Characterization and Gene Cloning of an Acidic Thaumatin-Like Protein (TLP 1), an Allergen from Sapodilla Fruit (Manilkara zapota)

Background: Allergy to sapodilla (Manilkara zapota) fruit ingestion is rare. An independent study from our
group has identified a basic thaumatin-like protein (TLP 2) as the major allergen. The present study was aimed
at identifying and characterizing additional allergens from sapodilla.
Methods: Allergic subjects were identified by case history, skin prick test (SPT) and allergen-specific IgE.
Sapodilla extract was fractionated using SP-Sepharose into 3 components (SP1, SP2 and SP3) which were
analyzed by native�SDS-PAGE, IgE-immunoblot, isoelectric focusing (IEF) and N-terminal sequencing. The
conserved regions of plant TLPs and the N-terminal sequence were used to design primers for PCR.
Results: SPT and ELISA confirmed a subject with oral allergy syndrome (OAS) to sapodilla and custard apple.
Two proteins (26.9 and 24.5 kDa; reducing conditions) were detected as allergens, of which the latter in SP2
has already been identified as basic TLP (TLP 2). The 26.9 kDa protein present in SP1 was identified as an
acidic TLP based on native PAGE, IEF and N-terminal sequencing. Presence of a basic β-1,3-glucanase in
SP3 was inferred by zymography. Sequence analysis of the genomic clone of the acidic TLP gene revealed
that it is intronless and non-glycosylated. Evolutionary relatedness to olive, grape and kiwi fruit allergenic TLPs
were inferred by phylogenetic analysis.
Conclusions: An acidic TLP (TLP 1) was identified as a new allergen in sapodilla. TLP 1 is a single polypeptide
(207 residues) belonging to the thaumatin family of the GH64-TLP-SF superfamily. Clinically, sapodilla
should be considered in the list of fruits causing OAS.