Glyko® Sialidase A™ [GK80040]

Price:
$ 256.00

Description

Releases α(2,3)-, α(2,6)-, α(2,8)-, and α(2,9)-linked N-acetylneuraminic acid from oligosaccharides and glycoproteins. Also capable of releasing N-glycolylneuraminic acid.

Source:
Recombinant gene from Arthrobacter ureafaciens, expressed in E. coli.

Specificity:
The enzyme releases α(2,3)-, α(2,6)-, α(2,8)-, and α(2,9)-linked N-acetylneuraminic acid from complex carbohydrates. The initial rate of hydrolysis of α(2,6) linkages is reported to be approximately twice that of α(2,3)-linked sialic acid however, in practice, this kinetic selectivity is of little consequence during extended incubations. Effective digestion of glycolipid substrates is facilitated by addition of a detergent, such as sodium taurodeoxycholate to the incubation.

Sialidase A is capable of releasing N-glycolylneuraminic acid (Neu5Gc, NGNA) in addition to N-acetylneuraminic acid (Neu5Ac, NANA) [1], although similarly to other sialidases [2] the activity is lower toward Neu5Gc than Neu5Ac.

The enzyme can also be used to remove sialic acid from gangliosides [3], glycosphingolipds (ceramide & oligosaccharide) with sialic acid.

May be used for exoglycosidase sequencing in conjunction with Sialidase S (GK80021), which is specific for α(2,3)-linked sialic acids.

Sialidases are also known as neuraminidases.

Ships with:
WS0049 5x Reaction Buffer B [250 mM sodium phosphate pH 6.0]

Reaction Buffer:
5X concentrated buffer which when diluted gives 50 mM sodium phosphate pH 6.0.

Formulation:
20 mM Tris HCl pH 7.5, containing 25 mM NaCl.

pH optimum:
6.0

Unit Definition:
One unit is defined as the amount of enzyme required to catalyze the release of 1 μmole of p-nitrophenol from p-nitrophenyl-α-D-N-acetylneuraminic acid per minute at 37° C, pH 5.5.

Size: 1 U (200 µl)

Concentration: ≥ 5 U/ml

Product Code: GK80040

References:

  1. Uchida Y, Tsukada Y, Sugimori T.  Enzymatic properties of neuraminidases from Arthrobacter ureafaciens.  1979 Nov;86(5):1573-85.
  2. Schauer R.  Chemistry, metabolism, and biological functions of sialic acids.   1982;40:131-234.
  3. Larsson EA, Olsson U, Whitmore CD, Martins R, Tettamanti G, Schnaar RL, Dovichi NJ, Palcic MM, Hindsgaul O.  Synthesis of reference standards to enable single cell metabolomic studies of tetramethylrhodamine-labeled ganglioside GM1.   2007 Feb 26;342(3-4):482-9
Tech Info

GK80040 Technical Data Sheet  

Publications

AN3001: Glycan Remodeling of Therapeutic Proteins using Galactosyltransferase and Sialyltransferases

FAQs

Q. Does GK80040 contain a purification tag, such as 6xHis?

A. No. Although GK80040 Sialidase A is expressed recombinantly, it does not contain a purification tag.

Q. I do not want to use the sodium phosphate reaction buffer supplied with GK80040. Can I use an acetate buffer? Is this good for digestion with multiple exoglycosidases?

A. We use an ammonium acetate buffer when we run multi-enzyme exoglycosidase digests that include GK80040 Sialidase A. We suggest a 10X reaction buffer of 500 mM ammonium acetate pH 5.5 w/ 0.05% azide. The reaction buffer can also be used diluted to 20X (25 mM ammonium acetate) and lower with purified glycans. This buffer works with most of our exoglycosidases in an overnight digestion:

Product Code  Product Name Source Specificity
GK80040 Sialidase A Recombinant gene from Arthrobacter ureafaciens, expressed in E.coli Releases non-reducing terminal α(2-3,6,8,9)-linked sialic acid
GK80021 Sialidase S Recombinant from Streptococcus pneumoniae, expressed in E. coli (6 x His tagged) Releases non-reducing terminal α(2-3)-linked sialic acid
GKX-5014

