- Email: [email protected]
Cloning sequencing of Lol pI, the major allergenic protein of rye-grass pollen
Volume 2"/9, number 2, 210-21~
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F ~ b r u a w 1991
,¢,. I~1 Fedel'#tion o1' European BIoehem!~al So
Cloning and sequencing of Lol pI, the major allergenic protein of ryegrass pelion Irwin J. Griffith t , Penelope M. Smith", Jeanne Pollock L, Piyada Thecrakulpisut =, Asit Avjioglu =,
Scan Davies =. Terryn Hough =, Mohan B, Singh=, Richard J, Simpson 3, Larry D, Ward = and R, Bruce Knox = hmtmLol¢lc Pharntaceulleal Corporation, One Kendall Square. Building 600, Cmnbrldg¢, MA ¢12139, USA, =Schao/ of Botany,
U#lverxity of Melbourne, ParkPille, Yirtar& 30.~2, ,4uJ~trr/lia and ~Joi~tl Prat¢#~ Structure Labor=/or)',. Ludwig ht.~lltute fm" Cum'er Rettar¢l~ (Melbourne Brtttleh) enlff The ~P'¢tlttr ~md Eli:¢t thlll hlslllt~te for Medical Rtsearch, Pctrkvlll¢, F/¢tortu 3052, d ustraliu
Received 5 December 1990 We have isolated a lull length eDNA tie)so encoding the major glycoprotein allerBen LoI pl, The clone was selected using a combination of immunological screening e r a eDNA expression library and PCR amplification ofLol pl.spccific transcripts, tolp[ expressed in bacteria as a fusion protein shows recognition by specific IgE antibodies present in ser=lof grass pollen.allergic subjects, Northern analysis has shown that the l.#lpl transcripts are expressedonly in pollen or rye.grass, Molecular cloning of Lolpl provides a molecular genetic approach to study the structure-function relationship of allergens,
Lolium preenne; Allergen; eDNA clonin B: Nucl¢otid¢ sequence: Amino acid sequence
1,INTRODUCTION Hayfever and seasonal allergic asthma due to grass pollen allergens are environmental diseases that afflict up to 25°'/0 of the population in cool temperate climates [1-3]. Although a degree of cross-reactivity has been described among antigens from different grass species [4-6], it is well established that rye-grass antigens are the most reactive allergens [7,8]. The most prominent allergen in rye-grass pollen is group I or Lol pl (IUIS nomenclature [91). This has beer= shown to be the major IgE-binding protein [7,8]. Elevated levels of Lol pI. specific IgE have been detected in sere of up to 95070 of grass pollen-allergic individuals, and thus of major clinical significance [7,8]. Other clinically significant allergens of rye-grass include LolplI, III, IV [9] and the newly identified Loi pIb [10]. Lol pl is an abundant acidic glycoprotein of Mr 35 kDa [8] and pI 5.5 [10], located in the cytosol of the pollen [10,11]. The amino acid sequence of this allergen has not been reported, although short sequences from • an N-terminal and an internal peptide fragment have been determined [12,13]. In this paper, we report the cloning, expression and sequencing of a full-length eDNA clone encoding l . o l p I .
2, MATERIALS AND METHODS 2.1. Plant materials attd alltibody probes Pollen of Loliunt perenne was collected from plants flowering in Melbourne, and stored in liquid nitrogen until used. MAbs 4.0,1, 21.3 and 3,2 are specific for Lolpl, and their preparation and characeeriza. lion haw been described elsewhere [4,10,14-16], IgE antibodies were from sere of grass pollen-allergic individuals, as determined by skin prick test and/or RAST (Radioallergosorbent test).
2.2, isolaliot) of pollen proteins and intntunoblotting Soluble proteins were extracted from rye.grass pollen by vigorous shaking in PBS (150 raM, pH 7,2) containing 1 mM PMSF, on ice for 3 h Conditions for ¢lectrophoresis and immunoblotfing with MAbs were essentially as described [16]. For lgE antihody binding, blots were incubated in allergic sere collected from at least 4 patients with high RAST scores for grass pollen, pooled and used diluted 1:5 in TBS/0.'~% BSA, The bound IgE was detected using ~2~i-lahelled anti. I~uman lgE (Kallestad, USA) essentially as described by Ford and Baldo [8],
2.3, Purification of Lol pin for N.term#tal sequencing Lolpl was isolated using preparative isoelectric focussing (Rotofor, BioRad, Richmond, CA) followed by SDS-PAGE (A, Avjioglu and M,B. Singh, unpublished data), The allergen was recovered from SDS-PAGE gels by electroftransfer (90 V/2 h; 4'C) onto polyvinylidene difluoride (PVDF) membrane, (Millipore, Bedford, MAt [17]. Proteins were visualized by staining with Coomassie brilliant blue R-250, washed extensively with deionized water it'll and sub. jected to sequence analysis [17,18]. Amino acid residues are given us. ing the one letter notation,
2.4, eDNA library and immunological screening Correspondence address: R.B. Knox, School of Botany, University of Melbourne Parkville, Vict0ria 3052, Australia
210
Poly (A ÷) mRNA was isolated essentially as described I19], eDNA was synthesized [20] and cloned into the EcoRl site of" the vector lambda.gt II. Immunological screening was done by plating tile
Published by Elsevier Science Publishers B, V,
Volume 2"/9, number 2
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eDNA library, and ~
K 1 94-67---- . .
2,$, Nu¢lenrldP sequlartnx
3.1. Identification of Lol pI by immunoblotting The recombinant clone (13R) was isolated using Lol pI-specific MAbs, which provides evidence of its rela-
3
4
5
43-.- tt
I
Lol Rl=lbI 30--
2,6. O/i~om~t'/¢ottde.v Oli$onucleolides used in this study are shown in "l'able 1 Anchor Primer tAP), Anchor Linker CAL) and Anchor Template (AT) ha.re been described previously [221, LpA-3 and LpA.S were derived from the sequence of the Lolpl eDNA clone 13R, Sequenelnll primers werce derived from tile internal sequence of PCR.ijenerated clones, All oligomers were purchased from Research Genetics (Huntsville, At,., USA),
3. RESULTS
2
D
The eDN/~ clone I ~R was hotated from the phalle, and subcloned Into pGEbl,3Z vectors (Promellal. DNA settaenee was determined by dt)uble.stranded sequeneln= carried out by Ih¢ dideo,~p chain tetmtn;t. tlon method I l l ] , usloll 1"7 DNA polyrnerase (Pharmacist), PCR amplified DNA was cloned Into M13 vector for didcoxy sequencinll usinll ~eqtlenas¢ (US Bloclt~mieals),
2.'7, Pol.vmPra~e chain reaction fPCR,l cloning Full length clones eneodlnll Lalp[ were obtained usin$ a modifica. tion [22] of tile Anchored PCR [23,24], Double stranded eDNA was synthesized from I/~g of total R N A with the e D N A Synthesis System (Life Technologies, Inc,, G:tithersberg, MD, USA)usirt8 oligo dT Its primer, blunt ended with T4 polymerase and blunt end llilated to self.annealed AT and A L primers, Linkered eDNA O/d from a 20/d reaction) was mixed with 100 pmol each of AP and LpA.S primers, IO ~ul of IOX reaction buffer (GeneAmp kit, US Biochemicals, Cleveland, OH, USA) and 0.5/d of 7". aquattcus polymerase. The mixture was brought to 100~d with deionized water and amplified for 25 cycles as described 1221, Briefly, the first 5 cycles consisted of denaturation at 94~C ror 1 rain, annealinl} at 45"C for i.S rain, and polymerization at "70"C for I .S min. The htst 20 cycles were as sta ted, except that annealing was at 55*C, 0.01 volume of the primary PCR was risen reamplified with oligomers AP and LpA.3 as above. LpA.3 js nested (internal) relative to oligonter LpA-5 used in the pritnary PCR, DNA from the secondary PCR was recovered by sequential chloroform, phenol and chloroform extractions, precipitated with 0.5 vol. 7.5 M AmoAc and I.$ vol. isopropanol. DNA from the pellet was digested with Xba I and Pst I, run on a preparative low melt gel, and ligated into Xba i/Pst I.digested M13.
