Identiﬁcation of Potential IgE-Binding Epitopes Contributing to the Cross-Reactivity of the Major Cupressaceae Pectate-Lyase Pollen Allergens (Group 1)

: Pectate-lyase allergens, the group 1 of allergens from Cupressaceae pollen, consist of glycoproteins exhibiting an extremely well-conserved three-dimensional structure and sequential IgE-binding epitopes. Up to 10 IgE-binding epitopic regions were identiﬁed on the molecular surface, which essentially cluster at both extremities of the long, curved β -prism-shaped allergens. Most of these IgE-binding epitopes possess very similar conformations that provide insight into the IgE-binding cross-reactivity and cross-allergenicity commonly observed among Cupressaceae pollen allergens. Some of these epitopic regions coincide with putative N -glycosylation sites that most probably consist of glycotopes or cross-reactive carbohydrate determinants, recognized by the corresponding IgE antibodies from allergic patients. Pectate-lyase allergens of Cupressaceae pollen offer a nice example of structurally conserved allergens that are widely distributed in closely-related plants ( Chamæcyparis , Cryptomeria , Cupressus , Hesperocyparis , Juniperus , Thuja ) and responsible for frequent cross-allergenicity.


ELISA Inhibition
The 9-mer peptides corresponding to the predicted epitope #1 (10GDSNWDQNR18) and epitope #10 (297RSTRDAFSN305) of Cup s 1 were synthesized using the Fmoc chemistry (Genscript USA Inc., Piscataway, NJ, USA). The capacity of synthetic peptides to inhibit the IgE-Cup s 1 interaction was checked by ELISA. Briefly, the wells were coated with 1 µg mL −1 of purified Cup s 1 in phosphate buffer saline (PBS, pH 7.5) [20], and after overnight incubation (4 • C), the wells were washed 3 times with PBS and then incubated (2 h, room temperature) with PBS containing 0.1% Tween 20 (v/v) and 1% BSA (w/v) (PBSTB). Then, 50 µL of 1:30 diluted CPAP serum in PBSTB, previously incubated with 1 mM or 2 mM of synthetic peptide, was added, and the plates were incubated (1 h 30, room temperature) under constant stirring. Then, mouse monoclonal anti-human IgE coupled to alkaline phosphatase (mAb anti human-IgE-AP) was added (diluted 1:500, Sigma-Aldrich, St. Louis, MO, USA) and 1 h incubation was performed. One hundred microliters of AP substrate, the 5-bromo-4-chloro-3-indolylphosphate (BCIP, Promega Corporation, Madison, WI, USA), was added, and after incubation (45 min in the dark), the reaction was stopped. Between each incubation step, 3 washes with PBS containing 0.1% Tween 20 (v/v) were performed, The absorbance at 405 nm was recorded on a Titertek Multiscan spectrophotometer (Labsystems, Thermo Fisher Scientific, Villebon-sur-Yvette, France). Each value is the mean of three separate experiments, and appropriate controls were performed under the same conditions.

IgE-Binding Epitope Mapping: SPOT Experiments
Overlapping 15-mer peptides, frameshifted by three residues, corresponding to the entire amino acid sequences of PL Cry j 1 (access number (AN): BAA05542.1), Cup a 1 (AN: CAB62551.1), Cup s 1 (AN: AAF72625.1), Jun o 1 (AN: CAC48400.1), and Jun v 1 (AN: AAF15427.1), were synthesized using the SPOT technique [21]. For these experiments, we used the Multipep automatic SPOT synthesizer (Intavis Bioanalytical instruments, Cologne, Germany). After different treatments to bind the peptides as described previously [22], the membranes were soaked overnight in tris-buffered saline (TBS) containing 2 mL blocking buffer (Roche-Diagnostics, Meylan, France) and 1 g sucrose. Then, membranes were incubated (2 h) with CPAP serum (1:10 v/v) in the presence of an anti-protease cocktail (Roche) in a moist chamber. Membranes were then soaked in a 1:4000 dilution of mAb anti human-IgE-AP for 1 h. After, the interacting peptide spots were stained for 30 min by adding BCIP. Then, the membranes were washed three times with deionized water and dried for scanning. Negative controls were obtained by using either TBS without serum or a serum from non-allergic patients to Cupressaceae pollen. Between each incubation step, 3 washes with TBS containing 0.1% (v/v) tween (TBSTw) were performed.

