Investigation Of Action Pattern Of A Novel Chondroitin Sulfate/Dermatan Sulfate 4-O-endosulfatase

Chondroitin sulfates (CS) and dermatan sulfates (DS) are the linear polysaccharides of anionic glycosaminoglycans. They are ubiquitously expressed on cell surfaces, in extracellular matrices, and in connective tissues. The sugar chains of CS and DS usually exist as co-polymeric hybrid chains (CS/DS), which not only play structural roles, but also possess a series of biological functions, which were achieved by the interactions between the specific functional modules of sugar chains and target proteins. Increasing studies have shown that the sulfation parterrn of CS/DS plays a key role in the various functions.CS/DS sulfatases are sequence consereative enzymes that selectively hydrolyze sulfate groups within CS/DS sugar chains. CS/DS sulfatases can be generally classified into exo-and endo-types. Exosulfatase prefers to remove the terminal sulfate groups of sugar chains, while endosulfatase exhibits their desulfonation activity against both internal and terminal sulfate groups. Therefore, CS/DS endodulfatases are important tools for the preparation of homogeneous oligosaccharides and polysaccharides, studies on the structure and function of CS/DS chains, and medical treatment of related diseases.Recently, we have reported a novel CS/DS 4-O-endosulfatase of Vibrio sp. FC509, the first CS/DS sulfatase identified from marine bacteria. The enzyme could specifically remove the 4-O-sulfate from various CS and DS sugar chains, with an efficiency of 17-65%. However, the desulfation pattern of this enzyme is still not very clear. Herein, a series of hexasaccharides with various sulfation patterns were prepared and their structures were identified. The desulfation pattern of the 4-O-endosulfatase was elucidated through analyzing the products from the digeastion of these structure-known oligosaccharides by this 4-O-endosulfatase.1. Preparation of unsaturated hexasaccharides with various sulfation patterns: The CS-A and CS-E polysaccharides were partially degraded using HCLase, and then the oligosaccharide products ranging from disaccharide to dodecasaccharide were fractionized using gel filtration. The hexasaccharide fractions of CS-A and CS-E were subfractionized using a anion-exchange column YMC-Pack Polyamine II to obtain hexasaccharides with various sulfation patterns. Obtained fractions were further purified using the YMC-Pack PA-G column. Purified hexasaccahride were labeled with 2-amino benzamide (2-AB) and analyzed by fluorescent HPLC for purity. Total 14 pure hexaccharides were obtained for sulfation pattern studies.2. Structure analysis of hexasaccharides with various sulfation patterns:Fourteen pure hexasaccharides were individually reacted with excess CSase ABC, and the products were labeled by 2-AB to analyze the disaccharides composition through HPLC. To identify the disaccharide units at the non-reducing end, the 2-AB-labeled hexasaccharide samples were degraded by CSase ABC, and then the products were further labeled with 2-AB for HPLC anlysis. To determine the disaccharide unit at the reducing end, the 2-AB-labeled hexasaccharide samples were further degraded using HCDase followed by HPLC anlysis. As a result, fourteen hexasaccharides were indicated to share thirteen sulfation patterns.3. Investigation of the action pattern of 4-O-endosulfatase:1) Using the 4-O-endosulfatase to treat the various hexasaccharides and octasaccharides with specific sulfation pattern, we found that the 4-O-sulfate group could be completely removed whatever the HexUAl-3GalNAc(4S) was located at the reducing end, non-reducing end, or internal of hexasacchrides; However, the 4-O-sulfate group of HexUAl-3GalNAc(4S,6S) could not be removed whatever the disulfated disaccharide structure is located at the non-reducing end or internal of hexasaccharides; when the octasaccharide A4’5HexUA<xl-3GalNAc(6S)pi-4GlcApi-3GalNAc(4S)pl-4GlcA(2S) pl-3GalNAc(6S)pl-4GlcApi-3GalNAc(6S) were treated with 4-O-endosulfatase, the 4-O-sulfate groups could not be removed, suggesting that the HexUA(2S)l-3GalNAc(6S) unit in the sugar chains might inhibited the activity of 4-O-endosulfatase.2) The hexasaccharide A4’5HexUAal-3GalNAc(4S)pi-4GlcA pi-3GalNAc(4S)pl-4GlcApi-3GalNAc(4S) was treated with the 4-O-endosulfatase in a time course, desulfation analysis showed that the enzyme 4-O-endosulfatase could gradually remove sulfate groups from the hexasaccharide, and produce a non-sulfated hexasaccharide as the final product. The intermediate with two sulfate groups was analyzed for the sequence and the results showed that the disaccharide at reducing end of the hexasaccharide was non-sulfated, which suggested that the 4-O-endosulfatase removed 4-O-sulfate groups initially from the reducing end to the non-reducing end.This study has provided insights into the desulfation pattern of the novel 4-O-endosulfatse, which is useful for the preparation of homogeneous oligosaccharides or polysaccharides, as well as the studies on the structure-function relationship of CS/DS sugar chains.