Tadahiro Kumagai

In-gel β-elimination and aqueous-organic partition for improved O- and sulfoglycomics.

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.

ABO Blood Group as a Model for Platelet Glycan Modification in Arterial Thrombosis

ABO blood groups have long been associated with cardiovascular disease, thrombosis, and acute coronary syndromes. Many studies over the years have shown type O blood group to be associated with lower risk of cardiovascular disease than non–type O blood groups. However, the mechanisms underlying this association remain unclear. Although ABO blood group is associated with variations in concentrations of circulating von Willebrand Factor and other endothelial cell adhesion molecules, ABO antigens are also present on several platelet surface glycoproteins and glycosphingolipids. As we highlight in this platelet-centric review, these glycomic modifications may affect platelet function in arterial thrombosis. More broadly, improving our understanding of the role of platelet glycan modifications in acute coronary syndromes may inform future diagnostics and therapeutics for cardiovascular diseases.

COG7 deficiency in Drosophila generates multifaceted developmental, behavioral and protein glycosylation phenotypes

ABSTRACT Congenital disorders of glycosylation (CDG) comprise a family of human multisystemic diseases caused by recessive mutations in genes required for protein N-glycosylation. More than 100 distinct forms of CDGs have been identified and most of them cause severe neurological impairment. The Conserved Oligomeric Golgi (COG) complex mediates tethering of vesicles carrying glycosylation enzymes across the Golgi cisternae. Mutations affecting human COG1, COG2 and COG4–COG8 cause monogenic forms of inherited, autosomal recessive CDGs. We have generated a Drosophila COG7-CDG model that closely parallels the pathological characteristics of COG7-CDG patients, including pronounced neuromotor defects associated with altered N-glycome profiles. Consistent with these alterations, larval neuromuscular junctions of Cog7 mutants exhibit a significant reduction in bouton numbers. We demonstrate that the COG complex cooperates with Rab1 and Golgi phosphoprotein 3 to regulate Golgi trafficking and that overexpression of Rab1 can rescue the cytokinesis and locomotor defects associated with loss of Cog7. Our results suggest that the Drosophila COG7-CDG model can be used to test novel potential therapeutic strategies by modulating trafficking pathways. Highlighted Article: A Drosophila model of congenital disorders of glycosylation diseases (mutation of Cog7) suggests that modulating secretory trafficking could provide a novel means of therapy.

Improved in-gel reductive β-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 biological samples. Similar approaches, that would allow in-depth analysis of the glycans carried by glycoproteins resolved by SDS-PAGE, require special considerations in order to maximize recovery and sensitivity when using mass spectrometry (MS) as the detection method. A major hurdle to be overcome in achieving high-quality data is the removal of gel-derived contaminants that interfere with MS analysis. The sample workflow presented here is robust, efficient, and eliminates the need for in-line HPLC clean-up prior to MS. Gel pieces containing target proteins are washed in acetonitrile, water, and ethyl acetate to remove contaminants, including polymeric acrylamide fragments. O-linked glycans are released from target proteins by in-gel reductive β-elimination and recovered through robust, simple clean-up procedures. An advantage of this workflow is that it improves sensitivity for detecting and characterizing sulfated glycans. These procedures produce an efficient separation of sulfated permethylated glycans from non-sulfated (sialylated and neutral) permethylated glycans by a rapid phase-partition prior to MS analysis, and thereby enhance glycomic and sulfoglycomic analyses of glycoproteins resolved by SDS-PAGE.

Modeling Congenital Disorders of N-Linked Glycoprotein Glycosylation in Drosophila melanogaster

Protein glycosylation, the enzymatic addition of N-linked or O-linked glycans to proteins, serves crucial functions in animal cells and requires the action of glycosyltransferases, glycosidases and nucleotide-sugar transporters, localized in the endoplasmic reticulum and Golgi apparatus. Congenital Disorders of Glycosylation (CDGs) comprise a family of multisystemic diseases caused by mutations in genes encoding proteins involved in glycosylation pathways. CDGs are classified into two large groups. Type I CDGs affect the synthesis of the dolichol-linked Glc3Man9GlcNac2 precursor of N-linked glycosylation or its transfer to acceptor proteins. Type II CDG (CDG-II) diseases impair either the trimming of the N-linked oligosaccharide, the addition of terminal glycans or the biosynthesis of O-linked oligosaccharides, which occur in the Golgi apparatus. So far, over 100 distinct forms of CDGs are known, with the majority of them characterized by neurological defects including mental retardation, seizures and hypotonia. Yet, it is unclear how defective glycosylation causes the pathology of CDGs. This issue can be only addressed by developing animal models of specific CDGs. Drosophila melanogaster is emerging as a highly suitable organism for analyzing glycan-dependent functions in the central nervous system (CNS) and the involvement of N-glycosylation in neuropathologies. In this review we illustrate recent work that highlights the genetic and neurobiologic advantages offered by D. melanogaster for dissecting glycosylation pathways and modeling CDG pathophysiology.