Glutathione Transferases: Probing for isoform specificity ...
A Sensitive Method for Probing Sialoglycans Using Azido ... · format of Western blotting.1 Using...
Transcript of A Sensitive Method for Probing Sialoglycans Using Azido ... · format of Western blotting.1 Using...
AbstractSialoglycans have numerous biological functions. Here we describe a method to directly probe the glycans on glycoproteins. Our approach consists of in vitro incorporation of clickable monosaccharides using various recombinant glycosyltransferases and glycosidases, followed by detection via click chemistry in a format of Western blotting.1 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 specifi cally probing high mannose glycan.
IntroductionFunctions of sialoglycansSialic acids, also called neuraminic acids, are negatively charged monosaccharides that are usually located at the non-reducing ends of glycans on secreted glycoproteins. They regulate the solubility, stability, and half-lives of the secreted proteins. Due to their unique positioning, sialic acids frequently act as receptors or ligands during cell-cell and cell-pathogen interactions.2,3 Accordingly, numerous lectins expressed by mammalian cells and pathogens are known to bind cell surface sialic acids.4
Types of sialoglycans Sialylation occurs on N-glycans and O-glycans on glycoproteins. N-glycans can be categorized as high-mannose, hybrid, and complex types. Hybrid and complex N-glycans frequently contain terminal sialic acids. There are 8 prototypes of O-glycans that can be terminated with sialic acids.5 The most common types are core-1 O-glycan (Galβ1-3GalNAc-O-S/T) and core-2 O-glycan (GlcNAcβ1-6(Gal β1-3)GalNAc-O-S/T). Tn antigen, consisting of a GalNAc residue linked to Ser or Thr residues (GalNAc-O-T/S), can also be sialylated as sialyl-Tn antigen (Siaα2-6GalNAc-O-T/S).6 Both Tn and Sialyl-Tn antigens are frequently found on tumor cells.7,8
Sialyltransferases Sialic acids are introduced to glycans through specifi c sialyltransferases.9 The human genome has:
six β-galactoside α-2,3-sialyltransferases (ST3Gal1-6), two β-galactoside α-2,6- sialyltransferases (ST6Gal1, 2), six N-acetylgalactosaminide α-2,6-sialyltransferases (ST6GalNAc1-6), six α-N-acetyl-neuraminide α-2,8-sialyltransferases (ST8Sia1-6).
Table 1. The substrate specifi cities of the glycosyltransferases used in this research.
Probing sialoglycans via in vitro incorporation of azido-sugar and click chemistryAs an example, we probed the sialoglycans on fetal bovine fetuin, by introducing azido-sialic acid using recombinant sialyltransferases.10 Fetal bovine fetuin contains three N-linked complex glycans and three O-linked glycans.11–13 The known O-glycans on the protein include a disialylated core-1 O-glycan (Siaα2-3Galβ1-3(Siaα2-6)GalNAc), a monosialylated core-1 O-glycan (Siaα2-3Gal β1-3GalNAc) and a disialylated hexasaccharide (Siaα2-3Gal β1-3 [Siaα2-3Gal β 1-4GlcNAc β1-6]GalNAc).11, 14, 15
Assay ProceduresAll azido sugars and click chemistry reagents are manufactured through Tocris Bioscience and R&D Systems.1. Deglycosylation (optional)To probe a glycosylation site that is already fully occupied, a deglycosylation step is required. For example, if a fully sialylated Gal residue is going to be probed, a desialylation step with a sialidase is then required. An oligosaccharide can be removed with a combination of glycosidases as well to expose hidden glycosylation sites. 2. Incorporation of clickable monosaccahrides via GlycosyltransferaseClickable monosaccharides are introduced to existing glycosylation sites or sites that are created by Step 1, i.e. azido sialic acid from CMP-azido-sialic acid is introduced by sialyltransferases, and GlcNAz from UDP-GlcNAz is introduced by GlcNAc transferases. Reaction conditions specifi ed by manufacture’s inserts for these enzymes are followed. The input concentrations for the donor substrates normally range from 10 to 100 µM. 3. Click chemistry reactionClick chemistry reagents, are directly added to the glycosyltransferase reactions and incubated at room temperature for appropriate time. For Copper dependent click chemistry, 1 mM ascorbic acid, 30 µM CuCl2, 10 to 100 µM alkyne conjugate are added to the reactions and incubated for 1 hour at room temperature. 4. SDS-PAGE and gel blotting The samples are resolved on SDS-PAGE gels, blotted to nitrocellulose membrane. The blots are blocked with 10% fat-free milk and washed thoroughly with 25 mM Tris, pH 7.6, 137 mM NaCl and 0.01% Tween. The blots are then probed with strep-HRP and visualized with chemiluminescence.
