@article {765, title = {Identification of multiple isomeric core chitobiose-modified high-mannose and paucimannose -glycans in the planarian. [Mass Spectrometry Facility - Glycomics (INT)]}, journal = {J Biol Chem}, volume = {293}, year = {2018}, month = {2018 May 04}, pages = {6707-6720}, abstract = {

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 -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 -linked oligosaccharides from the planarian revealed the presence of multiple isomeric high-mannose and paucimannose structures with unusual mono-, di-, and polygalactosylated ( = 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. -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 GalĪ²1-4Fuc-synthesizing enzyme from nematodes (GALT-1) that were expressed in the prepharyngeal, pharyngeal, and mesenchymal regions in 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 These results contribute greatly to our understanding of -glycan biosynthesis and suggest the presence of a GlcNAc-independent biosynthetic pathway in

}, issn = {1083-351X}, doi = {10.1074/jbc.RA117.000782}, author = {Subramanian, Sabarinath Peruvemba and Babu, Ponnusamy and Palakodeti, Dasaradhi and Subramanian, Ramaswamy} } @article {532, title = {Targeted, Site-specific quantitation of N- and O-glycopeptides using (18)O-labeling and product ion based mass spectrometry.}, journal = {Glycoconj J}, volume = {34}, year = {2017}, month = {2017 Feb}, pages = {95-105}, abstract = {

The site-specific quantitation of N- and O-glycosylation is vital to understanding the function(s) of different glycans expressed at a given site of a protein under physiological and disease conditions. Most commonly used precursor ion intensity based quantification method is less accurate and other labeled methods are expensive and require enrichment of glycopeptides. Here, we used glycopeptide product (y and Y0) ions and (18)O-labeling of C-terminal carboxyl group as a strategy to obtain quantitative information about fold-change and relative abundance of most of the glycoforms attached to the glycopeptides. As a proof of concept, the accuracy and robustness of this targeted, relative quantification LC-MS method was demonstrated using Rituximab. Furthermore, the N-glycopeptide quantification results were compared with a biosimilar of Rituximab and validated with quantitative data obtained from 2-AB-UHPLC-FL method. We further demonstrated the intensity fold-change and relative abundance of 46 unique N- and O-glycopeptides and aglycopeptides from innovator and biosimilar samples of Etanercept using both the normal-MS and product ion based quantitation. The results showed a very similar site-specific expression of N- and O-glycopeptides between the samples but with subtle differences. Interestingly, we have also been able to quantify macro-heterogeneity of all N- and O-glycopetides of Etanercept. In addition to applications in biotherapeutics, the developed method can also be used for site-specific quantitation of N- and O-glycopeptides and aglycopeptides of glycoproteins with known glycosylation pattern.

}, issn = {1573-4986}, doi = {10.1007/s10719-016-9733-8}, author = {Srikanth, Jandhyam and Agalyadevi, Rathinasamy and Babu, Ponnusamy} } @article {533, title = {A rapid, nonradioactive assay for measuring heparan sulfate C-5 epimerase activity using hydrogen/deuterium exchange-mass spectrometry.}, journal = {Methods Mol Biol}, volume = {1229}, year = {2015}, month = {2015}, pages = {209-19}, abstract = {

Heparin and heparan sulfate (HS) glycosaminoglycans have important roles in anticoagulation, human development, and human diseases. HS C5-epimerase, which catalyzes the epimerization of GlcA to IdoA, is a crucial enzyme involved in the biosynthesis of heparin-related biomolecules. Here, we describe a detailed method for measuring the total activity of HS C5-epimerase that involves the following steps: H/D exchange upon epimerization of the substrate with HS C5-epimerase, low-pH nitrous acid treatment of the substrate, the separation of low-pH nitrous acid-cleaved disaccharides using HPLC, and mass spectrometry analysis. This nonradioactive method is rapid and sensitive and, importantly, allows us to study the reversible nature of HS C5-epimerase.

}, keywords = {Animals, Biocatalysis, Carbohydrate Epimerases, Chromatography, Ion Exchange, Chromatography, Liquid, Deuterium Exchange Measurement, Disaccharides, Enzyme Assays, Glucuronic Acid, Heparitin Sulfate, Humans, Iduronic Acid, Mass Spectrometry, Sf9 Cells}, issn = {1940-6029}, doi = {10.1007/978-1-4939-1714-3_19}, author = {Babu, Ponnusamy and Victor, Xylophone V and Raman, Karthik and Kuberan, Balagurunathan} } @article {534, title = {Glycans in regeneration.}, journal = {ACS Chem Biol}, volume = {9}, year = {2014}, month = {2014 Jan 17}, pages = {96-104}, abstract = {

Glycans participate in many key cellular processes during development and in physiology and disease. In this review, the functional role of various glycans in the regeneration of neurons and body parts in adult metazoans is discussed. Understanding glycosylation may facilitate research in the field of stem cell biology and regenerative medicine.

}, keywords = {Animals, Extremities, Glycosylation, Humans, Hydra, Nerve Regeneration, Neurons, Polysaccharides, Regeneration, Regenerative Medicine}, issn = {1554-8937}, doi = {10.1021/cb400784j}, author = {Babu, Ponnusamy} } @article {535, title = {Unique, polyfucosylated glycan-receptor interactions are essential for regeneration of Hydra magnipapillata.}, journal = {ACS Chem Biol}, volume = {9}, year = {2014}, month = {2014 Jan 17}, pages = {147-55}, abstract = {

Cell-cell communications, cell-matrix interactions, and cell migrations play a major role in regeneration. However, little is known about the molecular players involved in these critical events, especially cell surface molecules. Here, we demonstrate the role of specific glycan-receptor interactions in the regenerative process using Hydra magnipapillata as a model system. Global characterization of the N- and O-glycans expressed by H. magnipapillata using ultrasensitive mass spectrometry revealed mainly polyfucosylated LacdiNAc antennary structures. Affinity purification showed that a putative C-type lectin (accession number Q6SIX6) is a likely endogenous receptor for the novel polyfucosylated glycans. Disruption of glycan-receptor interactions led to complete shutdown of the regeneration machinery in live Hydra. A time-dependent, lack-of-regeneration phenotype observed upon incubation with exogenous fuco-lectins suggests the involvement of a polyfucose receptor-mediated signaling mechanism during regeneration. Thus, for the first time, the results presented here provide direct evidence for the role of polyfucosylated glycan-receptor interactions in the regeneration of H. magnipapillata.

}, keywords = {Animals, Carbohydrate Sequence, Hydra, Lectins, C-Type, Molecular Sequence Data, Polysaccharides, Regeneration}, issn = {1554-8937}, doi = {10.1021/cb400486t}, author = {Sahadevan, Sonu and Antonopoulos, Aristotelis and Haslam, Stuart M and Dell, Anne and Ramaswamy, Subramanian and Babu, Ponnusamy} }