is similar Marchesi, 1984; Sahr et al., 1990; Winkelmann et al., 1990). to that identified by Winograd et al. (1991) for the a repeat * This work was supported by National Institutes of Health Grants 14 and 15 of Drosophila spectrin. All peptides containing the 158HL 33884 (to P. J. C.) and HL 38794 (to D. W. S.), an American intact a subunitNHz-terminal sequence,includinga Heart Association Research Fellowship (Southeastern Pennsylvania amino acid polypeptide comprisedof the intact NH,-terminal Affiliate) (to L. K.), and by partial support from a National Cancer sequence andthefirstrepeatunit of a-spectrin, showed Institute Cancer Core Grant CA 10815. The costs of publication of specific high affinity binding to spectrin dimers, purified P this article were defrayed in part by the payment of page charges. subunit, and a recombinant 0-spectrin polypeptide containing This article must therefore be hereby marked “advertisement” in the COOH-terminal repeats,816 and 17. accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Present address: Laboratory of Molecular Neurobiology, New York State Inst. for Basic Research in Developmental Disabilities, 1050 Forest Hill Rd., Staten Island, NY 10314. Tel.: 718-494-5160. To whom correspondence should he addressed: The Wistar Inst. of Anatomy and Biology, 3601 Spruce St,, Philadelphia, PA 19104. Tel.: 215-898-3859;Fax: 215-898-3868.

The abbreviations used are: HS, hereditary spherocytosis; HE, hereditary elliptocytosis; HPP, hereditary pyropoikilocytosis; IPTG, isopropyl I-thio-P-~-galactopyranoside; PMSF, phenylmethylsulfonyl fluoride; HPLC, high-performance liquid chromatography; PVDF, polyvinylidene difluoride.

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Recombinant a-Spectrin Polypeptides MATERIALS AND METHODS

Extraction of spectrin, purification of 6 monomer, and NH,-terminal sequence analysis was performed according to Speicher et al. (1992). Construction of a-Spectrin Expression Plasmids-The a 3 plasmid contains 4.3 kb from the 5' end of a-spectrin cDNA, including the translation initiation codon (Sahr et al., 1990). Specific oligonucleotide primers were designed to amplify, by polymerase chain reaction (Mullis et al., 1986) using Taq polymerase (Boehringer Mannheim) or Vent polymerase (New England Biolabs), the 1.92-kb cDNA fragment encoding the first 639 amino acids of a-spectrin, which closely correlates in size with the tryptic a1 80-kDa domain (Speicher et al., 1983a). The 5' end primer started with the first codon following the initiation codon, so that a blunt end was produced prior to ligation into the NcoI site of the pKK233-2 vector (Pharmacia LKB Biotechnology Inc.), which was filled in by Klenow fragment of DNA polymerase I. The 3' end primer contained base changes to create a termination codon, followed by the XbaI site, by changing the codons for amino acids 639 and 640: ATA CAG AAAinto ATC TAG AAA. The outlined scheme allowed blunt-end cloning into the pKK233-2 vector, with the initiation codon provided by the vector and the stop codon provided by the DNA fragment in correct orientation of the insert (clone pKK7). Expression of the authentic spectrin polypeptides was defined by Western blot analysis. COOH-terminal truncations andmutation of the valine at position 31, which exhibited secondary initiation activity, were obtained also by polymerase chain reaction. Mutagenesis of the Val3' GTG into Val3' GTC involved two subsequent polymerase chain reactions. In the first reaction, the desired change was introduced into two DNA fragments that overlapped the region to be mutated. Thesefragments were annealed through the overlapping region and extended, and the full-length fragment was amplified in the second polymerase chain reaction as described by Horton et al. (1989). Mutants were identified by restriction digestion with NarI, whose site was introduced 5' to the valine codon without changing the encoded amino acid sequence. The 5' end primer was the same as the 5'primer used for the pKK7 construct.In subsequent constructs, 3' end-specific primers were designed to introduce stop codons at the desired position of the spectrin repetitive unit (position 31, Speicher et al., 1993), followed by the XbaI restriction site. This scheme allowed unidirectional blunt end-XbaI cloning into the previously modified pKK233-2 vector. In total, three expression constructs encoding amino acids 1-158 (clone pKK21), 1-264 (clone pKK15), and 1-370 (clone pKK24), respectively, were generated. NH2-terminal deletions of the pKK21 clone (1-158)were performed using unique restriction sites in the construct. Deletion of the first 27 amino acids was obtained by cloning of the NarI-XbaI fragment, blunt-ended at its 5' end into the NcoI (end-filled with Klenow) and Xbal sites of the previously modified pKK233-2 vector (clone pKK10). The 1-49 deletion was obtained by cloning of the Hind111 fragment blunt-ended by mung bean nuclease of the pKK21 clone (50-158) into the SmaI site of the pGEX-2T vector (clone pGEX-2T-HindIII). This construction resulted in addition of three extra amino acids (GSP) after cleavage with thrombin. All plasmid constructs were verified by restriction enzyme digestions and by sequencing using Sequenase 2.0 (United States Biochemical Corp.). Other recombinant DNA procedures, including metabolic labeling (Tran3'S-label; ICN), were performed according to Maniatis et al. (1982). Expression and Purification of Recombinant a-Spectrin Polypeptides-Recombinant spectrin polypeptides were purified from cultures of JM105 cells. Overnight cultures were diluted 1:10 and grown to an optical density of 0.5-0.7 at 550 nm prior to induction with (IPTG) at a final concentraisopropyl 1-thio-6-D-galactopyranoside tion of 1 mM. Inductions were carried out for 3 h, and cells were collected by centrifugation a t 3,000 X g. The bacterial pellet from 500 ml of culture was resuspended in 10 ml of 50 mM Tris, pH 8.0, 25% sucrose and incubated on ice with 2 mg/ml lysozyme for 10-15 min. Then the solution was adjusted with MgCI2 to 7 mM, and DNase I was added (up to 0.24 mg/ml); the incubation continued for another 10 min. Cells were lysed by addition of 3 ml of lysis buffer (50 mM Tris, 0.5% sodium deoxycholate, 1%Nonidet P-40, and 10 mM EDTA, pH 8.0); at this point, phenylmethylsulfonyl fluoride (PMSF) was also added to a final concentration of 1 mM. The lysate was centrifuged at 20,000 X g for 1 h; all recombinant a-spectrin polypeptides remained soluble in the bacterial lysate supernatant. Polypeptides obtained from the 1.92-kb expression construct (clone pKK7) were

