JOURNAL OF BACTERIOLOGY, Aug. 1992, p. 5204-5210 Vol. 174, No. 160021-9193/92/165204-07$02.00/0Copyright © 1992, American Society for Microbiology

Cloning, Sequence Analysis, and Expression in Escherichiacoli of a Streptococcal Plasmin Receptor

RICHARD LoTrENBERG,l* CHRISTOPHER C. BRODER,1t MICHAEL D. P. BOYLE,2STEPHANIE J. KAIN,1t BRETT L. SCHROEDER,1 AND ROY CURTISS III3

Department ofMedicine, University of Florida, Gainesville, Florida 326101; Department ofMicrobiology,Medical College of Ohio, Toledo, Ohio 436992; and Department of Biology,

Washington University, St. Louis, Missouri 63130WReceived 17 April 1992/Accepted 10 June 1992

Plasmin(ogen) receptors are expressed by many gram-positive and gram-negative bacteria. We previouslyisolated a plasmin receptor from a pathogenic group A streptococcal strain (C. C. Broder, R. Lottenberg, G.0. von Mering, K. H. Johnston, and M. D. P. Boyle, J. Biol. Chem. 266:4922-4928, 1991). The gene encodingthis plasmin receptor, plr, was isolated from a A gtll library of chromosomal DNA from group A streptococcalstrain 64/14 by screening plaques with antibodies raised against the purified streptococcal plasmin receptorprotein. The gene was subcloned by using a low-copy-number plasmid and stably expressed in Escherichia coli,resulting in the production of an immunoreactive and functional receptor protein. The DNA sequence of thegene contained an open reading frame encoding 335 amino acids with a predicted molecular weight of 35,787.Upstream of the open reading frame, putative promoter and ribosomal binding site sequences were identified.The experimentally derived amino acid sequences of the N terminus and three cyanogen bromide fragments ofthe purified streptococcal plasmin receptor protein corresponded to the predicted sequence encoded byph-. Thededuced amino acid sequence for the plasmin receptor protein revealed significant similarity (39 to 54%identical amino acid residues) to glyceraldehyde 3-phosphate dehydrogenases.

Group A streptococci cause pharyngitis and invasiveinfections such as cellulitis and bacteremia (5). Certainbacterial surface structures and secreted products have beensuggested to contribute to tissue invasion. One of thesesecreted products, streptokinase, is a plasminogen activatorand converts the host zymogen plasminogen to the activeprotease, plasmin (47). We have reported that certain strainsof group A streptococci which produce streptokinase ex-press high-affinity surface receptors which are specific forhuman plasmin. Once bound to the bacteria, plasmin re-mains enzymatically active and cannot be inhibited byphysiological inhibitors (34). Although classically describedas the enzyme responsible for fibrin degradation, plasmin isa serine protease with trypsin-like specificity and has activityfor a broad range of substrates. Plasmin can degrade severalmammalian extracellular matrix proteins, such as fibronectinand laminin, and can enhance collagenase activity (32).Therefore, the ability to generate and capture active plasminmay contribute to the invasive propensity of certain strep-tococcal strains.The interaction of plasmin with group A streptococci has

high affinity (kd, 10-l M) and is specific for plasmin, with nosignificant binding demonstrated for structurally related pro-teins (10, 13). The receptor has minimal affinity for the nativezymogen, plasminogen with N-terminal glutamic acid, sug-gesting that the surface receptor recognizes conformation-ally dependent structures of the plasmin(ogen) molecule (8).We recently purified a plasmin-binding protein from thesurface of a pathogenic group A streptococcal strain (9). The

* Corresponding author.t Present address: Laboratory of Viral Diseases, National Insti-

tute of Allergy and Infectious Diseases, National Institutes ofHealth, Bethesda, MD 20892.

t Present address: Department of Neurology, Texas Tech Uni-versity Health Science Center, Lubbock, TX 79430.

-41,000-Mr protein was physicochemically, antigenically,and functionally distinct from the secreted plasmin(ogen)-binding and -activating protein, streptokinase, which wasisolated from the same strain (9). In this article, we reportthe cloning, DNA sequencing, and expression of the geneencoding the -41,000-Mr streptococcal plasmin receptor.

(This study was presented in part at the 91st GeneralMeeting of the American Society for Microbiology, Dallas,Tex., 5 to 9 May 1991 [34a].)

MATERIALS AND METHODS

Bacterial strains, plasmids, and media. Group A strepto-coccal strain 64/14 is an M-untypeable clinical isolate thatwas passaged in mice 14 times (45). Escherichia coli Y1090[AlacUl69 proA+ Alon araD139 strA supF trpC22::TnlO(Tetr) (pMC9)] was used in screening the expression library,and E. coli X6060 [F' (traD36 proAB lacIq AlacZMJS)::TnS(Kmr)IaraD139 A(ara leu)7697 AlacX74 AphoA20galE galKrecAl rpsE argE(Am) rpoB thil was used for transformationand gene expression. A low-copy-number plasmid pYA2204with a replicon derived from pREG153 (a low-copy-numberIncW vector derived from the R plasmid R388) was used(18). The plr was subcloned into the pUC9 lacZa multiplecloning site of pYA2204.

Streptococci were grown in Todd-Hewitt broth. E. coliY1090 for X infection was grown in 1% tryptone supple-mented with 0.5% yeast extract and 0.4% maltose. E. coliX6060 was grown in L broth supplemented with kanamycin(50 pg/ml).

