Structure-Activity Relationships of Bioengineered Heparin/Heparan Sulfates Produced in Different Bioreactors
Heparin and heparan sulfate are structurally-related carbohydrates with therapeutic applications in anticoagulation, drug delivery, and regenerative medicine. This study explored the effect of different bioreactor conditions on the production of heparin/heparan sulfate chains via the recombinant expression of serglycin in mammalian cells. Tissue culture flasks and continuously-stirred tank reactors promoted the production of serglycin decorated with heparin/heparan sulfate, as well as
... n sulfate, while the serglycin secreted by cells in the tissue culture flasks produced more highly-sulfated heparin/heparan sulfate chains. The serglycin produced in tissue culture flasks was effective in binding and signaling fibroblast growth factor 2, indicating the utility of this molecule in drug delivery and regenerative medicine applications in addition to its well-known anticoagulant activity. Molecules 2017, 22, 806 2 of 12 of approximately twenty enzymes in the Golgi  , although the regulators of the expression of these enzymes are not fully understood. These enzymes are involved in chain initiation, elongation, epimerization, and sulfation. While this structural heterogeneity provides an opportunity to fine-tune the biological activity for particular applications, the precise identification of structure-function relationships has been challenging  . However, certain structural features are known to be required for highly-specific interactions, such as a pentasaccharide structure containing an 3-O-sulfated glucosamine for binding to anti-thrombin III  , whereas other structures are less specific, such as a contiguous string of highly-sulfated disaccharides for binding to fibroblast growth factor (FGF) and downstream growth factor activation  . Heparin is sourced predominantly from animal tissues, particularly porcine intestinal mucosa and, to a lesser extent, bovine lung tissues due to concerns over bovine spongiform encephalopathy contamination  . Commercially-available heparan sulfates are synthesized as byproducts of heparin production or by selective de-sulfation of heparin [21, 22] . The production of heparan sulfate libraries from tissues is time consuming and technically challenging  . The growing demand for heparin and heparan sulfates for clinical applications has led researchers to explore alternative methods of production including chemoenzymatic synthesis , chemical synthesis , sulfation of polysaccharides  , and metabolic engineering  . A bioengineered heparin-like heparan sulfate was recently reported by the authors by expressing serglycin in mammalian cells  . This recombinant serglycin was decorated with chondroitin/dermatan sulfate in addition to heparin/heparan sulfate chains [1,28] similar to serglycin isolated from natural sources where it has been shown to be decorated with multiple types of glycosaminoglycan chains covalently attached to its eight glycosaminoglycan attachment sites     . The aim of this study was to explore the effect of different bioreactor conditions on the yield, structure, and activity of heparin/heparan sulfates produced by expressing serglycin in mammalian cells. Bioreactors, including tissue culture flasks, continuously-stirred tank reactors (CSTR), and shaker flasks, were investigated as each of these have been used for commercial scale production of bioactives  . Different bioreactors and culture conditions were found to change the structure of the heparin/heparan sulfate chains produced by the cells with the serglycin produced being effective at binding and signaling FGF-2. This supports the use of these bioreactors and our approach to produce heparin/heparan sulfates for use in the clinic as an anticoagulant, as well as future uses in drug delivery and regenerative medicine applications.