14789

purified by affinity chromatography as described by Speicher et al. (1983b) and yielded approximately 10 mgof purified recombinant polypeptides/liter of starting bacterial culture. The 18.7-kDa recombinant polypeptide (residues 1-158) expressed by the clone pKK21 was purified using conventional methods, which included ammonium sulfate precipitation(45-60% ammonium sulfate pellet of bacterial lysate supernatant), gel filtration chromatography using Sephacryl S-100 (Pharmacia), and anion exchange chromatography using DEAE-cellulose (Whatman, DE521 where the N 18.7kDa polypeptide was eluted at 85 mM NaCl, 20 mM Tris, and 1 mM EDTA, pH 7.9. The final yield was approximately 1-1.5 mg of purified polypeptide/liter of bacterial culture. The 01 polypeptide (residues 50-158), expressed as the glutathione S-transferase fusion protein, clone pGEX-2T-HindII1, was purified from bacterial lysate supernatant using a glutathione-Sepharose column (Pharmacia). The polypeptide wascleaved from the carrier protein by thrombin digestion in the elution buffer (50 mM Tris and 5 mM glutathione, pH 8.0) and purified by rechromatography on a glutathione-Sepharose column followed by high performance liquid chromatography (HPLC) gel filtration. For affinity purification using recombinant glutathione S-transferase 6-spectrin fusion protein, bacterial lysate supernatants containing a polypeptides were precipitated with 70% ammonium sulfate, resuspended in 50 mM Tris, 1mM EDTA, 1mM PMSF, 2 pg/ml leupeptin, 1pg/ml pepstatin A, pH 8.0, and dialyzed against phosphate-buffered saline, pH 7.4, containing 1 mM EDTA, 1 mM PMSF, 2 gg/ml leupeptin, and 1 +g/ml pepstatin A. Incubations of the bacterial lysates with the recombinant P-spectrin fusion protein were performed on ice for a period of 3-6 days at totalprotein concentrations ranging from 4.3 to 11.4 mg/ml. Purification was achieved by binding recombinant polypeptides a:@ complexes to a glutathione-Sepharose column at 4 "C, followed by a 30-45-min incubation at 37 "C in the same buffer. The (Y polypeptide(s) containing the high affinity tetramer binding site could bespecifically eluted from the column at 37 "C, since the head-to-head a:@binding affinity at 37 "C is about 32-fold lower at 37 "C than at 0 "C (DeSilva et al., 1992). Expression and Purification of P-Spectrin Fusion Protein-The DNA fragment encoding amino acid residues 1903-2137 of ,6-spectrin (Winkelmann et al., 1990)was obtained by polymerase chain reaction. The 5' end primer contained a BamHI restriction site, whereas the 3' end primer contained an EcoRJ site immediately following the stop codon. This design allowed unidirectional cloning into the BamHI and EcoRI restriction sitesof the pGEX-2T expression vector (Pharmacia). The lysis and purification of cells were performed according to Kennedy et al. (1991), with addition of the following protease inhibitors: 1 mM PMSF, 1 mM EDTA, 2 pg/ml leupeptin, and 1 pg/ ml pepstatin A. Gel Electrophoresis and Western Blotting-Linear polyacrylamjde gels (7%) or gradient gels(5-15%)wereused to separate proteins (Laemmli, 1970); gels were stained with Coomassie Brilliant Blue R250. Protein standards were purchased from Bio-Rad and from Diversified Biotech. Western blot analysis was performed according to Harlow and Lane (1988) using nitrocellulose or polyvinylidene difluoride (PVDF) membranes. Monoclonal antibody against the CUI80kDa domain (1:5,000 dilution) (Yurchenco et al., 1982) and rabbit polyclonal antibody (1:100,000 dilution) (Speicher et al., 1992) were used to detect recombinant 01 polypeptides. 6-Spectrin recombinant fusion protein was detected using rabbit polyclonal antibody purchased from ICN (1:4,000dilution). Blots were incubated with alkaline phosphatase-conjugated anti-mouse or anti-rabbit immunoglobulin G antibodies purified from goat and developed in 100 mM Tris, 100 mM NaCI, 5 mM MgCI,, 0.3 mg/ml nitro blue tetrazolium, and 0.15 mg/ml 5-bromo-4-chloro-3-indoyl phosphate, pH 9.5. The reaction was stopped by rinsing the membranes with distilled water. Protein concentrations were estimated using the bicinchoninic acid assay (Pierce Chemical Co.) using bovine serum albumin as a standard. Circular Dichroism (CD) Measurements-CD spectra were performed on a Jasco 5720 instrument at room temperature in a 0.2-mm path length cell. Isotonic KC1 buffer (10 mM Tris, 20 mM NaC1, 130 mM KC1, 1 mM 2-mercaptoethanol, and 30 p~ PMSF, pH 7.4) was used as a solvent. Protein concentrations used in measurements (0.23-0.50 mg/ml) were determined by duplicate amino acid analysis ofthe samples. Mean residue ellipticity ([Q]MR) is expressed in degree cm'/dmol. CD curve smoothing was done using software supplied from Jasco.

