Controlled site-selective protein glycosylation for precise glycan structure-catalytic activity relationships.
Glycoproteins occur naturally as complex mixtures of differently glycosylated forms which are difficult to separate. To explore their individual properties, there is a need for homogeneous sources of carbohydrate-protein conjugates and this has recently prompted us to develop a novel method for the site-selective glycosylation of proteins. The potential of the method was illustrated by site-selective glycosylations of subtilisin Bacillus lentus (SBL) as a model protein. A representative library of mono- and disaccharide MTS reagents were synthesized from their parent carbohydrates and used to modify cysteine mutants of SBL at positions 62 in the S2 site, 156 and 166 in the S1 site and 217 in the S1' site. These were the first examples of preparations of homogeneous neoglycoproteins in which both the site of glycosylation and structure of the introduced glycan were predetermined. The scope of this versatile method was expanded further through the combined use of peracetylated MTS reagents and careful pH adjustment to introduce glycans containing different numbers of acetate groups. This method provides a highly controlled and versatile route that is virtually unlimited in the scope of the sites and glycans that may be conjugated, and opens up hitherto inaccessible opportunities for the systematic determination of the properties of glycosylated proteins. This potential has been clearly demonstrated by the determination of detailed glycan structure-hydrolytic activity relationships for SBL. The 48 glycosylated CMMs formed display kcat/KM values that range from 1.1-fold higher than WT to 7-fold lower than WT. The anomeric stereochemistry of the glycans introduced modulates changes in kcat/KM upon acetylation. At positions 62 and 217 acetylation enhances the activity of alpha-glycosylated CMMs but decreases that of beta-glycosylated. This trend is reversed at position 166 where, in contrast, acetylation enhances the kcat/KMs of beta-glycosylated CMMs but decreases those of alpha-glycosylated. Consistent with its surface exposed nature changes at position 156 are more modest, but still allow control of activity, particularly through glycosylation with disaccharide lactose.
Davis
B G
BG
Department of Chemistry, University of Durham, UK. ben.davis@durham.ac.uk
Lloyd
R C
RC
Jones
J B
JB
D000107:Acetylation; D001407:Bacillus / Q000201:enzymology; D002384:Catalysis; D020650:Combinatorial Chemistry Techniques; D003545:Cysteine / Q000737:chemistry; D006023:Glycoproteins / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D006867:Hydrolases / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008698:Mesylates / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D011134:Polysaccharides / Q000737:chemistry / Q000494:pharmacology; D013329:Structure-Activity Relationship; D020860:Subtilisin / Q000737:chemistry / Q000378:metabolism
D006023:Glycoproteins; D008698:Mesylates; D011134:Polysaccharides; D006867:Hydrolases; D020860:Subtilisin; D003545:Cysteine