February 1991
20-'--
14-.-
T
21"3 40.1 3"2
Ig E
Fig. I, lmmunoblot analysis of lllE and MAb bindina to Lolpl pro. uins from rye.grass pollen, Mr (kDa) Is denoted on the left, 120 g of [}ellen proteins were loaded per lane. Lolp[ is denoted with an arrow. Lane I : SDS.separation of total rye.grass pollen proteins (staining is '~vith Co0massie brilliant blue R-2J0); lanes 2-5: bindinll of MAbs or IilE on Western blots of total rye.grass pellets proteins,
lionship to the natsve allergen On Western blots of pollen proteins, MAbs 3.2, 21.3 and 40.1 show strong affinity for a 35-kDa protein (Fig, 1). This protein showed high specificity for l e e antibodies from pooled sera of grass pollen-allergic individuals (Fig. 1). All the MAbs and IgE used in this study bound strongly to the Lolpl reference standard provided by NIAID (National Institutes of Health, Bethesda, MD; data not shown).
3.2. N.terminal sequence of Lol pl We purified Lol pI by two-dimensional gel electrophoresis involving preparative isoelectric focussing in the first dimension, followed by SDS-PAGE of the individual fractions. This procedure successfully Table 1
Oligonucleotide sequences Oligomer
Nucleotide sequence 5'p-AATGATCGATGCT ~ 5'GGGTCTAGAGGTACCGTCCG
Strand i i
AL AP AT
5~GGGTCTAGAGGTACCGTCCG~TCGATCATT
LpA-2 LpA-3 LpA-5 LpA-9 LpA-10
5'GGAGTCGTGGGGAGCAGTC 5'GGGAATTCCATGGCGAAOAAGGGC 5'CCCTGCAGTCATGCTCACTTGGCCGAGTA ~'CCCTGCAGATTATTIGAGATCTTGAG 5t GTGACAGCCTCGCCOG 5' CCGTCGACGTACTTCA
T-pA-iA
Nucleotldes . . . . . .
coding coding non.coding non-coding non-coding non-coding
671->689 442- > 458 810< -827 867 < -884 351 <-367 582 < -607
AL and AT are synthetic linker oligonucleotides containing Xba I, Kpn I and/or Cla I restriction sites [22]. The AP sequence is contained within the AT oligomer [22]. LpA-IA, -2, .3, -5, -9 and -10 correspond to the coding or non-coding sequence of Lol pl, as indicated. LpA-2 contains an Eco RI restriction site, while LpA-3 and-5 contaln Pstl restriction sites added for cloning purpose s, All restriction sites are underlined;
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Vol.rne 2"/9, ntimber 2
F~bru~ry 1~91
& H
~
~
S
V
L
L
-10 1 GT~GT~GC~CTGTTC~CCG~TTCCTG~GCAOCG¢~¢ATGGC~CGCG~GGTA¢CA¢C4 V v A ~ F A V F L a S ~ l{ G v A ~ v ~ 20 10 GG¢CCC~CATCACGGC¢GAGTACGGCG~C~G~¢76~CGCGAJ~AG¢ACCTGGTAT
~?~
]0 40 ~¢~G¢¢G~CCGGCGCCGG~CCC~GGACA~CGGCGGCGCGTGC~GTAC~GACG~C ~ ~ P ? G A G P K D ~ G G A CG ¥ K O V
~]~,
50 GO G~¢~GGCGCCGTTC~CGGC~T~ACCGGCTGCGGC~C~CCC¢¢~TC~¢~GGaCG~C D K A P r ~ G H T O C G N T P Z r K D
~%9
?0 80 CG?GGCTGCGGCTCCTGCTTCG~GATCAAGTGCACC~GC¢¢~GTCCTGCTCCGGCGAG R G C G S C ? ~ I K C T K ~ £ G ¢ S G ~
]5~
S0 100 GC?GTCACCGTCACAATC~CCG~CGACAATGAGG~GCCT~TCGCACCCT~CCACTTCGAC ^ v T v T x T a D . E £ e ~ A P ~ H r o 13R CTCTCGGGCCt,¢OCATTCGGGTCC~TGGCG~G~GGGCGAGGAGCAG~AGCTCCG¢~GC L S C H A F G ~ H ~ M K G ¢ £ O K L R ~
41~
499
i~0 140 GCCGGCGAGCTGGAGCTCC~GT?CAGGCGGGTC~GTGC~GTACCCGGACGGCACC~G A G E L E L Q F R R V K C K Y P D G T K
539
150 160 CCGACATTCCACGTCGAG~GGCTTCC~CCCC~CTACCTCGCTA~¢TGGTG~GT~C P T F ~ V B K A S N P H Y L A I L ~ K ~
599
I~0 180 GT~GACGGCGkCGGTGACGTGGTGGCGGTGGACATC~GG~G~GGGC~GGAT~GTGG V D G D G D V V A V ~ Z K ¢ K G K D K W
659
190 200 ATCGAGCTCAAGGAGTCGTGGGG~GCAGTCTGGAGGA?CGACACCCCCGAT~GCTGACG 2 E L K E S W G A V W R I D T P D K L T
719
2~0 220 GGCCCATTCACCGTCCGCTACACCACCGAGGGCGGCACCAP~TCCG~GTCGAGGATGTC G P F T V R Y T ? R G G ? K S E v E ~ v
7~9
230 240 ATCCCTGAGGGCTGG~GGCCGACACCTCCTACTCGGCC~G~GAGC~GAAGTGGAGTG ~ P E G W K A ~ T S ¥ S A K *
839
A~CTTCTTCCAATCAGCTT~TTTTGACTC~GATCTCA~T~TCCAGCCGCACATATA
899
T~CGAGGCGGTGAGACATACAAGCTCCTCCATGAGTATATTCATTCATGCCGTATAGAGA
959
GGAGAAAGATGCCTG~T~GAGTTTGAGGTCGACACCTTGTGAG~GTGTATATAGGAG
1019
G~CCC~TCTGGCTCCATCTTTCTTTGCTCGCACGGTGTACTGCT~GGTTATCTTCTA
1099
ACAGGCCAGATT~CCTACTATCT~TATATGC~CGTATGGTCATTTTCCCTAAAAAAA
1139
Lo!pt
hydropathlcity profile 100
5
o
4l
,,,I,,,
t~ I
200
I,,,,I,,,,I,,,,i
,J
I
~
-5 -4
,I.,
l
I II 100
212
1;~
. 200
3 2 1 0 -1 -2 -3 -4
Vol.m¢ 219. n.mbw .~
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separated Lolp[ (pl $.$) in sufficient quantifies for N. terminal protein sequence amtlysis, The sequence oh. rained was as follows: I A K V P P G P W I TAIF=.YGDKWLD A K ? T ..... This sequence is analogous to that repotcd by Certain et
at, [12]. 3.3, r$olatio~1of cDlVA clones The cDNA, expression librarywas screened for clones that expr~sed epitopes recognised by MAb 40,l. Screening of a total of S xlCP plaques yielded 18 immune.positive clones. These clones were plaquepurified, a~d screened for igE binding using sera from grass pollen-~tllcrgic subjects. Four clones were fo=nd to express proteins with affinity for l i e antibodies, Of these, the clone 13R. ( - 0.8 kb in size including poly (A)" tail), bound strongly to MAbs 40.1, 3,2 and 21 3, all known to preferentially recognise Lolpl[lO]. Thus, the clone e.xpresses an allergen as defined by recognition o f IgE from sere o f allergic individuals and was designated as a putative Lol pl clone,
1353 ~" 107 0 " 872="
3.4. Nucleotide sequence of Lol pl cDNA clone 13R, selected by expression screening, contains 764 nucleotides. There are 146 amino acids in the open reading frame ( O R F ) a , d the predicted M', is 16.2 kDa. The translated amino acid sequence terminates with the 28'amino acid sequence described by Esch and Klapper [I 3]. Since this clone exhibits strong Mab and IgE binding, the sequence contains both antigenic and allergenic ¢pitopcs. However, mature LolpI has an approximate Mr of 35 kDa, so the translated reading frame is apparently not full-length. Clone 13R also lacks the NHx-terminal sequence defined for Lol pl [12], confirming that it is a truncated clone. Full-length Lol pI clones were obtained by P C R of rye-grass pollen RNA. Primers based on synthetic linker sequences and Lol pl-specific sequences derived from the 3' end o f clone 13R were used to generate these clones. The nucleotide sequence of Lol pI is shown in Fig. 2a. There is an open reading frame (ORF) of 789 bp starting with an A T G initiation codon at position 33 and terminating with a TGA stop codon at position 822. This ORF, which has a 64°70 GC content, corresponds to a polypeptide of 263 amino acids (Mr 29.1 kDa). The proposed translation initiation site and its flanking sequences (nucleotides 29-37.) share 67070 identity with the
6 0 3 D-
Fig, 3. Tissue-specific expression of' Lol p! in rye-grass pollen, Nor. them blot analysis of total RNA from rye.grass pollen, seed, leaf. root and inflorescence using 13R cDNA as n probe.