Bioinformatics
Multiple amino acid sequence alignment of Jun a 1, Jun o 1, Cup s 1, Jun v 1, and Cry j 1 was carried out with CLUSTAL-X [23]. Molecular modeling of pectate-lyase allergens Cha o 1 (Chamaecyparis obtusa), Cry j 1 (Cryptomeria japonica), Cup s 1 (Cupressus sempervirens), Cup a 1 (C. arizonica), Jun o 1 (Juniperus oxicedrus), Jun v 1 (J. virginiana), pectate-lyase homologs from Metasequoia glyptostroboides (PL-Mg), and Taxodium distichum (PL-Td) was carried out with YASARA Structure [24], using the atomic coordinates of the PL allergen Jun a 1 of mountain cedar (Juniperus ashei) (RCSB Protein Data Bank code 1PXZ) [9] as a template. PROCHECK [25], ANOLEA [26], and the calculated QMEAN scores [27,28] were used to assess the geometric and thermodynamic qualities of the three-dimensional models. As an example, none of the residues of the Cup s 1 model occurred in the non-allowed regions in the Ramachandran plot. Using ANOLEA to evaluate the model, only 8 residues (over 345) of the Lol p 1 model exhibited an energy over the threshold value. Both residues are mainly located in the loop regions connecting the β-sheets and α-helices in the model. The calculated QMEAN score of the model gave a value of 0.717. Reliable values of 0.743 (Cha o 1), 0.730 (Cup a 1), 0.686 (Cry j 1), 0.705 (Jun o 1), and 0.718 (Jun v 1) were obtained for the QMEAN score of the different modeled PL allergens, respectively. Assuming that the putative N-glycosylation sites Asn-X-Thr/Ser are actually glycosylated, a classic bi-antennary high-mannose glycan chain with a tri-mannoside core (Man) 2 -(Man) 3 -(GlcNAc) 2 was modeled using the GlyProt server of Glycosciences (http://www.glycosciences.de/modeling/glyprot/php/main.php, accessed on 14 March 2021) and represented in CPK on the molecular surface of the Cupressaceae PL allergens. The PL allergens from the less closely related species, PL-Td, PL-Mg, and pectate-lyase homolog from Prunus persica (PL-Pp), were similarly modelled using various PL proteins as templates: the endo-xylogalactoronan hydrolase of Aspergillus tubingensis (PDB code 4C2L) [29], the endo-polygalacturonases of Erwinia carotovora (PDB code 1BHE) [30] and Colletotrichum lupini (PDB code 2IQ7) [31], the rhamno-galacturonase A of Aspergillus aculeatus (PDB code 1RMG) [32] and the exo-poly-β-D-galacturonidase of Thermotoga maritima (PDB code 3JUR) [33] for PL-Td and PL-Mg, respectively, and the PL allergen Jun a 1 (PDB code 1PXZ) [9] for PL-Pp. Rather satisfactory QMEAN scores of 0.500 (PL-Td), 0.5819 (PL-Mg), and 0.595 (PL-Pp) were calculated for the three protein PL models, respectively. The surface occupied by the SPOT identified sequential IgE-binding epitopic stretches of the modeled allergens that were differently colored and displayed on the molecular surface of the PL models with YASARA. Molecular cartoons were displayed with Chimera [34]. Electrostatic potentials of Cup s 1, Cry j 1, and Jun o 1 were calculated with YASARA and displayed on the molecular surface as red (electro-negatively charged) and blue (electropositively charged) patches. The root-mean-square deviation of atomic positions (rmsd, in Å) between the superposable Cα of pairwise superposed PL was calculated at the SuperPose web server (http://www.wishart.biology.ualberta.ca, accessed on 16 March 202) [35].
The surface occupied by the SPOT identified sequential IgE-binding epitopic stretches of the modeled allergens that were differently colored and displayed on the molecular surface of the PL models with YASARA. Molecular cartoons were displayed with Chimera [34]. Electrostatic potentials of Cup s 1, Cry j 1, and Jun o 1 were calculated with YASARA and displayed on the molecular surface as red (electro-negatively charged) and blue (electro-positively charged) patches. The root-mean-square deviation of atomic positions (rmsd, in Å) between the superposable Cα of pairwise superposed PL was calculated at the SuperPose web server (http://www.wishart.biology.ualberta.ca, accessed on 16 March 202) [35].

