LRRK2 (leucine-rich repeat kinase 2) is one of the causative gene products for familial Parkinson’s disease (PD), harboring a Ser/Thr kinase domain with unknown functions. Recently we identified several small GTPase Rab proteins including Rab10 as physiological substrates of LRRK2, and we found that LRRK2 mutations linked with PD abnormally increase the Rab10 phosphorylation in cells and tissues. We utilized Phos-tag SDS–PAGE for detecting the phosphorylation of endogenous Rab10. On Phos-tag SDS–PAGE gels, one can detect protein phosphorylation as a bandshift, which enabled us to detect the endogenous levels of Rab10 phosphorylation without generating phospho-specific antibodies. In this review, we summarized the recent Phos-tag analysis on Rab10 phosphorylation by LRRK2.

Post translational protein modification is recognized as an important mechanism for diversification of protein function. Research into signal transduction mechanisms focusing on regulation of transcriptional expression of genes and on the transport or degradation of proteins, is an intensive area of investigation. Covalent modification of opsin by retinal is a critical mechanism by which vitamin A participates in the process of vision. Retinoic acid (RA) is an oxidized form of retinal that is a potent inducer of HL60 cell differentiation, which can achieve complete remission in patients with acute promyelocytic leukemia. While one mechanism by which RA achieves its effects involves RA nuclear receptors, retinoylation (post-translational modification of proteins by RA) represents an alternate non-genomic mechanism. One metabolic pathway for retinoylation involves the intermediate formation of retinoyl-CoA and the transfer and covalent binding of the retinoyl moiety to protein. The regulatory subunit of cAMP-dependent protein kinase (proteinkinase A, PKA)(RIIα) is retinoylated at early time after RA treatment to HL60 cells. Once retinoylated, RIIα units are translocated to the nucleus where phosphorylation of nuclear proteins occurs. This promotes the phosphorylation of nuclear proteins by PKA and this may induce differentiation into granulocyte-like cells. We are currently identifying nuclear proteins phosphorylated by PKA, which are increased by RA treatment and determining the sites on RIIα that are modified. These efforts may clarify actions of RA that function in cooperation with the RA nuclear receptor and retinoylation.

O-linked β-N-acetylglucosamine (O-GlcNAc) modification is one of protein post-translational modifications in multicellular eukaryotes. This modification occurs selectively on serine and/or threonine residues of specific cytoplasmic and nuclear proteins and dynamically modulates their molecular functions. However, conventional methods for the evaluation of the physiological O-GlcNAcylation level (e.g., immune-purification, mass spectrometric analysis) of a specific protein need time-consuming and complicated steps. We therefore developed a rapid and easy method for the detection and quantification of an O-GlcNAcylated protein. Here, we describe the principal and experimental procedure of the novel affinity gel electrophoresis that separates O-GlcNAcylated and non-O-GlcNAcylated forms of proteins. The polyacrylamide-conjugated wheat germ agglutinin (WGA), which preferentially binds to N-acetylglucosamine residues, selectively induces retardation of the mobility of O-GlcNAcylated proteins during electrophoresis and thereby allows the visualization of both O-GlcNAc-modified and unmodified forms of specific proteins. Thus, our method, termed WGA-SDS-PAGE, provides a useful tool for quantitative monitoring of protein O-GlcNAcylation.

High-throughput quantitative and qualitative analytical procedures are required for glycoscientific research (e.g. metabolism of glycans, glyco-biomarker discovery, and quality control of glycoprotein pharmaceuticals). Among some analytical techniques, microchip electrophoresis is one of the candidate technique for high-throughput quantitative and qualitative glycoanalysis. We describe here a strategy for high-throughput analytical procedures for the relative quantitation of N-linked glycans by using and automated microchip electrophoresis system “MultiNA”, which is specialized for DNA/RNA analysis. We designed suitable labeling of glycans for fluorescence detection in “MultiNA” and optimized separation condition for labeled glycans without any hardware modification. 8-Aminopyrene-1,3,6-trisulfonate (APTS)-labeled N-glycans released from some glycoproteins were successfully separated based on the difference in branching type and terminal modifications within 100s. Furthermore, we also designed microchip lectin-
affinity electrophoresis for glyco-profiling. Thus, automated microchip electrophoresis provides high-throughput quantitative and qualitative glycoanalytical procedures and “MultiNA” will provide a new dimension of glycoscience in the field of biology.

Xerostomia is a symptom growing in the elderly, and associated with reduced salivary flow and increased salivary viscosity. Salivary mucins are responsible for the viscous property of the saliva. This study introduces aging alterations of a salivary gland by using submandibular glands of mice as a model. Supported molecular matrix electrophoresis (SMME) is a membrane electrophoresis to characterize mucins. Using SMME, we revealed that submandibular glands of the aged mice produced unknown mucin-like molecule that was absent in the glands of young mice. O-Glycosylations of the unknown mucin-like molecule are characterized by high ratio of sialoglycans to neutral glycans. The increase of sialoglycan-content may be attributed to the increased expression of the sialyl transferase genes, ST6GalNAc I and ST6GalNAc II, in the submandibular gland in the aged mice.