Glycobiology Updates

A Proof-of-Concept Analysis of Carbohydrate-Deficient Transferrin by Imaged Capillary Isoelectric Focusing and In-Capillary Immunodetection

Carbohydrate-deficient transferrin (CDT) is a reliable biomarker for chronic alcohol abuse. We developed a method for CDT analysis by capillary isoelectric focusing, followed by immunodetection directly in the capillary, in an automated fashion and on a single platform (Peggy Sue™; ProteinSimple, CA, USA). Transferrin glycoforms in serum samples, including disialo-transferrin, were separated and their apparent isoelectric points and relative percentages were determined. The relative CDT values (percent of total transferrin) matched expected values for both healthy and alcoholic samples. Because the method leveraged the sensitivity of an immunoassay, CDT was measured when serum samples were diluted up to 1200-fold, reducing the volume of serum required. Finally, the process is fully automated, with up to 96 samples analyzed per batch.

Differential Distribution of N- and O-Glycans and Variable Expression of Sialyl-T Antigen on HeLa Cells—Revealed by Direct Fluorescent Glycan Imaging

Cells are covered with glycans. The expression and distribution of specific glycans on the surface of a cell are important for various cellular functions. Imaging these glycans is essential to aid elucidation of their biological roles. Here, utilizing methods of direct fluorescent glycan imaging, in which fluorescent sialic acids are directly incorporated into substrate glycans via recombinant sialyltranferases, we report the differential distribution of N- and O-glycans and variable expression of sialyl-T antigen on HeLa cells. While the expression of N-glycans tends to be more peripheral at positions where cell-cell interaction occurs, O-glycan expression is more granular but relatively evenly distributed on positive cells. While N-glycans are expressed on all cells, sialyl-T antigen expression exhibits a wide spectrum of variation with some cells being strongly positive and some cells being almost completely negative. The differential distribution of N- and O-glycans on cell surface reflect their distinctive roles in cell biology.

Although chimeric antigen receptor (CAR) T-cell therapy has the potential to revolutionize the way we treat cancer, many challenges remain, not the least of which is cellular infiltration efficacy. A recent paper by Mondal et al (2019) has demonstrated that ex vivo fucosylation enhances the efficiency of CAR T-cell infiltration into bone marrow by 10 fold. Specifically, Fucosyltransferase 6/7-mediated fucosylation of cell surface type 2 sialylLacNAc drives the formation of sialyl Lewis X (sLe^X), which is normally decreased on CAR T-cells as a consequence of cell culture expansion. Stable sLe^X displayed on the cell membrane allows for enhanced binding to the endothelial lectin E-Selectin. This ligand-receptor interaction is crucial for the tethering and rolling of CAR T-cells in the vasculature. Finally, this paper underscores the fact that glycans represent a crucial yet underrated target for enhancing cell-based therapeutic intervention.

A major challenge to assessing the role of glycosylation in cellular function has been the lack of selective, easy-to-use reagents and assays. To address this issue, we have focused a great deal of effort on developing simple and accessible assays that all investigators can use to visualize terminal glycans. In order to drive this endeavor home, we have generated an easy to use protocol for visualizing O-GlcNAc in cultured cells. This assay does not require any expensive capital purchases. It can be completed using commonly available laboratory reagents. See how!

Did you know that Bio-Techne scientists have advanced the Glycobiology field with many peer-reviewed scientific publications? The end-result of our research and development enterprise is a broad array of solutions designed to fulfill your glycobiology research and development needs. With that in mind, this white paper was written to summarize peer-reviewed glycobiology research by Bio-Techne scientists. Our aim was to provide a quick and user-friendly gateway to our science and assay development. From radioactive sulfation assays to fluorescent visualization of glycans in situ, our scientists have worked at the leading edge of glycobiology for nearly a decade. Learn more!