14790

Recombinant a-Spectrin Polypeptides

RESULTS Expression and Function of thekDa NH2-terminal aI Polypep-

A

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kDa

B

1

2

tides-Spectrin self-association involves the NH2-terminal region of a-spectrin and COOH-terminal of P-spectrin. In 106.0. 106.0 order to study the role of the a subunit in this interaction the 80.0- - e < 80.0 NH2-terminal region of a-spectrin cDNA was subcloned for expression in Escherichia coli into the pKK233-2 expression 49.5. vector. The first construct was designedto express a polypep49.5 tide containing the NH2-terminal 639 residues of a-spectrin 32.5 with a termination codon TAG introduced at theposition of 27.5. codon 640 (Fig. lA). This recombinant polypeptide included 18.5. 32.5 all residues found in the a180-kDa domain previously shown 27.5 to associate with spectrin dimers (Morrow et al., 1980). Western blot analysis of IPTG-induced E. coli lysates, clone FIG.2. Characterization of NH,-terminal truncated specpKK7, revealedfive polypeptides immunoreactive with a trin polypeptides.A, proteins were separated by a 5-15% gradient monoclonal antibody against the a180-kDa tryptic domain of SDS-polyacrylamide gel (Laemmli, 1970) and stained with Coomassie spectrin (Speicher et al., 1983a). Recombinant polypeptides Blue. Lane 1, immunoaffinity-purified recombinant a-spectrin polywere immunoaffinity-purifiedfrom the bacterial lysate super- peptides from the pKK7 construct and are numbered 1-5. Lane 2, polypeptides bound to the recombinant COOH-terminal 0-spectrin natant. Analysis by SDS-gel electrophoresis revealedfive fusion protein at 4 "C and eluted at 37 "C.B, proteins were separated bands, designated as polypeptides 1-5 (Fig. 2A, lane I), which on 7% SDS gel. Lane 1, affinity-purified 8-spectrin fusion protein were characterized by amino acidsequencing of the NH2 eluted with 5 mM glutathione and 50 mM Tris, pH8.0. The -28-kDa terminus after blotting to PVDF membrane (Table I). The protein which copurified with the 8-spectrin fusion protein bound an largest polypeptide, 1, contained the authentic NH2-terminal anti-glutathione S-transferase antibody (Molecular Probes) and presequence of a-spectrin. The NH2-terminal amino acid se- sumably is a breakdown product from the fusion protein. Glutathione quences of polypeptides 2-5 indicated that they were products S-transferase alone did not bind recombinant a polypeptides (data not shown). Lane 2, Western blot analysis of lane 1 using a polyclonal of secondary translation initiation sites ratherthan of prote- anti-spectrin antibody (ICN) at a dilution of 1:4,000 and developed olysis. Polypeptides 3 and 4 arose by initiation at methionine using a goat anti-rabbit IgG conjugated with alkaline phosphatase. codon positions 70 and 108, respectively, whereas polypeptides 2 and 5 were initiated at positions 31 and 217, respectively, TABLE I by utilizing a valine codon GTG. Based upon migration on Identification of aZ domain recombinant polypeptides obtained from SDS gels and confirmed DNA sequence of the pKK7 conthe pKK7 clone by N-terminal sequencing struct, all five polypeptides presumably terminate at residue a1 domain Sequence Location" 639. Therefore, these bands represent a useful NH2-terminal wlweutide truncation series defined as follows: polypeptide 1, residues 1 MEQFPKETW... #1 1-639; polypeptide 2, residues 31-639; polypeptide 3, residues 2 MbLTRYQ... #31 70-639; polypeptide 4, residues 108-639; polypeptide 5, resi3 MEKVNI ... #70 dues 217-639. 4 MSELEKT ... #lo8 5 MbNQYANE... #217 These five polypeptides were studied for their ability to associate with spectrin dimers or purified B monomers using a Residue number as identified by Sahr et a l.(1990).

