improved-gel-reductive-elimination-for-comprehensive-o-linked-sulfo

Improved In-gel Reductive &#946;-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of ...

University of Georgia

Ishikawa Prefectural University

Autophagy is a cellular process that nonspecifically degrades cytosolic components and is involved in many cellular responses. We found that amino sugars with a free amino group such as glucosamine, galactosamine and mannosamine induced autophagy via an mTOR-independent pathway. Glucosamine-induced autophagy at concentrations of at least 500 microM to over 40 mM. In the presence of 40 mM glucosamine, autophagy induction was initiated at 6h and reached a plateau at 36 h. Glucosamine-induced autophagy could remove accumulated ubiquitin-conjugated proteins as well as 79-glutamine repeats. Therefore, orally administered glucosamine could contribute to the prevention of neurodegenerative diseases and promotion of antiaging effects.

Glucosamine induces autophagy via an mTOR-independent pathway.

Addition of fucose (Fuc) to glycoprotein N-linked glycans or in O-linkage directly to Ser/Thr residues modulates specific cell-cell interactions and cell signaling events. Vertebrates and invertebrates add Fuc in α6-linkage to the reducing terminal N-acetylglucosamine residue of N-glycans. In Drosophila and other invertebrates, Fuc can also be added in α3-linkage to the same residue. These difucosylated N-glycans are recognized by anti-horseradish peroxidase (anti-HRP) antisera, providing a well-established marker for insect neural tissue. To understand the mechanisms and consequences of tissue-specific glycan expression, we identified a single α3-fucosyltransferase (FucTA) that produces the anti-HRP epitope in Drosophila embryos. FucTA transcripts are temporally and spatially restricted to cells that express the anti-HRP epitope and are missing in a mutant that lacks neural α3-fucosylation. Transgenic expression of FucTA, but not of any other candidate α3-fucosyltransferase, rescues the anti-HRP epitope in the embryonic nervous system of this mutant. Mass spectrometric characterization of the N-glycans of Drosophila embryos overexpressing FucTA confirms that this enzyme is indeed responsible for the biosynthesis of difucosylated glycans in vivo. Whereas ectopic expression of FucTA in the larval wing disc produces mild wing notching, the heterochronic, pan-neural expression of FucTA in early differentiating neurons generates neurogenic and cell migration phenotypes; this latter effect is associated with reduced GDP-Fuc levels in the embryo and indicates that the diversion of fucosylation resources towards fucosylation of N-glycans has an impact on developmental signaling associated with O-fucosylation.

Neural-specific α3-fucosylation of N-linked glycans in the Drosophila embryo requires fucosyltransferase A and influences developmental signaling associated with O-glycosylation.

The past 25 years have seen significant advances in understanding the diversity and functions of glycoprotein glycans in Drosophila melanogaster. Genetic screens have captured mutations that reveal important biological activities modulated by glycans, including protein folding and trafficking, as well as cell signaling, tissue morphogenesis, fertility, and viability. Many of these glycan functions have parallels in vertebrate development and disease, providing increasing opportunities to dissect pathologic mechanisms using Drosophila genetics. Advances in the sensitivity of structural analytic techniques have allowed the glycan profiles of wild-type and mutant tissues to be assessed, revealing novel glycan structures that may be functionally analogous to vertebrate glycans. This review describes a selected set of recent advances in understanding the functions of N-linked and O-linked (non-glycosaminoglycan) glycoprotein glycans in Drosophila with emphasis on their relatedness to vertebrate organisms.

The N's and O's of Drosophila glycoprotein glycobiology.

Siglec-8 and Siglec-F are single pass transmembrane inhibitory receptors found on the surface of human and mouse eosinophils, respectively, but very little is known about their physiologic glycan ligands. This article reviews the latest knowledge on this topic and outlines the strategies being used to further define the production and glycobiochemical nature of these molecules in the lung.

The role of lung epithelial ligands for Siglec-8 and Siglec-F in eosinophilic inflammation.

The Golgi receives the entire output of newly synthesized cargo from the endoplasmic reticulum, processes it in the stack largely through modification of bound oligosaccharides, and sorts it in the trans-Golgi network. GRASP65 and GRASP55, two proteins localized to the Golgi stack and early secretory pathway, mediate processes including Golgi stacking, Golgi ribbon linking and unconventional secretion. Previously, we have shown that GRASP depletion in cells disrupts Golgi stack formation. Here we report that knockdown of the GRASP proteins, alone or combined, accelerates protein trafficking through the Golgi membranes but also has striking negative effects on protein glycosylation and sorting. These effects are not caused by Golgi ribbon unlinking, unconventional secretion or endoplasmic reticulum stress. We propose that GRASP55/65 are negative regulators of exocytic transport and that this slowdown helps to ensure more complete protein glycosylation in the Golgi stack and proper sorting at the trans-Golgi network.

