Fluorescent Glycan Labeling and Detection

Glycan Labeling and Detection Using Fluorescent Donor Substrates

Glycosylation is an abundant posttranslational modification that occurs on a large number of proteins and lipids. It is a dynamic process that produces structural differences that fine tune the functions of glycoconjugates. Glycosylation is known to be important for regulating protein folding, secretion, localization, stability, and activity. In addition, cell surface glycans are crucial for cell recognition and migration in processes such as immune cell trafficking. Glycosylation is primarily determined by the availability of specific substrates, and the presence of enzymes that regulate the addition and removal of glycan moieties. To allow researchers to more easily analyze protein glycosylation, Bio-Techne offers kits and individually packaged labeling enzymes and fluorescent-labeled donor substrates that make it possible to label and detect glycans using SDS-PAGE and a fluorescent imager.

Benefits of Fluorescent Glycan Labeling

• Fluorescent labels can be directly introduced on glycans in glycoproteins and glycolipids using various recombinant fucosyltransferases and sialyltransferases
• Fluorescent labeling of glycans allows protein glycosylation to be detected using SDS-PAGE and a fluorescent imager
• Fluorescent labels can be introduced into glycans of live cells for glycan imaging

Kits for Specific Glycan Labeling and Detection

Bio-Techne offers a variety of kits and individually packaged labeling enzymes and fluorescent donor substrates for specific glycan labeling and detection. These kits and reagents can be used to determine whether a protein contains N-glycans, O-glycans, heparan sulfate or O-GlcNAc modifications. When labeled N-glycans are released by PNGase F digestion, they can be revealed as glycan fingerprints by polyacrylamide gel electrophoresis.

N-Glycan Labeling and Detection

N-glycans are carbohydrate-based polymers attached to asparagine residues in the protein sequence, N-X-S/T (Asn-X-Ser/Thr), where X can be any amino acid except Pro. Researchers can determine if their protein is N-glycosylated by N-glycan-specific labeling using the strategy shown below. Our N-glycan Labeling and Detection Kit provides all of the reagents necessary for N-glycan labeling. Additionally, we offer individually packaged enzymes and fluorescent CMP sialic acid donors for N-glycan labeling and detection by SDS-PAGE.

O-Glycan Labeling and Detection

O-glycans usually refer to O-GalNAc and its derivatives. O-glycans are initiated by attaching a single GalNAc residue to a Ser/Thr residue on a protein via polypeptide GalNAc transferases. O-GalNAc can be further extended by other sugar residues to become different subtypes, such as Core-1 and Core-2 O-glycans. O-glycan-specific labeling is achieved through direct incorporation of conjugated sialic acids, such as Cy5-conjugated Neu5Ac (Cy5-Neu5Ac), or replacement of existing sialic acids with Cy5-Neu5Ac via an O-glycan specific sialyltransferase, such as ST3Gal1 or ST3Gal2. Labeled products are then separated by SDS-PAGE and detected using a FluorChem fluorescent imager. Our O-glycan Labeling and Detection Kit provides all of the reagents necessary for O-glycan labeling. Additionally, we offer individually packaged enzymes and fluorescent CMP sialic acid donors for O-glycan labeling and detection by SDS-PAGE.

