Autophagy contributes to the selective degradation of liquid droplets, including the P-Granule, Ape1-complex and p62/SQSTM1-body, although the molecular mechanisms and physiological relevance of selective degradation remain unclear. In this report, we describe the properties of endogenous p62-bodies, the effect of autophagosome biogenesis on these bodies, and the in vivo significance of their turnover. p62-bodies are low-liquidity gels containing ubiquitin and core autophagy-related proteins. Multiple autophagosomes form on the p62-gels, and the interaction of autophagosome-localizing Atg8-proteins with p62 directs autophagosome formation toward the p62-gel. Keap1 also reversibly translocates to the p62-gels in a p62-binding dependent fashion to activate the transcription factor Nrf2. Mice deficient for Atg8-interaction-dependent selective autophagy show that impaired turnover of p62-gels leads to Nrf2 hyperactivation in vivo. These results indicate that p62-gels are not simple substrates for autophagy but serve as platforms for both autophagosome formation and anti-oxidative stress.

In sharp contrast with analysis of N-glycan that can be prepared by PNGase F, O-glycan analysis remains challenging due to a lack of versatile and simple procedures, especially those mediating cleavage of O-glycans from proteins. Most N-glycans and O-glycans are modified with sialic acids at the non-reducing end and their glycosidic linkages are labile, making it difficult to measure glycans by mass spectrometric analysis. In addition, sialic acid residues present on glycan chains via α2,3-, α2,6-, and α2,8-linkages as structural isomers. In this study, we firstly established a direct and linkage-specific derivatization method for sialylated O-glycans on proteins via linkage-specific lactone-opening aminolysis. In this procedure, labile sialylated glycans were not only stabilized, but also allowed distinguishing between sialyl linkages. Furthermore, we revealed that general reductive β-elimination was not useful for O-glycan cleavages with undesirable degradations of resulting methyl amides. Using β-elimination in the presence of pyrazolone (PMP), with low pH despite alkali base concentration, SALSA-derivatized O-glycans could be cleaved with minimal degradations. Cleaved and PMP-labeled O-glycans could be efficiently prepared in an open reaction system at high temperature (evaporative BEP reaction) and detected by simple liquid-phase extraction. Moreover, in the evaporative BEP reaction by changing the alkali solution with LiOH, the lithiated O-glycans could be observed and provided a lot of fragment information reflecting the complex structure of the O-glycans. Direct sialic acid linkage-specific derivatization of O-glycans on glycoproteins is simple protocol containing in-solution aminolysis-SALSA and acetonitrile precipitation for removal of excess reagents. Evaporative β-elimination with pyrazolone makes possible intact O-linked glycan analysis just by liquid-phase extraction. These analytical methods established by the appropriate combination of direct-SALSA and evaporative β-elimination will facilitate O-glycomic studies in various biological samples.

Sialylation is a terminal glycosylated modification of glycoproteins that regulates critical biological events such as cell adhesion and immune response. Our previous study showed that integrin α3β1 plays a crucial role in regulating the sialylation of N-glycans. However, the underlying mechanism for the regulation remains unclear. This study investigated how sialylation is affected by focal adhesion kinase (FAK), which is a critical downstream signal molecule of integrin β1. We established a stable FAK knockout (KO) cell line using the CRISPR/Cas9 system in HeLa cells. The results obtained from lectin blot, flow cytometric analysis, and MS showed that the sialylation levels were significantly decreased in the KO cells compared with that in wild-type (WT) cells. Moreover, phosphatidylinositol 4-phosphate (PI4P) expression levels were also reduced in the KO cells due to a decrease in the stability of phosphatidylinositol 4-kinase-IIα (PI4KIIα). Notably, the decreased levels of sialylation, PI4P, and the complex formation between GOLPH3 and ST3GAL4 or ST6GAL1, which are the main sialyltransferases for modification of N-glycans, were significantly restored by the re-expression of FAK. Furthermore, the decreased sialylation and phosphorylation of Akt and cell migration caused by FAK deficiency all were restored by overexpressing PI4KIIα, which suggests that PI4KIIα is one of the downstream molecules of FAK. These findings indicate that FAK regulates sialylation via the PI4P synthesis pathway and a novel mechanism is suggested for the integrin-FAK-PI4KIIα-GOLPH3-ST axis modulation of sialylation in N-glycans.

Glycans present extraordinary structural diversity commensurate with their involvement in numerous fundamental cellular processes including growth, differentiation, and morphogenesis. Unlike linear DNA and protein sequences, glycans have heterogeneous structures that differ in composition, branching, linkage, and anomericity. These differences pose a challenge to developing useful software for glycomic analysis. To overcome this problem, we developed the novel Toolbox Accelerating Glycomics (TAG) program. TAG consists of three units: ‘TAG List’ creates a glycan list that is used for database searching in TAG Expression; ‘TAG Expression’ automatically annotates and quantifies glycan signals and draws graphs; and ‘TAG Pathway’ maps the obtained expression information to biosynthetic pathways. Herein, we discuss the concepts, outline the TAG process, and demonstrate its potential using glycomic expression profile data from Chinese hamster ovary (CHO) cells and mutants lacking a functional Npc1 gene (Npc1 knockout (KO) CHO cells). TAG not only drastically reduced the amount of time and labor needed for glycomic analysis but also detected and quantified more glycans than manual analysis. Although this study was limited to the analysis of N-glycans and free oligosaccharides, the glycomic platform will be expanded to facilitate the analysis of O-glycans and glycans of glycosphingolipids.