Exploiting enzyme specificities in digestions of chondroitin sulfates A and C: Production of well-defined hexasaccharides

被引:30
作者
Pomin, Vitor H. [1 ]
Park, Younghee [1 ]
Huang, Rongrong [1 ]
Heiss, Christian [1 ]
Sharp, Joshua S. [1 ]
Azadi, Parastoo [1 ]
Prestegard, James H. [1 ]
机构
[1] Univ Georgia, Complex Carbohydrate Res Ctr, Athens, GA 30602 USA
关键词
chondroitin sulfate; chondroitinase; enzymatic specificity; hyaluronidase; oligosaccharides; HEPARAN-SULFATE; TESTICULAR HYALURONIDASE; PROTEOGLYCAN EXPRESSION; MONOCLONAL-ANTIBODIES; DERMATAN SULFATE; SHARK CARTILAGE; AC LYASE; GLYCOSAMINOGLYCAN; BINDING; INFLAMMATION;
D O I
10.1093/glycob/cws055
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Interactions between proteins and glycosaminoglycans (GAGs) of the extracellular matrix are important to the regulation of cellular processes including growth, differentiation and migration. Understanding these processes can benefit greatly from the study of protein-GAG interactions using GAG oligosaccharides of well-defined structure. Materials for such studies have, however, been difficult to obtain because of challenges in synthetic approaches and the extreme structural heterogeneity in GAG polymers. Here, it is demonstrated that diversity in structures of oligosaccharides derived by limited enzymatic digestion of materials from natural sources can be greatly curtailed by a proper selection of combinations of source materials and digestive enzymes, a process aided by an improved understanding of the specificities of certain commercial preparations of hydrolases and lyases. Separation of well-defined oligosaccharides can then be accomplished by size-exclusion chromatography followed by strong anion-exchange chromatography. We focus here on two types of chondroitin sulfate (CS) as starting material (CS-A, and CS-C) and the use of three digestive enzymes with varying specificities (testicular hyaluronidase and bacterial chondroitinases ABC and C). Analysis using nuclear magnetic resonance and mass spectrometry focuses on isolated CS disaccharides and hexasaccharides. In all, 15 CS hexasaccharides have been isolated and characterized. These serve as useful contributions to growing libraries of well-defined GAG oligosaccharides that can be used in further biophysical assays.
引用
收藏
页码:826 / 838
页数:13
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