flags web

Bioengineered Heparin

Heparin, a highly sulfated glycosaminoglycan (GAG)1, is used extensively as an anticoagulant. Heparin, and its analogues, are used during surgery and dialysis, and are often used to coat indwelling catheters and other devices where the vascular system is exposed. Administered parenterally, often continuously due to its short half-life, over 0.5 billion doses are required per year. Currently obtained from mucosal tissue of meat animals, mainly porcine intestine, and to a lesser extent bovine lung, early stage heparin production is poorly controlled, due to the animal sourcing. This problem came into sharp focus in 2008 when the presence of contaminating over-sulfated chondroitin sulfate in the pig heparin supply resulted in approximately 80 deaths in the United States. This, coupled with the fact that only two doses are obtained per animal means that there is high demand for alternative and more controlled sources of heparin2,3.

Based on the related heparan sulfate (HS) biosynthetic pathway, and in collaboration with Prof. Robert Linhardt, we have devised a chemoenzymatic process that begins with bacterial synthesis of the backbone structure. Escherichia coli K5 synthesizes a polysaccharide capsule consisting of repeating units of [(→4) β-D-glucuronic acid (GlcA) (1→4) N-acetyl-α-D-glucosamine (GlcNAc) (1→)]n, known as heparosan4-6. The fermentation process involves the use of glucose as the sole carbon source and ammonium chloride as the sole nitrogen source. Using fed-batch fermentation, with exponential feeding, a growth rate of 0.12 h-1 was achieved with a yield of 15 g/L heparosan5. On a fundamental level, the ability of E. coli K5 to produce heparosan was further studied by deleting the eliminase gene, which resulted in a significant reduction in heparosan shedding into the medium and heparosan content in the capsule of the cells, indicating its pivotal role in heparosan synthesis and shedding by E. coli K56. Once extracted from the fermentation broth and purified the heparosan undergoes a series of chemoenzymatic modifications to make heparin (Figure BH-1)8,9.

Figure70

Figure BH-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 steps2.

We are further optimizing the various enzymatic reactions required for bioengineered heparin synthesis. Specifically, we have developed a high throughput microtiter-based approach that relies on a coupled bienzymic colorimetric assay for heparan sulfate and heparin O-sulfotransferases (OSTs) acting on polysaccharide substrates using arylsulfotransferase-IV and p-nitrophenylsulfate as a sacrificial sulfate donor10,11. This approach has led to a detailed assessment of OST reaction kinetics. An example is shown in Figure BH-2 and 3 for 6OST-3 kinetics10. Two-substrate Michaelis-Menten kinetic analysis was performed by varying PAPS (5-40 μM) and N-sulfoheparosan (12-100 μg/ml) concentrations, with and without 3 h pre-incubation of 6OST-3 with C5-epi, to further understand the influence of C5-epimerization on 6OST-3 activity. Under conditions where N-sulfoheparosan was preincubated with C5-epi, the primary Lineweaver-Burk plots suggested ternary complex kinetics, represented by coincident intersection (Figs. BH-2A and B).The secondary replots against the intercept values (Figs. BH-2C and D) afforded KM,PAPS= 9.8 μM, KM,NSH= 37 μg/ml, kcat = 0.2 s-1, and a kcat/KM,PAPS = 2.04 x 104 s-1M-1. Under conditions without C5-epi present, ternary complex mechanism kinetics was also observed (Figs. BH-3A and B). Secondary replots against the intercept values (Figs. BH-3C and D) afforded KM,PAPS= 39.2 μM, KM,NSH= 41 μg/ml, kcat = 0.04 s-1, and a kcat/KM,PAPS = 1.02x103 s-1M-1. These data ares summarized in Table BH-1.

Figure71

Figure BH-2. Dual-substrate kinetics of 6OST-3 on C5-epi treated N-sulfoheparosan. (A) Primary Lineweaver-Burk plot with respect to [PAPS]-1. (B) Primary Lineweaver-Burk plot with respect to [N-sulfoheparosan]-1. (C) Secondary replot of intercepts against [PAPS]-1. (D) Secondary replot of intercepts against [N-sulfoheparosan]-1.