Specific Detection of N- and O-Glycans on Different Glycoproteins

N-Glycan and O-Glycan Labeling of ACE-2 and CA125/MUC16. (A) Recombinant Human ACE-2 (Catalog # 933-ZN) was labeled on N-glycans (N) using Recombinant Human ST6Gal1 (Catalog # 7620-GT) and Cy3-Neu5Ac and on O-glycans (O) using Recombinant Human ST3Gal2 (Catalog # 7275-GT) and Cy3-Neu5Ac. The labeled samples were then treated with a 10-fold serial dilution of PNGase F starting with 100 ng using the PNGase F N-glycan Releasing Kit (Catalog # EA006). The digestions were separated on a 17% SDS-PAGE gel. The upper part of the image is the fluorescent image obtained using a FluorChem fluorescent imager and the lower part of the image is the TCE image of the gel. While all labeled N-glycans could be released by PNGase F treatment, no labeled O-glycans were released by PNGase F, demonstrating the specific labeling on N- and O-glycans by ST6Gal1 and ST3Gal2, respectively. (B) Recombinant Human CA125/MUC16 (Catalog # 5609-MU) was labeled on N-glycans (N) using Recombinant Human ST6Gal1 (Catalog # 7620-GT) and Cy5-Neu5Ac and on O-glycans (O) using Recombinant Human ST3Gal2 (Catalog # 7275-GT) and Cy5-Neu5Ac. The labeled samples were treated with 4-fold serial dilution of PNGase F starting with 640 ng using the PNGase F N-glycan Releasing Kit (Catalog # EA006). The digestions were separated on a 17% SDS-PAGE gel. The upper part of the image is the fluorescent image obtained using the FluorChem fluorescent imager and the lower part of the image is the TCE image of the gel. While all labeled N-glycans could be released by PNGase F treatment, no labeled O-glycans were released by PNGase F treatment, demonstrating the specific labeling on N- and O-glycans by ST6Gal1 and ST3Gal2, respectively.

N-Glycan and O-Glycan Labeling on Different Glycoproteins. (A) Recombinant Human CEACAM-7 (Catalog # 9010-CM), Recombinant Human CEACAM-8/CD66b (Catalog # 9639-CM), Recombinant Human MUC-1 (Catalog # 10332-MU), and Recombinant Human CA125/MUC16 (Catalog # 5609-MU) were labeled on N-glycans (N) using Recombinant Human ST6Gal1 (Catalog # 7620-GT) and Cy3-Neu5Ac and on O-glycans (O) using Recombinant Human ST3Gal1 (Catalog # 6905-GT) and Cy5-Neu5Ac and then separated on a 4-20% SDS-PAGE gel. Only N-glycans were detected on CEACAM-7 and CEACAM-8, and only O-glycans were detected on MUC-1, while both N- and O-glycans were detected on MUC16. The left side of the data is showing the TCE image of the gel and the right side is showing the fluorescent image obtained using a FluorChem fluorescent imager. (B) A collection of glycoproteins, including Recombinant SARS-CoV-2 Spike RBD (RBD; Catalog # 10500-CV), Recombinant Human Erythropoietin/EPO (Catalog # 287-TC), fetal bovine Fetuin, Recombinant Human ACE-2 (Catalog # 933-ZN), Recombinant Human CA125/MUC16 (Catalog # 5609-MU), Recombinant Human MUC-1 (Catalog # 10332-MU), Recombinant Human CD19 (Catalog # 9269-CD), Recombinant Human PD-1 (Catalog # 1086-PD), and Recombinant Human IL-6R alpha (Catalog # 227-SR) were labeled on O-glycans with Cy3-Neu5Ac using Recombinant Human ST3Gal2 (Catalog # 7275-GT; green) and on N-glycans with Cy5-Neu5Ac and Recombinant Human ST6Gal1 (Catalog # 7620-GT; red). The samples were then either left untreated or treated with PNGase F using the PNGase F N-glycan Releasing Kit (Catalog # EA006) and separated on a 17% SDS-PAGE gel. The left side of the figure is showing gel images of labeled samples without PNGase F treatment, and the right side of the figure is showing gel images of the samples after PNGase F treatment. The upper part of the image is the TCE image of the gel, while the lower part is the fluorescent image obtained using a FluorChem fluorescent imager. While MUC-1 primarily contains O-glycans and CD19 primarily contains N-glycans, the other proteins contain both N- and O-glycans at different levels. All labeled N-glycans in red except that on MUC-1 could be released by PNGase F treatment, further confirming the specific labeling. The nature of the labeling on MUC-1 by ST6Gal1 is not clear.

Glycan Labeling Reagents and Labeled Glycans

Labeled glycans can be used as substrates for glycosyltransferases and glycosidases. Enzymatic modifications of a labeled glycan will result in a mobility shift of the glycan when it is separated by polyacrylamide gel electrophoresis.

