Atopic Dermatitis Patients with Pet Dander Sensitization Mount IgE and T cell Responses to Mammalian Cystatins Including the Human Self-Protein

Background: Immediate as well as delayed-type hypersensitivity immune reactions to pet-borne allergens are commonly observed in atopic diseases. Further on in atopic dermatitis (AD), cross-reactivity to self-proteins is discussed to contribute to the disease. Human cystatin A and the cat allergen Fel d 3 belong to the cystatin family, an evolutionary conserved protein family. The objective of the present study was to assess cross-reactivity between mammalian cystatins and to analyze T cell responses to cystatin in AD patients sensitized to pet dander. Methods: cDNA coding for dog cystatin was cloned from dog skin. Sera of 245 patients with IgE-sensitization to cat and dog dander were tested for IgE-binding to recombinantly expressed feline, canine, and human cystatin, respectively. Of these, 141 were also diagnosed for AD. Results: Cystatin-specific IgE was detected in 14.7 %(36) of patients, of which 19 suffered from AD. Within the AD patients, 9 carried measurable IgE against all three cystatins. Cystatin-sensitized AD patients did not differ from non-cystatin sensitized patients in terms of disease severity, age or total IgE levels. T cell cytokine measurements showed elevated IL-4 levels after stimulation with feline and human cystatin. Conclusion: The humoral response suggests that next to Fel d3 also the homologous protein from dog might play a role in allergy. Further on, the human cystatin appears to be capable of driving a type2 immune response in sensitized AD patients and may therefore be considered a so-called autoallergen, as it has been proposed for other evolutionary conserved proteins.

piggyBac Transposition and the Expression of Human Cystatin C in Transgenic Chickens

A bioreactor can be used for mass production of therapeutic proteins and other bioactive substances. Although various methods have been developed using microorganisms and animal cells, advanced strategies are needed for the efficient production of biofunctional proteins. In microorganisms, post-translational glycosylation and modification are not performed properly, while animal cell systems require more time and expense. To overcome these problems, new methods using products from transgenic animals have been considered, such as genetically modified cow’s milk and hen’s eggs. In this study, based on a non-viral piggyBac transposition system, we generated transgenic bioreactor chickens that produced human cystatin C (hCST3). There were no differences in the phenotype or histochemical structure of the wild-type and hCST3-expressing transgenic chickens. Subsequently, we analyzed the hCST3 expression in transgenic chickens, mainly in muscle and egg white, which could be major deposition warehouses for hCST3 protein. In both muscle and egg white, we detected high hCST3 expression by ELISA and Western blotting. hCST3 proteins were efficiently purified from muscle and egg white of transgenic chickens using a His-tag purification system. These data show that transgenic chickens can be efficiently used as a bioreactor for the mass production of bioactive materials.

DMPC Phospholipid Bilayer as a Potential Interface for Human Cystatin C Oligomerization: Analysis of Protein-Liposome Interactions Using NMR Spectroscopy

Studies revolving around mechanisms responsible for the development of amyloid-based diseases lay the foundations for the recognition of molecular targets of future to-be-developed treatments. However, the vast number of peptides and proteins known to be responsible for fibril formation, combined with their complexity and complexity of their interactions with various cellular components, renders this task extremely difficult and time-consuming. One of these proteins, human cystatin C (hCC), is a well-known and studied cysteine-protease inhibitor. While being a monomer in physiological conditions, under the necessary stimulus—usually a mutation, it tends to form fibrils, which later participate in the disease development. This process can potentially be regulated (in several ways) by many cellular components and it is being hypothesized that the cell membrane might play a key role in the oligomerization pathway. Studies involving cell membranes pose several difficulties; therefore, an alternative in the form of membrane mimetics is a very attractive solution. Here, we would like to present the first study on hCC oligomerization under the influence of phospholipid liposomes, acting as a membrane mimetic. The protein–mimetic interactions are studied utilizing circular dichroism, nuclear magnetic resonance, and size exclusion chromatography.

The Influence of the Mixed DPC:SDS Micelle on the Structure and Oligomerization Process of the Human Cystatin C

Human cystatin C (hCC), a member of the superfamily of papain-like cysteine protease inhibitors, is the most widespread cystatin in human body fluids. Physiologically active hCC is a monomer, which dimerization and oligomerization lead to the formation of the inactive, insoluble amyloid form of the protein, strictly associated with cerebral amyloid angiopathy, a severe state causing death among young patients. It is known, that biological membranes may accelerate the oligomerization processes of amyloidogenic proteins. Therefore, in this study, we describe an influence of membrane mimetic environment—mixed dodecylphosphocholine:sodium dodecyl sulfate (DPC:SDS) micelle (molar ratio 5:1)—on the effect of the hCC oligomerization. The hCC–micelle interactions were analyzed with size exclusion chromatography, circular dichroism, and nuclear magnetic resonance spectroscopy. The experiments were performed on the wild-type (WT) cystatin C, and two hCC variants—V57P and V57G. Collected experimental data were supplemented with molecular dynamic simulations, making it possible to highlight the binding interface and select the residues involved in interactions with the micelle. Obtained data shows that the mixed DPC:SDS micelle does not accelerate the oligomerization of protein and even reverses the hCC dimerization process.

Structural Characterization of Covalently Stabilized Human Cystatin C Oligomers

Human cystatin C (HCC), a cysteine-protease inhibitor, exists as a folded monomer under physiological conditions but has the ability to self-assemble via domain swapping into multimeric states, including oligomers with a doughnut-like structure. The structure of the monomeric HCC has been solved by X-ray crystallography, and a covalently linked version of HCC (stab-1 HCC) is able to form stable oligomeric species containing 10–12 monomeric subunits. We have performed molecular modeling, and in conjunction with experimental parameters obtained from atomic force microscopy (AFM), transmission electron microscopy (TEM) and small-angle X-ray scattering (SAXS) measurements, we observe that the structures are essentially flat, with a height of about 2 nm, and the distance between the outer edge of the ring and the edge of the central cavity is ~5.1 nm. These dimensions correspond to the height and diameter of one stab-1 HCC subunit and we present a dodecamer model for stabilized cystatin C oligomers using molecular dynamics simulations and experimentally measured parameters. Given that oligomeric species in protein aggregation reactions are often transient and very highly heterogeneous, the structural information presented here on these isolated stab-1 HCC oligomers may be useful to further explore the physiological relevance of different structural species of cystatin C in relation to protein misfolding disease.