Programmed death ligand 1 (PD-L1) and programmed death protein 1 (PD-1) are well known immune checkpoint targets for cancer. Several biologics have been approved by the Food and Drug Administration for the treatment of cancer including anti-PD-1 monoclonal antibodies (mAbs) (Keytruda, Opdivo, Libtayo), anti-PD-L1 mAbs (Tecentriq, Bavencio, Imfinzi) and the anti-CTLA-4 monoclonal antibody (Yervoy). Although they have revolutionized cancer therapeutics, they are not effective in all contexts; thus, the imperative to maximize the efficacy of therapeutic mAbs. One approach to improve efficacy is to optimize the constant (Fc) region of the antibody. This region contains the site of N-glycosylation and is important for mediating antibody dependent cellular toxicity (ADCC). As such, it can have a dramatic impact on the efficacy of immune checkpoint therapy.Goletz et al. (2018) assessed the impact of “Glyco-optimization” on anti PD-L1 (Tecentriq) efficacy. They assessed the efficacy of PD-L1 variants that were non-glycosylated (Tecentriq) or N-glycosylated (and core fucosylated) or N-glycosylated (and defucosylated). Although antigen binding was identical in all mAbs, the defucosylated antibody exhibited increased binding to FCγIIIa receptors. This variant was also correlated with increased NK cell mediated ADCC against a PDL-L1+ cancer cell line and increased CD8 T cell activation relative to the other variants. This paper highlights the critical importance of glycosylation assessment as part of the development workflow for mAbs as therapeutics. Bio-Techne’s Simple Western™ platform is ideal for high throughput glycan characterization.

Cytotoxic T Lymphocytes (CTLs) induce apoptosis in target cells via the activity of pore forming perforin proteins and granzyme serine proteases at the immunological synapse. Granzymes enter target cells via transmembrane pores formed by oligomers of the cytotoxic perforin protein. One of the important unanswered questions in the field of immunology is how CTLs regulate the activity of perforin prior to storage in secretory granules. House et al. (2017) recently published data indicating that C-terminal glycosylation precludes premature perforin activity in CTLs. Specifically, they demonstrated that high mannose N-glycosylation or complex N-glycosylation at asparagine 549 (Asn549) on the perforin C-terminus inhibits oligomerization and pore formation. This paper highlights the critical, yet oft-unappreciated role of glycosylation in the functioning immune system. Furthermore, it also provides an excellent proof of concept of how glycoenzymes are a useful tool for assessing the role of glycosylation in biological function. Bio-Techne has the largest selection of high-quality R&D Systems glyco-enzymes on the market.

A recent review by de Jesus et al. (2018) discussed the role of O-GlcNAcylation in immune cell function and cancer. This post-translational modification (PTM) is characterized by a rapid and transient cycling of O-GlcNAc on and off the hydroxy residues of serine or threonine acceptors. Glycan attachment and removal are catalyzed by the enzymes O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. This PTM is active at the level of transcription, post-transcriptional, post translational cellular activity, and cellular signaling in several immune cell types, including neutrophils, B cells, T cells, natural killer cells, macrophages, and lymphoid or myeloid cancer cells. Of interest, O-GlcNAcylation has a role in leukemia oncogenesis. The inverse activity of OGA and OGT may serve to tune oncogenesis, which is enhanced when O-GlcNAcylation is suppressed and suppressed when O-GlcNAcylation is enhanced. Finally, a consistent theme in this review is how little we understand O-GlcNAcylation in this context. A major roadblock is “the limited availability of user-friendly, effective technology ideal for the detection of O-GlcNAcylation site(s) in target proteins.” Bio-Techne fills this void with the most selective and user-friendly tools for assessing O-GlcNAcylation on the market.

Transmissible Spongiform Encephalopathies (TSEs), are a collection of progressive and invariably fatal neurodegenerative diseases. As such, there is an imperative to mechanistically understand disease progression. TSEs, also known as prion diseases, are a consequence of the conversion of the normal prion protein (PrPc) to the misfolded prion protein (PrPsc). Prion diseases have been documented in rodents, cattle, and humans. Human PrP is a glycosylphosphatidylinositol (GPI) anchored glycoprotein with two C-terminal glycosylation sites, Asn-181 and Asn-197. Given the role of glycosylation in protein folding and quality control, it is an obvious target for study. Recently, Yi et al. (2018) published data indicating that PrP N-glycosylation plays an important role in PrP aggregation and cytotoxicity. Specifically, they observed altered PrP subcellular localization in cell lines expressing PrP N-glycosylation deficient mutants. They also observed increased proteinase K (PK) resistance and aggregation in unglycosylated mutants. Increased PK resistance is an important observation because PK resistance is a hallmark of the misfolded, infectious PrPsc. Finally, the authors observed increased cytotoxicity in the non-glycosylated PrP mutant. Taken together these data provide compelling evidence of the importance of glycosylation for PrP activity. Taken a step further, these data also suggest a potential target for therapeutic intervention. Looking for a way to automate prion glycosylation research? See how Simple Western is used in a PrPC degycosylation workflow.