-

Valine codon is present at thislocation in the spectrin sequence.

a HPLC gel filtration assay (DeSilvaet al., 1992). The largest polypeptide bound to spectrin dimers and 8-spectrin but the other polypeptides (2-5) did not. In addition, polypeptides 3 and 4 exhibited anomalous behavior on gel filtration, eluting as very broad peaks (data not shown). This suggested that these polypeptides did not renature properly or aggregated into nonspecific complexes. 1 4 ATY pKK24 As an alternative approach to demonstrate specific interaction between recombinant a polypeptides and P-spectrin, the COOH-terminal region of /3-spectrin was subcloned into 1 3 v NAA pKK15 the pGEX-2T vector and expressed as a glutathione S-transferase fusion protein. The expressed region covered 236 amino acids (residues 1902-2137 subcloned from the 08 cDNA clone) 1 v RAL pKK21 (Winkelmann et al., 1990)starting with residue 29 in the 16th repetitive unit (start of the spectrin conformational unit) FIG.1. Diagram of NHa- andCOOH-terminaltruncation series of recombinant spectrin polypeptides derived from the (Speicher and Marchesi, 1984; Speicher et al., 1993; Winograd a1 domain. A, top line: the a1 80-kDa domain according to Speicher et al., 1991) through to the COOH-terminal end of 8-spectrin. et al. (1983b); bottom line: the 1.92-kb cDNA construct(pKK7) The fusion protein was purified from the bacterial lysate encoding residues 1-639of erythrocytea-spectrin; block triangles show the startingpoints of alternatively initiated recombinantpoly- supernatant on a glutathione-Sepharosecolumn (2-3 mg/liter peptides as identified by NH2-terminal sequencing, numbered 1-5. B, of culture) (Fig. 2B). lines represent COOH-terminal truncated constructswith an authenLow temperature head-to-head binding of the recombinant tic NH2-terminal sequence. The COOH terminus in each case was /3 fusion protein and a polypeptides 1-5 was performed, since phased according to Speicher et al. (1993); top line, pKK24, residues 1-370; middle line, pKK15, residues 1-264; bottom line, pKK21, it was shown previouslythat univalent complementary polyresidues 1-158. Three letters at the end of each line represent the peptides containing the tetramer binding site would readily last threeamino acids (single letter code) included in the construct. associate at low temperatures and the binding activity was

Recombinant a-Spectrin Polypeptides much stronger at 0-4 "C than athigher temperatures (DeSilva et al., 1992). The P-spectrin fusion protein bound to the glutathioneSepharose was incubated a t 4 "C with the mixture of five polypeptides expressed using the pKK7 vector. The only a recombinant polypeptide which bound to theCOOH-terminal @ fusion protein was the polypeptide 1.This polypeptide could be eluted from the column by increasing the temperature to 37 "C (Fig. 2A, lane 2), whereas the P-spectrin fusion protein remained bound to theglutathione-Sepharose column regardless of the incubationtemperature. The same result was obtained when the a recombinant polypeptides were incubated with the fusion protein in solution and then applied to theglutathione-Sepharose column (not shown). This functional assay also provided a simple method for purification of the largest polypeptide directly from the bacterial lysate supernatant concentrated with ammonium sulfate. Polypeptides 2-5 did not bind to the P-spectrin fusion protein under any condition, indicating that NHp-terminal truncationof 30 or more residues abolished binding (e.g. polypeptide 2, residues 31-639). The head-to-head interaction of functional recombinant a and /3 polypeptides exhibited the previously observed temperature dependence (DeSilva et al., 1992), since the polypeptide 1 exhibited substantially weaker binding at 37 "C compared with 4 "C. Functional Analysis of COOH-terminal-truncated a Polypeptides-To identify a minimal functional region of the CY subunit involved in tetramer formation, a polypeptides were expressed which contained the NH2-terminal sequence of the molecule, but were further truncated at the COOH-terminal end. The last amino acid included in the constructs was the 30th residue in the 106-amino acid repeat which corresponds to the endof folding units (Speicher et al., 1993). As a result, the expression constructs covered an NH2-terminal partial conformational unit plus either one, two, or three additional full 106 residue conformational units (Fig. 1B). In these expression constructs, the secondary initiation codon in position 31 of the spectrin (Fig. l ) , GTG (Val), was replaced by the noninitiation codon, GTC, by site-directed mutagenesis (see "Materials and Methods"). Expression of the construct covering the NH2-terminal158 residues (pKK21) resulted in a single band, which correlated well with the expected molecular mass of 18.7 kDa (Fig. 3A). From the clone pKK15, two polypeptides were obtained: the larger corresponding to the predicted full-size product (31.0 kDa) and thesmaller corresponding to apparent initiation at the methionine codon, position 70 (23.7 kDa). A similar result was apparent with the clone pKK24 which resulted inexpression of two bands corresponding to the 43.2- and 30.4-kDa polypeptides estimated for the full-size product and apparent initiation at methionine 108, respectively. Products originating a t secondary initiation sites appeared to be more abundant with these constructs. However, no peptidescorresponding to the secondary initiation at position Val"' were observed, indicating that this initiation was effectively eliminated by the base change of G to C in all constructs. The binding properties of the 18.7-kDa polypeptide, as well as polypeptides containing oneand two additional repeatunits with the P-spectrin fusion protein were evaluated (Fig. 3B). Each a recombinant with an intact NHp-terminal bound to the P fusion protein. However, the a recombinant polypeptides in these samples which resulted from initiation at alternate sites (NH2-terminal truncations, see Fig. 3A) did not bind to the P-spectrin fusion proteinconsistent with the results described above from theNH2-terminal-truncated forms obtainedusing the pKK7clone. To confirm the location of the head-to-head association site within the NH2-terminalregion of the a 18.7-kDa poly-

