Role of the Drosophila YATA protein in the proper subcellular localization of COPI revealed by in vivo analysis.

yata mutants of Drosophila melanogaster exhibit phenotypes including progressive brain shrinkage, developmental abnormalities and shortened lifespan, whereas in mammals, null mutations of the yata ortholog Scyl1 result in motor neuron degeneration. yata mutation also causes defects in the anterograde intracellular trafficking of a subset of proteins including APPL, which is the Drosophila ortholog of mammalian APP, a causative molecule in Alzheimer's disease. SCYL1 binds and regulates the function of coat protein complex I (COPI) in secretory vesicles. Here, we reveal a role for the Drosophila YATA protein in the proper localization of COPI. Immunohistochemical analyses performed using confocal microscopy and structured illumination microscopy showed that YATA colocalizes with COPI and GM130, a cis-Golgi marker. Analyses using transgenically expressed YATA with a modified N-terminal sequence revealed that the N-terminal portion of YATA is required for the proper subcellular localization of YATA. Analysis using transgenically expressed YATA proteins in which the C-terminal sequence was modified revealed a function for the C-terminal portion of YATA in the subcellular localization of COPI. Notably, when YATA was mislocalized, it also caused the mislocalization of COPI, indicating that YATA plays a role in directing COPI to the proper subcellular site. Moreover, when both YATA and COPI were mislocalized, the staining pattern of GM130 revealed Golgi with abnormal elongated shapes. Thus, our in vivo data indicate that YATA plays a role in the proper subcellular localization of COPI.

Separation and analysis of charged isomers of monoclonal immunoglobulin G by ceramic hydroxyapatite chromatography.

Analysis of monoclonal antibody (MAb) heterogeneity caused by posttranslational modifications is important for pharmaceutical quality assurance of antibody drugs. In this study, by employing small ceramic hydroxyapatite (HAp) particles that were self-manufactured to have a 2.5-µm diameter, we attempted to separate and analyze MAb isomers. The MAb without N-linked oligosaccharides could be separated by 2.5-µm HAp chromatography as well as MAb with N-linked oligosaccharides. Hence, a variety of N-linked oligosaccharides do not appear to be involved in the separation mechanism of HAp chromatography. However, there is a difference in retention time between MAb with and without N-linked oligosaccharides, meaning that the presence of N-glycan could influence the retention time of HAp chromatography. Subsequently, the MAb fractions separated by 2.5-µm HAp chromatography were analyzed by isoelectric focusing, and seven isomers of the MAb having different isoelectric points (pI) were identified. The MAb isomers were eluted in order of lower pI isomers with sodium phosphate buffer, and enzyme-linked immunosorbent assay indicated the immunoreactivity of the fraction including the lowest pI isomers to be remarkably reduced. This study yielded details of the separation behavior of HAp chromatography owing to 2.5-µm HAp particles.

Scanning electron microscopy-based approach to understand the mechanism underlying the adhesion of dengue viruses on ceramic hydroxyapatite columns.

Although ceramic hydroxyapatite (HAp) chromatography has been used as an alternative method ultracentrifugation for the production of vaccines, the mechanism of virus separation is still obscure. In order to begin to understand the mechanisms of virus separation, HAp surfaces were observed by scanning electron microscopy after chromatography with dengue viruses. When these processes were performed without elution and with a 10-207 mM sodium phosphate buffer gradient elution, dengue viruses that were adsorbed to HAp were disproportionately located in the columns. However, when eluted with a 10-600 mM sodium phosphate buffer gradient, few viruses were observed on the HAp surface. After incubating the dengue viruses that were adsorbed on HAp beads at 37°C and 2°C, the sphericity of the dengue viruses were reduced with an increase in incubation temperature. These results suggested that dengue virus was adsorbed to the HAp surface by electronic interactions and could be eluted by high-salt concentration buffers, which are commonly used in protein purification. Furthermore, virus fusion was thought to occur with increasing temperature, which implied that virus-HAp adhesion was similar to virus-cell adhesion.

Purification of anti-Japanese encephalitis virus monoclonal antibody by ceramic hydroxyapatite chromatography without proteins A and G.

Antibody purification using proteins A and G has been a standard method for research and industrial processes. The conventional method, however, includes a three-step process, including buffer exchange, before chromatography. In addition, proteins A and G require low pH elution, which causes antibody aggregation and inactivates the antibody's immunity. This report proposes a two-step method using hydroxyapatite chromatography and membrane filtration, without proteins A and G. This novel method shortens the running time to one-third the conventional method for each cycle. Using our two-step method, 90.2% of the monoclonal antibodies purified were recovered in the elution fraction, the purity achieved was >90%, and most of the antigen-specific activity was retained. This report suggests that the two-step method using hydroxyapatite chromatography and membrane filtration should be considered as an alternative to purification using proteins A and G.