Controlled site-selective protein glycosylation for precise glycan structure-catalytic activity relationships

Glycoproteins occur naturally as complex mixtures of differently glycosylat ed forms which are difficult to separate. To explore their individual prope rties, there is a need for homogeneous sources of carbohydrate-protein conj ugates and this has recently prompted us to develop a novel method for the site-selective glycosylation of proteins. The potential of the method was i llustrated by site-selective glycosylations of subtilisin Bacillus lentils (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 S-2 site, 156 and 166 in the S-1 site and 217 in the S-1' site. These were the first examples of preparations of homogeneous neoglycoproteins in which both the site of gly cosylation and structure of the introduced glycan were predetermined. The s cope of this versatile method was expanded further through the combined use of peracetylated MTS reagents and careful pH adjustment to introduce glyca ns containing different numbers of acetate groups. This method provides a h ighly controlled and versatile route that is virtually unlimited in the sco pe of the sites and glycans that may be conjugated, and opens up hitherto i naccessible opportunities for the systematic determination of the propertie s of glycosylated proteins. This potential has been clearly demonstrated by the determination of detailed glycan structure-hydrolytic activity relatio nships for SBL. The 48 glycosylated CMMs formed display k(cat)/K-M values t hat range from 1.1-fold higher than WT to 7-fold lower than WT. The anomeri c stereochemistry of the glycans introduced modulates changes in k(cat)/K-M upon acetylation. At positions 62 and 217 acetylation enhances the activit y of alpha-glycosylated CMMs but decreases that of beta-glycosylated. This trend is reversed at position 166 where, in contrast, acetylation enhances the k(cat)/K(M)s of beta-glycosylated CMMs but decreases those of alpha-gly cosylated. Consistent with its surface exposed nature changes at position 1 56 are more modest, but still allow control of activity, particularly throu gh glycosylation with disaccharide lactose. (C) 2000 Published by Elsevier Science Ltd. All rights reserved.