consensus plant sequence AACAATGGC [25]. The most critical nucleotide, a purine at position - 3, is con. served. Sequence corresponding to the 13R clone starts at nucleotide 474. The predicted protein sequence has a hydrophobic putative signal peptide sequence of 23 amino acids (Fig. 2 a,b). This is indicative of a mature processed protein of 240 amino acids (Mr 26.6 kDa). The amino acid composition from the deduced sequence, rich in glycine and lysine, is in complete agreement with previous data obtained by amino acid analyses of LolpI protein [26,27]. There is a single potential Nglycosylation site having the characteristic Asn-xSer/Thr motif (residues 9-I l) in the hydrophilic region of the mature protein (Fig. 2b).
Fig. 2. eDNA, predicted amino acid sequence and hydropathicity profile of Lol pl from rye-grass pollen. (a) The nucleotlde and deduced amino acid sequence of Lol pl. The deduced amino acid sequence represented by the single letter code is shown below the DNA sequence, and begins at the first potential in.frame initiation codon. The open reading frame continues for 263 amino acids and ends with the TGA stop codon denoted by an asterisk. The putative signal peptlde is indicated by negative numbers. Underlined amino acids were identified by protein microsequencing (this report; and [12,13]). The arrow indicates the start of the 13R sequence, (b) Hydropathicity profile of predicted amino acid sequence based on the method of Kyte and Doolittle [35] with a window of '7 amino acids,
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A search of existin~ data.bases showed no simil=rlty between the deduced amino acid sequenceof Lol pl and any other known protein, The Lol pl nucleotide sequence was compared with that or the newly identified Lo/plb [10] and no sequence homolo=y was observed,
3, ~, E:cpr¢.~ionof ~ene for Lot pl i~ Vario,.~ryc.gra~ To determine the tissue specificity of Lolpl sene expression, we examined the level of specific transcripts in various: tissues. Northern blots probed with eDNA !3R showed hybridization to a single abundant transcript ( - 1350 bases) in pollen (Fig. 3), Hybridization was also observed to similar sized transcripts in i n . florescence, but no hybridization to any transcripts in rye.grass seed, leaf or root was observed (Fig. 3). 4, DISCUSSION In this report we describe the cloning of a eDNA se.quence encoding an allergen from rye-grass pollen. Several lines of evidence confirm that clone 13R corresponds to the major glycoprotein allergen, Lol p l . First, the clone was immunoselected from the lambdagt 11 e~pression library using a MAb that shows high Levels of binding to Lot pI. Second, the polypeptid¢ encoded by the cDNA clone was recognised by lgE antibodies from pooled sera of grass pollen-allergic individuals. Third, a segment of the deduced amino acid sequence of the clone corresponded to the 38-amino acid tryptic fragment of Lo/pl (YTTE G G T K S E F E D V I P E G W K A D T S Y S A K ) described by Esch and Klapper [13l. Finally, the pollen specificity of transcripts Lol p Ih
:
v,~ruary =we
hybtidizin$ wi~h L3R,. is coasistent ~ith tissuespe.eifici. ty of Lot pi anti,lens, as observed earlier by immunoblottinil [10} The Nderminal sequenceof' Lol p[, was not detected in the ORF of the I3R clone, However, full lenath clones obtained by PCR ampllflc~ttion, encoded an amino acid sequence which corresponded with the N. terminal sequence of Lol pl ([tZ]; ~,nd this p~per). The lonsest ORF is 789 nucleolides lens, which corresponds to a protein of 263 amino acids with a Leadersequence of 23 amino acids. The molecular mass for the mature form of/.,ol p[ (240 amino acids) was calculated to be 26,6 kDa, This would suggest that 24% of the 35 kDa molecular mass of tl~e pollen protein is due to post. translationa~ modifictttions. The Lo/pl signal peptide has some motifs common to otl~er plant signal peptides, especially those of secreted plant enzymes, e.g. amylase [28]. The motifs close to the cleavage site show some similarity wi~h those of signal peptides which direct proteins into the Lumen of the ER for glycosylation, e.g. V L L , . . F L OSAHG I in Lol pL a n d V L L . . . L S ASI..ASG t in alpha-~tmyiase [28]. The - l and -3 rule for the prediction of a leader se. quence cleavage site [29] is that amino acids at these positions should be sinai[ and neutral: O and A in Lol pl. The deduced amino • acid sequence o f the full length L,o/pl clone contains both the N-terminal sequence [12] and tryptic peptide fragment sequence [13] previously reported for Lolpl, as well as the N.terminal protein se. quence of Lo/pl reported here. The N-~erminal sequences ([12]; and this report) helped define the cleavage site between the leader sequence and the mature form of Lol pl.
~. 0 K L R $ A G ~- 5 K ~ Q E' R P,V K C K
Y?
D G ~ " ~ P "r H ~ " ~
R"~A
),54
L o l p Z! L o l p IXZ
Lol p ZA Lol p X! &el p ZZZ
&el p l& &el p IZ Lol p ~ !
0 o v v,
Iol,
v o,,,[],
o,
Fig. 4, Homologies of Lo/pl with Lo/pll and III.Alignment of deduced amino acid sequence of Lolpl with reported sequences of Lo/plI [26J and LoJ pill [27]. Identical residues are shown in bold letters, These residues and other similar residues in t~.e three sequences are indicated by boxes. The following residues were considered similar: A,S,T; D,IS; N,Q; R,K; I,L,M,V; F,Y,W.
