Scandinavian Journal of Clinical and Laboratory Investigation

ISSN: 0036-5513 (Print) 1502-7686 (Online) Journal homepage: http://www.tandfonline.com/loi/iclb20

The Structural Requirements of Epitopes with Ige Binding Capacity Demonstrated by Three Major Allergens from Fish, Egg and Tree Pollen S. Elsayed, J. Apold, E. Holen, H. Vik, E. Florvaag & T. Dybendal To cite this article: S. Elsayed, J. Apold, E. Holen, H. Vik, E. Florvaag & T. Dybendal (1991) The Structural Requirements of Epitopes with Ige Binding Capacity Demonstrated by Three Major Allergens from Fish, Egg and Tree Pollen, Scandinavian Journal of Clinical and Laboratory Investigation, 51:sup204, 17-31 To link to this article: http://dx.doi.org/10.3109/00365519109104592

Published online: 08 Jul 2009.

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Scand J Clin Lab Invest 1991; 51, Suppl. 204: 17-31

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The structural requirements of epitopes with IgE binding capacity demonstrated by three major allergens from fish, egg and tree pollen S. ELSAYED, J, APOLD, E. HOLEN, H. VIK, E. FLORVAAG & T. DYBENDAL Allergy Research Group, Laboratory of Clinical Biochemistry, University Hospital, University of Bergen, Bergen, Norway. Elsayed S, Apold J, Holen E, Vik H, Florvaag E, Dybendal T. The structural requirements of epitopes with IgE binding capacity demonstrated by three major allergens from fish, egg and tree pollen. Scand J Clin Lab Invest 1990; 51, Suppl. 204: 17-31. Three major allergens from cod fish, egg white and tree pollen, were characterized by studies on their allergenic and antigenic structures. The major allergen of cod fish, Allergen M "parvalbumins PI 4.75",is composed of 113 amino acid residues with a molecular weight of 12,328 daltons. It comprised three domains, AB, CD and EF, consisting of 3 helices interspaced by one loop. Each of the loops of the CD and E F domains each coordinates one Ca*'. The antigenicity and allergenicity of Allergen M was deduced from studying the modified protein and some particular synthetic peptides. Three sites were encompassing IgE binding epitopes namely peptides 33-44, 65-74 and 88-96. A novel peptide (49-64), of the CD-domain, was demonstrated to be allergenically/antigenically active and cross reactive with birch pollen allergen, which incidentally was used as a negative control. This site encompassed two repetitive sequences (D-E-D-K) and (D-E-LK), suggested to be mutually critical for the specificity of antibody binding. This hypothesis was reconfirmed by SPPS of several analogous peptides of region 39-64. Furthermore, peptide 88-103 of the EF-domain was similarly synthesized; it functioned as a monovalent hapten, blocking and not eliciting allergic reaction. Moreover, peptide 13-32 of domain AB, the non-calcium binding domain, was thoroughly tested. The results of PK inhibition showed clear activity and the peptide was found to function at the level of a divalent determinant. Ovalbumin (OA) is the most dominant of five major allergens of egg white and universally used as model protein. OA allergenic epitopes were shown to be mainly determined by the primary structure and depend on certain peptide chain length. The N-terminal decapeptide (OA 1-10) was shown to react with reaginic IgE. Direct skin test on egg allergic patients, showed no activity and the site was therefore concluded to encompasses one single Ig binding haptenic epitope. Peptide OA 323-339, was demonstrated to be valuable in studies of T-cell

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18 S. Elsayed, J. ApoM, E. Holen, H. Kk,E. Florvaag & T. Dybendal

recognition of protein antigens. Three analogous peptides of this region were prepared and clearly shown to be immunogenic in rabbits and to bind specific IgE from patients allergic to egg. OA 323-339 was concluded to encompass an allergenic and antigenic epitope which was recognized by human and rabbit B-lymphocytes. Eight peptides in the region 11-122 were similarly synthesized. A test battery was performed to study this region using rabbit polyclonal antibodies and human specific IgE. Some of these sites were involved in binding of particular Ig paratopes. Five immunogenic peptides from the major aliergens of tree pollen extracts (segment 23-38), were synthesized. The selection of those peptides was setteled using two algorithms for providing the optimal hydrophobicity. All the synthetic peptides and analogues from region 23-38, could inhibit the binding of specific IgE to the intact molecules. A minimal requirements of an allergenic epitope was clearly demonstrated to be the repetitive four amino acids peptides interspaced by 6 unrelated spacer arm. A certain minimal molecular size of 12-15 amino acids seemed necessary in all the epitopes synthesized for studying the biological activity and for a successful predictive algorithms of the helicity.

