The intrinsic nature of glycosylation, namely nontemplate encoded, stepwise elongation and termination with a diverse range of isomeric glyco-epitopes (glycotopes), translates into ambiguity in most cases of mass spectrometry (MS)-based glycomic mapping. It is arguable that whether one needs to delineate every single glycomic entity, which may be counterproductive. Instead, one should focus on identifying as many structural features as possible that would collectively define the glycomic characteristics of a cell or tissue, and how these may change in response to self-programmed development, immuno-activation, and malignant transformation. We have been pursuing this line of analytical strategy that homes in on identifying the terminal sulfo-, sialyl, and/or fucosylated glycotopes by comprehensive nanoLC-MS-product dependent MS analysis of permethylated glycans, in conjunction with development of a data mining computational tool, GlyPick, to enable an automated, high throughput, semi-quantitative glycotope-centric glycomic mapping amenable to even nonexperts. We demonstrate in this work that diagnostic MS ions can be relied on to inform the presence of specific glycotopes, whereas their possible isomeric identities can be resolved at MS level. Both MS and associated MS data can be acquired exhaustively and processed automatically by GlyPick. The high acquisition speed, resolution, and mass accuracy afforded by top-notch Orbitrap Fusion MS system now allow a sensible spectral count and/or summed ion intensity-based glycome-wide glycotope quantification. We report here the technical aspects, reproducibility and optimization of such an analytical approach that uses the same acidic reverse phase C18 nanoLC conditions fully compatible with proteomic analysis to allow rapid hassle-free switching. We further show how this workflow is particularly effective when applied to larger, multiply sialylated and fucosylated N-glycans derived from mouse brain. The complexity of their terminal glycotopes including variants of fucosylated and disialylated type 1 and 2 chains would otherwise not be adequately delineated by any conventional LC-MS/MS analysis.
Sialic acids are typically added to the end of glycoconjugates by sialyltransferases. Among the six ST8 α- N -acetyl-neuraminide α-2,8-sialyltransferases (ST8SIA) existing in adult brains, ST8SIA2 is a schizophrenia-associated gene. However, the in vivo substrates and physiological functions of most sialyltransferases are currently unknown. The ST8SIA3 is enriched in the striatum. Here, we showed that ablation of St8sia3 in mice ( St8sia3 -KO) led to fewer disialylated and trisialylated terminal glycotopes in the striatum of St8sia3 -KO mice. Moreover, the apparent sizes of several striatum-enriched G-protein-coupled receptors (GPCRs) (including the adenosine A 2A receptor (A 2A R) and dopamine D 1 /D 2 receptors (D 1 R and D 2 R)) were smaller in St8sia3 -KO mice than in WT mice. A sialidase treatment removed the differences in the sizes of these molecules between St8sia3 -KO and WT mice, confirming the involvement of sialylation. Expression of ST8SIA3 in the striatum of St8sia3 -KO mice using adeno-associated viruses normalized the sizes of these proteins, demonstrating a direct role of ST8SIA3. The lack of ST8SIA3-mediated sialylation altered the distribution of these proteins in lipid rafts and the interaction between D 2 R and A 2A R. Locomotor activity assays revealed altered pharmacological responses of St8sia3 -KO mice to drugs targeting these receptors and verified that a greater population of D 2 R formed heteromers with A 2A R in the striatum of St8sia3 -KO mice. Since the A 2A R-D 2 R heteromer is an important drug target for several basal ganglia diseases (such as schizophrenia and Parkinson’s disease), the present study not only reveals a crucial role for ST8SIA3 in striatal functions but also provides a new drug target for basal ganglia-related diseases.
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