Integration of Web-Based Tools to Visualize, Integrate, and Interpret Glycogene Expression and Glycomics Data.

Glycosylation is the most abundant and diverse post-translational modification occurring on proteins. Glycans play important roles in modulating cell adhesion, growth, development, and differentiation. Changes in glycosylation affect protein structure and function and contribute to disease processes. Therefore, understanding glycosylation patterns is key for the identification of targets for the diagnosis of diseases, cellular states, and therapy. Glycosylation is a non template-driven process governed by the action of numerous enzymes and substrate availability that varies among cell types and species. Therefore, qualitative and quantitative assessment of global glycosylation and individual glycans remains challenging because it requires integration of multiple complex data types. Glycan structure and quantity data are often integrated with assessments of gene expression to aid contextualization of observed glycosylation changes within biological processes. However, correlating glycogene expression to the glycan structure is challenging because transcriptional changes may not always concur with the final gene product; there is often a lack of information on nucleotide sugar pools, and the final glycan structure is the result of many different glycogenes acting in concert. To overcome these challenges, interactive online tools are emerging as key resources for facilitating the analysis and integration of glycomics and glycogene expression data. Importantly, these tools work in concurrence with glycan biosynthetic schemes and therefore provide a clear indication of the molecular pathways where the glycan and glycogene are involved. In this chapter, we describe the applications of four freely available online tools that can be used for integrated visualization, interpretation, and presentation of RNAseq and glycomics results.

Multinozzle Emitter for Improved Negative Mode Analysis of Reduced Native N-Glycans by Microflow Porous Graphitized Carbon Liquid Chromatography Mass Spectrometry.

Microflow porous graphitized carbon liquid chromatography (PGC-LC) combined with negative mode ionization mass spectrometry (MS) provides high resolution separation and identification of reduced native N-glycan structural isomers. However, insufficient spray quality and low ionization efficiency of N-glycans present challenges for negative mode electrospray. Here, we evaluated the performance of a recently developed multinozzle electrospray source (MnESI) and accompanying M3 emitter for microflow PGC-LC-MS analysis of N-glycans in negative mode. In comparison to a standard electrospray ionization source, the MnESI with an M3 emitter improves signal intensity, identification, quantification, and resolution of structural isomers to accommodate low-input samples.

The known unknowns of apolipoprotein glycosylation in health and disease.

Apolipoproteins, the protein component of lipoproteins, play an important role in lipid transport, lipoprotein assembly, and receptor recognition. Apolipoproteins are glycosylated and the glycan moieties play an integral role in apolipoprotein function. Changes in apolipoprotein glycosylation correlate with several diseases manifesting in dyslipidemias. Despite their relevance in apolipoprotein function and diseases, the total glycan repertoire of most apolipoproteins remains undefined. This review summarizes the current knowledge and knowledge gaps regarding human apolipoprotein glycan composition, structure, glycosylation site, and functions. Given the relevance of glycosylation to apolipoprotein function, we expect that future studies of apolipoprotein glycosylation will contribute new understanding of disease processes and uncover relevant biomarkers and therapeutic targets. Considering these future efforts, we also provide a brief overview of current mass spectrometry based technologies that can be applied to define detailed glycan structures, site-specific compositions, and the role of emerging approaches for clinical applications in biomarker discovery and personalized medicine.

Glycomic and glycotranscriptomic profiling of mucin-type O-glycans in planarian Schmidtea mediterranea.