Key words: Allergic diseases; parvalbumin; Allergen M; immunoglobulin E; ovalbumin; antigenic structure; primary structure; solid phase peptide synthesis; birch pollen allergens; allergenic epitopes; Ig paratopes. Reprints: S . Elsayed, Allergy Research Group, Laboratory of Clinical Biochemistry, University Hospital, N-5021 Bergen, Norway. INTRODUCTION Allergic diseases affect between 10-20 % of the population and usually start during infancy, childhood or adolescence. The most common allergic diseases are rhinoconjunctivitis, bronchial asthma, atopic dermatitis and gastric disorders of several types. The diseases not only interfere with the physical condition of the individuals but also complicates their social relations in an early phase of life. Allergic diseases also result in significant mortalities with rising numbers for every year [l]. Despite considerable advances in many areas of modern medicine, the fundamental therapeutic alternatives available to patients with allergic diseases have not been changed in the past decades. Many of the basic mechanisms that regulate immune reactions and in particular those regulating IgE re-

sponse are still not well known. IgE is found in very small concentrations in serum and is an important parameter in type I hypersensitivity due to its higher levels in allergic diseases. One of the special characteristics of the isotype is that it binds high affinity receptors (FC,RI) on mast cells and basophils and low affinity (FC,RII) on lymphocytes and other inflammatory cells like eosinophils, monocytes, platelets etc. [2]. The major counterparts of allergic reactions are specific IgE and allergens. The interaction between two IgE molecules and multivalent allergen on target cells is the initial process of the allergic reaction. Divalent haptenic allergens induce the bridging, while monovalent haptens can block single F(ab), paratope, leading to inhibition of further aggregation of IgE and release of mediators [3-41.

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Synthetic allergenic epitopes

Following the interaction of allergen with IgE on mast cells, mediator release triggers a marked inflammatory response including alteration of the vascular permeability and allowing the infiltration of many components of the immune system. A detailed understanding of the structure of the allergenic molecules which participate in the regulation process is a prerequisite. Many approaches for the study of allergen specific regulation of IgE synthesis have been tried: One of these is by using modified allergens (allergoids) that have reduced reactivity for IgE but retained IgG inducing capacity [5]. Another interesting approach is the use of synthetic monovalent haptenic peptides for blocking single paratopes of IgE and subsequent inhibition of mediator release [6]. In this overview the work done on the characterization and elucidation of the primary structure, antigenic structure, localization of the allergenic epitopes and the their possible applications will be discussed. FISH ALLERGENS Allergy to fish plays a classical role in the history of allergology. Cod is inhabiting the North Sea and mostly used in Scandinavia as fresh, salted or dried food. Allergy to fish is a common problem in the fish processing communities, particulary in children and young adults. Fish allergens may act as inhalant or food allergens [7]. They, unexpectedly, could be detected in dust samples collected from 20 class rooms of Norwegian schools located in different geographical areas [8]. Using gel filtration chromatography (GFC), ion exchange and isoelectric focusing (IEF) techniques, it was possible to isolate the major allergen of cod fish, designated Allergen M [9,10]. Allergen M belongs to a large group of vertebrates' muscle calcium binding proteins namely parvalbumins. Like the light chain of myosin and troponin C, these proteins belong to the calmodulin group, which is

19

found in mammalian muscles as well as fish [ll]. The protein was also found to be secreted by a hepatome cell line (5123tc) and was designated oncomodulin in recognition of its frequent expression in neoplastic and fetal placental tissues. The oncomodulin strongly resembled parvalbumin in their Ca2+ binding affinity and molecular structure [ 121. Allergen M (parvalbumin PI 4.75) is composed of 113 amino acid residues with a molecular weight of 12,328 daltons [13,141. The cleavage of Allergen M at its single arginyl peptide bond yielded two allergenically active fragments: TM1, 75 residues and TM2,38 residues respectively [ 151. The amino acid sequence of Allergen M and several other members of the parvalbumin have been elucidated. A high amino acid sequence homology was shown between Allergen M from cod and the corresponding parvalbumin of other fish species confirming the allergenic cross reactivity of fish [14]. One parvalbumin representative (carp PI 4.25) has been examined by X-ray diffraction analysis and the tertiary structure was resolved [16]. The molecule is composed of three homologous domains, AB, CD and EF; each comprises one third of the chain and is consisting of helices interspaced by one loop. The loops of the CD and EF domains coordinate, strongly but reversibly, one Ca2' each (Fig. 1). The three homologous domains were evolved by triplication of one single gene through evolution [17].