β(1-4) Galactosidase

Streptococcus pneumoniae

Releases non-reducing terminal β(1-4)-linked galactose
GKX-5013 β(1-3,4) Galactosidase Bovine testis Releases non-reducing terminal β(1-3,4)-linked galactose
GKX-5007

α(1-3,4,6)-Galactosidase

Green coffee bean Releases non-reducing terminal α(1-3,4,6)-linked galactose (alpha gal)
GKX-5003 β(1-2,3,4,6) Hexosaminidase Jack bean Releases non-reducing terminal β(1-2,3,4,6)-linked N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) residues
GKX-5023

β(1-2,3,4,6) Hexosaminidase

Jack bean Releases non-reducing terminal β(1-2,3,4,6)-linked GlcNAc and GalNAc residues, higher concentration than GKX-5003 for improved release of bisecting GlcNAc
GK80050

β N-Acetylhexosaminidase

Recombinant gene from Streptococcus pneumoniae, expressed in E. coli Cleaves all non-reducing terminal β-linked GlcNAc, but not bisecting β(1-4) GlcNAc. Does not release GalNAc
GKX-5010

α(1-2,3,6)-Mannosidase

Jack bean Releases non-reducing terminal α(1-2,3,6)-linked mannose, with kinetic preference for α(1-2,3)-linked mannose over α(1-6)-linked mannose. 
GKX-5009

α(1-2)-Mannosidase

Aspergillus saitoi Releases non-reducing terminal α(1-2)-linked mannose, high mannose N-glycan structures are digested to Man5 

 

Generally, 2 μl of each exoglycosidase is used in a 20-30 μl reaction with an overnight incubation (16 hours) at 37°C. A general example is shown below, please Contact Us for more details.

  • Prepare purified fluorescently labeled glycans from 100-500 µg of glycoprotein in 100 µl of water. Add 5ul of Sequencing Buffer (500 mM Ammonium Acetate pH 5.5 with 0.05% Azide). Aliquot into 5 tubes:  
Reagent (µl) Rxn 1 Rxn 2 Rxn 3 Rxn 4 Rxn 5
Labeled glycans in buffer 20 20 20 20 20
Water 8 6 4 2 0
Sialidase A*  [GK80040] - 2 2 2 2
β-Galactosidase  [GKX-5013 or GKX-5014] - - 2 2 2
β N-acetylhexoseaminidase  [GKX-5023] - - - 2 2
α-Mannosidase   [GKX-5010] - - - - 2

*If sialic acid linkage determination is needed, Sialidase S [GK80021] cleaves α(2-3)-linked sialic acid may also be used in a separate reaction from sialidase A, which cleaves α(2-3,6,8,9)-linked sialic acids.

  • Cap the tubes, vortex, and incubate overnight at 37°C.
  • If the glycans need to purified from the reaction prior to analysis by e.g. LC-MS, samples can be spun through Nanosep® 10K Omega spin filters to remove protein.
  • If injecting on UPLC, a 1 µl aqueous injection may be used.
  • If injecting > 1 µl on LC, measure volume and bring samples to 70% acetonitrile for e.g. 10 µl injection.

For data using these methods see our poster, Characterizing Low-Abundance Glycans on Therapeutic Proteins, and our WCBP 2016 presentation.

GKE-5006 α(1-2,3,4,6) fucosidase (bovine kidney) is unlikely to release core α1,6) fucose from the innermost GlcNAc of N-glycans if there is a fluorophore attached to GlcNAc. If possible, fucosidase digestion to remove core fucose should be performed on released N-glycans prior to labeling.

Acetate buffers work better than phosphate for injecting onto LC (phosphate phase separates in acetonitrile), and acetate is volatile so samples can be dried down if necessary.