Radioiodination of proteins. Human plasminogen isolatedfrom plasma by chromatography on lysine-Sepharose (35)and streptococcal protein G (Calbiochem, San Diego, Calif.)were labeled with 1251I (Amersham Corp., Arlington Heights,Ill.) by using a mild lactoperoxidase reaction with Enzymo-beads (Bio-Rad, Richmond, Calif.) (37). Plasmin was gener-

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ated from radiolabeled plasminogen as previously described(9).DNA manipulations. Streptococcal chromosomal DNA

was isolated by a modification of the procedure reported byHudson and Curtiss (25). Streptococci were grown overnightas standing cultures at 37°C in Todd-Hewitt broth supple-mented with 2.2 mM K2HPO4. Bacterial pellets were treatedwith lysozyme, mutanolysin, and pronase. The cells werelysed with sodium dodecyl sulfate (SDS). Genomic DNAwas further purified by two successive cesium chloridegradient centrifugations. Plasmid DNA was isolated by thealkaline lysis procedure (4) and purified by cesium chloridegradient centrifugation. Restriction enzyme digestions andreactions with DNA-modifying enzymes were performedaccording to the manufacturers' recommendations (Be-thesda Research Laboratories, Inc., Gaithersburg, Md., andPromega Corp., Madison, Wis.). A phage were isolated bythe plate lysate method (48) and then precipitated withpolyethylene glycol. DNA was purified by using an anion-exchange resin (Qiagen, Studio City, Calif.). DNA fragmentsof interest were recovered from agarose gels by usingGeneClean (Bio 101, La Jolla, Calif.). Other DNA manipu-lations were performed essentially as described by Maniatiset al. (36).

Preparation of the A gtll library. The streptococcal ge-nomic library was constructed as described by Huynh et al.(27). Chromosomal DNA was mechanically sheared to gen-erate fragments approximately 2 to 7 kb in length. The DNAwas treated with EcoRI methylase, treated with Klenowfragment of DNA polymerase to generate blunt ends, andligated with EcoRI linkers. Excess linkers were removed bydigestion with a high concentration of EcoRI. The DNA wasligated into EcoRI-generated A gtll arms and packaged intoX phage with Packagene (Promega Corp.).

Screening of the streptococcal library. The resulting non-amplified A gtll library was diluted in 10 mM Tris-2.5 mMMgSO4-0.01% gelatin-0.1 M NaCl, pH 7.5, and used toinfect E. coli Y1090, yielding a density of 200 to 400 plaquesper plate. The infected cells were mixed with 0.45% softagar, plated on 1.2% L agar supplemented with ampicillin (50FgIml), incubated at 42°C for 3 to 4 h to induce lysis, andoverlaid with nitrocellulose filters impregnated with 10 mMisopropylthiogalactoside to induce the lac promoter. Afterincubation at 37°C for approximately 16 h, the filters wereremoved, washed, and blocked in 100 mM Tris-300 mMNaCI-5 mM EDTA-0.05% Triton X-100-0.25% gelatin, pH7.4 (NET-gel). The filters were then incubated with murineantiplasmin receptor antibody (9) for approximately 18 h atroom temperature and then incubated with goat anti-mouseimmunoglobulin G (Cappel, Organon Teknika) for 3 to 4 h.Antigen-antibody complexes on washed filters were detectedwith 1"I-streptococcal protein G. Autoradiographs weregenerated by exposing the nitrocellulose filters to KodakXAR-5 film with intensifying screens at -70°C and thenusing automated film developing. Immunoreactive plaqueswere isolated and purified through two additional screenings.PAGE and protein blotting. SDS-polyacrylamide gel elec-

trophoresis (PAGE) was carried out with a 10% (wt/vol)polyacrylamide separating gel and a 4% (wt/vol) polyacryl-amide stacking gel according to the method of Laemmli (30).Gels for Western blotting (immunoblotting) were equili-brated, and separated proteins were transferred by methodsdescribed previously (9). For studies of plasmin binding,nitrocellulose membranes were blocked for 1 h at roomtemperature in NET-gel buffer. The blots were then incu-bated for 2 h at room temperature with 1"I-human plasmin

to identify plasmin-binding protein bands (9). The blots werewashed in NET-gel and subjected to autoradiography at-700C.

In vitro transcription-translation. Plasmid-encoded pro-teins were generated in vitro with a DNA-directed transcrip-tion-translation kit (Amersham). Protein products labeledwith [35S]methionine (specific activity, 1,000 Ci/mmol; Am-ersham) were subjected to SDS-PAGE and identified byfluorography with sodium salicylate (11).

Purification of the recombinant streptococcal plasmin recep-tor protein. E. coli X6060(pRL015) was lysed with a Frenchpressure cell. The resulting material was centrifuged at10,000 x g for 30 min, and the supernatant fluids weresubjected to ammonium sulfate fractionation. Successiveadditions of ammonium sulfate were performed at roomtemperature; the precipitates were removed by centrifuga-tion at 10,000 x g for 30 min, dialyzed against phosphate-buffered saline, and analyzed by SDS-PAGE and Westernblotting. At 55% ammonium sulfate saturation, the recombi-nant -41,000-Mr plasmin receptor protein remained in thesupernatant fluid, whereas the majority of E. coli proteinsprecipitated.DNA sequencing and analysis. DNA sequencing was per-

formed by dideoxy chain termination by using the Sequenasekit (United States Biochemical Corp., Cleveland, Ohio) witha-35S-ATP (specific activity > 1,000 Ci/mmol; Amersham)by following the instructions of the manufacturer. Sequenceswere determined by using the universal forward primersupplied with the kit, recessed M13 primers, and sequence-specific oligonucleotides synthesized by the University ofFlorida Interdisciplinary Center for Biotechnology ResearchDNA Core Laboratory. Sequences were analyzed with theGenetics Computer Group programs (University of Wiscon-sin-Madison) (14).Amino acid sequencing. Mutanolysin-extracted proteins (9)

from strain 64/14 were subjected to SDS-PAGE. The pro-teins were electrotransferred to a polyvinylidene difluoridemembrane (Immobilon-P; Millipore Corp., Bedford, Mass.)by using 10 mM 2-(N-morpholino)ethanesulfonic acid (pH6.0)-20% methanol as the transfer buffer. Protein bands werestained with Coomassie brillant blue. The -41,000-Mr pro-tein band, which had previously been shown to bind plas-min, was excised. Microsequencing by automated Edmanchemistry was performed with an Applied Biosystems model470A gas-phase sequencer with an on-line 120A PTH ana-lyzer (Washington University Protein Chemistry Laborato-ry). Cyanogen bromide fragmentation of the -41,000-Mrprotein was performed by immersing the polyvinylidenedifluoride membrane-bound protein in 70% formic acid andtreating with cyanogen bromide overnight at room tempera-ture. The fragments were separated by SDS-PAGE andtransferred to a polyvinylidene difluoride membrane as de-scribed above. Four peptides were identified and sequencedat the University of Florida Interdisciplinary Center forBiotechnology Research Protein Chemistry Core Facility byusing an Applied Biosystems model 470 sequencer with anon-line PTH analyzer.