Results
Cupressaceae PL allergens consist of a highly conserved family of pollen proteins with amino acid sequences that share high percentages of both identity (≥75%) and similarity (≥95%) (Figure 1).   According to their amino acid sequence similarities, the three-dimensional models built for Cup s 1, Cry j 1, Jun o 1, Cha o 1, Cup a 1, Jun v 1, and PL-Td from the atomic coordinates of Jun a 1 used as a template exhibit a closely related structural pattern made of three parallel β-sheets organized in a long, curved helical β-prism. Only Cup s 1, Cry j 1, and Jun o 1 structures are shown in Figure 2. o 1), which also belongs to the Cupressaceae, was included in the alignment for comparison.
According to their amino acid sequence similarities, the three-dimensional models built for Cup s 1, Cry j 1, Jun o 1, Cha o 1, Cup a 1, Jun v 1, and PL-Td from the atomic coordinates of Jun a 1 used as a template exhibit a closely related structural pattern made of three parallel β-sheets organized in a long, curved helical β-prism. Only Cup s 1, Cry j 1, and Jun o 1 structures are shown in Figure 2. However, they essentially differ by the degree of N-glycosylation due to the occurrence of two (Cup s 1, Jun a 1, Jun o 1, Jun v 1), three (Cup a 1), or four (Cha o 1, Cry j 1) N-glycosylation sites along their amino acid sequences (Figure 2A-C). In fact, all of these PL allergens consist of glycoallergens or cross-reactive carbohydrate determinants (CCDs), as previously demonstrated for Jun a 1 [11] and Cup a 1 [20]. The N-glycan chains decorating the molecular surface of PL allergens are well exposed and should be readily accessible to the corresponding cross-reacting anti-oligosaccharide IgE antibodies. Up to 10 different IgE-binding epitopic stretches were identified along the amino acid sequences of Jun a 1, Jun o 1, Cup s 1, Jun v 1, and Cry j 1 using the SPOT technique ( Figure 3). However, they essentially differ by the degree of N-glycosylation due to the occurrence of two (Cup s 1, Jun a 1, Jun o 1, Jun v 1), three (Cup a 1), or four (Cha o 1, Cry j 1) Nglycosylation sites along their amino acid sequences (Figure 2A-C). In fact, all of these PL allergens consist of glycoallergens or cross-reactive carbohydrate determinants (CCDs), as previously demonstrated for Jun a 1 [11] and Cup a 1 [20]. The N-glycan chains decorating the molecular surface of PL allergens are well exposed and should be readily accessible to the corresponding cross-reacting anti-oligosaccharide IgE antibodies. Up to 10 different IgE-binding epitopic stretches were identified along the amino acid sequences of Jun a 1, Jun o 1, Cup s 1, Jun v 1, and Cry j 1 using the SPOT technique ( Figure 3). However, some discrepancies occur between the Cupressaceae PL in the number and the extent of the sequential IgE-binding epitopic stretches along the amino acid sequences. In this respect, epitope #9 only occurs in Cup s 1 and Cry j 1 (Figure 4). However, some discrepancies occur between the Cupressaceae PL in the number and the extent of the sequential IgE-binding epitopic stretches along the amino acid sequences. In this respect, epitope #9 only occurs in Cup s 1 and Cry j 1 (Figure 4). Owing to the extremely conserved sequential and structural characteristics obse among PL allergens of the Cupressaceae, all of these molecules share IgE-bin epitopes arrayed on both extremities of the three-dimensional models ( Figure 5). Owing to the extremely conserved sequential and structural characteristics observed among PL allergens of the Cupressaceae, all of these molecules share IgE-binding epitopes arrayed on both extremities of the three-dimensional models ( Figure 5). Allergies 2022, 2, FOR PEER REVIEW 8 Figure 5. Localization of exposed IgE-binding epitopic regions on both faces of the molecular surface of Cup s 1 (A), Cry j 1 (B), and Jun o 1 (C). Exposed IgE-binding epitopes are numbered and colored red (epitope 1), pale blue (epitope 2), green (epitope 3), magenta (epitope 4), yellow (epitope 5), cyan (epitope 6), dark green (epitope 7), purple (epitope 8), pink-brown (epitope 9), and blue (epitope 10), respectively. Note that epitopes 4 and 9 are rarely exposed at the surface of the PL allergens.
