A general module assembly strategy for the generation of chondroitin sulfate polymers

Abstract

Chondroitin sulfate (CS) plays an array of astonishing roles in physiological processes via diverse sulfation patterns. However, structure–function relationships and mechanisms behind these amazing sulfation modifications are still unclear due to the lack of structurally defined CS. Here, a general module assembly strategy was reported to access abundant CS polymers that combined facile enzyme expression and a robust cofactor 3′-phosphoadenosine-5′-phosphosulfate (PAPS) generation assisted sulfation process. Soluble expression, up to 465 mg L⁻¹ and 497 mg L⁻¹, of two key sulfotransferases, chondroitin 4-O-sulfotransferase (C4ST) and N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6OST), was achieved in the Escherichia coli Origami B (DE3) system, which provided a toolbox for in vitro module assembly of CS polymers. CS-A and CS-E, two polymers with defined sulfation patterns, were further gram-scale synthesized with PAPS generation, achieving conversion rates up to 81% and 72%, respectively. Moreover, CS-C was converted from CS-E along with an endo-4-O-sulfatase reaction module with a sulfonation degree of 65%. The synthetic strategy provides an effective catalytic approach for the module assembly of CS with definite sulfation patterns. In addition, it also promotes the accessibility of complex sulfated glycans and facilitates answering questions underlying sulfation modification.