Strategies for Glycan Labeling. (A) Schemes for glycan labeling using Recombinant Human Fucosyltransferase/FUT8 (Catalog # 5768-GT) and GDP-Cy5-Fucose (Catalog # ES301) to generate Cy5-Fucose Labeled N2f (Catalog # GL304), followed by the step-wise synthesis of Cy5-Fucose Labeled Glycan G2f (Catalog # GL302), Cy5-Fucose Labeled xG2f, and Cy5-Fucose Labeled xxG2f using various glycosyltransferases, including Recombinant Human B4GalT1 (Catalog # 3609-GT), Recombinant Human B3GNT2 (Catalog # 3960-GT), or hydrolysis of xxG2f by F. keratolyticus Endo-beta-Galactosidase (Catalog # 8620-GH). Cy5-Fucose Labeled M2f was also generated using Recombinant Human Hexosaminidase/HEXA (Catalog # 6237-GH). (B) Products of the glycan labeling reactions and Endo-beta-Gal assay shown in part A were separated by SDS-PAGE and visualized using a FluorChem fluorescent imager.

Reagents for Glycan Labeling and Labeled Glycans

Glycan Labeling Reagents through Fucosylation

Fucosylation is a terminal modification. The labeling can be performed on free glycans as well as glycoproteins or glycans on the cell surface. Compared to labeling through sialylation, fucosyltransferases have much faster kinetics. For maximum labeling, existing fucose sugars can be removed by different fucosidases prior to labeling.

Strategy for Glycan Labeling through Fucosylation. Glycan labeling through fucosylation is achieved by direct incorporation of fluorescent-labeled fucose on free glycans or by replacing natural existing fucose sugars with flurorescent-labeled fucose (shown above). The samples are first treated with Fucosidase/FUCA1 to remove existing fucose moieties, followed by labeling with fluorescent-labeled fucose (GDP-F-Fucose) and a fucosyltransferase. The labeled products are then separated by SDS-PAGE and detected using a FluorChem fluorescent imager.

Reagents for Glycan Labeling through Fucosylation

Fluorescent Glycan Labeling with Recombinant Fucosyltransferases. (A) Fetal bovine fetuin (Fet) and asialofetuin (aFet) were labeled using Recombinant Human Fucosyltransferase 2/FUT2 (F2; Catalog # 7770-GT), Recombinant Human Fucosyltransferase 6/FUT6 (F6), Recombinant Human Fucosyltransferase 7/FUT7 (F7; Catalog # 6409-GT), or Recombinant Human Fucosyltransferase 9/FUT9 (F9; Catalog # 9347-GT), and GDP-Cy5-Fucose (Catalog # ES301) and then separated by SDS-PAGE. The left side of the data is showing the TCE image of the gel and the right side is showing the fluorescent image obtained using a FluorChem fluorescent imager. (B) Asialofetuin (aFet) was labeled using Recombinant Human Fucosyltransferase 2/FUT2 (F2; Catalog # 7770-GT) or Recombinant Human Fucosyltransferase 9/FUT9 (F9; Catalog # 9347-GT), and fetal bovine fetuin was labeled using Recombinant Human Fucosyltransferase 7/FUT7 (F7; Catalog # 6409-GT) or Recombinant Human Fucosyltransferase 6/FUT6 (F6), and either GDP-Alexa Fluor® 488-Fucose (488; custom order), GDP-Alexa Fluor® 555-Fucose (555; custom order), or GDP-Cy5-Fucose (Cy5; Catalog # ES301) and then separated by SDS-PAGE. The upper part of the image is the TCE image of the gel and the lower part of the image is the fluorescent image obtained using a FluorChem fluorescent imager.

Glycan Labeling Reagents through Sialylation

Sialylation is a common terminal modification. The labeling can be performed on free glycans as well as glycoproteins or glycans on the cell surface. For maximum labeling, existing sialic acid sugars can be removed by different neuraminidases prior to labeling.