These IgE-binding epitopic regions coincide with exposed areas that mainly consist of hydrophilic and charged residues with negative (epitopes 1 and 6) or positive (epitopes 3, 5, 8, and 10) net charges ( Figure 2D-F). In addition, IgE-binding epitopic regions 5 and 6 harbor a putative N-glycosylation site at 127NTS (except for Cry j 1) and 157NVT (except for Cry j 1), respectively, which are predicted to be actually glycosylated according to the GlyProt server ( Figure 2D-F). Although Cry j 1 exhibits four putative N-glycosylation sites, a single site 170NSS, which coincides with IgE-binding epitope 6, is predicted to be actually glycosylated ( Figure 5B). Some coalescence occurs between epitopic regions 1-2-3 and 7-8-9, respectively, which creates two patches of more extended IgE-binding epitopic regions at both ends of the allergen structure ( Figure 6). However, although closely linked to the exposed epitope 8, the potential epitope 9 is scarcely exposed on the surface of the allergens. Synthetic 9-mer peptides corresponding to the well-exposed epitopes 1 (10GDSNWDQNR18), 8 (240NNNYDQWNI248), and 10 (311SSGKTEETN319) of Cup s 1 inhibited (23% for epitope 1, 16% for epitope 8, and 36% for epitope 10, respectively) the interaction of Cup s 1 with IgE from allergic patient sera in ELISA inhibition experiments (Figure 7), which confirms some IgE-binding capacity of the epitopic regions identified , and Jun o 1 (C). Exposed IgE-binding epitopes are numbered and colored red (epitope 1), pale blue (epitope 2), green (epitope 3), magenta (epitope 4), yellow (epitope 5), cyan (epitope 6), dark green (epitope 7), purple (epitope 8), pink-brown (epitope 9), and blue (epitope 10), respectively. Note that epitopes 4 and 9 are rarely exposed at the surface of the PL allergens.
These IgE-binding epitopic regions coincide with exposed areas that mainly consist of hydrophilic and charged residues with negative (epitopes 1 and 6) or positive (epitopes 3, 5, 8, and 10) net charges ( Figure 2D-F). In addition, IgE-binding epitopic regions 5 and 6 harbor a putative N-glycosylation site at 127NTS (except for Cry j 1) and 157NVT (except for Cry j 1), respectively, which are predicted to be actually glycosylated according to the GlyProt server ( Figure 2D-F). Although Cry j 1 exhibits four putative N-glycosylation sites, a single site 170NSS, which coincides with IgE-binding epitope 6, is predicted to be actually glycosylated ( Figure 5B). Some coalescence occurs between epitopic regions 1-2-3 and 7-8-9, respectively, which creates two patches of more extended IgE-binding epitopic regions at both ends of the allergen structure ( Figure 6). However, although closely linked to the exposed epitope 8, the potential epitope 9 is scarcely exposed on the surface of the allergens. Figure 5. Localization of exposed IgE-binding epitopic regions on both faces of the molecular sur face of Cup s 1 (A), Cry j 1 (B), and Jun o 1 (C). Exposed IgE-binding epitopes are numbered and colored red (epitope 1), pale blue (epitope 2), green (epitope 3), magenta (epitope 4), yellow (epitope 5), cyan (epitope 6), dark green (epitope 7), purple (epitope 8), pink-brown (epitope 9), and blue (epitope 10), respectively. Note that epitopes 4 and 9 are rarely exposed at the surface of the PL allergens.