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Our resultsshow that the 28 amino acid styptic frnlp meat sequence YTTE GGTKS EFEDV IPEOW KADTS YSAK [13] is located at the COOH-trrminus or this alierilrn. We also round that the entire 97 amino acid sequences or Lo[ pit [301 lad Lol pHI []!1 have strains homology with the COOH-terminal end or Lol pI (FIg; 4). Lot pl has 41~% homology (3~% identity) with Lol pll and 44% homology (26% identity) with Lol p i l l while Lol PII and Lol pill have 73~ homololw (59% identity) which each other. However, despite strong homolol~y between amino acids 145 to 240 of Lol pl with the amino acid sequencesof Lo/pll and Lot pill, no serological cross.reactivity has been detected between Lolpl and Lolpll nor Lo/pill [321, Immune responsiveness to rye.grass pollen allergens inthe human population is significantly associated with the histocompatibility leukocyte antigen (HLA)-DR3 [33,34]. One interpretation of this finding is that HLADR3, or closely linked class II molecules, may be particularly effective at presenting certain allergenic determinants to T cells. Ansari et al. [30,31] predicted that the 28 amino acid tryptic fragment isolated by Esch and Klapper [13] may include an HLA.DR3 restricted T cell/la recognition site. Both Lo/pll and Lot pill have seq,uences homologous to this tryptic fragment and Ansari et al. proposed that the Lo/pI T.cdI determinant may be shared with Lol pll and Lolplll [30,31]. This may explain the concordant responsivenessof patients to Lol pl, Lo/ pll and Lol pill [33,34]. Now that the amino acid sequences of Lol pl, 11 and Ill are available it should be possible to experinaentally determine the T- and B.cell epitopes of these allergens usih~8 synthetic peptides based on their sequences. Acknowledgements: We tlmnk the Australian National Health and Medical Research Council, Asthma Foundation of Uictoria, lmmuLogic Pharmaceutical Corporation, and tl~e Australian Governmeat Department of Employment Education and Training for financial support for this project,
REFERENCES [1] Wuthrich, B (1989) Int. Arch, Allergy Appl, In'm~unol, 90, 3-10. [2] McNicholl, K. and Williams, M, (1973) Br, Med, I. 4, 16-20, [3] Fleming, D,M, and Crombie, D,L (1987) Br. Med. J, 294. 279-283. [4] Singh, M,B. and Knox, R,B, (1985) Int. Archs. Allergy Appl, Immunol, 78, 300-304, iS] Esch, R.E, and Klapper, D.G. (1987) J. Allergy Clin. lmmunol, 79, 489-495.
Febru#ry 1991
[61 Kmndrins, R...~l~ckm~to, V, trod Porter, O,J; tlgll•} Ins, Ards. All*rsy AppI. Immunol, till, 96=10~1, (?) Frddhoff, |,,R,, fihrlirh.Keul~ky, ~,, Gt'~tnh J,H., Meyer, D,A. ~nd M~r~h, D.G. (19Jl6) ]. Allerlly Clln, Immuaol. 6L JI6-)~L pt) Ford, D, •nd Bnldo. B,A, t19116) Ira, Arch, Allerl~y Appl, Ira. munol, 8i. 19;t-20~t. [9} M~nh, D,G., Ooodfdend, L., Khqh T,P,. Lowensleln, H. =~n~ PiitHs.Mil|s, T,A.E. (lgtilq Clin. AII~rlIY Ill, 201-209, [IOI SInllh, M.B,, Houl!h, T., Th~rc~kulpis~t, P., Avjiolllu, A,, De,vies, $,. Smith. P,M., 'r.ylor. P,E,, Simpson. R.J., W=trd, L,, M¢CMskey, J,. Pu~, R. an,~l Knox, R.~, (1990) Prec. Nat. Acrid. Sd. USA, i~ press, [I l) Staff, I.A., T~ylor, P,I~,, Smith. P,M,, Sinllh, M,19, And Kn©s. R,B, (1990) Hissed:hem J. 22. ~'16=;F~0, [12l Cott~tm, G.P,, Mot=tn, D.M, arid $tandrln ~, R, (191t6) Bioch=m, J, 234, 305-310. [1~,) Es¢lt, R,E,, Klapp~r, D,O, (191~9) Mol. Immunol. 26, ~S7-~.61, [14) Kahn, C,R, and Marsh, D,G. (1986) Mol. immunol, 23. 12~1-1288, [l~J Smart, I,J., Hcddlc. R.J., Zol=l, hi. and Bradley, J. (1911],)Ins Arch, Allergy AOPl, Immm'~ol. "/2, 24~-248 [16] Knox, R,B,, Sinsh, M.B., Hoallh, T, lind Theera,kuipisur. P, (1999) Adv, Blest. 95, i61-1?t.
[19] Ward, L,D., Reid, G,E,, Morit7, R,L, nnd Simpson, R,J, (1990) in: Cnrrent Research m Protein Chemistry (Villarran¢=, J,J. ed.) pp. i79-i93, Academic Press, New York, [18] Ward, L.D., Hon~., J,, Whlt©head, R,H. and Simpson, R.J, (1990) Electrophoresis, in press, [19] Herrin, D, and Mich~tds, A, (1994} Plant Mol Biol, Reporter 2,
24-29, [20] Gubler, U. and Hoffman, B.J, (1983) Gene 25, 263-269, [2l] ganger, F,, Nicklen, S, and Coulson. A,R, (19";?) Proc, Nat. Acad, Sci, U S A "/4. 5463-5468, [22] Rafnar, T,, Griffith, I,J,, Kuo, M..C,, Bond.J.F., Rollers, B,L, and Klapper, D.G. (1990) J, Biol, Chem,, in press, [23] Froltmnn, M,A., Dash, M,K. and Martin, G.R, (1981¢) Prec. Nat, Acad Sol, U S A 8~, 8998.9002, [24] Roux, K,H. and Dhanarajan; P. (1990) BioTech. 8.4B-57. [2S] Lutcke, H.A,, CL..,, K,C,, Mickel, F,S., Moss, K,A,. Kern, H,F, and Scheele, Q,A, (1987} EMBO J. 6, 43--48r [26] Johnson, P. and Marsh, D.G. (1966) Immunochemistry 3, I01-110, [27] Howlcth B.J. and Clarke, A.E. (1981) Biochem, J, 197.
695-'~O0, [28] Jones, R,L, and Robinson, D.G (1989) New Phytol, 111, 567-597, [29] Van Hcljne, G. (1984) J, Mol, Biol. 173, 243-251, [30] Ansari A.A,, Shenba$amarthl. P and Marsh, D,G. 0989) J. Biol, Chem, 264, llISl-ll185 [31] Ansari A.A,, Shenbagamurthi, P, and Marsh, D,G, (1989) Biochemistry 28, 8665-8670, [32] Ansari, A,A,, Kikhara, T.K. and Marsh, D,G, (1987) J. Ira. munol, 139, 4034-4041. [33] Freidhoff, L,R,, Ehrlich-Kautzky, E., Meyers, D.A,, Ansari, A.A., Bias, W.B, and Marsh, D,G. (1988) Tissue Antigens 31, 211-219. [34] Ansaril A,A,, Freidhoff, L.R,. Meyers. D,A,, Bias, W,B. and Marsh, D.G, (1989) Hum, Immunol. 25, 59-71, [35l Kyte, J and Doolittle, R,F, (1982) J, Mol, Biol. 157, 105-132.
NOTE ADDED IN PROOF Perez et al. (J. Blo[ Chem. (1990) 265, 16210-16215) have reported the cioning of two LoI pl isoforms. W e detected the amino acid polymorphisms defined by their two clones in our P C R analysts of rye.grass pollen. In addition, we have identified other potential polymorphic residues (data not shown), This c[early demonstrates that there are multiple Lo/pI isoforms.