SummaryUsing bovine fetal fetuin as a testing glycoprotein, we demonstrated the use of glycosyltransferases and clickable monosaccharides to probe glycan structure or epitope. We confi rmed that 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, the protein contains abundant sialyl core-1 glycan. In addition, we are able to detect sialyl Tn antigen on the protein, suggesting the high sensitivity and specifi city of this method for glycan epitope detection.
Principle of the Method
Results
Results
A Sensitive Method for Probing Sialoglycans Using Azido-sugars and Glycosyltransferases
Zhengliang L. Wu1*, Xinyi Huang1, Andrew J. Burton2, Karl A.D. Swift2 | 1 R&D Systems, Inc. 614 McKinley Place NE Minneapolis, MN 554132Tocris Bioscience, Tocris House, IO Centre, Moorend Farm Avenue, Bristol, BS11 0QL
RnDSy-lu-2945 Tocri-lu-2945
Trademarks and registered trademarks are the property of their respective owners. For research use or manufacturing purposes only. PS_Sialoglycan Detection_1747
References:1. Kolb, H.C. et al. (2001) Angew. Chem. Int. Ed. Engl.
40:2004.2. Varki, A. (1997) FASEB J. 11:248.3. Schauer, R. (2009) Curr. Opin. Struct. Biol. 19:507.4. Lehmann, F. et al. (2006) Cell Mol. Life Sci. 63:1331.5. Brockhausen, I. et al. (2009) O-GalNAc Glycans, in
Essentials of Glycobiology, A. Varki et al., Editors. Cold Spring Harbor (NY).
6. Julien, S. et al. (2012) Biomolecules 2:435.7. Ju, T. et al. (2014) Cancer Biomark. 14:63.
8. Yang, J.M. et al. (1994) Glycobiology 4:873.9. Harduin-Lepers, A. et al. (2005) Glycobiology 15: 805.10. Wu, Z.L. et al. (2015) Carbohydr. Res. 412: 1.11. Spiro, R.G. and V.D. Bhoyroo (1974) J. Biol. Chem.
249:5704.12. Baenziger, J.U. & D. Fiete (1979) J. Biol. Chem. 254:789.13. Green, E.D. et al. (1988) J. Biol. Chem. 263:18253.14. Ishii-Karakasa, I. (1997) Eur. J. Biochem. 247: 709.15. Edge, A.S. (1987) J. Biol. Chem. 262:16135.
Figure 5. Probing fetuin, D-fetuin and CD56 with various sialyltransferases. All samples were also treated with PNGase F under native condition and run side by side with the untreated samples. The glycans revealed by this method with great certainty are indicated. CD56 contained polysialic acid (PSA) attaching site that is recognized by ST8Sia4 (84).
