Uncovering The Catalytic Cleavage Mode Of Heparinase ? Using Synthetic Heparin Oligosaccharides By Chemoenzymatic Method

Heparin and Heparan sulfate(HS),belonging to glycosaminoglycans(GAGs).consist of disaccharide repeating units of 1-4-linked ?-D-glucuronic acid(GlcA)or a-L-iduronic acid(IdoA)and ?-D-glucosamine(GIcN).They are heterogeneous in molecular weight and fine structure due to variable modification of sulfation and isomerization at specific oligosaccharide fragments,which result in the diverse repertoire of biological activities and functions.The synthesis of heparin/HS oligosaccharides has been one of the most challenging fields in organic synthesis.Many domastic and overseas research groups have successfully developed man(?)new methods and strategies for tailored synthesis of heparin oligosaccharides with specific structures.However,the existing chemical synthesis strategies have many shortcomings,such as multi-step reaction,very low synthesis efficiency.In the previous studies,our group has successfully developed a multi-enzymatically catalytic method for synthesizing heparin/HS oligosaccharides by mimicking the biosynthetic pathway of heparin in vivo.In order to break through the restriction of enzyme recognition on substrates,the specific oligosaccharides were N-desulfated using a mild chemical modification to construct a more diverse library of heparin oligosaccharides.In addition,heparinase I,produced by Pedobacter heparinus,is a basic tool enzyme for characterizing the structure of heparin/HS and preparing low molecular weight heparin(LMWH)and oligosaccharides.The specificity of heparinase I was previously determined using heterogeneous heparin and its derivatives as substrates,which is not sysmetic enough for the application of the enzyme.Therefore,the present paper was focused on investigation of the degradation of heparinase I toward synthetic heparin oligosaccharides.It was proved for the first time that heparinase I was capable of cleaving various glycoside bonds of heparin oligosaccharides,and that the reactivity of oligosaccharide substrates was dependent on the modification mode of the oligosaccharides.Finally,the affinity of different oligosaccharides to heparinase I was analyzed by bio-layer interferometry(BLI).This study clearly elucidated the diverse catalytic cleavage mode of heparanase I toward heparin oligosaccharides,which laid a foundation for expanding the application of heparanise.The main results and conclusions of the study are as follows:1.Preparation of sugar donors and sulfate donorSugar donors UDP-GlcNTFA,UDP-GlcA and sulfate donors PAPS are required for synthesizing heparin oligosaccharides.They were synthesized by enzymatic or chemoenzymatic methods established in our laboratory,followed by purification by ion exchange column chromatography.The sugar donors and sulfate donor were prepared at gram scale with a purity of more than 95%.The enzymes used,including NahK,GlmU,PPA and KAST/APSK were expressed in E.coli and prepared by affinity column chromatography.2.Synthesis,purification and structure characterization of heparin oligosaccharides.The synthesis of oligosaccharide backbone was started from p-nitrophenol glucuronide(GIcA-PNP),with alternate addition of GIcNTFA or GlcA to the non-reducing end the oligosaccharides catalyzed by N-acetylglucosamine transferase(KfiA)and heparosan synthetase(PmHS2).Each step had a yield of up to 96%,with a purity of more than 90%after purification by C18 reversed-phase chromatography column.After completely removing trifluoroacetyl(TFA)group of GlcNTFA by LiOH,using PAPS as sulfate donor,the N-unsubstituted GlcN(GIcNH2)residues of oligosaccharides were converted to N-sulfated GlcN(GlcNS)catalyzed by N-sulfosyltransferase(NST).Under the catalysis of Cs-epimerase,the GlcA residues flanked by the GlcNS residues were converted to iduronic acid(IdoA),or under the co-catalysis of Cs-epimerase and 2-O-sulfotransferase(2-OST),the GlcA residues were converted to 2-O-sulfated iduronic acid(IdoA2S),and the yields of the two oligosaccharides were 40%and 80%,respectively.The purity of the two oligosaccharides were more than 92%.Finally,under the catalysis of 6-O-sulfotransferase 1/3(6-OST1/3),the GlcNS residues of oligosaccharides were modified as 6-O-sulfated GlcNS(GlcNS6S).Each of three resultant products had a yield of higher than 98%and the purity of higher than 95%.In addition,considering the restriction of enzyme recognition on substrates,a mild chemical method was introduced to completely remove N-sulfate groups of oligosaccharides to give four rare GlcNH2-containing oligosaccharides with the yields of 98%,70%,73%,65%,respectively.The purity of each oligosaccharide purified by ion exchange column chromatography was over 95%.ESI-MS and NMR analysis showed the structure of each of synthetic heparin oligosaccharide of different modification mode as expected.3.Study of the catalytic cleavage mode of heparinase IA serial of structurally defined heparin and HS oligosaccharides were synthesized and then used as substrates for the cleavage of heparinase I under different conditions to investigate the catalytic specificity.The results were as follows:(1)heparinase I has been shown to be unable(?)cleave the glycoside bond sites between GlcNH2 and non-sulfated GlcA(including GlcNH2-IdoA2S or GlcNH2-IdoA2S or GlcNH:26S-IdoA or GlcNH26S-IdoA2S).Combining with the literature that reported that-GlcNAc6S-IdoA2S could be cleaved by enzyme,we conclude that N-substitution of GlcN(GlcNS or GlcNAc)is necessary for heparinase I cleavage.In addition to the primary glycoside bonds between-GlcNS6S-IdoA2S-that was cleaved by heparinase I,it was found,for the first time,that the glycoside bonds between-GlcNS6S-GlcA2S-,-GlcNS-IdoA2S-,-GIcNS-GlcA2S,-GlcNS6S-IdoA-,GlcNS6S-GlcA-could also be cleaved by heparinase I,which indicated that 2-O-sulfated uronic acid or 6-O-sulfated GlcN is not necessary for heparinase I cleavage.(2)The six substrates were degraded by heparanase I with quite different cleavage rates.Firstly,the more highly sulfated oligosaccharide substrates showed higher cleavage rate by heparanase I than lower sulfated substrates.In addition,heparanase I preferencially acted on oligosaccharides containing IdoA residues than those containing GlcA residues.The enzymatic cleavage products containing unsaturated glucuronic acid(?UA)were prepared by HPLC and were identified by ESI-MS to confirm the cleavage sites of six heparin oligosaccharides.(3)BLI results showed that the difference in affinity of different oligosaccharides to heparinase ?.Penta-NS had the lowest affinity consistent with that it was not acted by heparinase ?.The affinities of Penta-NS6S-IdoA2S and Hexa-NS6S-GlcA2S to heparinase ? were higher than those of Penta-NS-IdoA2S and Hexa-NS-GlcA2S respectively,which suggested that the high cleavage efficiency of hypersulfated substrates is related to their high affinity.Penta-NS6S-IdoA had higher affinity than Penta-NS6S confirming that heparin oligosaccharides containing IdoA were more preferencial substrates for heparinase I over those containing GlcA.However,Hexa-NS6S-GlcA2A with lower reactivity had stronger affinity with the enzyme than Penta-NS6S-IdoA2S,indicating oligosaccharide substrates with longer chains were more closely bound to the enzyme,which could not help the cleavable sequence of oligosaccharide access to enzymatic active center.It was proved that the catalytic efficiency of heparanase I toward different heparin oligosaccharides could not simply attribute to affinity difference between them,but the global conformation formed by size,uronic acid types and net negative charge on the surface.In a word,insight into the catalytic cleavage mode of heparanase I on synthetic heparin oligosaccharides has important reference significance for more accurate characterization of heparin/HS structure as well as establishment of more reliable LMWH production process.