Interactome of Ambrosia artemisifolia pollen allergens and nanoplastics in simulated lung fluids

Interaction of nanoplastics (NP) with proteins produce a complex. Surrounded by protein coat - corona, NP develop new biological properties such as escaping from immune system, prolonged persistence in circulation and interference with cellular and molecular processes. After initial binding of molecules with higher affinity and formation of hard corona (HC), soft corona (SC) finalise biofilm NP encapsulation. The pollen of Ambrosia artemisifolia is widespread cause of respiratory allergic reactions, a single plant may produce about a billion grains of pollen per season. Ambrosia a. allergen was chosen as model system for investigation of NPs interactions with respiratory allergens. Ambrosia pollen extract (APE) was prepared by a short protocol, mimicking environmental exposure (ie. rainfall) and/or lung fluid extraction. APE was incubated in vitro with NPs of respirable size (nPP and nPET, 100 nm). Corona formation and composition were analysed by SDS PAGE, Schiff glycoprotein staining and immunoblotting with patients" sera. The profile of proteins bound to NPs with the highest affinity to be embedded into corona due to Vroman effect was analysed by mass spectrometry. These proteins are the most likely to be carried to respiratory system by NPs and to induce sensitization/allergic response. In SC of both nPET and nPP several main bands from APE are present, profile is similar to the bulk and extract profiles. HC shows different protein profile from SC for both NPs. There is enrichment of band at around 26-30 kDa (Mw corresponds to Amb a 4 allergen). This heavily glycosylated allergen consists of several isoforms. Schiff staining of glycoproteins confirmed that there is a glycopeptide in HC of both nPET and nPP. It seems that this glycopeptide, representing fragments of several proteins, Mw around 5 kDa preferentially binds to the surface of NPs. IgE-reactive protein at around 16 kDa in soft corona (and bulk) of both nPP and nPET was identified as nsLTP (Amb a 6) and plastocyanin (Amb a 3). In shotgun proteomics of Ambrosia elatior the major hit according to the number of peptides detected was PCC13-62-like protein, so far never shown at the protein level. Plant basic secretory protein, PBSP, also detected with high confidence in the extracts, was just a predicted protein, too. These two proteins have been detected in HC of nPP particles, major IgE-reactive band at 34 kDa. Specific adsorption of PCC13-62-like protein to NPs allows us to suggest novel protein PCC13-62-like protein to be lead biocorona component of NPs in the simulated lung fluids and a novel protein of Ambrosia pollen identified at the proteome level in this study. Acknowledgment. Funding from Horizon 2020, No965173, IMPTOX, by Ministry of Science, Innovation and Technological Development, Republic of Serbia, grant No451-03-66/2024-03/200168