Mapping of the Immunodominant Regions of Shrimp Tropomyosin Pan b 1 by Human IgE-Binding and IgE Receptor Crosslinking Studies

Abstract

Background: Epitope mapping of an allergen is generally done by IgE-binding assays with short synthetic peptides, but this provides little information about which domains are responsible for IgE receptor crosslinking on effector cells. Our aim was to map the immunodominant regions of shrimp tropomyosin by both IgE-binding and IgE-receptor crosslinking studies. Methods: Five overlapping fragments covering Pandalus borealis tropomyosin were cloned, expressed in Escherichia coli and characterized by circular dichroism spectroscopy, native PAGE and bis(sulfosuccinimidyl) suberate-crosslinking. IgE binding was detected by Western blot, indirect ELISA and inhibition ELISA, and IgE receptor crosslinking was investigated by basophil activation test and skin prick test with Norwegian shrimp allergic adults. Results: The N- and C-terminal fragments of tropomyosin showed the highest amount of secondary structure. Western blot studies showed preferential binding to the terminal fragments, while indirect and inhibition ELISA studies showed binding to all fragments, but with individual variations. Basophil CD63 expression was upregulated by all fragments at high concentrations (1 µg/ml) and showed individual variations comparable to ELISA results. A mixture of the fragments with equal molar ratios induced comparably strong CD63 activation as for tropomyosin. Skin prick test studies showed positive responses to the terminal and middle fragments and increased responses to the fragment mixture compared to whole tropomyosin. Conclusions: The terminal and middle fragments of tropomyosin had the highest IgE reactivity, but overall no clear immunodominant region was observed in this study. These results correlated well with previous studies with short peptides. Dividing shrimp tropomyosin into five fragments did not reduce the allergenicity of the protein.

Allergen Valency, Dose, and FcεRI Occupancy Set Thresholds for Secretory Responses to Pen a 1 and Motivate Design of Hypoallergens

“…For the small fraction that do form dimers based upon SDS-PAGE analysis, it is intriguing to note that the coiled-coil orientation of two identical epitopes in any pair of dimers renders their distance very close (<2 nm). Our preparations showed reduced allergenicity by comparison to an earlier study (51), presumably due to differences in design, affinity purification and/or concentration steps that will be critical to carry forward for potential clinical development.…”

“…While pepsin-cleaved fragments of natural Pen a 1 are potent initiators of degranulation, it has been reported that short, recombinant Pen a 1-derived polypeptides encompassing a single predominant IgE binding epitope have diminished capacity (51). As shown in Figure 5A, we designed and expressed five sequential Pen a 1-recombinant polypeptides (FR1 1–79 , FR2 68–127 , FR3 121–181 , FR4 172–236 , FR5 224–284 ), each 60–79 amino acids in length.…”

“…It makes sense to consider these structural features for the design of hypoallergenic allergy vaccines (73). Improved safety profiles might be expected after reengineering Pen a 1 to have reduced or abolished IgE binding (13) or by lowering the crosslinking capacity (14, 51). Mutagenesis and epitope deletion strategies have been shown to reduce allergenicity in the shrimp allergen, Met e 1 (74).…”

Tropomyosins in mosquito and house dust mite cross‐react at the humoral and cellular level

“…Proliferative responses of splenocytes from mice immunized with either rAed a 10.01 or rAed a 10.02 to different concentrations of all recombinant tropomyosins were assessed ( Figure 5A,B). rAed a 10.01-immunized mice showed stronger proliferative responses to Nevertheless, the T cell-activating regions Aed a 10.01 [21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40] , Aed a 10.01 111-130 , Aed a 10.01 [161][162][163][164][165][166][167][168][169][170][171][172][173][174][175][176][177][178][179][180] and Aed a 10.01 221-270 corresponded to T cell-activating regions in shrimp tropomyosin previously identified in humans. 19 Furthermore, these regions are highly conserved within the amino acid sequences of all tropomyosins and show 55%-100% of sequence identity (70%-100% of similarity)…”

News in Cellular Allergology: A Review of the Human Mast Cell and Basophil Granulocyte Literature from January 2013 to May 2015

“…Purified natural and recombinant Pan b 1 at 0.5 μg/ml (15 μ M ) activated basophils to a similar degree as 1 μg/ml of shrimp extract [58]. BAT with molecular allergen Ara h 2 is good at identifying true peanut-allergic Mediterranean patients and distinguishing them from peach-allergic patients [59].…”