These IgE-binding epitopic regions coincide with exposed areas that mainly consis of hydrophilic and charged residues with negative (epitopes 1 and 6) or positive (epitopes 3, 5, 8, and 10) net charges ( Figure 2D-F). In addition, IgE-binding epitopic regions 5 and 6 harbor a putative N-glycosylation site at 127NTS (except for Cry j 1) and 157NVT (except for Cry j 1), respectively, which are predicted to be actually glycosylated according to the GlyProt server ( Figure 2D-F). Although Cry j 1 exhibits four putative N-glycosylation sites, a single site 170NSS, which coincides with IgE-binding epitope 6, is predicted to be actually glycosylated ( Figure 5B). Some coalescence occurs between epitopic regions 1-2-3 and 7-8-9, respectively, which creates two patches of more ex tended IgE-binding epitopic regions at both ends of the allergen structure ( Figure 6) However, although closely linked to the exposed epitope 8, the potential epitope 9 is scarcely exposed on the surface of the allergens. Synthetic 9-mer peptides corresponding to the well-exposed epitopes 1 (10GDSNWDQNR18), 8 (240NNNYDQWNI248), and 10 (311SSGKTEETN319) of Cup s 1 inhibited (23% for epitope 1, 16% for epitope 8, and 36% for epitope 10, respectively) the interaction of Cup s 1 with IgE from allergic patient sera in ELISA inhibition experiments (Figure 7), which confirms some IgE-binding capacity of the epitopic regions identified Synthetic 9-mer peptides corresponding to the well-exposed epitopes 1 (10GDSNWDQNR18), 8 (240NNNYDQWNI248), and 10 (311SSGKTEETN319) of Cup s 1 inhibited (23% for epitope 1, 16% for epitope 8, and 36% for epitope 10, respectively) the interaction of Cup s 1 with IgE from allergic patient sera in ELISA inhibition experiments (Figure 7), which confirms some IgE-binding capacity of the epitopic regions identified by the SPOT technique. No inhibition occurred with an unrelated 9-mer peptide RTTADRQTA corresponding to an IgE-binding epitope of the Mal d 3 fruit allergen [36], used as a negative control.
Allergies 2022, 2, FOR PEER REVIEW by the SPOT technique. No inhibition occurred with an unrelated 9-mer pep RTTADRQTA corresponding to an IgE-binding epitope of the Mal d 3 fruit allergen used as a negative control. According to these structural similarities, most of the identified potential contin IgE-binding epitopes are similarly distributed on the molecular surfaces of the PL a gens ( Figure 5A-C). Looking at the conformation of the surface occupied by the served IgE-binding epitope 1 in closely related PL allergens, it reveals an overall formation all the more similar as it corresponds to an identical or very close amino sequence ( Figure 8C). In this respect, epitope 1 from Cup s 1, Cup a 1, Cry j 1, Jun o 1 v 1, and Cha o 1, which possess an identical or almost identical (Cha o 1) sequence hibit a very similar conformation. Conversely, epitope 1 from PL-Td and PL-Mg, w differ from other epitopes by a divergent amino acid sequence, exhibit a quite diffe overall conformation. Surprisingly, PL-Pp from peach (Punus persica), a member o distantly related Rosaceae family, is more like the PL allergens from cypresses (Cup Cup a 1) and junipers (Jun o 1, Jun v 1) than the more closely related PL allergens drawn redwood (PL-Mg) and bald cypress (PL-Td). All the three-dimensional models built for PL allergens from Cupressaceae including Cup s 1 from Cupressus sempervirens, Cup a 1 from C. arizonica, Cry j 1 from Cryptomeria japonica, Jun o 1 from Juniperus oxycedrus, Jun v 1 from J. virginiana, Chao 1 from Chamaecyparis obtusa, PL-Td from Taxodium distichum, and PL-Mg from Metasequoia glyptostroboides and PL-Pp from Prunus persica (Rosaceae), exhibit a very similar fold and are, therefore, readily superposable ( Figure 8A,B).
According to these structural similarities, most of the identified potential continuous IgE-binding epitopes are similarly distributed on the molecular surfaces of the PL allergens ( Figure 5A-C). Looking at the conformation of the surface occupied by the conserved IgE-binding epitope 1 in closely related PL allergens, it reveals an overall conformation all the more similar as it corresponds to an identical or very close amino acid sequence ( Figure 8C). In this respect, epitope 1 from Cup s 1, Cup a 1, Cry j 1, Jun o 1, Jun v 1, and Cha o 1, which possess an identical or almost identical (Cha o 1) sequence, exhibit a very similar conformation. Conversely, epitope 1 from PL-Td and PL-Mg, which differ from other epitopes by a divergent amino acid sequence, exhibit a quite different overall conformation. Surprisingly, PL-Pp from peach (Punus persica), a member of the distantly related Rosaceae family, is more like the PL allergens from cypresses (Cup s 1, Cup a 1) and junipers (Jun o 1, Jun v 1) than the more closely related PL allergens from drawn redwood (PL-Mg) and bald cypress (PL-Td). In addition to the partial conformational similarity of epitope 1 observed between the Cupressaceae PL allergens and PL-Pp, the alignment of amino acid sequences of Cup s 1 and PL-Pp suggests that additional conformational similarities would occur between both allergens (Figure 9). Especially, the amino acid sequence stretches of PL-Pp corresponding to epitopes 3, 4, 7, 9, and 10 of Cup s 1 display more than 50% identity, which suggests additional conformational epitopic similarities between both proteins. In addition to the partial conformational similarity of epitope 1 observed between the Cupressaceae PL allergens and PL-Pp, the alignment of amino acid sequences of Cup s 1 and PL-Pp suggests that additional conformational similarities would occur between both allergens ( Figure 9). Especially, the amino acid sequence stretches of PL-Pp corresponding to epitopes 3, 4, 7, 9, and 10 of Cup s 1 display more than 50% identity, which suggests additional conformational epitopic similarities between both proteins.