215
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,¢,. I~1 Fedel'#tion o1' European BIoehem!~al So
Cloning and sequencing of Lol pI, the major allergenic protein of ryegrass pelion Irwin J. Griffith t , Penelope M. Smith", Jeanne Pollock L, Piyada Thecrakulpisut =, Asit Avjioglu =,
Scan Davies =. Terryn Hough =, Mohan B, Singh=, Richard J, Simpson 3, Larry D, Ward = and R, Bruce Knox = hmtmLol¢lc Pharntaceulleal Corporation, One Kendall Square. Building 600, Cmnbrldg¢, MA ¢12139, USA, =Schao/ of Botany,
U#lverxity of Melbourne, ParkPille, Yirtar& 30.~2, ,4uJ~trr/lia and ~Joi~tl Prat¢#~ Structure Labor=/or)',. Ludwig ht.~lltute fm" Cum'er Rettar¢l~ (Melbourne Brtttleh) enlff The ~P'¢tlttr ~md Eli:¢t thlll hlslllt~te for Medical Rtsearch, Pctrkvlll¢, F/¢tortu 3052, d ustraliu
Received 5 December 1990 We have isolated a lull length eDNA tie)so encoding the major glycoprotein allerBen LoI pl, The clone was selected using a combination of immunological screening e r a eDNA expression library and PCR amplification ofLol pl.spccific transcripts, tolp[ expressed in bacteria as a fusion protein shows recognition by specific IgE antibodies present in ser=lof grass pollen.allergic subjects, Northern analysis has shown that the l.#lpl transcripts are expressedonly in pollen or rye.grass, Molecular cloning of Lolpl provides a molecular genetic approach to study the structure-function relationship of allergens,
Lolium preenne; Allergen; eDNA clonin B: Nucl¢otid¢ sequence: Amino acid sequence
1,INTRODUCTION Hayfever and seasonal allergic asthma due to grass pollen allergens are environmental diseases that afflict up to 25°'/0 of the population in cool temperate climates [1-3]. Although a degree of cross-reactivity has been described among antigens from different grass species [4-6], it is well established that rye-grass antigens are the most reactive allergens [7,8]. The most prominent allergen in rye-grass pollen is group I or Lol pl (IUIS nomenclature [91). This has beer= shown to be the major IgE-binding protein [7,8]. Elevated levels of Lol pI. specific IgE have been detected in sere of up to 95070 of grass pollen-allergic individuals, and thus of major clinical significance [7,8]. Other clinically significant allergens of rye-grass include LolplI, III, IV [9] and the newly identified Loi pIb [10]. Lol pl is an abundant acidic glycoprotein of Mr 35 kDa [8] and pI 5.5 [10], located in the cytosol of the pollen [10,11]. The amino acid sequence of this allergen has not been reported, although short sequences from • an N-terminal and an internal peptide fragment have been determined [12,13]. In this paper, we report the cloning, expression and sequencing of a full-length eDNA clone encoding l . o l p I .
2, MATERIALS AND METHODS 2.1. Plant materials attd alltibody probes Pollen of Loliunt perenne was collected from plants flowering in Melbourne, and stored in liquid nitrogen until used. MAbs 4.0,1, 21.3 and 3,2 are specific for Lolpl, and their preparation and characeeriza. lion haw been described elsewhere [4,10,14-16], IgE antibodies were from sere of grass pollen-allergic individuals, as determined by skin prick test and/or RAST (Radioallergosorbent test).
2.2, isolaliot) of pollen proteins and intntunoblotting Soluble proteins were extracted from rye.grass pollen by vigorous shaking in PBS (150 raM, pH 7,2) containing 1 mM PMSF, on ice for 3 h Conditions for ¢lectrophoresis and immunoblotfing with MAbs were essentially as described [16]. For lgE antihody binding, blots were incubated in allergic sere collected from at least 4 patients with high RAST scores for grass pollen, pooled and used diluted 1:5 in TBS/0.'~% BSA, The bound IgE was detected using ~2~i-lahelled anti. I~uman lgE (Kallestad, USA) essentially as described by Ford and Baldo [8],
2.3, Purification of Lol pin for N.term#tal sequencing Lolpl was isolated using preparative isoelectric focussing (Rotofor, BioRad, Richmond, CA) followed by SDS-PAGE (A, Avjioglu and M,B. Singh, unpublished data), The allergen was recovered from SDS-PAGE gels by electroftransfer (90 V/2 h; 4'C) onto polyvinylidene difluoride (PVDF) membrane, (Millipore, Bedford, MAt [17]. Proteins were visualized by staining with Coomassie brilliant blue R-250, washed extensively with deionized water it'll and sub. jected to sequence analysis [17,18]. Amino acid residues are given us. ing the one letter notation,
2.4, eDNA library and immunological screening Correspondence address: R.B. Knox, School of Botany, University of Melbourne Parkville, Vict0ria 3052, Australia
210
Poly (A ÷) mRNA was isolated essentially as described I19], eDNA was synthesized [20] and cloned into the EcoRl site of" the vector lambda.gt II. Immunological screening was done by plating tile
Published by Elsevier Science Publishers B, V,
Volume 2"/9, number 2
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eDNA library, and ~
K 1 94-67---- . .
2,$, Nu¢lenrldP sequlartnx
3.1. Identification of Lol pI by immunoblotting The recombinant clone (13R) was isolated using Lol pI-specific MAbs, which provides evidence of its rela-
3
4
5
43-.- tt
I
Lol Rl=lbI 30--
2,6. O/i~om~t'/¢ottde.v Oli$onucleolides used in this study are shown in "l'able 1 Anchor Primer tAP), Anchor Linker CAL) and Anchor Template (AT) ha.re been described previously [221, LpA-3 and LpA.S were derived from the sequence of the Lolpl eDNA clone 13R, Sequenelnll primers werce derived from tile internal sequence of PCR.ijenerated clones, All oligomers were purchased from Research Genetics (Huntsville, At,., USA),
3. RESULTS
2
D
The eDN/~ clone I ~R was hotated from the phalle, and subcloned Into pGEbl,3Z vectors (Promellal. DNA settaenee was determined by dt)uble.stranded sequeneln= carried out by Ih¢ dideo,~p chain tetmtn;t. tlon method I l l ] , usloll 1"7 DNA polyrnerase (Pharmacist), PCR amplified DNA was cloned Into M13 vector for didcoxy sequencinll usinll ~eqtlenas¢ (US Bloclt~mieals),
2.'7, Pol.vmPra~e chain reaction fPCR,l cloning Full length clones eneodlnll Lalp[ were obtained usin$ a modifica. tion [22] of tile Anchored PCR [23,24], Double stranded eDNA was synthesized from I/~g of total R N A with the e D N A Synthesis System (Life Technologies, Inc,, G:tithersberg, MD, USA)usirt8 oligo dT Its primer, blunt ended with T4 polymerase and blunt end llilated to self.annealed AT and A L primers, Linkered eDNA O/d from a 20/d reaction) was mixed with 100 pmol each of AP and LpA.S primers, IO ~ul of IOX reaction buffer (GeneAmp kit, US Biochemicals, Cleveland, OH, USA) and 0.5/d of 7". aquattcus polymerase. The mixture was brought to 100~d with deionized water and amplified for 25 cycles as described 1221, Briefly, the first 5 cycles consisted of denaturation at 94~C ror 1 rain, annealinl} at 45"C for i.S rain, and polymerization at "70"C for I .S min. The htst 20 cycles were as sta ted, except that annealing was at 55*C, 0.01 volume of the primary PCR was risen reamplified with oligomers AP and LpA.3 as above. LpA.3 js nested (internal) relative to oligonter LpA-5 used in the pritnary PCR, DNA from the secondary PCR was recovered by sequential chloroform, phenol and chloroform extractions, precipitated with 0.5 vol. 7.5 M AmoAc and I.$ vol. isopropanol. DNA from the pellet was digested with Xba I and Pst I, run on a preparative low melt gel, and ligated into Xba i/Pst I.digested M13.