Nucleotide sequence accession number. The nucleotidesequence of plr has been assigned GenBank accessionnumber M95569.

RESULTS

Cloning and expression of the streptococcal plasmin recep-tor gene. The focus of this study was to characterize the geneencoding the -41,000-Mr plasmin receptor protein of group

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5206 LOTIENBERG ET AL.

A streptococcal strain 64/14 (9). Strain 64/14 produces atleast two distinct proteins that display the ability to bindhuman plasmin(ogen). In addition to the -41,000-Mr plasminreceptor, these bacteria secrete an -47,000Mr plasminogenactivator protein, streptokinase. Previous studies from ourlaboratory have shown that these two proteins are antigen-ically and functionally distinct (9). Consequently, a mono-specific polyclonal antiplasmin receptor antibody prepara-tion, rather than plasmin, was used to screen a group Astreptococcal A gtll expression library generated from strain64/14 chromosomal DNA. Approximately 13,000 recombi-nant plaques were screened by using a plaque-lift assay asdescribed in Materials and Methods, and three plaques withstrong immunoreactivity were identified and designatedSPR4, SPR8, and SPR17. After two rounds of plaque puri-fication, protein products from phage lysates of the threeclones were collected and analyzed by SDS-PAGE andWestern blot analysis. Each positive clone produced animmunoreactive product with an Mr of -41,000 not presentin control lysates (data not shown), indicating that theprotein product was not fused with ,-galactosidase.

In order to express the streptococcal plasmin receptorprotein (Plr) stably in E. coli, subcloning with a plasmidvector was carried out. After digestion of the DNA fromSPR4 with EcoRI, agarose gel electrophoresis analysis re-vealed 2.4- and 2.7-kb DNA fragments in addition to the Xarms. We attempted to subclone each of the EcoRI frag-ments into the EcoRI site of the low-copy-number plasmidpYA2204. We were unable to isolate recombinant plasmidsthat carried the 2.4-kb fragment. However, the 2.7-kb frag-ment was successfully subcloned, yielding pRLO15. Whenthis plasmid was transformed into E. coli X6060, an-41,000-Mr protein that was recognized by the antiplasminreceptor antibody by Western blotting was observed. Thisprotein was not produced by strain X6060 alone or by X6060transformed with the vector alone (Fig. 1).The -41,000-Mr protein expressed from pRL015 was also

analyzed for the ability to bind plasmin. Lysates of X6060(pRL015) were subjected to SDS-PAGE, transferred to anitrocellulose membrane, and reacted with 1"I-plasmin.Several proteins contained in lysates of X6060(pRL015)demonstrated the ability to bind plasmin. Control lysates ofX6060 alone and X6060(pYA2204) also demonstrated similarplasmin-binding protein bands. Therefore, the streptococcalprotein was separated from E. coli plasmin-binding proteinsin order to determine whether the recombinant protein hadplasmin-binding activity. Supernatant fluids from lysatesobtained by French pressure cell treatment of X6060(pYA2204) as a control and X6060(pRL015) were subjectedto ammonium sulfate precipitation as described in Materialsand Methods. The supernatant fluids resulting from a 55%ammonium sulfate cut were analyzed by SDS-PAGE andWestern blotting. This manipulation resulted in the precipi-tation of all plasmin-binding proteins in the control lysate,whereas the -41,000-Mr protein produced by X6060(pRL015) remained in the supernatant fluid. This immunor-eactive protein demonstrated the ability to bind 1"I-plasmin(data not shown).

In vitro transcription-translation studies were performed.SDS-PAGE analysis of the protein products revealed thatboth pYA2204 (the vector) and pRL015 encoded severalproteins with Mrs of <30,000; however, pRL015 produced amajor protein product with an Mr of -41,000 not observedfor the control. A minor -33,000Mr protein, which mayrepresent degradation of the -41,000-Mr protein or anadditional streptococcal encoded product was also identified

A BKD

110-

84-

47-

33-

24-

16-1 2 3 1 2 3

FIG. 1. Expression of a streptococcal plasmin receptor proteinin E. coli. Parallel reducing SDS-10% polyacrylamide gels wereelectrophoresed. One gel was stained with Coomassie brillant blueto detect proteins (A). The proteins in the second gel were trans-ferred to a nitrocellulose membrane (B). The membrane wasblocked, reacted with mouse antiplasmin receptor antibody, andprobed with 125I-streptococcal protein G as described in Materialsand Methods. Lanes 1 contain mutanolysin-extracted streptococcalplasmin receptor protein; lanes 2 contain a whole-cell lysate ofX6060(pYA2204); and lanes 3 contain a whole-cell lysate ofX6060(pRL015).

(Fig. 2). The results of this experiment supported the ideathat pRL015 contains the structural gene for the streptococ-cal plasmin receptor protein.DNA sequence and characterization of the plasmin receptor

gene. The restriction map of the 2.7-kb EcoRI insert ofpRL015 is depicted in Fig. 3. DNA sequence analysis of theends of the 2.7-kb fragment did not reveal evidence of anopen reading frame. Therefore, subclones for sequencing

KD

116-84-58-

48.5-

36.5-26.6-

A BFIG. 2. Identification of pRL015-encoded proteins. Expression

of genes contained on plasmids was carried out in a DNA-directedtranscription-translation system as described in Materials and Meth-ods. The [35S]methionine-labeled protein products were analyzed byreducing SDS-10% PAGE and identified by fluorography. Lane A,pYA2204; lane B, pRL015.