O-Glycans on cell surfaces play important roles in cell-cell, cell-matrix and receptor-ligand interaction. Therefore, glycan-based interactions are important for tissue regeneration and homeostasis. Free-living flatworm Schmidtea mediterranea, because of its robust regenerative potential, is of great interest in the field of stem cell biology and tissue regeneration. Nevertheless, information on the composition and structure of O-glycans in planaria is unknown. Using mass spectrometry and in silico approaches, we characterized the glycome and the related transcriptome of mucin-type O-glycans of planarian S. mediterranea. Mucin-type O-glycans were composed of multiple isomeric, methylated, and unusually extended mono- and disubstituted O-N-acetylgalactosamine structures. Extensions made of hexoses and 3-O-methyl hexoses were the glycoforms observed. From glycotranscriptomic analysis, 60 genes belonging to five distinct enzyme classes were identified to be involved in mucin-type O-glycan biosynthesis. These genes shared homology with those in other invertebrate systems. Although a majority of the genes involved in mucin-type O-glycan biosynthesis were highly expressed during organogenesis and in differentiated cells, a few select genes in each enzyme class were specifically enriched during early embryogenesis. Our results indicate a unique temporal and spatial role for mucin-type O-glycans during embryogenesis and organogenesis and in adulthood. In summary, this is the first report on O-glycans in planaria. This study expands the structural and biosynthetic possibilities in cellular glycosylation in the invertebrate glycome and provides a framework towards understanding the biological role of mucin-type O-glycans in tissue regeneration using planarians.

Serotonin is essential for eye regeneration in planaria Schmidtea mediterranea.

Planaria is an ideal system to study factors involved in regeneration and tissue homeostasis. Little is known about the role of metabolites and small molecules in stem cell maintenance and lineage specification in planarians. Using liquid chromatography and mass spectrometry (LC-MS)-based quantitative metabolomics, we determined the relative levels of metabolites in stem cells, progenitors, and differentiated cells of the planarian Schmidtea mediterranea. Tryptophan and its metabolic product serotonin are significantly enriched in stem cells and progenitor population. Serotonin biosynthesis in these cells is brought about by a noncanonical enzyme, phenylalanine hydroxylase. Knockdown of Smed-pah leads to complete disappearance of eyes in regenerating planaria, while exogenous supply of serotonin and its precursor rescues the eyeless phenotype. Our results demonstrate a key role for serotonin in eye regeneration.

Identification of multiple isomeric core chitobiose-modified high-mannose and paucimannose N-glycans in the planarian Schmidtea mediterranea.

Cell surface-associated glycans mediate many cellular processes, including adhesion, migration, signaling, and extracellular matrix organization. The galactosylation of core fucose (GalFuc epitope) in paucimannose and complex-type N-glycans is characteristic of protostome organisms, including flatworms (planarians). Although uninvestigated, the structures of these glycans may play a role in planarian regeneration. Whole-organism MALDI-MS analysis of N-linked oligosaccharides from the planarian Schmidtea mediterranea revealed the presence of multiple isomeric high-mannose and paucimannose structures with unusual mono-, di-, and polygalactosylated (n = 3-5) core fucose structures; the latter structures have not been reported in other systems. Di- and trigalactosylated core fucoses were the most dominant glycomers. N-Glycans showed extensive, yet selective, methylation patterns, ranging from non-methylated to polymethylated glycoforms. Although the majority of glycoforms were polymethylated, a small fraction also consisted of non-methylated glycans. Remarkably, monogalactosylated core fucose remained unmethylated, whereas its polygalactosylated forms were methylated, indicating structurally selective methylation. Using database searches, we identified two potential homologs of the Galß1-4Fuc-synthesizing enzyme from nematodes (GALT-1) that were expressed in the prepharyngeal, pharyngeal, and mesenchymal regions in S. mediterranea. The presence of two GALT-1 homologs suggests different requirements for mono- and polygalactosylation of core fucose for the formation of multiple isomers. Furthermore, we observed variations in core fucose glycosylation patterns in different planarian strains, suggesting evolutionary adaptation in fucose glycosylation. The various core chitobiose modifications and methylations create >60 different glycoforms in S. mediterranea. These results contribute greatly to our understanding of N-glycan biosynthesis and suggest the presence of a GlcNAc-independent biosynthetic pathway in S. mediterranea.