Mammalian Brain Glycoproteins Exhibit Diminished Glycan Complexity Compared To Other Tissues | Communications

We noted a low abundance of galactose containing N-glycans (10–15%) and an even smaller amount containing sialic acid (1–3%). Riley, N. M., Hebert, A. S., Westphall, M. & Coon, J. Capturing site-specific heterogeneity with large-scale N-glycoproteome analysis. Chameleon Duo can be used to perform two-colour visible and near-infrared detection. On the contrary, known complex and paucimannose N-glycans were not sensitive to Endo H treatment; these glycans were present at the same relative intensity after the secondary PNGase F treatment (Fig. 2013; 52 (23517332): 3797-3806. Overview of affinity tags for protein Protoc. 3C), and no structures corresponding to these glycans were detected in the Endo H spectra (Fig. Research Resource Identification (RRID) (if available)|. USA 114, 11163–11168 (2017). New tools for content innovation and data sharing: enhancing reproducibility and rigor in biomechanics research. Quartu, M. Mammalian brain glycoproteins exhibit diminished glycan complexity compared to other tissues | Communications. Polysialylated-neural cell adhesion molecule (PSA-NCAM) in the human trigeminal ganglion and brainstem at prenatal and adult ages. Core M2 glycans have only been reported in the brain, where the key synthetic enzyme MGAT5B is highly enriched, and regulate remyelination, astrocyte activation, and oligodendrocyte differentiation 97, 98, 99, 100, 101.

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Bhattacharyya, R., Bhaumik, M., Raju, T. & Stanley, P. Truncated, Inactive N-Acetylglucosaminyltransferase III (GlcNAc-TIII) Induces Neurological and Other Traits Absent in Mice That Lack GlcNAc-TIII. 1 M NaOH solution and incubated overnight (12–16 h) at 45 °C. Microbiol 4, 2146–2154 (2019). Chameleon duo pre stained protein ladder model. For further analysis, individual glycans were categorized by monosaccharide composition or shared structural characteristics such as branching (Supplementary Note 1, Supplementary Data 2), and the abundance of these groups were compared between regions. SiRNA knockdown validation 101: incorporating negative controls in antibody research.

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7F), the sole enzyme responsible for the synthesis of core-2 O-Man glycans 66, and these structures were several-fold more abundant in this region. Though not as pronounced as the differences observed in plasma, these results illustrate that brain protein glycosylation shows some sex-dependence and underscore the importance of analyzing both sexes separately. 5) and incubated at room temperature for 90 min in the dark. Bartels, M. F. Protein O-Mannosylation in the Murine Brain: Occurrence of Mono-O-Mannosyl Glycans and Identification of New Substrates. GeneCards®: The Human Gene Database||A searchable, comprehensive database of annotated/predicted human genes; integrates genomic, transcriptomic, proteomic, genetic, clinical, and functional data from many web sources|||. Grant, M. K. O., Shapiro, S. L., Ashe, K. H., Liu, P. & Zahs, K. A Cautionary Tale: Endogenous Biotinylated Proteins and Exogenously-Introduced Protein A Cause Antibody-Independent Artefacts in Western Blot Studies of Brain-Derived Proteins. Thirstrup K. - Dächsel J. C. - Oppermann F. S. - Williamson D. S. - Smith G. P. - Fog K. - Christensen K. V. - Bakkenist C. J. Chameleon duo pre stained protein ladder test. Zetterberg H. - Koel-Simmelink M. - Teunissen C. E. - Lund-Johansen F. - Browning M. D. - Acharya P. - Quinlan A.

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L. P. -K., S. D. S., and A. S. -G. are employees of LI-COR Biosciences, and S. H., A. R., and A. are employees of Abcam Plc. Online 21, 6 (2019). Smale S. T. - Verhoef L. G. Antibody validation for Western blot: By the user, for the user. - Mattioli M. - Ricci F. - Li Y. Core 2 O-GalNAc glycans, defined by the addition of GlcNAc to the GalNAc of the core 1 structure, were highest in the cortex and cerebellum. The intensity of individual N-glycans isolated from the cortex using PNGase F (Fig. In SialoGlyco Chemistry and Biology I vol. 42, D490–D495 (2014). 2001; 116 (11463466): 55-63. 2003; 4 (12952525): 117. Endogenous, purified, tagged, or overexpressed target protein|. Neurochemistry 84, 557–565 (2003). The Mannose Receptor in the Brain.

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Arvin N. E. - Dawod M. - Kennedy R. T. - Zhu Z. 33, 10037–10047 (2013). Structural assignment of glycans was based on MS/MS results, enzyme sensitivity (PNGase F, Endo H), previously confirmed structures 35, 37, 58, and deductive reasoning when able. 1% for 30 min protected from light. Minimum information about a protein affinity reagent (MIAPAR) Biotech. C. - Considerations when quantitating protein abundance by J. Physiol.