Figure72

Figure BH-3. Dual-substrate kinetics of 6OST-3 in the absence of C5-epi treated N-sulfoheparosan. (A) Primary Lineweaver-Burk plot with respect to [PAPS]-1. (B) Primary Lineweaver-Burk plot with respect to [N-sulfoheparosan]-1. (C) Secondary replot of intercepts against [PAPS]-1. (D) Secondary replot of intercepts against [N-sulfoheparosan]-1.

Table BH-1. Detailed kinetic parameters of 6OST-3.

Figure53

A major challenge lies in quantifying the specific activity of glucuronyl-C-5 epimerase (Glce), the first enzyme that acts on the polysaccharide substrate, N-sulfoheparosan (Figure BH-1). Glce catalyzes the conversion of glucuronic acid to the isomer iduronic acid. Because glucuronic and iduronic acids have the same molecular weight and very minor chemical differences, Glce activity determination has been difficult. We have devised a novel approach that employs hydrogen-deuterium exchange and NMR spectroscopy and LC-MS to quantify Glce specific activity and to determine the reaction kinetics. Both real-time and discontinuous NMR spectroscopy is being used to obtain Glce reaction kinetics and elucidate the reaction mechanism of this complex epimerization reaction.

References

  1. F. Zhang, B. Yang, M. Ly, K. Solakyildirim, Z. Xiao, Z. Wang, J. M. Beaudet, A. Y. Torelli, J. S. Dordick, and R. J. Linhardt (2011), "Structural characterization of heparins from different commercial sources", Anal. Bioanal. Chem. 401, 2793-2803.
  2. D. Vaidyanathan , A. Williams , J.S. Dordick, M.A.G. Koffas, and R.J. Linhardt (2016), "Engineered heparins as new anticoagulant drugs", Bioeng Transl Med. November 2016
  3. U. Bhaskar, E. Sterner, A.M. Hickey, A. Onishi, F. Zhang, J.S. Dordick, R.J. Linhardt (2012), “Engineering of routes to heparin and related polysaccharides”, Appl. Microbiol. Biotechnol. 93, 1-16.
  4. Z. Wang, J.S. Dordick, and R.J. Linhardt (2011), "Escherichia coli K5 heparosan fermentation and improvement by genetic engineering", Bioengin. Bugs 2, 1-5.
  5. Z. Wang, M. Ly, F. Zhang, W. Zhong, A. Suen, A.M. Hickey, J.S. Dordick, and R.J. Linhardt (2010), "E. coli K5 fermentation and the preparation of heparosan, a bioengineered heparin precursor", Biotechnol. Bioeng. 107, 964-973.
  6. M. Ly, Z. Wang, T.N. Laremore, F. Zhang, W. Zhong, D. Pu, D.V. Zagorevski, J.S. Dordick, and R.J. Linhardt (2011), "Analysis of E. coli K5 capsular polysaccharide heparosan", Anal. Bioanal. Chem. 399, 737-745.
  7. A.M. Hickey, U. Bhaskar, R.J. Linhardt, and J.S. Dordick (2013), “Effect of eliminase gene (elmA) deletion on heparosan production and shedding in Escherichia coli K5” J. Biotechnol. 165, 175-177.
  8. J. Suwan, A. Torelli, A. Onishi, J.S. Dordick, and R.J. Linhardt (2012), "Addressing endotoxin issues in bioengineered heparin", Biotechnol. Appl. Biochem. 59, 420-428.
  9. U. Bhaskar, G. Li, L. Fu, A. Onishi, M. Suflita, J.S. Dordick, and R.J. Linhardt (2014), "Combinatorial one-pot chemoenzymatic synthesis of heparin", Carbohydr. Polym. 122, 399-407.
  10. E. Sterner, L. Li, P. Paul, J.M. Beaudet, J. Liu, R.J. Linhardt, and J.S. Dordick (2013), “Assays for determining heparan sulfate and heparin O-sulfotransferase activity and specificity”, Anal. Bioanal. Chem. 406, 525-536.
  11. P. Paul, J. Suwan, J. Liu, J. S. Dordick, and R. J. Linhardt (2012), "Recent advances in sulfotransferase enzyme activity assays", Anal. Bioanal. Chem. 403, 1491-1500.