February 1991
20-'--
14-.-
T
21"3 40.1 3"2
Ig E
Fig. I, lmmunoblot analysis of lllE and MAb bindina to Lolpl pro. uins from rye.grass pollen, Mr (kDa) Is denoted on the left, 120 g of [}ellen proteins were loaded per lane. Lolp[ is denoted with an arrow. Lane I : SDS.separation of total rye.grass pollen proteins (staining is '~vith Co0massie brilliant blue R-2J0); lanes 2-5: bindinll of MAbs or IilE on Western blots of total rye.grass pellets proteins,
lionship to the natsve allergen On Western blots of pollen proteins, MAbs 3.2, 21.3 and 40.1 show strong affinity for a 35-kDa protein (Fig, 1). This protein showed high specificity for l e e antibodies from pooled sera of grass pollen-allergic individuals (Fig. 1). All the MAbs and IgE used in this study bound strongly to the Lolpl reference standard provided by NIAID (National Institutes of Health, Bethesda, MD; data not shown).
3.2. N.terminal sequence of Lol pl We purified Lol pI by two-dimensional gel electrophoresis involving preparative isoelectric focussing in the first dimension, followed by SDS-PAGE of the individual fractions. This procedure successfully Table 1
Oligonucleotide sequences Oligomer
Nucleotide sequence 5'p-AATGATCGATGCT ~ 5'GGGTCTAGAGGTACCGTCCG
Strand i i
AL AP AT
5~GGGTCTAGAGGTACCGTCCG~TCGATCATT
LpA-2 LpA-3 LpA-5 LpA-9 LpA-10
5'GGAGTCGTGGGGAGCAGTC 5'GGGAATTCCATGGCGAAOAAGGGC 5'CCCTGCAGTCATGCTCACTTGGCCGAGTA ~'CCCTGCAGATTATTIGAGATCTTGAG 5t GTGACAGCCTCGCCOG 5' CCGTCGACGTACTTCA
T-pA-iA
Nucleotldes . . . . . .
coding coding non.coding non-coding non-coding non-coding
671->689 442- > 458 810< -827 867 < -884 351 <-367 582 < -607
AL and AT are synthetic linker oligonucleotides containing Xba I, Kpn I and/or Cla I restriction sites [22]. The AP sequence is contained within the AT oligomer [22]. LpA-IA, -2, .3, -5, -9 and -10 correspond to the coding or non-coding sequence of Lol pl, as indicated. LpA-2 contains an Eco RI restriction site, while LpA-3 and-5 contaln Pstl restriction sites added for cloning purpose s, All restriction sites are underlined;
211
FEB$ L~TTERS
Vol.rne 2"/9, ntimber 2
F~bru~ry 1~91
& H
~
~
S
V
L
L
-10 1 GT~GT~GC~CTGTTC~CCG~TTCCTG~GCAOCG¢~¢ATGGC~CGCG~GGTA¢CA¢C4 V v A ~ F A V F L a S ~ l{ G v A ~ v ~ 20 10 GG¢CCC~CATCACGGC¢GAGTACGGCG~C~G~¢76~CGCGAJ~AG¢ACCTGGTAT
~?~
]0 40 ~¢~G¢¢G~CCGGCGCCGG~CCC~GGACA~CGGCGGCGCGTGC~GTAC~GACG~C ~ ~ P ? G A G P K D ~ G G A CG ¥ K O V
~]~,
50 GO G~¢~GGCGCCGTTC~CGGC~T~ACCGGCTGCGGC~C~CCC¢¢~TC~¢~GGaCG~C D K A P r ~ G H T O C G N T P Z r K D
~%9
?0 80 CG?GGCTGCGGCTCCTGCTTCG~GATCAAGTGCACC~GC¢¢~GTCCTGCTCCGGCGAG R G C G S C ? ~ I K C T K ~ £ G ¢ S G ~
]5~
S0 100 GC?GTCACCGTCACAATC~CCG~CGACAATGAGG~GCCT~TCGCACCCT~CCACTTCGAC ^ v T v T x T a D . E £ e ~ A P ~ H r o 13R CTCTCGGGCCt,¢OCATTCGGGTCC~TGGCG~G~GGGCGAGGAGCAG~AGCTCCG¢~GC L S C H A F G ~ H ~ M K G ¢ £ O K L R ~
41~
499
i~0 140 GCCGGCGAGCTGGAGCTCC~GT?CAGGCGGGTC~GTGC~GTACCCGGACGGCACC~G A G E L E L Q F R R V K C K Y P D G T K
539
150 160 CCGACATTCCACGTCGAG~GGCTTCC~CCCC~CTACCTCGCTA~¢TGGTG~GT~C P T F ~ V B K A S N P H Y L A I L ~ K ~
599
I~0 180 GT~GACGGCGkCGGTGACGTGGTGGCGGTGGACATC~GG~G~GGGC~GGAT~GTGG V D G D G D V V A V ~ Z K ¢ K G K D K W
659
190 200 ATCGAGCTCAAGGAGTCGTGGGG~GCAGTCTGGAGGA?CGACACCCCCGAT~GCTGACG 2 E L K E S W G A V W R I D T P D K L T
719
2~0 220 GGCCCATTCACCGTCCGCTACACCACCGAGGGCGGCACCAP~TCCG~GTCGAGGATGTC G P F T V R Y T ? R G G ? K S E v E ~ v
7~9
230 240 ATCCCTGAGGGCTGG~GGCCGACACCTCCTACTCGGCC~G~GAGC~GAAGTGGAGTG ~ P E G W K A ~ T S ¥ S A K *
839
A~CTTCTTCCAATCAGCTT~TTTTGACTC~GATCTCA~T~TCCAGCCGCACATATA
899
T~CGAGGCGGTGAGACATACAAGCTCCTCCATGAGTATATTCATTCATGCCGTATAGAGA
959
GGAGAAAGATGCCTG~T~GAGTTTGAGGTCGACACCTTGTGAG~GTGTATATAGGAG
1019
G~CCC~TCTGGCTCCATCTTTCTTTGCTCGCACGGTGTACTGCT~GGTTATCTTCTA
1099
ACAGGCCAGATT~CCTACTATCT~TATATGC~CGTATGGTCATTTTCCCTAAAAAAA
1139
Lo!pt
hydropathlcity profile 100
5
o
4l
,,,I,,,
t~ I
200
I,,,,I,,,,I,,,,i
,J
I
~
-5 -4
,I.,
l
I II 100
212
1;~
. 200
3 2 1 0 -1 -2 -3 -4
Vol.m¢ 219. n.mbw .~
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Febru=rv 1991
separated Lolp[ (pl $.$) in sufficient quantifies for N. terminal protein sequence amtlysis, The sequence oh. rained was as follows: I A K V P P G P W I TAIF=.YGDKWLD A K ? T ..... This sequence is analogous to that repotcd by Certain et
at, [12]. 3.3, r$olatio~1of cDlVA clones The cDNA, expression librarywas screened for clones that expr~sed epitopes recognised by MAb 40,l. Screening of a total of S xlCP plaques yielded 18 immune.positive clones. These clones were plaquepurified, a~d screened for igE binding using sera from grass pollen-~tllcrgic subjects. Four clones were fo=nd to express proteins with affinity for l i e antibodies, Of these, the clone 13R. ( - 0.8 kb in size including poly (A)" tail), bound strongly to MAbs 40.1, 3,2 and 21 3, all known to preferentially recognise Lolpl[lO]. Thus, the clone e.xpresses an allergen as defined by recognition o f IgE from sere o f allergic individuals and was designated as a putative Lol pl clone,
1353 ~" 107 0 " 872="
3.4. Nucleotide sequence of Lol pl cDNA clone 13R, selected by expression screening, contains 764 nucleotides. There are 146 amino acids in the open reading frame ( O R F ) a , d the predicted M', is 16.2 kDa. The translated amino acid sequence terminates with the 28'amino acid sequence described by Esch and Klapper [I 3]. Since this clone exhibits strong Mab and IgE binding, the sequence contains both antigenic and allergenic ¢pitopcs. However, mature LolpI has an approximate Mr of 35 kDa, so the translated reading frame is apparently not full-length. Clone 13R also lacks the NHx-terminal sequence defined for Lol pl [12], confirming that it is a truncated clone. Full-length Lol pI clones were obtained by P C R of rye-grass pollen RNA. Primers based on synthetic linker sequences and Lol pl-specific sequences derived from the 3' end o f clone 13R were used to generate these clones. The nucleotide sequence of Lol pI is shown in Fig. 2a. There is an open reading frame (ORF) of 789 bp starting with an A T G initiation codon at position 33 and terminating with a TGA stop codon at position 822. This ORF, which has a 64°70 GC content, corresponds to a polypeptide of 263 amino acids (Mr 29.1 kDa). The proposed translation initiation site and its flanking sequences (nucleotides 29-37.) share 67070 identity with the
6 0 3 D-
Fig, 3. Tissue-specific expression of' Lol p! in rye-grass pollen, Nor. them blot analysis of total RNA from rye.grass pollen, seed, leaf. root and inflorescence using 13R cDNA as n probe.