FIG.1. Schematic representation of the Allergen M molecule suggested on the basis of the X-ray diffraction analysis of a model parvalbumin from carp (PI 4.25). The numbers on the arrows show the locations of the enzymic tryptic hydrolysis peptides. These sites were the biologically active and their activity was later confirmed by SPPS and subsequently biological testing.

20 S. Elsayed, J. Apold, E. Holen, H. Kk, E. Florvaag & T. Dybendal

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b

lo FIG. 2. Crossed immunoelectrophoresis CIE pattern of cod

fish Allergen M as visualized by Coomassie blue stain

(a); (b) is a schematic drawing of the precipitation lines.

The immunological structure of Allergen M was intensively studied using several approaches: The first approach used was by modifying certain amino acid residues and studying the reactivity of the modified derivatives. The second approach was to examine the immunochemical reactivity of a large number of overlapping peptides obtained by limited and selective tryptic hydrolyses. The third was by solid phase peptide synthesis (SPPS) of the segments encompassing sites of reactivity as result of pre-examining the native peptides “1. The antigenicity and allergenicity of the modified proteins and purified native or synthetic peptides were investigated by crossed immunoelectrophoresis (CIE) (Fig. 2), and crossed radio immuno-electrophoresis (CRIE), rocket line immunoelectrophoresis (RLIE) and quantitative precipitation inhibition (QPI). The allergenicity of the derivatives was examined by the analysis of the specific IgE binding or by inhibition

technique respectively [19]. Polymerization of Allergen M and modification of particular amino acids (AA) (Tyr-30 and Arg-75) in isolated and purified peptides indicated that Y-30 was part of the reactive site where as R-75 did not contribute to the activity. Unchelating of the two Ca” ions or masking of R-75, with subsequent perturbation of the molecular conformation, decreased the allergenicity by 25 % [20,21]. The results obtained indicated that three sites were assigned to encompass IgE binding epitopes: 1. Residue No. 33-44 on the junction between the AB and CD domains; 2. Residue 65-74 on the corresponding junction between CD and EF domains; 3. Residues 88-96 on the binding loop of the EF domain [13,14,19,22].

Synthetic allergenic epitopes 21

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ALLERGEN M SYNTHETIC PEPTIDES The reason for starting manual Merrifield SPPS [23] was the difficulties encountered in isolating the CD-domain, from the native enzymic hydrolysis preparation, in a homogeneous form and with a yield sufficient for examining the biological activities. A novel allergenic peptide from the CD-domain, peptide 49-64 was prepared by mimicking the amino acid sequence of this site, Fig. 3 1241. This peptide could deflect the Allergen M precipitation line in RLIE. It also was distinctly shown to cross react with birch allergen line, which incidentally was used as a negative control (Fig. 4). The hexadecapeptide encompassed two repetitive sequences (D-E-D-K) and (D-E-LK). It was tentatively suggested that the two tetrapeptides located at each termini are mutually critical for the specificity of antibody binding. Evidence was derived from the SPPS of four series of analogous peptides with the two terminal tetrapeptides and with unrelated spacer arm. The covalent structure of the analogous peptides is illustrated in Fig. 5. Similar immunochemical reactivities were demonstrated for the four synthetic analoges including the terminal tetrapeptides. The immunological reactivity of the region 41- 64 of Allergen M is determined by three tetrapeptides D-E-GK, D-E-D-K and D-E-GK, Fig. 6. The tetrapeptide arrangement is repeated in an elegant systematic order in three sites interspaced by 6 amino acid residues in a segment of 24 residues. The reactivity of these peptides was independent of the sequence of the two spacer arms [25]. The second Cat* coordination peptide 88-103 of the EF-domain (Fig. 7), was similarly synthesized and its biological activity was elucidated by in vitro and in vivo test systems. The inhibition of specific IgE binding and PK-test allowed to suggest that the immunological reactivity of this site was compatible with a monovalent haptenic function, blocking and not eliciting allergic

FIG. 3. The amino acid sequence of the first allergenic synthetic hexadecapeptide reported. The activity was also dependent on the chain length. The peptide on the second line was inactive both in IgE and IgG systems.