Strategies for O-Glycan and N-Glycan Labeling through Sialylation. (A) O-glycan-specific labeling is achieved through replacement of natural sialic acids with fluorescent-labeled sialic acid (for ex. Cy5-Neu5Ac). The samples are first desialylated with Recombinant C. perfringens Neuraminidase (Neu), which specifically removes alpha 2-3- and alpha 2-6-linked sialic acids prior to labeling. Cy5-Neu5Ac is then introduced using an O-glycan specific sialyltransferase, such as ST3Gal1, ST3Gal2, or S6GalNAc2. (B) N-glycan-specific labeling is achieved through direct incorporation of fluorescent-conjugated sialic acids, such as Cy3-conjugated Neu5Ac, or replacement of natural existing sialic acids with Cy3-Neu5Ac. To achieve maximum labeling, the samples are first desialylated with Recombinant C. perfringens Neuraminidase (Neu), which specifically removes alpha 2-3- and alpha 2-6-linked sialic acids prior to labeling. Fluorescent-labeled sialic acid is then introduced using an N-glycan specific sialyltransferase. Following labeling, the products are separated by SDS-PAGE and detected using a FluorChem fluorescent imager.

Reagents for Glycan Labeling through Sialylation

Fluorescent Glycan Labeling with Recombinant Sialyltransferases. (A) Fetal bovine fetuin (Fet) and asialofetuin (aFet) were labeled using Recombinant Human ST3Gal1 (31; Catalog # 6905-GT), Recombinant Human ST6Gal1 (61; Catalog # 7620-GT), or Recombinant Human ST6Gal4 (6A4), and the indicated fluorescent-labeled sialic acids and then separated by SDS-PAGE. The upper part of the image is the TCE image of the gel and the lower part of the image is the fluorescent image obtained using a FluorChem fluorescent imager. (B) Hela cells were labeled on O-glycans using Recombinant Human ST3Gal1 (Catalog # 6905-GT; red) and on N-glycans using Recombinant Human ST6Gal1 (Catalog # 7620-GT; green).

Heparan Sulfate/Hyaluronan Labeling Reagents

Heparan sulfate (HS) is a linear sulfated polysaccharide and hyaluronan (HA) is a non-sulfated polysaccharide. Enzymatic labeling has the benefits of adding fluorophores specific to the non-reducing ends, and therefore does not interfere with the normal biological functions of the molecules. For HS labeling, maximum labeling can be achieved by heparinase treatment prior to labeling.

Strategy for Hyaluronan or Heparan Sulfate Labeling. Hyaluronan or heparan sulfate labeling is achieved by treating the polysaccharides with Hyaluronan Synthase (HAS) or EXT1/2, the labeling enzymes for hyaluronan or heparan sulfate, respectively, and adding UDP-Azido-GlcNAc with UDP-GlcA. This is followed by a click chemistry reaction to replace the azide group with a fluorophore. The labeled products can then separated by SDS-PAGE and detected using a FluorChem fluorescent imager.

Reagents for Heparan Sulfate and Hyaluronan Labeling

Fluorescent Labeling of Hyaluronan and Heparan Sulfate. (A) Hyaluronan was labeled with Cy5 using Recombinant P. multocida Hyaluronan Synthase/HAS (Catalog # 9585-GT) and then digested with Recombinant Human Hyaluronidase 3/HYAL3 and Recombinant Human Hyaluronidase 1/HYAL1 (Catalog # 7358-GH) and analyzed by SDS-PAGE. The upper part of the image is the fluorescent image obtained using a FluorChem fluorescent imager and the lower part of the image is the TCE image of the gel. (B) Hela cells were labeled on heparan sulfate with Cy5 using Recombinant Human Exostosin 1/2 (Catalog # 8567-GT; red). Nuclei were counterstained with DAPI (Catalog # 5748; blue).