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Pvul Hind IIIEcoRI Sal

00.2 kb

Hincl HinclPvul \ EcoRV BamH EcoRMr.

pIr b

FIG. 3. Physical and genetic map of the 2.7-kb EcoRI fragment subcloned in pRL015. Restriction enzyme recognition sites are indicated.The location ofplr is denoted by the hatched box. The arrow indicates the orientation of the open reading frame.

were generated to locate the gene. The 1.7-kb PvuII-EcoRIfragment representing the right side of the DNA insert (Fig.3) was ligated with pYA2204 which had been digested withSmaI and EcoRI. Sequence analysis of each end of the insertDNA was performed, and an open reading frame to the rightof the PvuII site was identified. By using pRL015, syntheticoligonucleotides were used to extend the sequence upstreamand downstream of the PvuII site. A 1,008-bp open readingframe that began with ATG and terminated with a TAAcodon was revealed. The G+C content for the open readingframe was 40.5%. The sequence of the open reading frameand upstream region are shown in Fig. 4.

-351 ATAATAGTTCTGTTGAAAGGTTGTTGCAGATGACTGTAAGTAATCTTTTCACAATAGGTAGGGAGCATT

-10 RBS70 CCCTCTAATAATATTCT M GATTTTCATAAGGAGGAAATCACTAATGGTAGTTAAAGTTGGTATTAAC

M V V K V G I N

139 GGTMTCGGTCGTATCGGACGTCTTGCATTCCGCCGTATTCAAAACATCGAAGGTGTTGAAGTAACTCGTG F G R I G R L A F R R I Q N I EG V E V T R

208 ATCAATGACCTTACAGATCCAAATATGCTTGCACACTTGTTGAAATACGATACAACTCAAGGTCGTTTTI N D L T D P N N L A H L L K Y D T T 0 G R F

277 GATGGAACAGTTGAAGTTAAAGAAGGTGGA 1 GAAGTAAACGGAAACTTCATCAAAGTM1CTGCTGAAD G T V E V K E G G F E V N G N F I K-V S A E

346 CGTGATCCAGAAAACATCGACTGGGCAACTGATGGGGTTGAAATCGTTCTTGAAGCAACTGGTM1CTMR D P E N I D W A T D G V E I V L E A T G F F

415 GCTAAAAAAGAAGCAGCTGAAAAACACTTACATGCTAACGGTGCTAAAAAAGTTGTTATCACAGCTCCTA K K E A A E K H L H A N G A K K V V I T A P

484 GGTGGAAACGATGTTAAAACAGTTGTTTTCAACACTAACCACGACATTCTTGACGGTACTGAAACAGTTG G N D V K T V V F N T N H D I L D G T E T V

553 ATCTCAGGTGCTTCATGTACTACAAACTGTMTAGCTCCTATGGCTAAAGCTCTTCACGATGCATTCGGTI S G A S C T T N C L A PM A K A L.H.D A F G

622 ATTCAAAAAGGTCTTATGACTACAATCCACGCTTACACTGGTGACCAAATGATCCTTGACGGACCACACI 0 K GLMM T T I H A Y T G D Q M I L D G P H

691 CGTGGTGGTGACCTTCGTCGTGCACGCGCTGGTGCTGCAAACATCGTTCCTAACTCAACTGGTGCTGCTR G G D L R R A R A G A A N I V P N S T. G A A

760 AAAGCTATCGGTCTTGTTATCCCAGAACTTAACGGTAAACTTGACGGTGCTGCACAACGTGTTCCTGTTKA I G L V I P E L N G K L D G A A Q R V P V

829 CCAACTGGATCAGTAACTGAGTTGGTTGTAACTCTTGACAAAAACGTMTCTGTTGACGAAATCAACTCTP T G S V T E L V V T L D K N V S V D E I N S

898 GCTATGAAAGCTGCTTCAAACGATAGCTTCGGTTACACTGAAGATCCAATCGTTTCTTCAGATATCGTAA M K A A S N D S F G Y T E D P I V S S D I V

967 GGCGTATCATACGGTTCATTGTTTGACGCAACTCAAACTAAAGTAATGGAAGTTGACGGATCACAATTGG V S Y G S L F D A T Q T K V M E V D G S Q L

1036 GTTAAAGTTGTATCATGGTATGACAACGAAATGTCTTACACTGCTCAACTTGTACGTACTCTTGAGTACV K V V S W Y D N E M S Y T A Q L V R T L E Y

1105 TTCGCAAAAATTGCTAAATAATTAGTTATAACGAAGAGAGCTTGGMATGF A K I A K *

FIG. 4. Nucleotide sequence of the plasmin receptor gene. Theputative -35 and -10 regions and ribosomal binding site (RBS) areindicated. The first ATG triplet denotes the start of the open readingframe, and an in-frame termination codon is indicated by theasterisk. The predicted amino acid sequence of the protein is shownin single-letter code below the nucleotide sequence. The experimen-tally determined amino acid sequence for the purified wild-typestreptococcal protein is underlined. Dotted underlines representambiguous amino acid determinations. The DNA sequence wasdetermined for both strands as described in Materials and Methods.

N-terminal amino acid sequencing of the -41,000-M,plasmin receptor protein from strain 64/14 was performed,and an unambiguous sequence was obtained for 51 residues.Amino acid sequences were obtained for four peptides (Mrsof 3,000 to 16,000) generated by cyanogen bromide treatmentof the '41,,OOO-Mr protein. The sequence of one of thesepeptides had identity with the N-terminal sequence of theintact protein. The sequence for another peptide overlappedwith 14 residues of the N-terminal sequence and allowedassignment of an additional 23 residues. The deduced aminoacid sequence of the open reading frame exhibited completeidentity with 74 amino acid residues of the native protein,indicating that valine following the ATG initiation codonrepresents the N terminus of the receptor protein. Thesequences of two additional peptides (13 and 27 residues)were also determined and found to correspond to residues160 to 173 and 186 to 216, respectively (Fig. 4). Thus, 114 ofthe predicted 335 amino acid residues encoded byplr havebeen confirmed by amino acid sequencing of the nativestreptococcal protein.