consensus plant sequence AACAATGGC [25]. The most critical nucleotide, a purine at position - 3, is con. served. Sequence corresponding to the 13R clone starts at nucleotide 474. The predicted protein sequence has a hydrophobic putative signal peptide sequence of 23 amino acids (Fig. 2 a,b). This is indicative of a mature processed protein of 240 amino acids (Mr 26.6 kDa). The amino acid composition from the deduced sequence, rich in glycine and lysine, is in complete agreement with previous data obtained by amino acid analyses of LolpI protein [26,27]. There is a single potential Nglycosylation site having the characteristic Asn-xSer/Thr motif (residues 9-I l) in the hydrophilic region of the mature protein (Fig. 2b).
Fig. 2. eDNA, predicted amino acid sequence and hydropathicity profile of Lol pl from rye-grass pollen. (a) The nucleotlde and deduced amino acid sequence of Lol pl. The deduced amino acid sequence represented by the single letter code is shown below the DNA sequence, and begins at the first potential in.frame initiation codon. The open reading frame continues for 263 amino acids and ends with the TGA stop codon denoted by an asterisk. The putative signal peptlde is indicated by negative numbers. Underlined amino acids were identified by protein microsequencing (this report; and [12,13]). The arrow indicates the start of the 13R sequence, (b) Hydropathicity profile of predicted amino acid sequence based on the method of Kyte and Doolittle [35] with a window of '7 amino acids,
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Volume 279, number Z
A search of existin~ data.bases showed no simil=rlty between the deduced amino acid sequenceof Lol pl and any other known protein, The Lol pl nucleotide sequence was compared with that or the newly identified Lo/plb [10] and no sequence homolo=y was observed,
3, ~, E:cpr¢.~ionof ~ene for Lot pl i~ Vario,.~ryc.gra~ To determine the tissue specificity of Lolpl sene expression, we examined the level of specific transcripts in various: tissues. Northern blots probed with eDNA !3R showed hybridization to a single abundant transcript ( - 1350 bases) in pollen (Fig. 3), Hybridization was also observed to similar sized transcripts in i n . florescence, but no hybridization to any transcripts in rye.grass seed, leaf or root was observed (Fig. 3). 4, DISCUSSION In this report we describe the cloning of a eDNA se.quence encoding an allergen from rye-grass pollen. Several lines of evidence confirm that clone 13R corresponds to the major glycoprotein allergen, Lol p l . First, the clone was immunoselected from the lambdagt 11 e~pression library using a MAb that shows high Levels of binding to Lot pI. Second, the polypeptid¢ encoded by the cDNA clone was recognised by lgE antibodies from pooled sera of grass pollen-allergic individuals. Third, a segment of the deduced amino acid sequence of the clone corresponded to the 38-amino acid tryptic fragment of Lo/pl (YTTE G G T K S E F E D V I P E G W K A D T S Y S A K ) described by Esch and Klapper [13l. Finally, the pollen specificity of transcripts Lol p Ih
:
v,~ruary =we
hybtidizin$ wi~h L3R,. is coasistent ~ith tissuespe.eifici. ty of Lot pi anti,lens, as observed earlier by immunoblottinil [10} The Nderminal sequenceof' Lol p[, was not detected in the ORF of the I3R clone, However, full lenath clones obtained by PCR ampllflc~ttion, encoded an amino acid sequence which corresponded with the N. terminal sequence of Lol pl ([tZ]; ~,nd this p~per). The lonsest ORF is 789 nucleolides lens, which corresponds to a protein of 263 amino acids with a Leadersequence of 23 amino acids. The molecular mass for the mature form of/.,ol p[ (240 amino acids) was calculated to be 26,6 kDa, This would suggest that 24% of the 35 kDa molecular mass of tl~e pollen protein is due to post. translationa~ modifictttions. The Lo/pl signal peptide has some motifs common to otl~er plant signal peptides, especially those of secreted plant enzymes, e.g. amylase [28]. The motifs close to the cleavage site show some similarity wi~h those of signal peptides which direct proteins into the Lumen of the ER for glycosylation, e.g. V L L , . . F L OSAHG I in Lol pL a n d V L L . . . L S ASI..ASG t in alpha-~tmyiase [28]. The - l and -3 rule for the prediction of a leader se. quence cleavage site [29] is that amino acids at these positions should be sinai[ and neutral: O and A in Lol pl. The deduced amino • acid sequence o f the full length L,o/pl clone contains both the N-terminal sequence [12] and tryptic peptide fragment sequence [13] previously reported for Lolpl, as well as the N.terminal protein se. quence of Lo/pl reported here. The N-~erminal sequences ([12]; and this report) helped define the cleavage site between the leader sequence and the mature form of Lol pl.
~. 0 K L R $ A G ~- 5 K ~ Q E' R P,V K C K
Y?
D G ~ " ~ P "r H ~ " ~
R"~A
),54
L o l p Z! L o l p IXZ
Lol p ZA Lol p X! &el p ZZZ
&el p l& &el p IZ Lol p ~ !
0 o v v,
Iol,
v o,,,[],
o,
Fig. 4, Homologies of Lo/pl with Lo/pll and III.Alignment of deduced amino acid sequence of Lolpl with reported sequences of Lo/plI [26J and LoJ pill [27]. Identical residues are shown in bold letters, These residues and other similar residues in t~.e three sequences are indicated by boxes. The following residues were considered similar: A,S,T; D,IS; N,Q; R,K; I,L,M,V; F,Y,W.