14791

95.5-

55.0-

.

36.029.078.4-

'

12.4-

FIG. 3. The COOH-terminal truncated a1 domain recombinant peptides. A, Immunoblot analysis. Bacterial lysates were prepared from uninduced cultures (lanes I, 3, and 5 ) and from IPTGinducedcultures (lanes 2, 4, and 6 ) and separatedon10% SDSpolyacrylamide gel. Arrows indicate full-lengthpolypeptides predicted from the cDNA sequence: 18.7 kDa (clone pKK2.1, residues 1-158) (lane 2 ) ; 31.0 kDa (clone pKK15, residues 1-264) (lane 4 ) ; 43.2 kDa (clone pKK24, residues 1-370) (lane 6 ) . After transfer to a PVDF membrane, the polyclonal anti-spectrin antibody(Speicher etal., 1992) was used to detect recombinant spectrin polypeptides and was developed using alkalinephosphatase-conjugated anti-rabbit antibody. B, association of recombinant a-spectrin polypeptides with the (3-spectrin fusion protein. Bacterial lysate supernatants containing recombinant a polypeptides were concentrated with 70% ammonium sulfate and incubated separately with 1.8 mg of affinity-purified pspectrin fusion protein on ice for 5 days in phosphate-buffered saline containing 1 mM EDTA, 1 mM PMSF, 2 pg/ml leupeptin, and 1 pg/ ml pepstatin A, at a total protein concentration of 7.0-11.4 mg/ml. Then each incubation mixture was applied to a 1-ml reduced glutathione-Sepharose column, followed by an extensive wash with the same buffer at 0 "C. a polypeptides were eluted from the column after a 30-45-min incubation of the column at 37 "C and washed with the same buffer at 37 "C. The (3-spectrin fusion protein remained bound to the column regardless of the temperature used. Proteins were separated on 5-15% SDS-polyacrylamide gels and stained with Coomassie Blue. Lanes l , 3, and 5 represent bacterial lysates concentrated withammoniumsulfate containing recombinant a polypeptides. Lanes 2, 4, and 6 represent the recombinant a polypeptides which associated withthe COOH-terminal (3-fusionprotein at 0 "C and were eluted at 37 "C. In each case, the molecular mass estimated from the gel corresponds to the full-length a polypeptide. Authenticity of the polypeptides were confirmed by Western blot analysis (not shown). Lanes I and 2, clone pKK2.1; lanes 2 and 3, clone pKK15; and lanes 4 and 5 , clone pKK24.

peptide, two additional NH2-terminal truncations of the 1158 polypeptide were constructed and expressed in E. coli. Bacterial lysate containing the CY polypeptide residues 28-158 (clone pKK10) showed no binding to the P-spectrin fusion protein, but the level of expression was very low. The a polypeptide 50-158 (clone GEX-2T-HindIII) expressed initially as a glutathione-S-transferase fusion protein and then cleaved by thrombin also did not bindto theP-spectrin fusion protein. The purified polypeptide showed a CD spectrum almost identical to thatof native spectrindimers, CY monomer, and a 18.7-kDa polypeptide (Fig. 4). Therefore, loss of function of the 50-158 polypeptide was not theresult of loss of ahelicity. Association of the 18.7-kDa Polypeptide with Spectrin Dimers-The purified 18.7-kDa recombinant polypeptide was studied for its ability to bind spectrin dimers inan HPLC gel filtration binding assay (DeSilva et al., 1992). The recombinant polypeptide was combined with purified spectrin dimers over a range of molar ratios, and samples were analyzed by HPLC gel filtration immediately following incubation at 37 "C for 45 min (Fig. 5). Thetetramer peak decreased asthe amount of the recombinant 18.7-kDa polypeptide added to the incubation was increased, with a corresponding increase of the dimer peak. The tetramer and dimer peak fractions

