Even though amyloid aggregates were discovered many years ago the mechanism of their formation is still a mystery. Because of their connection to many of untreatable neurodegenerative diseases the motivation for finding a common aggregation path is high. We report a new high heat induced fibrillization path of a model protein β-lactoglobulin (BLG) when incubated in glycine instead of water at pH 2. By combining atomic force microscopy (AFM), transmission emission microscopy (TEM), dynamic light scattering (DLS) and circular dichroism (CD) we predict that the basic building blocks of fibrils made in glycine are not peptides, but rather spheroid oligomers of different height that form by stacking of ring-like structures. Spheroid oligomers linearly align to form fibrils by opening up and combining. We suspect that glycine acts as an hydrolysation inhibitor which consequently promotes a different fibrillization path. By combining the known data on fibrillization in water with our experimental conclusions we come up with a new fibrillization scheme for BLG. We show that by changing the fibrillization conditions just by small changes in buffer composition can dramatically change the aggregation pathway and the effect of buffer shouldn't be neglected. Fibrils seen in our study are also gaining more and more attention because of their pore-like structure and a possible cytotoxic mechanism by forming pernicious ion-channels. By preparing them in a simple model system as BLG we opened a new way to study their formation.

• MeSH
• amyloid * chemie   MeSH
• glycin farmakologie   MeSH
• laktoglobuliny * chemie   MeSH
• mikroskopie atomárních sil metody   MeSH
• voda MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND: Beta-lactoglobulin (BLG) is a bovine lipocalin in milk with an innate defense function. The circumstances under which BLG is associated with tolerance of or allergy to milk are not understood. OBJECTIVE: Our aims were to assess the capacity of ligand-free apoBLG versus loaded BLG (holoBLG) to protect mice against allergy by using an iron-quercetin complex as an exemplary ligand and to study the molecular mechanisms of this protection. METHODS: Binding of iron-quercetin to BLG was modeled and confirmed by spectroscopy and docking calculations. Serum IgE binding to apoBLG and holoBLG in children allergic to milk and children tolerant of milk was assessed. Mice were intranasally treated with apoBLG versus holoBLG and analyzed immunologically after systemic challenge. Aryl hydrocarbon receptor (AhR) activation was evaluated with reporter cells and Cyp1A1 expression. Treated human PBMCs and human mast cells were assessed by fluorescence-activated cell sorting and degranulation, respectively. RESULTS: Modeling predicted masking of major IgE and T-cell epitopes of BLG by ligand binding. In line with this modeling, IgE binding in children allergic to milk was reduced toward holoBLG, which also impaired degranulation of mast cells. In mice, only treatments with holoBLG prevented allergic sensitization and anaphylaxis, while sustaining regulatory T cells. BLG facilitated quercetin-dependent AhR activation and, downstream of AhR, lung Cyp1A1 expression. HoloBLG shuttled iron into monocytic cells and impaired their antigen presentation. CONCLUSION: The cargo of holoBLG is decisive in preventing allergy in vivo. BLG without cargo acted as an allergen in vivo and further primed human mast cells for degranulation in an antigen-independent fashion. Our data provide a mechanistic explanation why the same proteins can act either as tolerogens or as allergens.

• MeSH
• alergie na mléko farmakoterapie   imunologie   MeSH
• laktoglobuliny * chemie   farmakokinetika   farmakologie   MeSH
• leukocyty mononukleární imunologie   MeSH
• lidé MeSH
• mastocyty imunologie   MeSH
• mléko chemie   MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• skot MeSH
• železo * chemie   farmakokinetika   farmakologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• duodenum sekrece   MeSH
• laktoglobuliny analýza   MeSH
• nemoci slinivky břišní diagnóza   MeSH

• MeSH
• laktoglobuliny chemie   MeSH
• lékové interakce MeSH
• thiouracil chemie   MeSH

• MeSH
• fluorescenční spektrometrie MeSH
• ionty MeSH
• koncentrace vodíkových iontů MeSH
• laktoglobuliny biosyntéza   MeSH
• teplota MeSH
• vápník MeSH

• MeSH
• biomedicínský výzkum MeSH
• diabetes mellitus patofyziologie   MeSH
• laktoglobuliny biosyntéza   MeSH
• pankreas fyziologie   patofyziologie   MeSH
• pankreatická šťáva analýza   MeSH
• pankreatitida patofyziologie   MeSH

• MeSH
• alergie etiologie   MeSH
• dieta MeSH
• dítě MeSH
• dospělí MeSH
• infekce imunologie   MeSH
• kojenec MeSH
• kojení fyziologie   MeSH
• laktoglobuliny MeSH
• lidé MeSH
• mateřské mléko cytologie   MeSH
• průjem kojenců MeSH
• těhotenství MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• kojenec MeSH
• lidé MeSH
• těhotenství MeSH
• ženské pohlaví MeSH

• MeSH
• chronické selhání ledvin komplikace   MeSH
• dialýza ledvin MeSH
• laktoglobuliny izolace a purifikace   MeSH
• lidé MeSH
• pankreatitida enzymologie   MeSH
• Check Tag
• lidé MeSH

• MeSH
• karcinom diagnóza   MeSH
• laktoglobuliny analýza   diagnostické užití   MeSH
• lidé MeSH
• nádory slinivky břišní diagnóza   MeSH
• nemoci slinivky břišní diagnóza   MeSH
• pankreas MeSH
• pankreatitida diagnóza   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• srovnávací studie MeSH

• MeSH
• cholecystokinin diagnostické užití   MeSH
• duodenum MeSH
• laktoglobuliny analýza   MeSH
• lidé MeSH
• nádory slinivky břišní diagnóza   enzymologie   MeSH
• pankreas analýza   sekrece   MeSH
• pankreatitida diagnóza   enzymologie   MeSH
• sekretin diagnostické užití   MeSH
• Check Tag
• lidé MeSH