In silico approaches for the identification of potential allergens among hypothetical proteins from Alternaria alternata and its functional annotation.

Direct exposure to the fungal species Alternaria alternata is a major risk factor for the development of asthma, allergic rhinitis, and inflammation. As of November 23rd 2020, the NCBI protein database showed 11,227 proteins from A. alternata genome as hypothetical proteins (HPs). Allergens are the main causative of several life-threatening diseases, especially in fungal infections. Therefore, the main aim of the study is to identify the potentially allergenic inducible proteins from the HPs in A. alternata and their associated functional assignment for the complete understanding of the complex biological systems at the molecular level. AlgPred and Structural Database of Allergenic Proteins (SDAP) were used for the prediction of potential allergens from the HPs of A. alternata. While analyzing the proteome data, 29 potential allergens were predicted by AlgPred and further screening in SDAP confirmed the allergic response of 10 proteins. Extensive bioinformatics tools including protein family classification, sequence-function relationship, protein motif discovery, pathway interactions, and intrinsic features from the amino acid sequence were used to successfully predict the probable functions of the 10 HPs. The functions of the HPs are characterized as chitin-binding, ribosomal protein P1, thaumatin, glycosyl hydrolase, and NOB1 proteins. The subcellular localization and signal peptide prediction of these 10 proteins has further provided additional information on localization and function. The allergens prediction and functional annotation of the 10 proteins may facilitate a better understanding of the allergenic mechanism of A. alternata in asthma and other diseases. The functional domain level insights and predicted structural features of the allergenic proteins help to understand the pathogenesis and host immune tolerance. The outcomes of the study would aid in the development of specific drugs to combat A. alternata infections.

Functional significance of mouse seminal vesicle sulfhydryl oxidase on sperm capacitation in vitro.

During ejaculation, cauda epididymal spermatozoa are suspended in a protein-rich solution of seminal plasma, which is composed of proteins mostly secreted from the seminal vesicle. These seminal proteins interact with the sperm cells and bring about changes in their physiology, so that they can become capacitated in order for the fertilization to take place. Sulfhydryl oxidase (SOX) is a member of the QSOX family and its expression is found to be high in the seminal vesicle secretion (SVS) of mouse. Previously, it has been reported to cross-link thiol-containing amino acids among major SVS proteins. However, its role in male reproduction is unclear. In this study, we determined the role of SOX on epididymal sperm maturation and also disclosed the binding effect of SOX on the sperm fertilizing ability in vitro. In order to achieve the above two objectives, we constructed a Sox clone (1.7 kb) using a pET-30a vector. His-tagged recombinant Sox was overexpressed in Shuffle Escherichia coli cells and purified using His-Trap column affinity chromatography along with hydrophobic interaction chromatography. The purified SOX was confirmed by western blot analysis and by its activity with DTT as a substrate. Results obtained from immunocytochemical staining clearly indicated that SOX possesses a binding site on the sperm acrosome. The influence of SOX on oxidation of sperm sulfhydryl to disulfides during epididymal sperm maturation was evaluated by a thiol-labeling agent, mBBr. The SOX protein binds onto the sperm cells and increases their progressive motility. The effect of SOX binding on reducing the [Ca2+]i concentration in the sperm head was determined using a calcium probe, Fluo-3 AM. The inhibitory influence of SOX on the sperm acrosome reaction was shown by using calcium ionophore A32187 to induce the acrosome reaction. The acrosome-reacted sperm were examined by staining with FITC-conjugated Arachis hypogaea (peanut) lectin. Furthermore, immunocytochemical analysis revealed that SOX remains bound to the sperm cells in the uterus but disappears in the oviduct during their transit in the female reproductive tract. The results from the above experiment revealed that SOX binding onto the sperm acrosome prevents sperm capacitation by affecting the [Ca2+]i concentration in the sperm head and the ionophore-induced acrosome reaction. Thus, the binding of SOX onto the sperm acrosome may possibly serve as a decapacitation factor in the uterus to prevent premature capacitation and acrosome reaction, thus preserving their fertilizing ability.