Q. Can GK80040 be used with detergents and reductants?

A. The enzyme can be used with sodium dodecyl sulfate (SDS) and β-mercaptoethanol (βME) at certain levels.

The GK80110 Enzymatic Deglycosyaltion Kit uses SDS and bME in the denaturing protocol.

The 5x Denaturation Solution used in the GK80110 kit is 2% sodium dodecylsulfate (SDS) and 1 M βME, and 2.5μl is used in a reaction volume of around 50μl giving a final reaction concentration of 0.1% SDS, 50mM bME. Denaturation Solution and 5x Incubation Buffer are added to the glycoprotein substrate and the mixture is heated to 100°C for 5 minutes. After cooling, 2.5μl of a Detergent Solution (15% NP-40, final reaction concentration 0.75%) is added to counteract the inhibitory effect of SDS on PNGase F. This is followed by the addition of N-Glycanase, Sialidase A (GK80040) and O-Glycanase (GK80090), with incubation for 3 hours at 37°C.

Product Citations
  1. Ayako Kondo, Kazuo Takahashi, Hisateru Yamaguchi, Yuri Yoshida, Tomohiro Mizuno, Kazuki Nakajima, Hiroki Hayashi, Shigehisa Koide, Daijo Inaguma, Midori Hasegawa, Yoshiyuki Hiki, Yukio Yuzawa. Mass spectrometry-based approach for development of biomarkers in IgA nephropathy: a pilot trial. Fujita Medical Journal.2018; 4(2):36-41.
  2. Szabo Z, Thayer JR, Reusch D, Agroskin Y, Viner R, Rohrer J, Patil SP, Krawitzky M, Huhmer A, Avdalovic N, Khan SH, Liu Y, Pohl C. High Performance Anion Exchange and Hydrophilic Interaction Liquid Chromatography Approaches for Comprehensive Mass Spectrometry-Based Characterization of the N-Glycome of a Recombinant Human Erythropoietin.  2018 Apr 6;17(4):1559-1574.
  3. Yin H, An M, So PK, Wong MY, Lubman DM, Yao Z. The analysis of alpha-1-antitrypsin glycosylation with direct LC-MS/MS 2018 Feb 5. doi: 10.1002/elps.201700426. [Epub ahead of print]
  4. Ibáñez-Vea M, Huang H, Martínez de Morentin X, Pérez E, Gato M, Zuazo M, Arasanz H, Fernández-Irigoyen J, Santamaría E, Fernandez-Hinojal G, Larsen MR, Escors D, Kochan G. Characterization of Macrophage Endogenous S-Nitrosoproteome Using a Cysteine-Specific Phosphonate Adaptable Tag in Combination with TiO2 Chromatography.  2018 Mar 2;17(3):1172-1182.
  5. Won-Suk Song, Han-Gyu Park, Da-Hee Ann, Yoon-Yi Jeong, Mi-Young Koo, Yun-Gon Kim. Quantitative Analysis of Core-Fucosylated N-glycome according to Serum AFP Level for the Diagnosis of Hepatocellular Carcinoma. KSBB Journal. Vol.32 No.4(Wn.163), 2017.12, 279-285
  6. Matsuda C, Shiota Y, Sheikh AM, Okazaki R, Yamada K, Yano S, Minohata T, Matsumoto KI, Yamaguchi S, Nagai A. Quantification of CSF cystatin C using liquid chromatography tandem mass spectrometry.  2018 Mar;478:1-6
  7. Epp A, Hobusch J, Bartsch YC, Petry J, Lilienthal GM, Koeleman CAM, Eschweiler S, Möbs C, Hall A, Morris SC, Braumann D, Engellenner C, Bitterling J, Rahmöller J, Leliavski A, Thurmann R, Collin M, Moremen KW, Strait RT, Blanchard V, Petersen A, Gemoll T, Habermann JK, Petersen F, Nandy A, Kahlert H, Hertl M, Wuhrer M, Pfützner W, Jappe U, Finkelman FD, Ehlers M.  Sialylation of IgG antibodies inhibits IgG-mediated allergic reactions.   2017 Jul 18. pii: S0091-6749(17)31101-6. doi: 10.1016/j.jaci.2017.06.021. [Epub ahead of print]
