SmartEnzymes™

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GlycINATOR® (EndoS2)

GlycINATOR (EndoS2) is an IgG-specific endoglycosidase that hydrolyzes all glycoforms present at the Fc-glycosylation site. The enzyme acts on the chitobiose core and leaves the core GlcNAc intact.

 

Available in three different product formats:

Product Information

GlycINATOR (EndoS2) is an endoglycosidase from Streptococcus pyogenes that specifically hydrolyzes glycans at the Fc glycosylation site of IgG. The enzyme has been demonstrated to remove all glycoforms on IgG, including high-mannose, hybrid, complex, and bisecting type glycans (Sjögren et al. 2015). Deglycosylates all human IgG subclasses and IgG from many different species, including mouse, rat, monkey, sheep, goat, cow, and horse. It has been reported to have limited activity on glycans from alpha-1-acid glycoprotein (Sjögren et al. 2013). The enzyme is expressed in E. coli, contains a His-tag, and the molecular weight is 92 kDa.

The enzyme is used to reduce the complexity of antibody-based therapeutics or to study core afucosylation and N-glycan site occupancy. The rapid and mild reaction conditions ensure a minimized risk for sample prep-induced modifications (Sjögren et al. 2015).


Sjögren, J. et al., 2013. EndoS2 is a unique and conserved enzyme of serotype M49 group A Streptococcus that hydrolyses N-linked glycans on IgG and α1-acid glycoprotein. The Biochemical Journal, 455(1), pp.107–118.


Sjögren, J. et al., 2015. EndoS and EndoS2 hydrolyze Fc-glycans on therapeutic antibodies with different glycoform selectivity and can be used for rapid quantification of high-mannose glycans. Glycobiology, 25(10), pp.1053–1063.

Key Characteristics

  • Human IgG1-4, Fc-fusion proteins, IgG from mouse, rabbit, rat, monkey, sheep, goat, cow and horse
  • 30 min reaction
  • Requires native IgG fold
  • All Fc glycoforms of IgG

Applications

  • Level of afucosylation
    Afucosylation of the IgG glycans has substantial impact on the effector functions of the antibody and can be studied using GlycINATOR.
  • Reduce Sample Complexity
    The glycans can quickly be removed from native IgG using GlycINATOR for subsequent intact mass analysis.
  • GlyCLICK
    Site-specific conjugation of native IgG using enzymatic remodelling of the Fc glycans

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Product Formats

GlycINATOR® Enzyme

Supplied as lyophilized powder. The enzyme is available in 2000 unit vials for deglycosylation of up to 2 mg IgG.

GlycINATOR® LE

Lyophilized for endotoxin-sensitive assays. This product formulation has low levels of endotoxins per vial (< 0.2 EU). Available in 2000 unit vials.

Immobilized GlycINATOR®

Immobilized on agarose, allowing for IgG deglycosylation without contaminating the final preparation with GlycINATOR enzyme.
 


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FAQ and Support

Popular FAQ

Typically, GlycINATOR is the first choice since it displays enzymatic activity on all glycoforms present on IgG, including complex type, high mannose, bisected and hybrid type glycans. IgGZERO does not hydrolyze high mannose glycans. IgGZERO deglycosylates some species faster as compared to GlycINATOR, for example goat IgG.

We can only guarantee optimal deglycosylation for one-time use. We do not have a cleaning or regeneration protocol to provide. However, depending on the antibody and the following application the column can be reused. We do not recommend using with different antibodies due to the risk for contamination of carry-over from previous sample. If column reuse is desired store the column in 10-20% ethanol at +4-8°C.

IgGZERO (EndoS) and GlycINATOR (EndoS2) are both IgG-specific endoglycosidases that hydrolyses Fc glycans to the innermost GlcNAc. The GlycINATOR enzyme has a broader substrate selectivity and therefore much higher enzymatic activity on high mannose and some bisected and hybrid glycans that can occur on mAbs. IgGZERO deglycosylates some species faster as compared to GlycINATOR, for example goat IgG.

In both products the enzyme is immobilized on agarose beads for IgG-specific hydrolysis of the Fc glycans to the innermost GlcNAc. The deGlycIT contains immobilized IgGZERO (EndoS) and Immobilized GlycINATOR contains GlycINATOR (EndoS2). The GlycINATOR enzyme has much higher enzymatic activity for high mannose and some bisected and hybrid glycans that can occur on mAbs.

Yes, the GlycINATOR enzyme binds specifically to the Fc region and therefore will not deglycosylate N-linked glycans in the Fab region.

 

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