Recombinant a-Spectrin Polypeptides

14792

and, therefore, containsa functional tetramer binding site. Association of the 18.7-kDa Polypeptide with (3 MonomersMetabolically labeled recombinant 18.7-kDa polypeptide was incubated with the purified P-spectrinmonomeron ice at approximately 1:l and 1:2 molar ratios of labeled polypeptide to (3-spectrin. Fractions were analyzed by SDS-polyacrylamide gel electrophoresis and by autoradiography of dried Coomassie-stained gel (Fig. 6). The interaction of the 18.7kDa polypeptide with the native p subunit was observed a t 0 "C, even after a short incubation on ice (1.5 h). By 36 h all of the recombinant polypeptide was incorporated into the complex, indicating that the recombinantpolypeptide is fully functional. Based on three independent experimentsat 30 "C a KO= 6.6 f 1.2 X lo5M-' was obtained. DISCUSSION

- ~ . ~ ~ ~ ~ + ~ * ~ . ~ ~ ~ ~ ~ ~ ~ l , . , ~ , ~ , ~ . l ~ , , , , , ~ , , l , ~ ~ . ~ l ~ ~ ~ ~ , ~ . , , l , , , , ~ , ~ ~ ~ " ~ 190 0 W L rnm1 260 0

FIG.4. Spectra of purified erythrocyte spectrin and a subdimer (0.31 mg/ml); . . . ., CY monomer (0.30 unit peptides. -,

mg/ml); - - - -, CY 18.7-kDa (1-158) polypeptide (0.23 mg/ml); - . . - ., CY 13-kDa (50-158) polypeptide (0.50 mg/ml). All proteins were dialyzed into isotonic buffer and protein concentrations were determined by amino acid analysis prior to CD measurements. Mean residue ellipticity [Q]MR is expressed in degree cm'/dmol. Ellipticity values obtained were: -23,120 for the CY 18.7-kDapolypeptide; -27,340 for the a monomer; -28,360 for the dimer; and -29,115 for the CY 13 kDa.

iri

A

B

C

E T D

Spectrin self-association, or the head-to-head interaction, involves the NH2-terminalregion of the a and COOH-terminal region of (3-spectrin and is affected by many hereditary anemia mutations. Multiple HE mutations have been identified in the NH2-terminal region of the N subunit, but recent reports of mutations in theCOOH-terminal region of (3spectrin have provided an interesting insight into our understanding of this interaction (Eberet al., 1988; Tse et al., 1990; Pothier et al., 1990; Yoon et al., 1991; Tse et al., 1991; Gallagher et al., 1991). In this study, we have shown that recombinantpolypeptides containing the NH2 terminus of a-spectrin retained the ability to interact with spectrin dimers and (3 monomers, as well as recombinant polypeptides containing the COOH terminus of (3-spectrin. The head-to-head interactionwas retained also by a recombinant N polypeptide containing the first158 residues of the subunit, which bound to spectrin dimers at elevated temperatures (37 "C) and to(3 monomers and P recombinant polypeptides at high and low temperatures (37, 30, and 4 "C), which is consistentwith recent studiesby DeSilva et al. (1992). The association constantdetermined for this polypeptide compares favorably with values obtained at 30 "C by Morrow et al. (1980), Eberet al. (1988), andDeSilva et al. (1992). The functional interaction was lost with the deletion of the first 27 residues of a-spectrin as shown by the loss of binding activity of the recombinant polypeptide encompassing resi-

P

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80 90

Time (min)

FIG.5. Association of the 18.7-kDa recombinant polypeptide with spectrin dimers. Aliquots of spectrin (50 pg each) were combined with purified CY 18.7-kDa recombinant polypeptide at the following molar ratios of dimer:recombinant polypeptide: 1:0 ( A ) , 1:l ( B ) , 1:3 ( C ) , 1:6 (D), and 1:12 ( E ) . The final dimer concentration was 1.19 mg/ml in all samples. Samples were incubated a t 37 "C for 45 min and separated by HPLC gel filtration (Speicher et al., 1992). Positions of spectrin tetramer (T), dimer (D), and unbound recombinant polypeptide ( P ) are indicated by uertical lines. Formation of dimer-polypeptide complexes was detected by analysis of dimer peak fractions on Coomassie-stained SDS-polyacrylamide gels (data not shown). were analyzed on Coomassie-stained gel, and the 18.7-kDa polypeptide was detected in the dimer peak fractions (not shown), indicating that in fact the 18.7-kDa polypeptide associated with spectrin dimers. Appreciable amounts of the polypeptide were not associated with spectrin dimers after incubation at 0 "C as expected.At equimolar or submolar ratios essentially all of the recombinant polypeptide associated with dimers (not shown). These experiments showed that the 18.7-kDa recombinant polypeptide was able to compete with native spectrin dimers for the tetramer binding