O-GlcNAc/O-GalNAc Labeling Reagents

O-GlcNAc is only found in intracellular proteins and is particularly abundant in nuclear proteins. O-GlcNAc is introduced by O-GlcNAc transferase/OGT and removed by O-GlcNAcase/OGA. O-GlcNAc can be labeled either directly using either B3GALNT2 or B4GalT1 (Y285L) with UDP-Azido-GalNAc followed by a click chemistry reaction or through tandem labeling using B4GalT1 and ST6Gal1 with a fluorophore-labeled sialic acid donor. The tandem labeling method is more convenient as it does not require the click chemistry reaction that is required for direct labeling.

Strategies for O-GlcNAc Labeling. O-GlcNAc can be labeled either (A) directly using B3GALNT2 or B4GalT1 (Y285L) with UDP-Azido-GalNAc followed by a click chemistry reaction to replace the azide group with a fluorophore, or (B) by tandem labeling using B4GalT1 and ST6Gal1 with a fluorophore-labeled sialic acid donor. Following the labeling reaction, the products can then separated by SDS-PAGE and detected using a FluorChem fluorescent imager.

“Speaking about detection of O-GlcNAc via tandem glycan labeling – This is such a smart and efficient way to detect O-GlcNAc.”
- Peng Wu | The Scripps Research Institute

Reagents for O-GlcNAc Labeling

O-GlcNAc Detection Using the Tandem Labeling or Direct Labeling Methods. (A) Nuclear extracts of HEK293 cells were tandem labeled on O-GlcNAc using Recombinant Human B4GalT1 (Catalog # 3609-GT) and Recombinant Human ST6Gal1 (Catalog # 7620-GT). Lane 1 contains a protein Western marker, lanes 4 and 5 contain samples that were treated with Recombinant Human O-GlcNAc Transferase/OGT (Catalog # 8446-GT) first and then labeled for O-GlcNAc, lane 6 contains a sample that was labeled with Recombinant Human ST6Gal1 only. The left side of the figure is the fluorescent image obtained using a FluorChem fluorescent imager and the right side of the figure is the TCE image of the gel. (B) O-GlcNAc was detected on C3H10T1/2 mesenchymal stem cells by direct labeling using Recombinant Human B4GalT1 (Y285L) (Catalog # 7040-GT) and UDP-Azido-GalNAc (Catalog # ES103). Nuclei were counterstained with DAPI (Catalog # 5748; blue).

References

1. Kofsky, J.M. et al. (2023) Glycosyltransferases as versatile tools to study the biology of glycans. Glycobiology. [Online ahead of print].
2. Wu, Z.L. & J.M. Ertelt (2022) Endoglycosidase assay using enzymatically synthesized fluorophore-labeled glycans as substrates to uncover enzyme substrate specificity. Commun. Biol. 5:501.
3. Wu, Z.L. & J.M. Ertelt (2021) Assays for hyaluronidases and heparinase using nonreducing end fluorophore-labeled hyaluronan and heparan sulfate proteoglycan. Glycobiology 31:1435.
4. Wu, Z.L. et al. (2020) Detecting substrate glycans of fucosyltransferases with fluorophore-conjugated fucose and methods for glycan electrophoresis. Glycobiology 30:970.
5. Wu, Z.L. et al. (2020) Fluorescent detection of O-GlcNAc via tandem glycan labeling. Bioconjug. Chem. 31:2098.
6. Wu, Z.L. et al. (2020) Differential distribution of N- and O-glycans and variable expression of sialyl-T antigen on Hela cells – revealed by direct fluorescent glycan imaging. Glycobiology 30:454.
7. Wu, Z.L. et al. (2019) Direct fluorescent glycan labeling with recombinant sialyltransferases. Glycobiology 29:750.
8. Wu, Z.L. et al. (2018) Imaging specific cellular glycan structures using glycosyltransferases via click chemistry. Glycobiology 28:69.
9. Wu, Z.L. et al. (2016) Probing sialoglycans on fetal bovine fetuin with azido-sugars using glycosyltransferases. Glycobiology 26:329.

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