Discussion
Pectate-lyase allergens of Cupressaceae pollen consist of a homogenous family of structurally conserved glycoproteins. Their 38 kDa-polypeptide chains exhibit a similar three-dimensional fold made of three parallel β-sheets organized in a long, curved β-prism structure. However, they differ by the degree of glycosylation due to the occurrence of two (Cup s 1, Jun a 1, Jun o 1, Jun v 1), three (Cup a 1), or four (Cha o 1, Cry j 1) putative N-glycosylation sites along their polypeptide chains.
Using the SPOT technique, up to 10 sequential IgE-binding epitopic stretches were identified along the amino acid sequences of the PL allergens. In spite of a few discrepancies from one allergen to another, they occur at the same locations in the amino acid sequences of the different allergens. Moreover, the observed localization was remarkably reproducible for each of the assayed PL allergens. Most of these epitopic regions cluster at both ends of the β-prism structure to form more extended epitopic areas that are reminiscent of discontinuous IgE-binding epitopes. They essentially consist of hydrophilic and charged residues susceptible to create hydrogen bonds and electrostatic interactions with the corresponding paratope of IgE antibodies. Along this line, the PL allergen Jun a 1 from the mountain cedar has been reported to contain both linear and conformational IgE-binding epitopes [13]. Two of the IgE-binding epitopes identified in Cupressus and Juniperus PL allergens, namely epitopes 5 and 6, contain a N-glycosylation site, which is predicted to be actually glycosylated according to the GlyProt server and could thus correspond to glycotopes ( Figure 2G-I). However, grafted oligosaccharides do not prevent the induction of IgE antibodies against the epitopic region corresponding to the glycosylation site. Carbohydrate moieties of the Cupressaceae allergens were extensively investigated [11,20], and their role as IgE-binding determinants/glycotopes has been clearly demonstrated for Cup a 1 from Arizona cypress pollen [4,[37][38][39] and Jun a 1 from mountain cedar pollen [11].

Discussion
Pectate-lyase allergens of Cupressaceae pollen consist of a homogenous family of structurally conserved glycoproteins. Their 38 kDa-polypeptide chains exhibit a similar three-dimensional fold made of three parallel β-sheets organized in a long, curved β-prism structure. However, they differ by the degree of glycosylation due to the occurrence of two (Cup s 1, Jun a 1, Jun o 1, Jun v 1), three (Cup a 1), or four (Cha o 1, Cry j 1) putative N-glycosylation sites along their polypeptide chains.
Using the SPOT technique, up to 10 sequential IgE-binding epitopic stretches were identified along the amino acid sequences of the PL allergens. In spite of a few discrepancies from one allergen to another, they occur at the same locations in the amino acid sequences of the different allergens. Moreover, the observed localization was remarkably reproducible for each of the assayed PL allergens. Most of these epitopic regions cluster at both ends of the β-prism structure to form more extended epitopic areas that are reminiscent of discontinuous IgE-binding epitopes. They essentially consist of hydrophilic and charged residues susceptible to create hydrogen bonds and electrostatic interactions with the corresponding paratope of IgE antibodies. Along this line, the PL allergen Jun a 1 from the mountain cedar has been reported to contain both linear and conformational IgE-binding epitopes [13]. Two of the IgE-binding epitopes identified in Cupressus and Juniperus PL allergens, namely epitopes 5 and 6, contain a N-glycosylation site, which is predicted to be actually glycosylated according to the GlyProt server and could thus correspond to glycotopes ( Figure 2G-I). However, grafted oligosaccharides do not prevent the induction of IgE antibodies against the epitopic region corresponding to the glycosylation site. Carbohydrate moieties of the Cupressaceae allergens were extensively investigated [11,20], and their role as IgE-binding determinants/glycotopes has been clearly demonstrated for Cup a 1 from Arizona cypress pollen [4,[37][38][39] and Jun a 1 from mountain cedar pollen [11].