92
66
55
29
21
4336
32 6A4
B D-fetuin
31 32 35 61 62 6A2
6A4
81 84–
31 32 35 61 62
6A2
6A4 81 84–
D D-fetuin/O-glycosidase
31 32 35 61 62 6A2
6A4
81 84–
31 32 35 61 62
6A2
6A4 81 84–
32 6A481
84
C D-fetuin/PNGase F31 32 35 61 62 6A
2
6A4
81 84
31 32 35 61 62
6A2
6A4 81 84
32
61
6A4
A
Enzymes
Fetuin
Fetuin
31 32 35 61 62 6A2
6A4
81 84–
31 32 35 61 62
6A2
6A4 81 84–
Enzymes
Fetuin
Siaα2-3Galβ1-3GalNAc-O-S/T Galβ1-3GalNAc-R Galβ1-3GalNAc-O-S/T Galβ1-4GlcNAc-R
A D-fetuin Fetuin B MGAT1 probing
ProteinStaining
ProteinStaining
Strep-HRP
Strep-HRP
Con
trol
MG
AT1
GC
NT1
B3G
NT6
92
6655
2921
4336
92
6655
29
21
4336
12.46.8
92
6655
2921
4336
Con
trol
MG
AT1
GC
NT1
B3G
NT6
NA
Fetu
in
D-f
etui
n
RNas
e B
NA
Fetu
inD
-fet
uin
RNas
e B
Mar
ker
Galβ1-3GalNAc-O-S/T
GalNAc-O-S/T
Manα-3-R
92
6655
2921
4336
92
6655
2921
4336
Fetuin
ProteinStaining
Strep-HRP
D-fetuinFetuin +PNGaseF
D-fetuin +PNGaseF
CD56 +PNGaseFCD56
32 61 6A2
6A4
84 32 61 6A2
6A4
84 32 61 6A2
6A4
84 32 61 6A2
6A4
84 32 61 6A2
6A4
84 32 61 6A2
6A4
84
Siaα2-3Galβ1-3GalNAc-O-S/T Galβ1-4GlcNAc-R
PSA
Figure 1. Strategies for glycan detection using glycosiltransferases and clickable monosaccharides. A) O-glycan detection. GlcNAc transferases, B3GNT6 and GCNT1, that are strictly specifi c for the precursor glycans are used for further confi rmation of the glycans. The processes to the right side through core-1 are sensitive to O-glycosidase treatment. The processes to the left side are resistant to O-glycosidase treatment. B) N-glycan detection. MGAT1 is a GlcNAc transferase specifi c for high mannose and hybrid N-glycans. ST6Gal1 is a sialyltransferase specifi c for terminal galactose on N-glycans.
Figure 2. Probing fetal bovine fetuin with sialyltransferases. Upper panels, protein staining. Lower panels, strep-HRP detection. (A) Probing untreated fetuin. (B) Probing neuraminidase treated fetuin. (C) Samples in (B) were further treated with PNGase F under native conditions. (D) Probing neuraminidase and O-glycosidase treated fetuin. Some of the glycans labeled by this method can be ascertained based on the specifi cities of the labeling enzymes. For example, enzyme 6A4 strictly recognizes Siaα2-3Galβ1-3GalNAc-O-S/T. Enzymes 32, 61, 6A4, 81, 84 exhibited self-labeling and are also indicated.Key: 31, ST3Gal1; 32, ST3Gal2; 35, ST3Gal5; 61, ST6Gal1; 62, ST6Gal2; 6A2, ST6GalNAc2; 6A4, ST6GalNAc4; 81, ST8Sia2; 84, ST8Sia4; Fet, fetuin; Neu, C. perfringens neuraminidase.
Figure 4. Specifi cities of ST6GalNAc enzymes. (A) Probing pre-treated fetuin samples with ST6GalNAc enzymes. Sp. Galactosidase was used to remove galactose from asialo-fetuin. All three samples contained glycan substrate for ST6GalNAc1. In contrast, only fetuin contained glycan substrate for ST6GalNAc4. The glycans revealed by this method with great certainty are indicated. 1, 2, 4, 5, 6 refer to ST6GalNac1,2,4,5, and 6, respectively. (B) Strategies used for glycan probing in (A).
