Uncovering The Catalytic Mode Of Hs NDST4 And Development Of High-Throughput Glycosyltransferase Assay

Heparin(HP)has been the drug of choice to treat thrombotic disorders for more than 50 years and the market demand grows continuously.At present,the production of HP raw materials still relies on animal tissue extraction which is undermined by limited source and instable quality.The establishment of a safe and efficient method to synthesize HP in vitro instead of the existing production technique has been the direction of researchers’ efforts.enzymatic synthesis of active HP oligosaccharides has made significant breakthroughthrough more than 10 years of accumulation.However,there are still some bottlenecks restricting the large-scale preparation of HP drugs by enzymatic synthesis:the substrate specificity and catalytic mode of the crucial tool enzyme have not been totally demonstrated;N-sulfated glucosamine(GlcNS)cannot be directly linked to oligosaccharide backbone due to the strict substrate specificity of glycosyltransferase(GT)and the directed evolution of GT has been limited by a lack of sensitive screening methods for the isolation of novel natural GTs or their active variants.In order to improve the efficiency and application potential of HP oligosaccharide synthesis system,we carried out the work in two parts:study on the catalytic mode of N-Deacetylase/N-sulfotransferase 4(NDST4);establishment of yeast nucleoside diphosphatase(YND1)coupledhigh-throughput screening system for leloir-GT activity.1.The catalytic mode of N-Deacetylase/N-sulfotransferase 4(NDST4)N-Deacetylase/N-sulfotransferase(NDST).responsible for heparan sulfate(HS)N-sulfation.is a key enzyme directing further modifications including O-sulfation and C5-epimerazation.The arrangement of the sulfate groups in the HS sugar chains determines its ability to bind to the protein factor,which in turn affects the biological activity of the proteoglycans containing the sugar chains.Therefore,the systematic study of the catalytic mode of NDST is not only important to reveal the mechanism of HS biosynthesis,but also can directly guide the enzymatic synthesis of HP oligosaccharides in vitro.There are four NDST isozymes in human cells,which have different substrate specificity to regulate the N-sulfation modification of heparan sulfate.The modification mode of NDST1 has been fully elucidated.which has obtained great benefit from establishment of HS oligosaccharide synthesis system and the development of in vitro gene recombinant expression technique.However.the catalytic models of the three other NDSTs have not been fully studied.In this paper,the recombinant NDST4 was expressed by insect baculovirus system and a series of structure defined oligosaccharides were used to similate the N-sulfation modification of HP biosynthesis in vitro.The substrate specificity and other catalytic information of NDST4 were obtained by analyzing the structure of the reaction product.The results showed that:(1)the recombinant NDST4 alone did not possess N-deacetylase activity;(2)NDST4 had strong N-sulfotransferase activity;(3)unlike the dominating role of NDST1 in forming domain structures in HS.which carries out the modification in a highly ordered fashion.NDST4 did not show directionality.Based on the above conclusions,NDST4 is not a key enzyme in forming the alternative arrangement of highsulfuric acid region(NS domain)and low sulfuric acid region(NAc domain)during biosynthesis of HS.Besides,NDST4 has application potential as a tool enzyme in catalyzing N-sulfation of HP backbone.2.Establishment of yeast nucleoside diphosphatase(YND1)coupled high-throughput screening system for leloir-GT activityGlycosyltransferases(GTs)play critical roles as biological catalysts in the synthesis and modification of structurally defined oligosaccharides and glycoconjugates.The vast majority of GTs requires a nucleoside-activated monosaccharide as a donor,known as Leloir-GT.However,the rigorous substrate specificity limits the ability of natural GT,resulting in the contradiction of "sugar chain and its significant application value"and "enzymatic synthesis capacity defects".Using enzymatic synthesis of HP oligosaccharide as an example,the tool enzyme in HP backbone synthesis,Pasteurella multocida heparosan synthase 2(pmHS2).has strict monosaccharide donor selectivity which limits the efficiency of the existing system.The use of protein engineering to modify pmHS2 is an effective way to break the bottleneck of iduronic acid(IdoA)transformation.The success of directed evolution relies heavily on the development of efficient high throughput screening platforms.Most of the current Leloir-GTs activity assays are based on mass spectrometry and chromatographic separation methods,which do not meet the high throughput requirements for GT molecular directed evolution.In view of above problems,a high throughput screening method for Leloir-GT activity was established on the basis of soluble recombinant expression of yeast nucleoside diphosphatase(YND1).We have established a simple,sensitive and rapid high-throughput assay method by coupling the apyrase activity of YND1 toward UDP.the Leloir-GT reaction and phosphomolybdenum blue color reaction,andthree model GTs that are widely used as transferases for the synthesis of glycosaminoglycan backbone were used to validate the feasibility and discriminability of this method.The study content and results includes:(1)tothe Saccharomyces cerevisiae membrane-bond protein YND1 was expressed in E.coli in a soluble and active form by substituting the hydrophobic transmembrane region of YND1(Gene ID:856722)with three groups of glycine-serine repeating base(502F-51 8H/GSGSGS);(2)the enzymatic properties of recombinant YND1 showed that the optimum reaction condition of YND1 was consistent with that of most Leloir-GT,which therefore support our prediction that YND1 could be employed as a useful tool in glycotechnology applications;(3)the GT reaction was coupled with recombinant YND1 based on the optimized reaction condition.A rapid,simple and accurate method to detect GT activity was established.Using KfiA,pmHS2 and KfoC as model Leloir-GTs,the validity of the method was verified;(4)YND1 coupled GT assay could significantly promote the GT reaction and improve the efficiency of oligosaccharide synthesis by eliminating the potential inhibition of by-product N DP.