According to their amino acid sequence conservation, PL allergens from the Cupressaceae and other closely related families of Taxodiaceae (PL-Mg, PL-Td) are readily superposed with a rms of 0.36-Å, 0.34-Å, and 0.31-Å for the couples Cup s 1/Cry j 1, Cup s 1/Cha o 1, and Cup s 1/Jun o 1, respectively. With other less closely related PL-Td and PL-Mg, rms of 16.59-Å and 19.41-Å were measured for the superposed couples Cup s 1/PL-Td and Cup s 1/PL-Mg, respectively ( Figure 3A,B).
The IgE-binding epitopes of the Cupressaceae PL allergens and, especially, the IgEbinding epitopes 1, 2, and 3, are nicely conserved in other closely related PL allergens from Cupressaceae, e.g., in Cup a 1 from Cupressus arizonica (Hesperocyparis arizonica) and Cha o 1 from Chamaecyparis obtusa ( Figure 3C). Pectate lyase PL-Td from the western red cedar (Taxodium distichum), which also belongs to the Cupressaceae family, readily cross-reacts with Cup s 1 from Cupressus sempervirens pollen [37]. Some IgE-binding epitope similarity also occurs with a PL from Pinus sitchensis (access. ABK 2501), which belongs to the closely related Pinaceae family, with identity percentages ranging from 15% (epitope 7) up to 60% (epitopes 1 and 6). This IgE-binding epitope relatedness could extend the IgE-binding cross-reactivity of Cupressaceae to other closely related conifers.
A rather frequent cross-allergenicity between Cupressaceae pollen and peach fruit has been reported in patients allergic to cypress from Mediterranean countries defining a pollen food-associated syndrome [40]. Besides allergens from the recently described Gibberellin-regulated protein family shown to be totally or partly responsible for such cross-allergenicity in peach and cypress pollen [19,41], a 45-kDa protein was also reported as a cross-reactive allergen [40]. In this respect, pectate-lyase from peach fruit (access BAF43572, ≥42 kDa) exhibits some IgE-binding epitope similarity to Cup s 1 with identity percentages from 25% (epitopes 6 and 9) up to 67-70% (epitopes 1 and 7), which could also participate in the reported cross-allergenicity ( Figure 4).
All PL allergens from the Cupressaceae (Cha o 1, Cry j 1, Cup s 1, Jun o 1, Jun v 1) and Taxodiaceae (PL-Mg, PL-Td), contain two (Cup s 1, Jun o 1, Jun v 1, PL-Td), three (Cup a 1, PL-Mg), four (Cry j 1), or five (Cha o 1) putative N-glycosylation sites, and thus correspond to often highly glycosylated allergens. The N-oligosaccharide linked to one of the three N-glycosylation sites of Cup a 1 has been characterized as a mixture of complex glycans containing β1,2-Xyl and α1,3-Fuc linked to the core βMan and the first core GlcNAc residues, respectively [20]. A monoclonal antibody mAb 5E6 that specifically recognizes this Cup a 1 glycotope was further characterized [39]. Comparison between the native glycosylated Cup a 1 and a non-glycosylated recombinant Cup a 1 expressed in E. coli allowed researchers to discriminate between IgE from cypress allergic patients that recognize the carbohydrate epitopes and those IgE that interact with peptidic epitopes [42]. Moreover, IgE that recognize carbohydrate epitopes are able to induce histamine release from basophils, suggesting they display a functional role in the cypress allergy and should be responsible for some cross-reactivity and cross-allergenicity between closely related Cupressaceae species.
According to their widespread distribution in plant organs, e.g., in pollens and fruits, pectate-lyases appear as structurally conserved pan-allergens responsible for both the sensitization and allergenic reaction in susceptible individuals. Consequently, they display a high degree of clinically relevant IgE-binding cross-reactivity. This cross-allergenicity is of paramount importance for allergic patients, since the pollination periods of the various species of Cupressaceae follow one another, thus extending the exposure to cross-reactive allergens from February to July in Mediterranean countries.