Identification, characterization and purification of porcine Quiescin Q6-Sulfydryl Oxidase 2 protein

Abstract

BackgroundPost-spermiogenesis membrane surface modifications rely on molecules present in the reproductive tracts. Two isoforms (isoform 1 and 2) from Quiescin Q6-Sulfydryl Oxidase protein family have been identified in the male reproductive tract of rodent species. However, unlike isoform 1, scarce information is available for isoform 2, likely due to its lower expression level and lack of proper purification methods to obtain sufficient protein quantity for further assays.ResultsThis study demonstrated the presence of short and long forms of Quiescin Q6-Sulfydryl Oxidase 2 in boar, likely representing the secretory (short form) and transmembrane (long form) forms of Quiescin Q6-Sulfydryl Oxidase 2. Immunohistochemistry studies revealed the presence of Quiescin Q6-Sulfydryl Oxidase 2 in a broad range of porcine tissues; the pronounced vesicle-contained Quiescin Q6-Sulfydryl Oxidase 2 at the apical region of epididymis and seminal vesicles epithelium suggested its involvement in sperm physiology and its participation in semen formation. The majority of porcine Quiescin Q6-Sulfydryl Oxidase 2 could be purified via either antibody affinity column or be salted out using 10%–40% ammonium sulfate. Higher amount of low molecular weight Quiescin Q6-Sulfydryl Oxidase 2 observed in the seminal vesicle likely represents the secretory form of Quiescin Q6-Sulfydryl Oxidase 2 and reflects an exuberant secretory activity in this organ.ConclusionsWe demonstrated for the first time, the presence of Quiescin Q6-Sulfydryl Oxidase 2 in porcine species; moreover, two forms of Quiescin Q6-Sulfydryl Oxidase 2 were identified and exhibited distinct molecular weights and properties during protein purification processes. This study also provided feasible Quiescin Q6-Sulfydryl Oxidase 2 purification methods from slaughterhouse materials that could potentially allow obtaining sufficient amount of Quiescin Q6-Sulfydryl Oxidase 2 for future functional investigations.

Counteracting Cisplatin-Induced Testicular Damages by Natural Polyphenol Constituent Honokiol

“…Indirect immunofluorescent staining was carried out as described earlier [ 36 ]. Tissue sections were deparaffinized with 100% xylene and rehydrated with 100–80% ethanol.…”

Nanoparticulated Honokiol Mitigates Cisplatin-Induced Chronic Kidney Injury by Maintaining Mitochondria Antioxidant Capacity and Reducing Caspase 3-Associated Cellular Apoptosis

“…After mice were euthanized, kidneys were first weighted before fixation and procedures for paraffin-embedding. Five μm kidney sections were deparaffinized in xylene, rehydrated and underwent antigen retrieval, as described earlier [22]. Sections were stained with hematoxylin and eosin (H and E) for general morphological evaluation or with Masson’s Trichrome stain to assess the level of fibrosis.…”

“…Immuno-blotting experiments were performed as preciously described [22]. Proteins were separated by sodium dodecyl sulfate- polyacrylamide gel (SDS-PAGE) and subsequently blotted onto an Immobilon-P polyvinylidene difluoride (PVDF) membrane (Millipore, Burlington, MA, USA).…”

Honokiol, a Polyphenol Natural Compound, Attenuates Cisplatin-Induced Acute Cytotoxicity in Renal Epithelial Cells Through Cellular Oxidative Stress and Cytoskeleton Modulations

“…To evaluate the effects of cisplatin and HNK on the protein expression level of Occludin and E-cadherin, immune-blotting experiments were performed as preciously described ( Kuo et al, 2017 ). Proteins were separated by SDS-PAGE and wet-blotted onto an Immobilon-P PVDF membrane (Millipore, MA, United States).…”

“…Non-specific signals were minimized with blocking buffer (5 mM Tris, 250 mM sucrose, pH 7.4 with 0.05% v/v Tween-20 [TBST], supplemented with 5% milk powder). Primary antibody (E-Cadherin 1:250, Occludin 1:500, GADPH 1:10000) and secondary antibody (1:5000) were subsequently used as previously described ( Kuo et al, 2017 ). Protein signals were visualized by chemiluminescence (Merck Ltd., TW) and were detected with ChemiDoc TM XRS+ system (Bio-Rad).…”