  8. Lloyd KA, Steen J, Amara K, Titcombe PJ, Israelsson L, Lundström SL, Zhou D, Zubarev RA, Reed E, Piccoli L, Gabay C, Lanzavecchia A, Baeten D, Lundberg K, Mueller DL, Klareskog L, Malmström V, Grönwall C. Variable domain N-linked glycosylation and negative surface charge are key features of monoclonal ACPA: Implications for B-cell selection.  2018 Jun;48(6):1030-1045. 
  9. Zhao K, van der Spoel A, Castiglioni C, Gale S, Fujiwara H, Ory DS, Ridgway ND. 19q13.12 microdeletion syndrome fibroblasts display abnormal storage of cholesterol and sphingolipids in the endo-lysosomal system.  2018 Jun;1864(6 Pt A):2108-2118.
  10. Stavenhagen K, Kayili HM, Holst S, Koeleman CAM, Engel R, Wouters D, Zeerleder S, Salih B, Wuhrer M. N- and O-glycosylation Analysis of Human C1-inhibitor Reveals Extensive Mucin-type O-Glycosylation.  2018 Jun;17(6):1225-1238.
  11. Pagan JD, Kitaoka M, Anthony RM. Engineered Sialylation of Pathogenic Antibodies In Vivo Attenuates Autoimmune Disease.  2018 Jan 25;172(3):564-577.
  12. Reesink HL, Bonnevie ED, Liu S, Shurer CR, Hollander MJ, Bonassar LJ, Nixon AJ.   Galectin-3 Binds to Lubricin and Reinforces the Lubricating Boundary Layer of Articular Cartilage.   2016 May 9;6:25463.
  13. Chachadi VB, Bhat G, Cheng PW.  Glycosyltransferases involved in the synthesis of MUC-associated metastasis-promoting selectin ligands.   2015 Sep;25(9):963-75.
  14. Tharakaraman K, Raman R, Viswanathan K, Stebbins NW, Jayaraman A, Krishnan A, Sasisekharan V, Sasisekharan R.  Structural determinants for naturally evolving H5N1 hemagglutinin to switch its receptor specificity.   2013 Jun 20;153(7):1475-85.
  15. Cohen M, Elkabets M, Perlmutter M, Porgador A, Voronov E, Apte RN, Lichtenstein RG.  Sialylation of 3-methylcholanthrene-induced fibrosarcoma determines antitumor immune responses during immunoediting.   2010 Nov 15;185(10):5869-78.
  16. Stefanich EG, Ren S, Danilenko DM, Lim A, Song A, Iyer S, Fielder PJ.  Evidence for an asialoglycoprotein receptor on nonparenchymal cells for O-linked glycoproteins.   2008 Nov;327(2):308-15.
  17. Larsson EA, Olsson U, Whitmore CD, Martins R, Tettamanti G, Schnaar RL, Dovichi NJ, Palcic MM, Hindsgaul O.  Synthesis of reference standards to enable single cell metabolomic studies of tetramethylrhodamine-labeled ganglioside GM1.   2007 Feb 26;342(3-4):482-9.
  18. Malakhov MP, Aschenbrenner LM, Smee DF, Wandersee MK, Sidwell RW, Gubareva LV, Mshin VP, Hayden FG, Kim DH, Ing A, Campbell ER, Yu M, Fang F.  Sialidase fusion protein as a novel broad-spectrum inhibitor of influenza virus infection.   2006 Apr;50(4):1470-9.
  19. Zhang L, Bukreyev A, Thompson CI, Watson B, Peeples ME, Collins PL, Pickles RJ.  Infection of ciliated cells by human parainfluenza virus type 3 in an in vitro model of human airway epithelium.   2005 Jan;79(2):1113-24.
Safety

Product Safety Documentation for GK80040:

Product/Part No. Description
GK80040 Glyko® Sialidase A™
WS0049 5x Reaction Buffer B