Fig 1.
Hydrolysis and transglycosylation reactions using SG-oxazoline as donor substrates by Endo-S mutants.
(A) Hydrolysis reactions were performed using trastuzumab and Endo-S mutants at a 50:1 weight ratio. (B) Transglycosylation reactions were carried out using deglycosylated trastuzumab as the acceptor and 5 molar eq of donor oxazoline.
Fig 2.
Transglycosylation reactions by Endo-S variants with significantly reduced or no detectable hydrolysis activity.
Reactions were carried out using deglycosylated trastuzumab as the acceptor and 5 molar eq of donor oxazoline.
Fig 3.
Hypothetical model of the one-pot transglycosylation using SGP as the donor substrate.
SGP is reversibly converted to the active intermediate and GlcNAc-peptide mainly by Endo-M (1). The intermediate can be irreversibly hydrolyzed by rapidly by hydrolytic variants of Endo-M and Endo-S or slowly without them (2). If hydrolysis (2) is negligibly slow, the intermediate may be made available for conjugation to the antibody (3) by Endo-S.
Fig 4.
Transglycosylation reactions by variants of Endo-S, Endo-M, and their combinations.
(A) Transglycosylation using a mutant alone or a wild-type paired with either a wild-type or a mutant enzyme. (B) Transglycosylation using a pair of mutants. Reactions were carried out using deglycosylated trastuzumab as the acceptor. SGP or SG-Asn was used as the donor substrate at 300 molar eq.
Fig 5.
ESI-MS (after deconvolution) of the heavy chain of deglycosylated trastuzumab (A) and one-pot transglycosylated trastuzumab using SGP (B) or SG-Asn (C) as the donor substrate.
Fig 6.
Transglycosylation reactions by pairs of Endo-S and Endo-CC variants.
Reactions were carried out using deglycosylated trastuzumab as the acceptor at 37°C or 28°C with 300 molar eq of donor oxazoline.
Fig 7.
Mutated sites that contributed to improved activity.
The catalytic domain of Endo-S (Protein Data Bank code 4NUY, gray), and complex glycan (cyan) from its complex with Endo-F3 (Protein Data Bank code 1EOM, not shown) were approximately placed by superimposing the two catalytic domains. The side chains of key catalytic residues (magenta) and mutated sites that contributed to improved activity (green) are shown in stick form.
Fig 8.
Schematic representation of one-pot transglycosylation reactions.
(A) Scheme proposed by Li et al. Wild-type Endo-M efficiently hydrolyzes SGP. Hydrolyzed sialoglycan is subsequently converted to SG-oxazoline in situ to provide the active donor substrate for transglycosylation catalyzed by the Endo-S D233Q mutant. (B) Scheme proposed in this study. The Endo-M N175Q mutant converts SGP to the active intermediate, which is then conjugated to the deglycosylated antibody by an Endo-S mutant.