FIG.4. Rocket line imrnunoelectrophoresis patterns of octapeptide 57-64, in the first well and hexadecapeptide 49-64 in the second well of plate (a), using Allergen M line in the secondary gel and rabbit anti Allergen M in the antibody gel. In plate (b) birch pollen extract BV line was applied in the secondary gel and rabbit anti BV in the antibody gel. In plates (c) and (d) four different serum proteins were applied in the wells using anti Allergen M or anti BV in the antibody gels. A deflection of Allergen M line by peptide 49-64 was illustrated in plate (a). The same peptide could deflect one of the 4 line of BV extract. No deflection was seen for the control proteins, emphasizing the specificity of the reaction.

reaction. Despite 37.5 % homology of this peptide with the CD (49-64) peptide, it had no cross reactivity with birch pollen allergen as it lacked the characteristic terminal tetrapeptides [26]. Domain AB of Allergen M (residues No.13-32), the non-calcium binding domain, was also produced and thoroughly tested. It was homologous with the two previously

, v -

17

27

El 77 1s

2s

@ @ @ @

ES 7s 19

29

g v , ~

E9 79

~

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1x1

'"'I

Synthetic allergenic epitopes 23 1

2

3

4

5

6

7

8

9

1

0

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FIG. 9. The covalent structure of OA 1-10. Lysine was available as esterified to the resin and replaced Cys-11 of the original sequence. This replacement was examined in connection with peptide 11-19 and found of no effect on the activity 144,451.

turbing the conformation of the allergenic and antigenic determinants of OA were examined. An extract (protein content 8.43 %) was subjected to IEF, in SDS/ PAGE, showed 26 spots visualized by staining with Coomassie blue. OA was purified using a TSK-2000 HPLC column (LKB, Bromma, Sweden), and the purified protein gave high inhibition of specific IgE binding. The cleavage of OA with cyanogen bromide resulted in 4 fractions, all capable of binding specific IgE. Thermal denaturation of OA had no direct effect on the antigenic reactivity. The reactivity of the denatured protein was similar to those of the native protein. Carboxymethylation of OA gave a product with only 20 % of the inhibition reactivity. Treatment with trypsin did not abolish the allergenic and antigenic reactivities. Limited pepsin hydrolysis destroyed the antigenic structure of the molecule. This indicated that OA allergenic epitopes are mainly determined by the primary structure (sequential/continuous epitopes) and dependent on certain peptide chain length [37]. OVALBUMIN SYNTHETIC PEPTIDES The N-terminal decapeptide OA 1-10 (Fig. 9) was synthesized by SPPS, purified by gel filtration chromatography (GFC) and reversed phase (RP) HPLC. The antigenicity of the purified peptide was examined by quantitative precipitation inhibition using nephelometric methods. The allergenic activity was examined both in vitro and in vivo. OA 1-10, could react with functional structures on reaginic IgE from sera of

323

321

321

329

331

333

335

337

339

The covalent structures of the three OA peptides 323-339, 323-338 and 324-336. Peptide analoges in the second and third rows were 1 and 4 AA shorter than the first one. Truncation of OA 323-339 was done to investigate the effects of the chain length on the activity. FIG. 10.

individuals allergic to egg, inhibiting its further binding to OA. Direct skin test on two patients allergic to egg showed no activity. It was concluded that the decapeptide of the N-terminal of OA encompassed a single Ig binding (haptenic) epitope [38]. Furthermore, the synthetic peptides of OA were valuable in studies of T-lymphocytes recognition of protein antigens [39-411. A correlation between peptide Ia interaction and MHC restriction was demonstrated using OA 323-339. This peptide was found responsible for 25-35 % of BALBc mouse T-cell response to the intact OA molecule [41]. It was suggested that OA 323-339 is closely related to the peptide that is naturally created by the antigen presenting cell (APC) during processing of OA [42,43]. It is reasonable that the same determinant is the one recognized by B-cells leading to the synthesis of antibodies involved in the allergic response. Three analogous peptides mimicking the primary structure of this region of OA [36] were manually prepared by SPPS (Fig. 10). The synthetic preparations of the sites 323-339,323-338 and 324-336, were purified by GFC and R P HPLC. The precision and linearities of the synthesis were deduced from the amino acid composition prior each stage of coupling and the amino acid sequence determination on the ultimate chain. The peptides were conjugated to carrier bovine serum albumin (BSA) through a carbodiimide link prior immunization. This region of OA was clearly shown to be im-

