GH18 endo-β-N-acetylglucosaminidases use distinct mechanisms to process hybrid-type N-linked glycans

Abstract

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Mechanism of antibody-specific deglycosylation and immune evasion by Streptococcal IgG-specific endoglycosidases

“…Even when bound in the glycan binding sites of endoglycosidases that are not protein-specific, N-linked glycans adopt diverse structures that are decidedly unlike the parallel-branched glycans in EndoS and EndoS2. This is the case for the N-linked glycans that are the substrates for EndoBT-3987 38,39 (Supplementary Fig. 34), EndoF 3 40 , as well as many others.…”

“…We also studied the interaction of catalytically-inactive EndoBT-3987 D312A/E314 , a GH18 family ENGase that hydrolyzes HM glycans from myriad glycoproteins but is not specific for IgG antibodies or any other protein (Supplementary Fig. 16) 38,39 . Quantitative lineshape analysis of methyl-TROSY spectra showed that EndoS2 D186L binds Rituximab-Fc with a dissociation constant of 3.1 µM (Table 1, entry 1), similar to the previously reported K D of ∼9 µM for the binding of EndoS2 D186L to IgG1-CT obtained by surface plasmon resonance 28 .…”

Mechanism of antibody-specific deglycosylation and immune evasion by Streptococcal IgG-specific endoglycosidases

“…Even when bound in the glycan binding sites of endoglycosidases that are not protein-specific, N -linked glycans adopt diverse structures that are decidedly unlike the parallel-branched glycans in EndoS and EndoS2. This is the case for the N -linked glycans that are the substrates for EndoBT-3987 42 , 43 , EndoF 3 44 , as well as many others (Supplementary Fig. 35 ).…”

“…We also studied the interaction of catalytically inactive EndoBT-3987 D312A/E314L , a GH18 family ENGase that hydrolyzes HM glycans from myriad glycoproteins but is not specific for IgG antibodies or any other protein (Supplementary Fig. 17 ) 42 , 43 . Quantitative line shape analysis of methyl-TROSY spectra showed that EndoS2 D186L binds Rituximab-Fc with a dissociation constant of 3.1 µM (Table 1 , entry 1), similar to the previously reported K D of ∼9 μM for the binding of EndoS2 D186L to IgG1-CT obtained by surface plasmon resonance 28 .…”

N-glycan breakdown by bacterial CAZymes

“…Research into the structure–function relationship for GH18 enzymes that hydrolyse N-glycans from a number of different species has made huge progress over the last few years ( Figure 5 ). This is largely due to solving new product–complex crystal structures but also scanning mutagenesis to understand the contribution of the different residues in the active sites [ 22 , 25 , 26 ]. A number of observations and trends are emerging from the comparison of structural data of different enzymes.…”