Several putative regulatory sequences upstream of thestructural gene were identified. A potential ribosomal bind-ing sequence was found at position -9 (AAGGAGG). Puta-tive -35 (TTCACA) and -10 (TCTAAT) regions wereidentified upstream of the ribosomal binding site with aspacer of 19 bp. An additional subclone with the HindIII-BamHI fragment of pRL015 placed in the opposite orien-tation to the plasmid-encoded promoter expressed the re-ceptor protein in E. coli (data not shown), indicating thatthe upstream region served as a functional promoter in E.coli.

Sequence homologies. The deduced amino acid sequence ofPlr was compared with deduced amino acid sequences forgenes entered in the EMBL (release 26.0) and GenBank(release 67.0) data bases by using the TFASTA programbased on the algorithm of Lipman and Pearson (33). Glycer-aldehyde 3-phosphate dehydrogenases (GAPDHs) of bacte-rial origins (7, 46, 51) exhibited the greatest homology withPlr. The gram-positive Bacillus subtilis GAPDH demon-strated the highest score. As shown in Fig. 5, the sequencesshowed 56% identity and 73% similarity.Hydropathy plots of Plr and B. subtilis GAPDH were

determined as described by Kyte and Doolittle (29) (Fig. 6).Plr and B. subtilis GAPDH showed similar patterns overallexcept for differences in the C-terminal portion of themolecules. Common cell wall-spanning and membrane-an-choring motifs have been identified for several gram-positivesurface proteins (17). However, no similar regions wereidentified for Plr. No significant amino acid sequence homol-ogy between Plr and streptokinase, the other well-character-ized plasmin(ogen)-binding protein, was identified, support-ing our previous biochemical and immunological analyses(9).

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Plr VVKVGINGFGRIGRLAFRRIQNIEGVEVTRINDLTDPNLALLKYDTTQ 50

GAPDH AVKVGINGFGRIGRNVFRAALNNPEVEVVAVNDLTDANMLAHLLQYDSVH 50

Plr GRFDGTVEVKEGGFEVN6NFIKVSAERDPENIDWATDGVEIVLEATGFFA 100

GAPOH GKLDAEVSVDGNNLVVNGKTIEVSAERDPAKLSWGKQGVEIVVESTGFFT 100

Plr KKEAAEKHLHANGAKKVVIiAPGGNDVKTWVFNTNHDILD6GTE.TVISGA 149

GAPDH KRADLKHLEIA.GAKKVIISAPANEEDITIVMGVNEOKYiDAANHDVISA 149

Plr SCTTNCLAPMAKALHDAFGIQKGLMTTIHAYTGDQMILDGPHRGGDLRRA 199

GAPDH SCTTNCLAPFAKiLNDKFGIKRGTTHSY1NQ ILDLPHK. .DYRRA 197

Plr RAGAANIVPNSTGAAKAIGLVIPELNGKLD6MQRVPVPTGSVTELVVTL 249

GAPDH RAANIIPTSTGAAVSLVLPELKGKLNG VPTPNVSLVDLVAEL 247

P1 r DKNVSVDEINSAWKAASNDS .... FGYTEDPIVSSDIVGVSYGSLFDATQ 295

GAPDH NQEVTAEEVNAALKEMEGDLKGILGYSEEPLVSGDYNGNKNSSTIDALS 297

P1r TKVMEVDGSQLVKVYSVYDNEMSYTAQLVRTLEYFAKIAK 335

GAPOH TMVME...GSMISOYDNESGYSNRVVDLAMYIAKKGL 334

FIG. 5. Comparison of deduced amino acid sequences of Plrfrom group A streptococcal strain 64/14 and GAPDH from B.subtilis (51). Alignment was performed by using the Gap programfrom the Genetics Computer Group (41). Vertical lines indicateidentical amino acids. Double and single dots indicate related aminoacids with high and low similarities, respectively.

DISCUSSION

Surface molecules of bacteria have been extensively stud-ied for their roles in transport of nutrients, evasion of hostdefenses, and cellular metabolism. Recently, our laboratoxyisolated a group A streptococcal surface protein that dem-onstrated high affinity for human plasmin while displayingminimal reactivity with the native zymogen, Glu-plasmino-gen (8). This plasmin-binding surface protein has been puri-fied to homogeneity (9). The isolated plasmin receptor pro-tein was functionally and antigenically distinct from theplasminogen activator protein, streptokinase, secreted bythe same strain (9).

In this study, we isolated and analyzed plr, the geneencoding the group A streptococcal plasmin receptor. Byscreening a X gtll expression library with antiplasmin recep-tor antibodies, we identified a plasmin receptor gene withina 2.7-kb streptococcal DNA fragment. This fragment wassubcloned into a low-copy-number plasmid, and the receptorprotein was stably expressed in E. coli under the control of

putative streptococcal promoter elements. The recombinantreceptor protein demonstrated immunoreactivity and plas-min-binding activity. We determined the nucleotide se-quence forplr and upstream elements of the structural gene.An open reading frame of 1,008 bp was identified. The 40.5%G+C content ofplr was slightly higher than the 35 to 39%reported for group A streptococcal chromosomal DNA (20).The deduced amino acid sequence was identical for 74 aminoacid residues at the N terminus as well as three cyanogenbromide fragments obtained from the native streptococcalprotein. The amino acid sequence obtained for the strepto-coccal receptor protein revealed that the initial methionineresidue is cleaved.The putative ribosomal binding sequence and -10 and