Regulation of protein glycosylation and sorting by the Golgi matrix proteins GRASP55/65.

Endoplasmic reticulum (ER) α-glucosidase I is an enzyme that trims the distal α1,2-linked glucose (Glc) residue from the Glc3Man9GlcNAc2 oligosaccharide following its addition to nascent glycoproteins in the initial step of processing. This reaction is critical to the subsequent processing of N-glycans and thus defects in α-glucosidase I gene in human cause congenital disorder of glycosylation (CDG) type IIb. We identified the Caenorhabditis elegans α-glucosidase I gene (F13H10.4, designated agl-1) that encodes a polypeptide with 36% identity to human α-glucosidase I. The agl-1 cDNA restored the expression of complex-type N-glycans on the cell surface of α-glucosidase I-defective Chinese hamster ovary Lec23 cells. RNAi knockdown of agl-1 [agl-1(RNAi)] produced worms that were visibly similar to wild-type, but lifespan was reduced to about half of the control. Analyses of N-glycosylation in agl-1(RNAi) animals by western blotting and mass spectrometry showed reduction of paucimannose and complex-type glycans and dramatic increase of glucosylated oligomannose glycans. In addition, a significant amount of unusual terminally fucosylated N-glycans were found in agl-1(RNAi) animals. ER stress response was also provoked, leading to the accumulation of large amounts of triglucosylated free oligosaccharides (FOSs) (Glc3Man4-5GlcNAc1-2) in agl-1(RNAi) animals. Acceleration of ER-associated degradation in response to the accumulation of unfolded glycoproteins and insufficient interaction with calnexin/calreticulin in the ER lumen likely accounts for the increase of FOSs. Taken together, these studies in C. elegans demonstrate that decreased ER α-glucosidase I affects the entire N-glycan profile and induces chronic ER stress, which may contribute to the pathophysiology of CDG-IIb in humans.

Deficiency of α-glucosidase I alters glycoprotein glycosylation and lifespan in Caenorhabditis elegans.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is a widely used technique for protein separation, and in-gel tryptic digestion of resolved protein bands has enhanced the resolution of protoeomic analysis. To augment this technology and expand its usefulness for glycoproteomics, we have developed and improved methods to release and recover O-linked glycans from proteins resolved in SDS-PAGE gels for subsequent analysis by mass spectrometry (MS). Gel pieces containing target proteins are washed to remove contaminants. O-linked glycans are released through reductive β-elimination by hydrating gel pieces in base and adding reductant. Following straightforward sample cleanup, this simple treatment of glycoprotein gel pieces produces material suitable for MS analysis. We have applied this method to the analysis of mucin-type glycoproteins that are expected to carry high densities of sialylated and sulfated O-linked glycans. However, the strongly acidic nature of the sulfate moiety suppresses MS signal intensities, hampering detection and quantitative analysis. To enhance detection, we present an improved method for sulfoglycomics. A mixture of sulflo-, sialo-, and neutral glycans were permethylated and partitioned into a water-dichloromethane (DCM) solvent mixture. Sulfated glycans were selectively recovered from the aqueous phase, while neutral and sialylated glycans remained in the DCM phase. When applied to the analysis of human mucin salivary glycans, this partition method generated material of sufficient quality to identify more than 60 glycan structures by NSI-MS (LTQ-Orbitrap) in positive and negative ion modes. Also, nearly 100% of the sulfated O-linked glycans were recovered in the aqueous phase, demonstrating the feasibility of in-depth sulfoglycomic analysis using SDS-PAGE resolved proteins.

Article	Year
Glucosamine induces autophagy via an mTOR-independent pathway. Biochemical and biophysical research communications\| PubMed ID: 20045674	2010
Neural-specific α3-fucosylation of N-linked glycans in the Drosophila embryo requires fucosyltransferase A and influences developmental signaling associated with O-glycosylation. Glycobiology\| PubMed ID: 20688784	2010
The N's and O's of Drosophila glycoprotein glycobiology. Glycoconjugate journal\| PubMed ID: 22936173	2013
The role of lung epithelial ligands for Siglec-8 and Siglec-F in eosinophilic inflammation. Current opinion in allergy and clinical immunology\| PubMed ID: 23160308	2013
Regulation of protein glycosylation and sorting by the Golgi matrix proteins GRASP55/65. Nature communications\| PubMed ID: 23552074	2013
Deficiency of α-glucosidase I alters glycoprotein glycosylation and lifespan in Caenorhabditis elegans. Glycobiology\| PubMed ID: 23836288	2013
In-gel β-elimination and aqueous-organic partition for improved O- and sulfoglycomics. Analytical chemistry\| PubMed ID: 23937624	2013

Toshihiko Katoh

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