time (hrs.) a:p ratio

0 1:l

1.5

36

36

1:l

1:l

1:2

FIG.6. Association of the 18.7-kDa polypeptide with 8spectrin. Metabolically labeled (35S) recombinant polypeptide was combined with purified p monomers a t a total protein concentration of 1.1mg/ml at 0 "C.Aliquots of eachincubation were injected separately onto the HPLC gel filtration column immediately following each incubation. Aliquots from the B-spectrin peak (lanes I , 3, 5 , and 7) and from the 18.7-kDa peak (lanes 2, 4 , 6, and 8) were electrophoresed on a 5-15% SDS-polyacrylamide gel, dried, and exposed for 7 days a t -70 "C. Incubation times and molar ratios of substrates are site indicated below each lane.

14793

Polypeptides a-Spectrin Recombinant dues 28-158. Similarly, the pKK7polypeptide 2, residues 31639, did not have a functional binding site. It was possible to demonstrate that, in a polypeptide which lacked the first 49 amino acid residues (50-158), the loss of function was not due to the loss of local folding (see Fig. 4). Loss of the first 6 residues in a-spectrin toproduce the a1 80-kDa domain does not affect interaction with P-spectrin (Morrow et al., 1980), whereas the further loss of the next 10 residues, the a1 79kDa peptide, decreases bindingaffinity by about 50% (Speicher et al., 1993). Our results extend these earlierobservations as follows: ( a ) further truncationsby deleting the first 27, 30, or 49 residues totally abolishes detectable head-tohead association with the complementary p subunit; ( b ) the presence of one adjacent repeat unit is sufficient to stabilize the conformation of the NHz-terminal binding site; (c) the presence or absence of additional repeats (repeats 2-6) does not affect binding affinity;and ( d ) the NH2-terminalbinding site region has a lower helical content than full repeats;at least when it is not complexed with the complementary p site (compare CD spectra for the 1-158 recombinant with the 50158 recombinant). Therefore, it seems likely that most, if not all, of the region encompassed by residues 7-49 are directly involved in head-to-head association with p-spectrin. However, synthetic peptides encompassing the regions of interest (residues 7-28 and 7-53) did not show anyhead-to-head binding activityand did not appear to have appreciable native structure (data not shown). Although direct participation of the first full conformational repeat, residues 50-158, in the binding site is also a possibility, it seems more likely that the presence of this region acts as a template for folding of the less ordered terminal binding site region. The phasing of the recombinant spectrin polypeptides in this study was based on the conformational model of spectrin (Speicher and Marchesi, 1984; Speicher et al., 1993), where the conformational unit begins and ends around residue 30 of a repeat unit. Our current results support this model, since all polypeptides with this phasing apparently have a native conformation. In contrast,polypeptides 3 and 4 (clone pKK7), which are initiated internally at positions 47 and 86 of the firstrepeat unit, showed anomalous gel filtration profiles suggesting non-native conformation. The phasing used here was also shown to give polypeptides a native conformation and resistance to proteolysis for repeats 14 and 15 of Drosophila a-spectrin (Winograd et al., 1991). Correct phasing may be important for properfolding of recombinant polypeptides, although it may also be possible for some polypeptides with alternativeinitiation orstart points to refold in an apparently native conformation. For example, the alternatively initiated polypeptide 5, residues 217-639, contained a partial conformational unit on its amino-terminal end and was apparently native at least judging by its elution profile on gel filtration columns. The location of the spectrin self-association site within the first 49 residues of a-spectrin is in agreement with a recently proposed model of the interaction (Tse et al., 1990,1991; Speicher et al., 1993). The model by Tse et al. (1990) is based on the location of recently identified HE mutations at the COOH terminus of P-spectrin which induce a conformational change in the NHz-terminal region of the a subunit. The proposed interaction would involve formation of a repeatunit consisting of helices 1 and 2 from the COOH terminus of pspectrin and helix 3 from the NH2 terminusof a-spectrin. A