-35 regions for pir are consistent with sequences reportedfor other streptococcal genes and multiple B. subtilis genes(40). The AAGGAGG sequence located 9 bp upstream of theATG start codon corresponded to ribosomal binding se-quences reported for streptococci and is identical to thoseidentified for the B. subtilis and Bacillus stearothennophilusGAPDH genes (7, 51). Upstream regions ofplr correspondedto the consensus E. coli -35 and -10 sequences (TIGACAand TATAAT, respectively) as well as those reported forother streptococcal genes. The spacing of the 19 bp betweenthe putative -35 and -10 regions is greater than the 17 bpreported for E. coli and B. subtilis (40); however, spacingintervals reported for streptococci range from 13 to 21 bp(16).The deduced amino acid sequence for Plr was compared

with published sequences for other proteins. Plr exhibitssignificant similarity to the glycolytic enzyme GAPDH,reported for a number of prokaryotic and eukaryotic organ-isms. The best match was with B. subtils (56% identical and73% conserved amino acid residues). GAPDH from strepto-cocci has not been isolated or characterized, and the rela-tionship of the plasmin receptor to the glycolytically activeenzyme remains to be defined. However, the extensiveamino acid homology and similar hydropathy plots for Plrand B. subtilis GAPDH strongly suggest that Plr is a memberof the GAPDH family of proteins. Furthermore, preliminaryanalysis of the recombinant protein revealed that Plr hasGAPDH enzymatic activity (data not shown).GAPDH is a key enzyme involved in glucose metabolism

and has been the subject of many genetic studies. Multiplecopies of GAPDH genes have been identified for mammals,with many described as pseudogenes (44). Multiple GAPDHgenes have also been identified for E. coli, Trypanosomabrucei, Saccharomyces cerevisiae, and Drosophila melano-gaster (1, 24, 39, 50). E. coli and T. brucei each have two

1 00 200 300

I I I I I II II I I I I I I I I I I I I I I I II I I I I

0.5

P I r

- O . 5

0.5

GAP DH-n .svV. v1

FIG. 6. Hydropathy patterns of the amino acid sequences of Plr and B. subtils GAPDH. The scale corresponds to amino acid residuepositions.

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STREPTOCOCCAL PLASMIN RECEPTOR 5209

GAPDH genes with significant differences in deduced aminoacid sequence (1, 39); however, the translated product of thesecond E. coli GAPDH gene has not been reported. One ofthe trypanosomal isoenzymes is localized in the glycosome,a specialized metabolic organelle, while the other GAPDH isfound in the cytoplasm (31).

In addition to its usual intracellular location, GAPDH hasbeen identified on the surface of hematopoietic cells andSchistosoma mansoni, an invasive parasite (3, 19). Allen andHoover characterized a membrane-associated 37,000Mrprotein expressed by the erythroleukemic cell line K562 (2).Peptide mapping and molecular cloning studies revealed theprotein to be homologous to GAPDH (3). A similar findinghas been reported for the blood fluke responsible for abdom-inal schistosomiasis (19). A 37,000Mr surface immunogen ofS. mansoni was characterized by isolating the cDNA encod-ing the protein. The deduced amino acid sequence hadsignificant homology to that of human GAPDH. Like Plr,neither of these surface proteins had domains correspondingto previously described membrane-anchoring structures (6,15). Interestingly, Hekman et al., while studying the expres-sion of recombinant plant GAPDH in E. coli, were able totarget the protein to the outer membrane by geneticallyfusing the signal sequence of E. coli OmpA to Ricinuscommunis GAPDH (22). Instead of accumulating the plas-mid-encoded protein in the periplasm as anticipated, recom-binant GAPDH integrated in the outer membrane of E. coli.This finding suggested that the plant cytoplasmic GAPDHhas certain primary or secondary structures which allow foranchoring into cell membranes.The mechanism for surface localization of Plr is not

known. The details for anchoring of other gram-positivesurface proteins have not been elucidated; however, certainamino acid motifs have been suggested to serve as mem-brane- and/or cell wall-spanning domains (17). Group Astreptococcal M proteins are attached to the cell membranevia the C-terminal region containing a conserved amino acidmotif and a high density of hydrophobic amino acids (43).Although other streptococcal and staphylococcal proteinshave corresponding domains, no similar regions were iden-tified for Plr.Comparisons of deduced amino acid sequences for GAP-

DHs from a variety of organisms reveal a conserved familyof proteins (7, 23). Biochemical investigations of GAPDHhave focused on glycolytic activity and delineation of spe-cific domains involved in catalysis or quarternary structure(12, 21, 42, 49). No role for the leukemic cell or schistosomalmembrane-associated GAPDHs has been determined. How-ever, alternative functions for GAPDHs have been de-scribed. These include bundling of microtubules, proteinkinase activity, and uracil DNA glycosylase activity (26, 28,38). The intriguing similarity of Plr to GAPDH adds furthersupport to the existence of a family of GAPDH-relatedproteins with diverse biological functions.

ACKNOWLEDGMENTSWe thank Henry Baker, Jorge Galan, Paul Gulig, and Dena

Minning-Wenz for many helpful suggestions; Hong Wang for devel-oping methods for partially purifying the recombinant protein; andCarroll Kissam for preparing the manuscript.

This research was supported by National Institutes of Healthgrants HL 41898 and DE 06673.

ADDENDUM IN PROOFAt the 92nd General Meeting of the American Society for

Microbiology, 26-30 May 1992, V. Pancholi and V. A.

Fischetti communicated the identification of a novel multi-functional protein (MF6) expressed on the surface of Mserotype 6 group A streptococci (Abstr. 92nd Gen. Meet.Am. Soc. Microbiol., abstr. no. B-252, 1992).

REFERENCES1. Alefounder, P. R., and R. N. Perham. 1989. Identification,

molecular cloning and sequence analysis of a gene clusterencoding the class II fructose 1,6-biphosphate aldolase, 3-phos-phoglycerate kinase and a putative second glyceraldehyde3-phosphate dehydrogenase of Escherichia coli. Mol. Micro-biol. 3:723-732.

2. Allen, R. W., and B. A. Hoover. 1985. Characterization of theprocessed form of a ubiquitous protein displaying a varia-ble membrane organization in erythroid cells. Blood 65:1048-1055.

