Glycosaminoglycans (GAGs) are critical components of the stem cell niche and consist of long chain polymers of recurring disaccharide units usually composed of either D-glucosamine or D-galactosamine, and D-glucuronic acid or L-iduronic acid that when coupled to a core protein result in the formation of proteoglycans (PGs). Perhaps the most widely used of the GAG-based drugs is the highly sulfated anticoagulant heparin, which is used during surgery and dialysis, as a treatment of deep-vein thrombosis, and is used to coat indwelling catheters and other devices where the vascular system is exposed. Heparin is isolated from animal tissue, primarily pig intestines although it can be available from bovine and ovine intestines and other organs. Due to the animal source, the early stages of heparin production, e.g., generation of the raw heparin, occurs under poorly controlled, non-cGMP conditions, and purposeful contamination with related polysulfated polysaccharides was determined to result in the death of over 80 people in the U.S. in 2008. This has prompted the call for a non-animal sourced heparin.
In collaboration with Prof. Robert Linhardt, we have developed a biomanufacturing approach to a bioengineered heparin (Figure 1). Using the E. coli capsular polysaccharide heparosan, a series of chemical and enzymatic transformations are being optimized to generate a chemically and biologically equivalent (to USP) heparin. We have gained an improved understanding of the various sulfotransferases in the biosynthesis of heparin. Specifically, we developed a high-throughput microtiter-based colorimetric assay for elucidating the kinetic mechanism of heparin O-sulfotransferases (OSTs) acting on polysaccharide substrates using a given OST with arylsulfotransferase-IV and p-nitrophenylsulfate as sacrificial sulfate donor to regenerate the PAPS OST substrate.
Figure 1. Biosynthetic pathway of heparin. The biosynthetic pathway includes the biosynthesis of polysaccharide backbone as well as the modification steps. The synthesis is initiated with a tetrasaccharide linkage region that contains xylose-galactose-galactose-glucuronic acid. The backbone is synthesized by HS polymerase. The backbone polysaccharide is then modified via five enzymatic modification steps. Relevant References: Bhaskar et al. Angew. Chem. Int. Ed. 58, 5962-5966 (2019); Zhang et al. Anal. Bioanal. Chem. 401, 2793-2803 (2011); Vaidyanathan et al. Bioeng Transl Med. 2, 17-30 (2017); Bhaskar et al. Appl. Microbiol. Biotechnol. 93, 1-16 (2012); Wang et al. Bioengin. Bugs 2, 1-5 (2011); Wang et al. Biotechnol. Bioeng. 107, 964-973 (2010); Ly et al. Anal. Bioanal. Chem. 399, 737-745 (2011); Hickey et al. J. Biotechnol. 165, 175-177 (2013); Bhaskar et al. Carbohydr. Polym. 122, 399-407 (2014); Fu et al. J. Med. Chem. 60, 8673-8679 (2017).
More recent work has focused on the mechanistic understanding of glucuronosyl C5-epimerase (Glce), the first enzyme that acts on the polysaccharide substrate N-sulfoheparosan (NSH). The chemoenzymatic synthesis of heparin, through a multi-enzyme process, represents a critical challenge in providing a safe and effective substitute for this animal sourced anticoagulant drug. D-Glucuronyl C5-epimerase (C5-epi) is an enzyme acting on a heparin precursor, N-sulfoheparosan, catalyzing the reversible epimerization of D-glucuronic acid (GlcA) to L-iduronic acid (IdoA). Apparent steady-state kinetic parameters for both the forward and the pseudo-reverse reactions of C5-epi have been determined for the first time using polysaccharide substrates directly relevant to the chemoenzymatic synthesis and biosynthesis of heparin [Vaidyanathan et al. Glycobiology, 30, 847-858, 2020] (Figure 2). The forward reaction shows unusual sigmoidal kinetic behavior and the pseudo-reverse reaction displays non-saturating kinetic behavior. The atypical sigmoidal behavior of the forward reaction was probed using a range of buffer additives. Surprisingly, the addition of 25 mM each of CaCl2 and MgCl2 resulted in a forward reaction exhibiting more conventional Michaelis-Menten kinetics. The addition of 2-O-sulfotransferase, the next enzyme involved in heparin synthesis, in the absence of 3’-phosphoadenosine 5’-phosphosulfate (PAPS), also resulted in C5-epi exhibiting a more conventional Michaelis-Menten kinetic behavior in the forward reaction accompanied by a significant increase in apparent Vmax. This study provides critical information for understanding the reaction kinetics of C5-epi, which may result in improved methods for the chemoenzymatic synthesis of bioengineered heparin.
Figure 2. Offline and real-time NMR changes in the H-1 anomeric signal of IdoA produced using C5-epimerase. (A) Spectral overlay using the offline experiments using the engineered enzyme with NSH as the substrate where the IdoA H-1 peak increases as a function of time. The colored lines represent: blue, 15 min; red, 30 min; green, 45 min; purple, 60 min; and yellow, 90 min. (B) Real-time 1D 1H-time spectrum showing an increase in signal corresponding to IdoA H-1 as a function of time (Topspin 3.2.7, contour mode). (C) Michaelis-Menten profiles for engineered C5-epi (0.5 mg/mL), acting on NSH with no additive (black), with 2OST (0.5 mg/mL) added as an additive without PAPS (red) or with added 25 mM CaCl2 + 25 mM MgCl2 (green). Error bars (n = 3) are within the plot symbols. [Vaidyanathan et al. Glycobiology, 30, 847-858, 2020]
We further elucidated the processibility of C5-epimerase on NSH substrate (Figure 3). Using full length NSH, containing different amounts of N-acetylglucosamine (GlcNAc) residues, we demonstrated that C5-epimerase specificity depends on polysaccharide sequence, particularly the location of GlcNAc residues within the chain [Vaidyanathan et al. Biochemistry, 59, 2576-2584, 2020]. We leveraged deuterium exchange and the novel β-glucuronidase heparanase BP, which cleaves at the GlcA residue to obtain sequence information. Specifically, liquid chromatography-mass spectrometry and gel permeation chromatography of partial/complete heparanase BP digestion products from various NSH substrates treated with C5-epimerase provides information on C5-epimerase activity and action pattern. This study provides insight into optimizing the large-scale production of bioengineered heparin.
Figure 3. Full length N-sulfoheparosan (NSH), a direct substrate for the production of bioengineered heparin containing different amounts of N-acetylglucosamine (GlcNAc) residues, is used as substrate for glucuronyl C5-epimerase. The specificity and action pattern of the enzyme was determined as a function of the polysaccharide sequence and the presence of GlcNAc residues. [Vaidyanathan et al. Biochemistry, 59, 2576-2584, 2020]
Current Collaborator:
Robert Linhardt – Rensselaer Polytechnic Institute