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High-mannose N-glycans are often considered immature precursor structures but comprise the majority of all N-glycans in the brain. 7I), consistent with the general restricted presence of O-mannose glycans to the brain and a few other tissues 37, 38, 75. Sato, C. & Kitajima, K. Sialic Acids in Neurology. The reaction tube was washed with an additional 100 μL of 5% acetic acid which was added to the column, followed by 1 mL of 5% acetic acid, and the entire flow-through was placed in a speed vacuum to remove the organic solvents and lyophilized prior to permethylation as described below. Van Waalwijk van Doorn L. J. Peer review information. Proper application of antibodies for immunohistochemical detection: antibody crimes and how to prevent them. Five of the top 10 most abundant N-glycans in the brain were high-mannose structures, including the most abundant, Man5GlcNAc2 (Man-5), which comprised nearly half of the total glycan signal in the brain (Fig. Sequencing was performed on Illumina HiSeq 2500 instrument resulting in approximately 30 million of 50 bp reads per sample. We detected a small amount (1–2%) of O-glycans containing the NeuGc form of sialic acid, consistent with prior studies 37, 57. In a third unique case, the peak at m/z: 2040 was partially Endo H sensitive, indicating a mixture of hybrid and non-hybrid glycans present at this mass. 29, R229–R231 (2019). Plasma N-glycan profiling was performed as described previously 73.

We hypothesize that this restricted repertoire of protein glycans arises from their tight regulation in the brain. The participation of sialic acids in microglia–neuron interactions. The cortex, hippocampus, striatum, and cerebellum have overall similar glycomes; however, we identified several glycans, glycan classes, and glycosylation enzymes that differ significantly between brain regions, emphasizing the need to study these regions independently. Utilizing MALDI-TOF glycomics, MS/MS, lectin blotting, and RNA sequencing, we have generated a comprehensive map of the predominant N- and O-linked protein glycans across multiple brain regions and both sexes of mice. In contrast to brain N-glycans, which had a large amount of fucose (~30%) and a paucity of sialic acid (~2%), few brain O-glycans were fucosylated (~10%), while the majority were sialylated (~90%). Grouped expression analysis of 30 general tissue types showed that the brain is the only region with a significantly down-regulated gene set, and the only region which is significantly different when comparing differences in both directions (Fig. The majority of CDGs present with neurologic symptoms including intellectual disability, seizures, and structural abnormalities, illustrating the particular importance of glycosylation in the brain 16. A quasi-quantitative dual multiplexed immunoblot method to simultaneously analyze ATM and H2AX phosphorylation in human peripheral blood mononuclear cells. A guide to choosing fluorescent Methods. Translate the article.

Proteoglycans, composed of a core protein modified by various glycosaminoglycan (GAG) chains, have also been a focus, and are known to be temporally and spatially regulated throughout brain development, serving as guidance cues during cell migration and axon pathfinding 24, 25, 26. Each column was placed in a 15 mL glass tube, and glycopeptides were eluted using 2 mL of 20% 1-propanol, 2 mL of 40% 1-propanol, and 2 mL of 100% 1-propanol. Defining antibody validation for Western blotting. Freeze, H. H., Eklund, E. A., Ng, B. 76 1–64 (Elsevier, 2019). 354 known glycosyltransferases, glycosylhydrolases, sulfotransferases, and glycan-related genes IDs from humans were used as input into the GENE2FUNC platform of FUMA, which utilizes the GTEx v8 data of both 30 general tissue types, with all brain regions summarized as one tissue type, and 54 specific tissue types that include 13 individual brain regions. Schmitz, B., Peter-Katalinic, J., Egge, H. Monoclonal antibodies raised against membrane glycoproteins from mouse brain recognize N-linked oligomannosidic glycans. These products typically do not have pictures or detailed descriptions. Mice from both sexes were used in this study and were 12 weeks old at the time of tissue harvest, sample size specified for each experiment.

Inhibition of the streptavidin–biotin interaction by Biochem. Validation strategy|. 2012; 709 (22122927): 21-31. Neumeister V. - Gilda J. E. - Ghosh R. - Cheah J. X.

Development 140, 4970–4981 (2013). Neuroscience 164, 1685–1694 (2009). Damerell, D. The GlycanBuilder and GlycoWorkbench glycoinformatics tools: updates and new developments. Commercial antibodies: the good, bad, and really ugly.

For glycomic analyses, statistical analysis of individual and groups of glycans was performed with Microsoft Excel Version 16. Linnartz, B., Kopatz, J., Tenner, A. Sialic Acid on the Neuronal Glycocalyx Prevents Complement C1 Binding and Complement Receptor-3-Mediated Removal by Microglia.