3. Allen, R. W., K. A. Trach, and J. A. Hoch. 1987. Identificationof the 37-kDa protein displaying a variable interaction with theerythroid cell membrane as glyceraldehyde-3-phosphate dehy-drogenase. J. Biol. Chem. 262:649-653.

4. Birnboim, H. C. 1983. A rapid alkaline extraction method for theisolation of plasmid DNA. Methods Enzymol. 100:243-255.

5. Bisno, A. L. 1991. Group A streptococcal infections and acuterheumatic fever. N. Engl. J. Med. 325:783-793.

6. Blobel, G. 1980. Intracellular protein topogenesis. Proc. Natl.Acad. Sci. USA 77:1496-1500.

7. Branlant, G., and C. Branlant. 1985. Nucleotide sequence of theEscherichia coli gap gene. Eur. J. Biochem. 150:61-66.

8. Broder, C. C., R. Lottenberg, and M. D. P. Boyle. 1989.Mapping of the human plasmin domain recognized by theunique plasmin receptor of group A streptococci. Infect. Im-mun. 57:2597-2605.

9. Broder, C. C., R. Lottenberg, G. 0. von Mering, K. H. Johnston,and M. D. P. Boyle. 1991. Isolation of a prokaryotic plasminreceptor: relationship to a plasminogen activator produced bythe same micro-organism. J. Biol. Chem. 266:4922-4928.

10. Broeseker, T. A., M. D. P. Boyle, and R. Lottenberg. 1988.Characterization of the interaction of human plasmin with itsspecific receptor on a group A streptococcus. Microb. Pathog.5:19-27.

11. Chamberlain, J. P. 1979. Fluorographic detection of radioactiv-ity in polyacrylamide gels with the water-soluble fluor, sodiumsalicylate. Anal. Biochem. 98:132-135.

12. Corbier, C., S. Clermont, P. Billard, T. Skarzynski, C. Branlant,A. Wonacott, and G. Branlant. 1990. Probing the coenzymespecificity of glyceraldehyde-3-phosphate dehydrogenases bysite-directed mutagenesis. Biochemistry 29:7101-7106.

13. DesJardin, L. E., M. D. P. Boyle, and R. Lottenberg. 1989.Group A streptococci bind human plasmin but not other struc-turally related proteins. Thromb. Res. 55:187-193.

14. Devereux, J., P. Haeberli, and 0. Smithies. 1984. A comprehen-sive set of sequence analysis programs for the VAX. NucleicAcids Res. 12:387-395.

15. Ferguson, M. A. J., and A. F. Williams. 1988. Cell-surfaceanchoring of proteins via glycosyl-phosphatidylinositol struc-tures. Annu. Rev. Biochem. 57:285-320.

16. Ferretti, J. J., and R. Curtiss III. 1987. Compilation of nucleo-tide sequences that signal the initiation of transcription andtranslation in streptococci (appendix C), p. 293. In J. J. Ferrettiand R. Curtiss III (ed.), Streptococcal genetics. AmericanSociety for Microbiology, Washington, D.C.

17. Fischetti, V. A., V. Pancholi, and 0. Schneewind. 1991. Commoncharacteristics of the surface proteins from gram-positive cocci,p. 290-294. In G. M. Dunny, P. P. Cleary, and L. L. McKay(ed.), Genetics and molecular biology of streptococci, lacto-cocci, and enterococci. American Society for Microbiology,Washington, D.C.

18. Galan, J. E., J. F. Timoney, and R. Curtiss III. 1988. Expressionand localization of the Streptococcus equi M protein in Esche-nichia coli and Salmonella typhimurium, p. 34-40. In D. G.Powell (ed.), Proceeding of the Fifth International Conferenceof Equine Infectious Diseases. University Press of Kentucky,Lexington, Ky.

VOL. 174, 1992

on May 20, 2021 by guest

http://jb.asm.org/

Dow

nloaded from

5210 LOTiENBERG ET AL.

19. Goudot-Crozel, V., D. CailHol, M. Djabali, and A. J. Dessein.1989. The major parasite surface antigen associated with humanresistance to schistosomiasis is a 37-kD glyceraldehyde-3P-dehydrogenase. J. Exp. Med. 170:2065-2080.

20. Hardie, J. M. 1986. Genus Streptococcus, p. 1043-1071. In J. G.Holt, P. H. A. Sneath, N. S. Mair, and M. E. Sharpe (ed.),Bergey's manual of systematic bacteriology. Williams & Wil-kins, Baltimore.

21. Harris, J. I., and M. Waters. 1976. Glyceraldehyde-3-phosphatedehydrogenase. Enzymes 13:1-49.

22. Hekman, W. E., D. T. Dennis, and J. A. Miernyl. 1990.Secretion of Ricinus communis glyceraldehyde-3-phosphate de-hydrogenase by Eschenichia coli. Mol. Microbiol. 4:1363-1369.

23. Hensel, R., P. Zwickl, S. Fabry, J. Lang, and P. Palm. 1989.Sequence comparison of glyceraldehyde-3-phosphate dehydro-genases from the three urkingdoms: evolutionary implication.Can. J. Microbiol. 35:81-85.

24. Holland, J. P., L. Labieniec, C. Swimmer, and M. J. Holland.1983. Homologous nucleotide sequences at the 5' termini ofmessenger RNAs synthesized from the yeast enolase and glyc-eraldehyde-3-phosphate dehydrogenase gene families. J. Biol.Chem. 258:5291-5299.

25. Hudson, M. C., and R. Curtiss HI. 1990. Regulation of expres-sion of Streptococcus mutans genes important to virulence.Infect. Immun. 58:464-470.

26. Huitorel, P., and D. Pantaloni. 1985. Bundling of microtubulesby glyceraldehyde-3-phosphate dehydrogenase and its modula-tion by ATP. Eur. J. Biochem. 150:265-269.