The amyloid beta (Aβ) cascade hypothesis is the most prominent mechanistic description of the progression of Alzheimer's Disease (AD). According to this hypothesis, the deposition of Aβ is an early salient event that precedes several events, including the formation of neurofibrillary tangles (NFTs) in the brains of people with AD. The amyloidogenic beta amyloid (Aβ 1-42) is generated by the sequential cleavage of amyloid precursor protein (APP) by beta secretase and the gamma secretase protein complex. An incredible amount of effort has focused on the cellular trafficking of APP, beta secretase, and gamma secretase in order to understand how to preclude the secretion and deposition of Aβ. A review by Kizuka et al. (2018) summarizes what is known about the relationship between glycosylation and AD pathogenesis. Studies have shown that N-glycosylation and sialylation are important for APP transport and secretion. Glycosylation has also been shown to have an important role in the pace of beta secretase production. Removal of N-glycans from the beta secretase-beta site APP cleaving enzyme 1 (BACE-1) has been shown to slow the maturation of the protein. Interestingly, BACE-1 itself was found to regulate glycosylation via its proteolytic action. Gamma secretase is a complex of four proteins including presenilin 1/2, nicastrin, anterior pharynx -defective-1, and presenilin enhancer 2. Although Nicastrin is the only N-glycosylated protein, at this point it isn’t clear what role glycosylation plays in nicastrin function. The activity of alpha secretases, a disintigrin and metalloprotease domain (ADAM) proteins, preclude the generation of the Aβ 1-42 as part of the non-amyloidogenic pathway. ADAM subcellular trafficking has been shown to be altered by mutation of the second of four N-glycan sites. Finally, NFTs are formed by the aggregation of the cytosolic Tau protein. Although there is evidence that Tau N-glycosylation is altered in the context of AD, more studies are needed to confirm published findings. Taken together it is clear that we are only beginning to understand the role of glycosylation in the context of AD progression. Bio-Techne has the largest selection of high-quality of solutions for investigating the glycosylation of AD-related proteins.

Glycans are basic molecules in all living organisms. However, they are not well characterized due to the unavailability of specific and high-sensitivity detection reagents. We describe here a method for enzyme-based glycan imaging using glycosyltransferases. Azido-sugars are incorporated into target glycans followed by click chemistry-based conjugation with fluorescent reporters. Since glycosyltransferases have well defined substrate specificities, this method offers precise detection of target glycans. Examples include imaging of O-GlcNAc, O-GalNAc (Tn antigen), T antigen, sialyllactosamine (sLN), hyaluronan (HA), and heparan sulfate epitopes in different cell lines. This highly specific technique will facilitate the detection and characterization of biologically important glycans.

We describe a novel technique for probing terminal glycans on glycoproteins. Our approach consists of in vitro incorporation of specially developed azido-monosaccharides and various recombinant glycosyltransferases and glycosidases, followed by detection via click chemistry and traditional chemiluminescence. The technique uses SDS-PAGE and protein transfer in a manner similar to Western blot. It is very specific and does not require expensive equipment such as mass spectrometry. To demonstrate its utility, we probed for both N- and O-linked sialoglycans. Using fetal bovine fetuin as an example, we demonstrated: (I) The non-reducing end Gal residues on both N-glycans and O-glycans on the protein are fully sialylated, while the O-linked GalNAc residues are not; (II) The protein contains abundant sialyl core-1 glycan; (III) The protein also contains sialyl Tn antigen. We also present a method for specifically probing high mannose glycans.

We have developed a novel technique capable of probing terminal monosaccharides on proteins using SDS-PAGE. This technique does not require expensive equipment and is more specific than comparable methods. Additionally, it offers any laboratory the ability to gain a greater understanding of the glycan profiles of their protein of interest without significant