24 S. Elsayed, J. ApoM, E. Holen, H. Ilik, E. Florvaag & T. Dybendal

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Residues

Peptide

AA sequence

11-19

C F D V F K E L K

20-33

V H H A N E N I F Y C P I A

34-46

I M S A L A M V Y L G A K

47-55

D S T R T Q I N K

56-70

V V R F D K L P G F G D S I E

71-84

A Q C G T S V N V H S S L R

85-92

D I L N Q I T K

105-122

L Y A E E R Y P I L P E Y L Q C V K

FIG. 11. Eight peptides in the region 11-122 of OA were synthesized and their biological activity was examined. The covalent structure of the 8 peptides varied in length from 8 to 18 AA.

munogenic in rabbits; It gave antigen precipitation with rabbit anti OA 323-339/BSA in CIE and similarly bound specific IgE from patients allergic to egg in CRIE. These were confirmed by QPI and specific IgE inhibitions. OA 323-339 was concluded to encompass an allergenic and antigenic epitope which was recognized by human and rabbit B-cell. OA peptides in the region 11-122 were similarly, synthesized [45]. All the synthetic peptides were purified firstly by GFC and followed by either ion exchange or RP HPLC (Fig. 11). A test battery of experiments was performed by using rabbit polyclonal antibodies and human specific IgE. Some of the sites were possibly involved in binding of particular Ig paratopes. The immunogenicity of the peptides and their antigenic capacity were also investigated as shown in Fig. 12 [44,45].

TREE POLLEN ALLERGENS Extracts of tree pollen of Betulaceae family (including birch, hazel, alder and others) contain large numbers of protein antigens. Very few of these were demonstrated to be major allergens capable to react with specific IgE antibodies from allergic patients [46-511. The major allergens of birch, alder and hazel, have been studied in details [46-531. The major allergen of birch pollen BV45 (Bet v I), was previously isolated by GFC and eluated in the molecular weight region 15 - 29 Kd [48,49]. Further purification of this fraction on an SP-Trisacryl M cation exchange matrix allowed 6 peaks of which the fourth (BV4A4) and sixth (BV4A6) included the most dominant IgE-binding birch pollen isoallergens, Bet v I and Bet v I1 (Fig. 13). Final purification using peptide Micro Separation System, Applied Biosystem, USA, revealed two sharp peaks with high degree of homogeneities. The criteria of the homogeneities was

Synthetic allergenic epitopes 25

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OA-peptide AA-residue

Purification GFC HPLC

Antigenicity CIE QPI

Mergenicity in vim in vivo

Prim. Structure Comp. Sequence

Mr. Recovery

11-19 880921

t

t

+

-

20-33 881006

t

t

t

-

1612 32.6 %

34-46

t

t

t

-

1350 24 %

47-55 881028

t

+

t

-

1087 48.4 %

56-70 890120

t

t

+

-

1660 91.4 %

71-84 890508

t

t

+

-

1441 19.4 %

85-92 900403

t

t

-

-

105-122 9oO405

t

t

-

-

1109 43.5 %

881111

926 75.2 % 2028 89.6 %

+ = Experiment was done - = Experiment was so far not done FIG. 12. The peptides of the region 11-122of OA. The battery of experiments performed including the purification, examining the antigenicity and allergenicity, AA composition and finally the elucidation of the AA sequence.

settled by automatic N-terminal AA sequence analysis shown in Fig. 14, [54]. Hazel major allergen HIa, Internat. Nomenclature Cor a1 [%], was isolated, purified and 56 residues of its N-terminal amino acid sequence were determined [56-581. High cross reactivity was recently reported between alder, birch and hazel major allergens [59]. The helical and P-sheet potentials (P) of this protein segment were calculated from the P, and P, values of the segments under consideration by different algorithms and segment 29-39 was suggested to prefer a helical conformational state [60,61]. N-terminal AA sequence analysis of the two fractions allowed 51 cleavages with correct identification of phenyl thiohydantoin (F’TH) AA [54]. The sequence data of the two isoallergens showed large