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Our resultsshow that the 28 amino acid styptic frnlp meat sequence YTTE GGTKS EFEDV IPEOW KADTS YSAK [13] is located at the COOH-trrminus or this alierilrn. We also round that the entire 97 amino acid sequences or Lo[ pit [301 lad Lol pHI []!1 have strains homology with the COOH-terminal end or Lol pI (FIg; 4). Lot pl has 41~% homology (3~% identity) with Lol pll and 44% homology (26% identity) with Lol p i l l while Lol PII and Lol pill have 73~ homololw (59% identity) which each other. However, despite strong homolol~y between amino acids 145 to 240 of Lol pl with the amino acid sequencesof Lo/pll and Lot pill, no serological cross.reactivity has been detected between Lolpl and Lolpll nor Lo/pill [321, Immune responsiveness to rye.grass pollen allergens inthe human population is significantly associated with the histocompatibility leukocyte antigen (HLA)-DR3 [33,34]. One interpretation of this finding is that HLADR3, or closely linked class II molecules, may be particularly effective at presenting certain allergenic determinants to T cells. Ansari et al. [30,31] predicted that the 28 amino acid tryptic fragment isolated by Esch and Klapper [13] may include an HLA.DR3 restricted T cell/la recognition site. Both Lo/pll and Lot pill have seq,uences homologous to this tryptic fragment and Ansari et al. proposed that the Lo/pI T.cdI determinant may be shared with Lol pll and Lolplll [30,31]. This may explain the concordant responsivenessof patients to Lol pl, Lo/ pll and Lol pill [33,34]. Now that the amino acid sequences of Lol pl, 11 and Ill are available it should be possible to experinaentally determine the T- and B.cell epitopes of these allergens usih~8 synthetic peptides based on their sequences. Acknowledgements: We tlmnk the Australian National Health and Medical Research Council, Asthma Foundation of Uictoria, lmmuLogic Pharmaceutical Corporation, and tl~e Australian Governmeat Department of Employment Education and Training for financial support for this project,
REFERENCES [1] Wuthrich, B (1989) Int. Arch, Allergy Appl, In'm~unol, 90, 3-10. [2] McNicholl, K. and Williams, M, (1973) Br, Med, I. 4, 16-20, [3] Fleming, D,M, and Crombie, D,L (1987) Br. Med. J, 294. 279-283. [4] Singh, M,B. and Knox, R,B, (1985) Int. Archs. Allergy Appl, Immunol, 78, 300-304, iS] Esch, R.E, and Klapper, D.G. (1987) J. Allergy Clin. lmmunol, 79, 489-495.
Febru#ry 1991
[61 Kmndrins, R...~l~ckm~to, V, trod Porter, O,J; tlgll•} Ins, Ards. All*rsy AppI. Immunol, till, 96=10~1, (?) Frddhoff, |,,R,, fihrlirh.Keul~ky, ~,, Gt'~tnh J,H., Meyer, D,A. ~nd M~r~h, D.G. (19Jl6) ]. Allerlly Clln, Immuaol. 6L JI6-)~L pt) Ford, D, •nd Bnldo. B,A, t19116) Ira, Arch, Allerl~y Appl, Ira. munol, 8i. 19;t-20~t. [9} M~nh, D,G., Ooodfdend, L., Khqh T,P,. Lowensleln, H. =~n~ PiitHs.Mil|s, T,A.E. (lgtilq Clin. AII~rlIY Ill, 201-209, [IOI SInllh, M.B,, Houl!h, T., Th~rc~kulpis~t, P., Avjiolllu, A,, De,vies, $,. Smith. P,M., 'r.ylor. P,E,, Simpson. R.J., W=trd, L,, M¢CMskey, J,. Pu~, R. an,~l Knox, R.~, (1990) Prec. Nat. Acrid. Sd. USA, i~ press, [I l) Staff, I.A., T~ylor, P,I~,, Smith. P,M,, Sinllh, M,19, And Kn©s. R,B, (1990) Hissed:hem J. 22. ~'16=;F~0, [12l Cott~tm, G.P,, Mot=tn, D.M, arid $tandrln ~, R, (191t6) Bioch=m, J, 234, 305-310. [1~,) Es¢lt, R,E,, Klapp~r, D,O, (191~9) Mol. Immunol. 26, ~S7-~.61, [14) Kahn, C,R, and Marsh, D,G. (1986) Mol. immunol, 23. 12~1-1288, [l~J Smart, I,J., Hcddlc. R.J., Zol=l, hi. and Bradley, J. (1911],)Ins Arch, Allergy AOPl, Immm'~ol. "/2, 24~-248 [16] Knox, R,B,, Sinsh, M.B., Hoallh, T, lind Theera,kuipisur. P, (1999) Adv, Blest. 95, i61-1?t.
[19] Ward, L,D., Reid, G,E,, Morit7, R,L, nnd Simpson, R,J, (1990) in: Cnrrent Research m Protein Chemistry (Villarran¢=, J,J. ed.) pp. i79-i93, Academic Press, New York, [18] Ward, L.D., Hon~., J,, Whlt©head, R,H. and Simpson, R.J, (1990) Electrophoresis, in press, [19] Herrin, D, and Mich~tds, A, (1994} Plant Mol Biol, Reporter 2,
24-29, [20] Gubler, U. and Hoffman, B.J, (1983) Gene 25, 263-269, [2l] ganger, F,, Nicklen, S, and Coulson. A,R, (19";?) Proc, Nat. Acad, Sci, U S A "/4. 5463-5468, [22] Rafnar, T,, Griffith, I,J,, Kuo, M..C,, Bond.J.F., Rollers, B,L, and Klapper, D.G. (1990) J, Biol, Chem,, in press, [23] Froltmnn, M,A., Dash, M,K. and Martin, G.R, (1981¢) Prec. Nat, Acad Sol, U S A 8~, 8998.9002, [24] Roux, K,H. and Dhanarajan; P. (1990) BioTech. 8.4B-57. [2S] Lutcke, H.A,, CL..,, K,C,, Mickel, F,S., Moss, K,A,. Kern, H,F, and Scheele, Q,A, (1987} EMBO J. 6, 43--48r [26] Johnson, P. and Marsh, D.G. (1966) Immunochemistry 3, I01-110, [27] Howlcth B.J. and Clarke, A.E. (1981) Biochem, J, 197.
695-'~O0, [28] Jones, R,L, and Robinson, D.G (1989) New Phytol, 111, 567-597, [29] Van Hcljne, G. (1984) J, Mol, Biol. 173, 243-251, [30] Ansari A.A,, Shenba$amarthl. P and Marsh, D,G. 0989) J. Biol, Chem, 264, llISl-ll185 [31] Ansari A.A,, Shenbagamurthi, P, and Marsh, D,G, (1989) Biochemistry 28, 8665-8670, [32] Ansari, A,A,, Kikhara, T.K. and Marsh, D,G, (1987) J. Ira. munol, 139, 4034-4041. [33] Freidhoff, L,R,, Ehrlich-Kautzky, E., Meyers, D.A,, Ansari, A.A., Bias, W.B, and Marsh, D,G. (1988) Tissue Antigens 31, 211-219. [34] Ansaril A,A,, Freidhoff, L.R,. Meyers. D,A,, Bias, W,B. and Marsh, D.G, (1989) Hum, Immunol. 25, 59-71, [35l Kyte, J and Doolittle, R,F, (1982) J, Mol, Biol. 157, 105-132.
NOTE ADDED IN PROOF Perez et al. (J. Blo[ Chem. (1990) 265, 16210-16215) have reported the cioning of two LoI pl isoforms. W e detected the amino acid polymorphisms defined by their two clones in our P C R analysts of rye.grass pollen. In addition, we have identified other potential polymorphic residues (data not shown), This c[early demonstrates that there are multiple Lo/pI isoforms.
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