| Glycosaminoglycans (GAGs) are a type of heteropolysaccharides, which mainly exist in connective tissue, and are a linear biomacromolecule. According to the disaccharides composition, GAGs could be divided into different types, including chondroitin sulfate (CS)/dermatan sulfate (DS), heparin (HP)/heparan sulfate (HS) and hyaluronic acid (HA). GAGs play key important roles in various biological processes, including anticoagulation, central nervous system development and repair, regulating cell signal transduction.At present, HP are mainly purified from porcine intestinal mucosa, and a large amount of heparinoid (HPND), is produced during the purification. HPND mainly contains HS/HP, DS, and a small amount of CS. Due to the high similarity of physical and chemical properties of various GAGs in HPND, it is difficult to separate them from each other for higher value applications. Sulodexide (SLDX) is an antithrombotic drug composed of HS/HP and DS. SLDX is widely used due to its better antithrombotic effect and little side effect. However, it is only provided by original manufacturer although its patent has expired, and thus its price is very high. In this study several commercial glycosaminoglycan-degrading enzymes were cloned and expressed, and were used for the purification of HS/HP from HPND. We tried to prepared SLDX by combining various fractions of the purified HS/HP and original HPND, and further compared the bioactivities of the generic drugs and the original drug.1. Cloning, expression and purification several glycosaminoglycan-degrading enzymes:In this study, using Flavobacterium heparinum genome as template, three Heparinases (Ⅰ, Ⅱ and Ⅲ) were prepared through gene amplification, expression vector construction, strain induction, protein expression and purification. The expression quantity of Heparinase I is 300-320 mg/L and the enzyme activity is 90-100 IU/mg; The expression quantity of Heparinase Ⅲ is 240-260 mg/L and the activity is 80-90 IU/mg. CSaseABC was purified from Proteus vulgaris NCTC 4636, and the enzyme activity is 4000 IU/L. HCLase was cloned and expressed from Vibrio sp. FC509 found by our lab, and its activity against HA and CS-A is 450,000 IU/mg and 202,000 IU/mg, respectively.2. Analysis and imitation of soludexide:the molecular weight and disaccharide composition analysis showed that SLDX had a very wide molecular mass distribution, and mainly contained HS/HP (-80%) and DS (-20%). The HS/HP of SLDX is composed of 6 types of disaccharides with different structures, among of which trisulfated disaccharide (HexUA(2S)-GlcN(NS,6S)) and nonsulfated disaccharide (HexUA-GlcNAc) accounted for higher proportions,38.03% and 19.35%, respectively. In contrast, the DS mainly contains HexUAα1-3GalNAc(4S). Based on these results, we isolated high pure HS/HP from HPND by enzymatic method, further explore the preparation of SLDX by combining the different fractions of purified HS/HP and HPND.2. Comparison of the bioactivities of SLDX and generic drug:The activity of anti-Factor Xa and Factor Ⅱa of generic drugis 62.29 IU/mg and 58.11 IU/mg, respectively, while that of SLDX is 74.23 IU/mg and 60.54 IU/mg. Correspondingly, the ratio of anti-Factor Xa to Factor Ⅱa for SLDX and generic drug is 1.22 and 1.07, respectively. The in vivo study showed that the activity of LPL release for SLDX and generic drug is 174.3 mU/ml and 164.9 mU/ml, respectively.In this study, we have succeed to clone, express and purify servel GAG-degrading enzymes with high activity and application value, and on this basis have preliminarily exploited HPND to prepare a generic drug which is very similar to SLDX in composition and bioacticity. |
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