,-----

I I

I

0

10

20

30 40 50 Fraction Number

60

70

80

FIG. 13. The elution diagram of the major allergen of birch BV45 (Bet vI) using SP-Trisacryl M cation exchange matrix. Six peaks were obtained, of which the fourth and sixth included the two isoallergens in question.

homologies (Fig. 14) with the major hazel pollen allergen, Cor a I [56], the reported major birch pollen allergen Ag 23 [58] and

26 S. Elsayed, J. Apold, E. Holen, H. Hk,E. Florvaag & T. Dybendal AA Sequence of Hazel and Birch Isoallergens

1 2 3 4 5 6 7 8 9 10 G V F N Y E A E T T (BV4AS4890628)BetvI V F N Y E T E T T (BV4AS6890403)Bet v I I

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G V F N Y E A E T T (Hazelcoral) G V F N Y E A E T T (Ag23) G V F N Y E T E T T (cDNA sequence) 11 12 13 14 15 16 17 18 19 20 S V I P A A W L W K (BV4AS4890628)BetvI S V I P A A M L F K (BV4AS6890403)BetvII

S V I S V I S V I

P A A X L F K (Hazelcoral) P A A R L F K ( A g 2 3 ) P A A R L F K (cDNAsequence)

21 22 23 24 25 26 21 28 29 30 F I L D G D N L F (BV4AS4890628)BetvI A F I L D G D & L F (BV4AS6890403)BetvII

X

S Y V L D G D K L L (Hazelcoral) A F I L D G D N L F (Ag23) A F I L D G D N L F (cDNA sequence)

31 32 33 34 35 36 37 38 39 40 P K V A P Q A X T S. (BV4AS4890628)BetvI (BV4AS6890403)BetvII P K V A P Q X Q

s

P K V A P Q A L T S (HazelCora1) P K V A P - - - - - (Ag23) P K V A P Q A I S S (cDNA sequence) 41 42 43 44 45 46 41 48 49 50 V E N I Y E B G G W (BV4AS4890628)BetvI y X X p (BV4AS6890403)BetvII V X N X Y V E N V G G N X X P (Hazelcoral) K E N N P W L T A Y (Ag23) V E N I E G N G G P (cDNAsequence)

51 52 53 54 55 56 57 58 59 60

G - - - - - - - - K - - - - - - - - -

(BV4AS4890628)BetvI (BV4AS6 890403)Bet v I1

x x

(Hazel Cor a 1) (Ag 23 1 (cDNA sequence)

L/IX

x

L/I

_ _ - _ -

G T I

K K I

FIG. 14. Comparative N-terminal sequence analysis of the isoallergens of birch and hazel. The f i s t two structures were taken from ref. 54, the third from ref. 56, the fourth from ref. 58 and the last one from ref. 62.

Synthetic allergenic epitopes 27

1-

25 D G

D

K

L

L

P

K

V

A

35 (K)

2-

D

G

D

L

E P

K

V

A

(K)

3-

D

G

D

N N

L

E

P

K

V

A

(K)

D

G

D

K

L

L

P

K

V

A

P

Q

A

L

(K)

G

D

K

L

L

P

K

V

A

P

Q

A

L

(K)

L

4-

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5-

V

L

D

30

FIG. 15. The covalent structures of the synthetic peptides from hazel(l,4 and 5) and birch (2 and 3) major allergens. The synthesis were initiated on a K-esterified resin and all the chains have therefore an extra K at the carbonyl terminal. The underlined AA are the difference between hazel and birch in this region of the chain.

the cDNA sequence of the allergen from cloning birch pollen allergen genes [62]. The sequence homologies support that BV allergens are derived from a gene family expressing several isologous allergens of which two with 13 variable residues in a segment of 51 AA. The antigenicity of the two fractions, Bet v I and Bet v 11, was demonstrated by CIE giving single symmetrical antigenic precipitation [54]. The specific IgE-binding capacity of the two isoallergens was supported by Immunoautoradiography of the plates provided from fused rocket immunoelectrophoresis and by specific IgE binding inhibition.