number of HE mutations are located in the first 49 residues of a-spectrin (resulting in aI/74 and aI/78 phenotypes; reviewed by Palek and Lambert, 1990; see also Speicher et al., 1993) and presumably affect the bindingsite directly by disrupting helix 3. Mutations located in the COOH-terminal region of P-spectrin affect p17 helices 1 and 2, leaving helix 3 of the partial repeat unit at the NH2 terminusof a-spectrin unprotected for trypsin cleavage and can result in the same aI/74 phenotype. This data is in agreement with proteolytic footprinting studies and the refined conformational model of the interaction by Speicher et al. (1993). Mutations located further within the a-spectrin molecule (a2, -4, and -5 repeat units) may affect dynamics of the closedopen dimer equilibrium and hence could shift equilibrium of spectrin tetramers into dimers (DeSilva et al., 1992; Speicher et al., 1993). The ability to make abundantly,purefunctional recombinant polypeptides, encoding one to several repeatunits, should facilitate further characterization of the spectrin tetramer binding site. It may also be possible to identify interactions between neighboring spectrin repeat units within the a chain. Acknowledgments-We would like to thank Dr.Laszlo Otvos, Jr., Gyorgyi I. Szendri, and Emma Lang (The Wistar Institute, Philadelphia, PA) for performing the CD measurements andhelp in interpretation of this data. We also would like to thank Dr. Bernard Forget (Yale University, New Haven, CT) for providing /3-spectrin cDNA clones. REFERENCES Bennett, V., and Lambert, S. (1991)J. Clin. Inuest. 87,1483-1489 Desilva, T. M., Peng, K., Speicher, K.D., and Speicher,D. W. (1992)Biochemistry 31, 10872-10878 Eber, S. W., Morris, S. A., Schroter, W., and Gratzer, W. B. (1988)J. Clin. Inuest. 81,523-530 Galla her, P. G., Tse, W. T., Costa, F., Scarpa, A., Boivin, P., Delaunay, J., ancfForget, B. G. (1991)J . Biol. Chern. 266, 15154-15159 Harlow, E., and Lane, D. (1988)Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY Hodon, R. M., Hunt, H. D., Ho, S. N., Pullen, J. K., and Pease, L. R. (1989) Gene (Amst.)77,61-68 Kennedy, S., Warren, S., Forget, B., and Morrow, J. (1991)J. CellBiol. 115(1), 267-277 Knowles, w. J., Morrow, J. S., Speicher, D. W., Zarkowsky, H. S., Mohandas, N., Mentzer, W. C., Shohet, S. B., and Marchesi, V. T. (1983)J. Clin. Inuest.

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Morrow, J. S., Speicher, D. W., Knowles, W. J., Hsu, C. J., and Marchesi, V. T. (1980)Proc. Natl. Acad. Sci. U. S. A. 77,6592-6596 Mullis, K., Faloona, F., Scharf, S., Saiki, R., Horn, G., and Erlich, H. (1986) Cold Sprin Harbor Symp. Quant. Biol. 51,263-273 Palek, J., a n f l a m b e r t , S. (1990)Semin. Hematol. 27(4), 290-332 Pothier, B., Alloisio, N., Marechal, J., Morle, L., Ducluzeau, M. T., Caldani, C., Philippe, N., and Delaunay, J. (1990)Blood 76,2061-2069 Sahr, K. E., Laurila,P.,Kotula, L., Scarpa, A. L., Coupal, E., Leto, T. L., Linnenhach, A. J., Winkelmann, J. C., Speicher, D. W., Marchesi, V. T., Curtis, P. J., and Forget, B. G. (1990)J. Bid. Chem. 265,4434-4443 Spelcher, D. W., and Marchesi, V. T. (1984)Nature 311,177-180 Speicher, D. W.,Morrow, J. S., Knowles, W. J., and Marchesi, V. T. (1982)J . Biol. Chem. 257,9093-9101 Speicher, D. W., Davis, G., and Marchesi, V. T. (1983a)J. Biol. Chem. 258,

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Speicher, D. W., Davis, G., Yurchenco, P. D., and Marchesi, V. T. (1983b)J . Biol. Chem. 258,14931-14937 Speicher, D. W., Weglarz, L., and DeSilva, T. M. (1992)J. Biol. Chem. 267, 14775-1 - _ . A7R9 Speicher, D. W., DeSilva, T. M. Speicher, K. D. Ursitti, J. A., Hembach, P., and Weglarz, L. (1993)J. Biol.' Chem. 268,422'7-4235 Tse, W. T., Lecomte, M. C., Costa, F. F., Garbarz, M., Feo, C., Boivin, P., Dhermy D. and For et B G (1990)J. Cltn. Inuest. 86,909-916 Tse, W. T.', G h a her, G., Pothier, B., Costa, F. F., Scarpa, A., Delaunay, J., and Forget, B. (1991)Blood 78,517-523 Winkelmann, J. C., Chang, J. G., Tse, W. T., Scarpa, A. L., Marchesi, V. T., and Forget, B. G. (1990)J. Biol. Chem. 265, 11827-11832 Winograd, E., Hume, D., and Branton, D. (1991)Proc. Natl. Acad. Sci. L! S. A. 88. I ~ ~ R R - I ~ ~ W Yoon, S.-H., Yu, H., Eber, S., and Prchal, J. T. (1991)J. Biol. Chem. 266, 8490-8494 Yurchenco, P. D., Speicher, D. W., Morrow, J. S., Knowles, W. J., and Marchesi, V. T. (1982)J. Biol. Chem. 257,9102-9107 . V

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