27. Huynh, T. V., R. A. Young, and R. W. Dav'is. 1985. Construc-tion and screening cDNA libraries in X gtlO and X gtll, p. 49-78.In D. M. Glover (ed.), DNA cloning. IRL Press, Oxford.

28. Kawamoto, R. M., and A. H. Caswell. 1986. Autohosphorylationof glyceraldehydephosphate dehydrogenase and phosphoryla-tion of protein from skeletal muscle microsomes. Biochemistry25:656-661.

29. Kyte, J., and R. F. Doolittle. 1982. Simple method for displayingthe hydropathy character of a protein. J. Mol. Biol. 157:105-132.

30. Laemmli, U. K. 1970. Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature (London)227:680-685.

31. Lambeir, A.-M., A. M. Loiseau, D. A. Kuntz, F. M. Vellieux,P. A. M. Michels, and F. R. Opperdoes. 1991. The cytosolic andglycosomal glyceraldehyde-3-phosphate dehydrogenase fromTrypanosoma brucei. Eur. J. Biochem. 198:429-435.

32. Liotta, L. A., R. H. Goldfarb, and R. Brundage. 1981. Effect ofplasminogen activator (urokinase), plasmin and thrombin onglycoprotein and collagenous components of basement mem-brane. Cancer Res. 41:4629-4636.

33. Lipman, D. J., and W. R. Pearson. 1985. Rapid and sensitiveprotein similarity searches. Science 227:1435-1441.

34. Lottenberg, R., C. C. Broder, and M. D. P. Boyle. 1987.Identification of a specific receptor for plasmin on a group Astreptococcus. Infect. Immun. 55:1914-1928.

34a.Lottenberg, R., C. C. Broder, M. B. Streiff, and M. D. P. Boyle.1991. A group A streptococcal plasmin receptor demonstrateshomology with glyceraldehyde-3-phosphate dehydrogenase,B-30, p. 30. Abstr. 91st Gen. Meet. Am. Soc. Microbiol. 1991.American Society for Microbiology, Washington, D.C.

35. Lottenberg, R., F. R. Dolly, and C. S. Kitchens. 1985. Recurrent

thromboembolic disease and pulmonary hypertension associ-ated with severe hypoplasminogenemia. Am. J. Hematol. 19:181-193.

36. Maniatis, T., E. F. Fritsch, and J. Sambrook 1982. Molecularcloning: a laboratory manual. Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.

37. McCoy, H. E., C. C. Broder, and R Lottenberg. 1991. Strep-tokinases produced by pathogenic group C streptococci demon-strate species-specific plasminogen activation. J. Infect. Dis.164:515-521.

38. Meyer-Siegler, K., D. J. Mauro, G. Seal, J. Wurzer, J. K.DeRiel, and M. A. Sirover. 1991. A human nuclear uracil DNAglycosylase is the 37-kDa subunit of glyceraldehyde-3-phos-phate dehydrogenase. Proc. Natl. Acad. Sci. USA 88:8460-8464.

39. Michels, P. A. M., M. Marchand, L. Kohl, S. Allert, R. K.Wierenga, and F. R. Opperdoes. 1991. The cytosolic and glyco-somal isoenzymes of glyceraldehyde-3-phosphate dehydroge-nase in Trypanosoma brucei have a distant evolutionary rela-tionship. Eur. J. Biochem. 198:421-428.

40. Moran, C. P., Jr., N. Lang, S. F. J. LeGrice, G. Lee, M.Stephens, A. L. Sonenshein, J. Pero, and R Losick. 1982.Nucleotide sequences that signal the initiation of transcriptionand translation in Bacillus subtilis. Mol. Gen. Genet. 186:339-346.

41. Needleman, S. B., and C. Wunsch. 1970. A general methodapplicable to the search for similarities in the amino acidsequence of two proteins. J. Mol. Biol. 48:443-453.

42. Olsen, K. W. 1991. Structural basis of the thermal stability ofglyceraldehyde-3-phosphate dehydrogenases. Int. J. Pept. Pro-tein Res. 22:469-475.

43. Pancholi, V., and V. A. Fischetti. 1989. Identification of anendogenous membrane anchor-cleaving enzyme for group Astreptococcal M protein. J. Exp. Med. 170:2119-2133.

44. Piechaczyk, M., J. M. Blanchard, S. Riaad-El Sabouty, C. Dani,L. Marty, and P. Jeanteur. 1984. Unusual abundance of verte-brate 3-phosphate dehydrogenase pseudogenes. Nature (Lon-don) 312:469-471.

45. Reis, K. J., M. Yarnall, E. M. Ayoub, and M. D. P. Boyle. 1984.Effect of mouse passage on Fc receptor expression by group Astreptococci. Scand. J. Immunol. 20:433-439.

46. Schlaepfer, B. S., W. Portmann, C. Branlant, G. Branlant, andH. Zuber. 1990. Nucleotide sequence of the glyceraldehyde-3-phosphate dehydrogenase from Bacillus megateium. NucleicAcids Res. 18:6422.

47. Siefring, G. E., and F. J. Castellino. 1976. Interaction ofstreptokinase with plasminogen. J. Biol. Chem. 251:3913-3920.

48. Silhavy, T. J., M. L. Berman, and L. W. Enquist. 1984.Experiments with gene fusions. Cold Spring Harbor Labora-tory, Cold Spring Harbor, N.Y.

49. Soukri, A., A. Mougin, C. Corbier, A. Wonacott, C. Branlant,and G. Branlant. 1989. Role of the histidine 176 residue inglyceraldehyde-3-phosphate dehydrogenase as probed by site-directed mutagenesis. Biochemistry 28:2586-2592.

50. Tso, J. Y., X.-H. Sun, and R. Wu. 1985. Structure of twounlinked Drosophila melanogaster glyceraldehyde-3-phosphatedehydrogenase genes. J. Biol. Chem. 260:8220-8228.

51. Viaene, A., and P. Dhaese. 1989. Sequence of the glyceralde-hyde-3-phosphate dehydrogenase gene from Bacillus subtilis.Nucleic Acids Res. 17:1251.

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