THE SYNTHETIC PEPTIDES O F TREE POLLEN ALLERGENS Five immunogenic peptides (Fig. 15), from the major allergens of tree pollen extracts (AA residues 23-38), were synthesized by Merrifield SPPS [63]. The primary structures of these peptides were deduced from the known N-terminal amino acid sequence of the major allergen of hazel H1 a [56] and birch Bet v l [54,62]. In a segment of 16 AA (23-38), two residues were the difference between birch and hazel, underlined in Fig. 14. The selection of the peptides was performed on the basis of optimal hydrophobicity using similar algorithms as before [60,61]. The segment 29-34 of these pep-

tides was predicted to prefer helical rather than @sheet conformational state. The immunogenicity of the R P HPLC purified peptides was tested in rabbits using conjugated peptide/BSA through a carbodiimide spacer arm. The results indicated that all constituents of the conjugate could elicit an immune response. The peptides gave precipitation lines in CIE when using rabbit antibodies against the conjugated preparation [63]. All the synthetic peptides a n d analogues from region 23-38, could inhibit, in dose/response patterns, the binding of specific IgE to the intact molecules. Similar results were shown for the shorter segment 25-34 suggesting that the C-terminal tetrapeptide did not contribute to the activity. The allergenicity of these peptides and their analoges are currently examined by PK-inhibition, histamine release and provocation tests [64]. .The N-terminal peptide, AA 1-16 from Bet v I has the following covalent structure: G V F N Y E A E T T S V I P A A

It was similarly synthesized, purified and its biological activity is currently investigated [65]. Preliminary data showed that it gave approximately 15 % specific IgE using individual reaginic sera and one serum pool of patients allergic to birch pollen. Further studies are necessary to ascertain the biological activity of the N-terminal region of birch pollen allergen.

28 S.Ekayed, J. Apold, E. Holen, H. Kk, E. Florvaag & T. Dybendal

by human and rabbit B-lymphocytes.

CONCLUDING REMARKS

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1. The results obtained from Allergen M indicated that three sites were assigned to encompass IgE binding epitopes:

a. Residues 33-44 on the junction between the AB and CD domains; b. Residues 65-74 on the corresponding junction between CD and EF domains; c. Residues 88-96 on the binding loop of the E F domain. 2. The hexadecapeptide 49-64 encompassed two repetitive sequences (D-E-D-K) and (D-E-L-K). This allowed to suggested that the two tetrapeptides located at each termini are mutually critical for the specificity of antibody binding. The hypothesis was confirmed by synthesis of several analogous peptides.

3. The second CaiZ coordipation peptide 88-103 of the EF-domain of Allergen M, was similarly synthesized and its biological activity was elucidated. The immunological reactivity of this site was compatible with a monovalent haptenic function. 4. The peptide of the AB-loop of Allergen M was concluded to be reactive in both IgE and IgG test systems, and function at the level of a divalent determinant. 5. Ovalbumin peptide 1-10 reacted with reaginic IgE and inhibited its further binding to OA. Direct skin test on two patients allergic to egg showed no activity. It was concluded that the decapeptide of the N-terminal of OA was a haptenic epitope. 6. OA 323-339 was clearly shown to be immunogenic in rabbits; it was concluded to encompass an allergenic and antigenic epitope which was recognized

7. Tree pollen allergens peptide analoges from the region 23-38 would inhibit the binding of specific IgE to the intact molecules. Similar results were shown for the shorter segment 25-34 suggesting that the C-terminal tetrapeptide did not contribute to the activity. 8. The N-terminal peptide of birch major allergen 1-16 was also synthesized and is currently investigated. 9. A minimal requirements of an allergenic epitopes was clearly demonstrated to be repetitive four amino acids peptides interspaced by 6 unrelated residues. 10. A certain molecular size of 12-15 amino acids seemed necessary for the immunoglobulin antibody binding and for a successful predictive algorithms of the helicity.

ACKNOWLEDGEMENTS The present studies were supported by the Research Fund of the Norwegian Asthma and Allergy Federation, the Norwegian Research Council for Science and the Humanities. The Technical assistance of Ms. Judit M. Eriksen and Ms. Lena Stavseng is sincerely acknowledged. REFERENCES Thompson RA. The current status of allergy immunotherapy (hyposensitization). Allergy 1989; 44:369-79. 2. Vercelli D, Geha RS. The IgE system. Annals of Allergy 1989; 6 3 4-11. 3. Ishizaka K. Twenty years with IgE: From the identification of IgE to regulatory factors for IgE response. J Immunol 1985; 135: i-x. 4. Ishizaka T, Ishizaka K. Activation of mast cells for mediator release through IgE receptors. Prog Allergy 1984, 34: 188. 1.

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