Figure 3. Probing fetal bovine fetuin with GlcNAc transferases. (A) Probing fetuin and D-fetuin with different GlcNAc transferases. (B) Probing different samples with MGAT1. RNase B is not visible in the protein staining as it lacks a tryptophan residue. NA, 1918 H1N1 neuraminidase; D-fetuin, desialylated fetuin. GCNT1 reveals core-1 O-glycan; B3GNT6 reveals Tn antigen (GalNAc-O-S/T); MGAT1 reveals high mannose glycans (indicated with arrows).
N
NN
Glycosidase treatment
click chemistry reaction in the presence of Cu (+1)
N 3Azido-sugar
incorporation
B3GNT6
Sensitive to O-glycosidaseResistant to O-glycosidase
ST6GalNAc2ST6GalNAc2,4
ST6GalNAc2
Sialidase
Sialidase
Disialyl core-1
Disialyl hexasaccharide
Monosialyl core-1
SialidaseS/T
sTn
S/TS/T
ST3Gal1, 2
ST6Gal1
ST3Gal1, 2
GCNT1
core-1
N
N 3
Sialidase
Sialidase
MGAT1
Complex
NN
High mannose Hybrid
Tn
N 3
S/T
N 3
N 3
S/T
N 3
S/T
N 3
S/TS/T
S/T
S/T
S/TS/T
S/T
N 3
S/T
N
N 3 N 3 N 3
N
N 3
N 3
N
N 3
A B
NA Fetuin
1 2 4 5 6 1 2 4 5 6 1 2 4 5 6MAF AF/Gal
ProteinStaining
Strep-HRP
Siaα2-3Galβ1-3GalNAc-O-S/T
GalNAc-O-S/T
ST6GalNAc1,2
β-Galactosidase
Disialyl core-1
Monosialyl core-1
sTn
ST6GalNAc1,2,4
ST6GalNAc1,2
Sialidase
Sialidase
Sialidase
N3
N3
N3
S
S S
S S
SSS
B
Enzymes Codes Recognized substrate (acceptor site in red)
ST3Gal1 31 Galβ1-3GalNAc-R
ST3Gal2 32 Galβ1-3GalNAc-R
ST3Gal5 35 Ganglioside lactosylceramide (Galβ1-Glcβ1-Cer)
ST6Gal1 61 Galβ1-4GlcNAc-R
ST6Gal2 62 Not clear
ST6GalNAc1 6A1
Galβ1-3GalNAc-O-S/T
Siaα2-3Galβ1-3GalNAc-O-S/T
GalNAc-O-S/T
ST6GalNAc2 6A2
Galβ1-3GalNAc-O-S/T
Siaα2-3Galβ1-3GalNAc-O-S/T
GalNAc-O-S/T
Enzymes Codes Recognized substrate (acceptor site in red)
ST6GalNAc4 6A4 Siaα2-3Galβ1-3GalNAc-O-S/T
ST6GalNAc5 6A5Ganglioside GM1b
(Siaα2,3Galβ1,3GalNAcβ1,4Gal1,4Glcβ1-Cer)
ST6GalNAc6 6A6 Ganglioside GD1a, GT1b and GM1b
ST8Sia1 81 Siaα2-3Galβ1-4GlcCer
ST8Sia4 84 neural cell adhesion molecules
MGAT1 Manα-3- of N-linked glycan
GCNT1 core-1 O-glycan (Galβ1-3GalNAc-O-S/T)
B3GNT6 Tn antigen (GalNAc-O-S/T)
Advantages of the Method• GREAT SPECIFICITY. Labeling is achieved through enzymes that are known to be selective for substrate
recognition.• SUPERIOR SENSITIVITY. Labeling is through covalent bonding via click chemistry. • DIRECT. No need for releasing glycans from conjugation.• SIMPLE. Only common laboratory equipment for Western blotting is needed.• FAST. Results can be obtained in a day.• Applicable to glycan analysis on biotherapeutics.
N3GlcNAcGalNAc
Sialic acid
GalactoseMannose
Asparagine
S Serine
Azide
Biotin
N
Key for the Principle of the Method, Figures 1 and 4.