Exploiting Substrate Specificities of 6- O-Sulfotransferases to Enzymatically Synthesize Keratan Sulfate Oligosaccharides

Yunfei Wu; Gaël M Vos; Chin Huang; Digantkumar Chapla; Anne L M Kimpel; Kelley W Moremen; Robert P de Vries; Geert-Jan Boons

doi:10.1021/jacsau.3c00488

Exploiting Substrate Specificities of 6- O-Sulfotransferases to Enzymatically Synthesize Keratan Sulfate Oligosaccharides

JACS Au. 2023 Oct 13;3(11):3155-3164. doi: 10.1021/jacsau.3c00488. eCollection 2023 Nov 27.

Authors

Yunfei Wu¹, Gaël M Vos¹, Chin Huang^{2

3}, Digantkumar Chapla², Anne L M Kimpel¹, Kelley W Moremen^{2

3}, Robert P de Vries¹, Geert-Jan Boons^{1

2

4

5}

Affiliations

¹ Department of Chemical Biology and Drug Discovery, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Universiteitsweg 99, Utrecht 3584 CG, The Netherlands.
² Complex Carbohydrate Research Center, University of Georgia, 315 Riverbend Road, Athens, Georgia 30602, United States.
³ Department of Biochemistry, University of Georgia, Athens, Georgia 30602, United States.
⁴ Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, Utrecht 3584 CH, The Netherlands.
⁵ Department of Chemistry, University of Georgia, Athens, Georgia 30602, United States.

Abstract

Keratan sulfate (KS) is a glycosaminoglycan that is widely expressed in the extracellular matrix of various tissue types, where it is involved in many biological processes. Herein, we describe a chemo-enzymatic approach to preparing well-defined KS oligosaccharides by exploiting the known and newly discovered substrate specificities of relevant sulfotransferases. The premise of the approach is that recombinant GlcNAc-6-O-sulfotransferases (CHST2) only sulfate terminal GlcNAc moieties to give GlcNAc6S that can be galactosylated by B4GalT4. Furthermore, CHST1 can modify the internal galactosides of a poly-LacNAc chain; however, it was found that a GlcNAc6S residue greatly increases the reactivity of CHST1 of a neighboring and internal galactoside. The presence of a 2,3-linked sialoside further modulates the site of modification by CHST1, and a galactoside flanked by 2,3-Neu5Ac and GlcNAc6S is preferentially sulfated over the other Gal residues. The substrate specificities of CHST1 and 2 were exploited to prepare a panel of KS oligosaccharides, including selectively sulfated N-glycans. The compounds and several other reference derivatives were used to construct a microarray that was probed for binding by several plant lectins, Siglec proteins, and hemagglutinins of influenza viruses. It was found that not only the sulfation pattern but also the presentation of epitopes as part of an O- or N-glycan determines binding properties.