Elsevier

Carbohydrate Polymers

Volume 233, 1 April 2020, 115847
Carbohydrate Polymers

Mass spectrometric evidence for the mechanism of free-radical depolymerization of various types of glycosaminoglycans

https://doi.org/10.1016/j.carbpol.2020.115847Get rights and content

Highlights

  • Free-radical degradation mechanism of GAGs was explored using HILIC-FTMS.

  • Fragments generated by free-radical degradation maintain basic structural units.

  • High-sulfate levels resist free-radical degradation.

  • Oligomers with modified reducing ends were related to linkage and sulfate level.

Abstract

Glycosaminoglycans (GAGs) are large, complex carbohydrate molecules that interact with a wide range of proteins involved in physiological and pathological processes. Several naturally derived GAGs have emerged as potentially useful therapeutics in clinical applications. Natural polysaccharides, however, generally have high molecular weights with a degree of polydispersity, making it difficult to investigate their structural properties. In this study, we establish a free-radical–mediated micro-reaction system and use hydrophilic interaction chromatography (HILIC)–Fourier transform mass spectrometry (FTMS) to profile the degraded products of various types of GAGs, heparin, chondroitin sulfate A, NS-heparosan, and oversulfated chondroitin sulfate (OSCS), to reveal the free-radical degradation mechanism of GAGs. The results show that the bulk fragments of GAGs generated by free-radical degradation can maintain their basic structural units and sulfate substituents. In addition, an abundance of oligomers modified with oxidation at their reducing ends or by dehydration also appeared. We discovered that these modifications were related in terms of the degree of sulfation and the α- or β-linkage of HexNY (Y = SO3 or Ac), and especially that the different linkage of the disaccharide unit is the main factor in modification. In addition, the method based on micro-free-radical reaction and HILIC-FTMS is both effective and sensitive, thus suggesting its broad practical value for the structural characterization and in the biological structure-function studies of GAGs.

Introduction

Glycosaminoglycans are a family of linear, sulfated, negatively charged polysaccharides that have molecular weights of roughly 10–100 kDa (Booth & Thomason, 1991; Gandhi & Mancera, 2008). Based on differences among the repeating disaccharide units comprising GAGs, they can be categorized into four main groups: chondroitin sulfate (CS)/dermatan sulfate (DS), heparin (HP)/heparan sulfate (HS), hyaluronan (HA), and keratan sulfate (KS). In vivo, most GAGs are linked to the core protein as proteoglycans (PGs), while HA exists only as a GAG. The characterization and identification of GAGs in biological samples can reveal the relationship between their structures and their biological functions (Chen et al., 2013; Li, Ly, & Linhardt, 2012). In addition to their physiological and pathophysiological roles, these naturally complex polysaccharides are active biological and pharmaceutical agents (Li, Ly et al., 2012; Silva et al., 2019). For this reason, several GAG-based drugs are already in clinical use. For example, the naturally occurring anticoagulant heparin is a heterogeneous, polydisperse, highly sulfated polysaccharide belonging to the GAG family. The low-molecular weight heparins (LMWHs) have become the drugs of choice for the treatment of deep vein thrombosis, pulmonary embolism, arterial thrombosis, and unstable angina (Quader, Stump, & Sumpio, 1998), and they have been shown to improve the survival of cancer patients (Furukawa, Okada, & Shinohara, 2017; Green, Hull, Brant, & Pineo, 1992). In addition, the EULAR Recommendations for the Treatment of Knee OA, published in 2003, listed oral CS as both evidence 1A and strength of recommendation A, representing the highest level for a therapeutic strategy (Jordan et al., 2003; Richy et al., 2003; Uebelhart, 2008). Currently, GAGs have a wide range of medical applications, from topical moisturizers to anticoagulants used in most surgical procedures, and exhibit great pharmaceutical value (DeAngelis, 2012; Yip, Smollich, & Gotte, 2006).

The fundamental biological, pathological, pharmacological, and therapeutic roles of GAGs have challenged researchers to devise new processes to prepare these critical polysaccharides, as well as novel methods to decode their fine complex structures, procedures that are needed to establish their structure-activity relationships. The direct analysis of intact GAGs is difficult due to their relatively high molecular mass and polydispersity. As a consequence, controlled enzymatic or chemical depolymerization is often required to lower their molecular weights and simplify their polydisperse mixtures prior to analysis. Natural GAGs are high-molecular-weight polymers with complex structures. Only a few types of GAGs have specific cleavage enzymes, and if their structural or chemical groups change, the original enzymes will lose their specific effect on the modified GAGs. Therefore, it is desirable to establish a widely applicable GAG degradation method. Chemical degradation methods include acid or alkali degradation, oxidant degradation, free-radical degradation, and others. Acid or alkali degradation is difficult to control and often results in damage to GAG structure. Different preparation methods result in products that differ from each other structurally. One of the most commonly used processes of GAG chemical depolymerization is the process carried out by free-radical attack (mainly hydroxyl). In recent years, reactive oxygen species (ROS) produced by chemical agents or radiation has been used to degrade sulfated polysaccharides (Kjellen & Lindahl, 1991; Ofman, Slim, Watt, & Yorke, 1997; Vismara et al., 2010; Zhao, Yang, Li, Zhang, & Linhardt, 2013). Free-radical degradation, which is based on the Fenton reaction and is a common chemical degradation method in GAG analysis, differs from enzymatic degradation and can be applied to all polysaccharides without selectivity. Vismara et al. (2007), (2010) has described a mechanism for this reaction. With the catalysis of transition metal ions (Cu+, Fe2+, and others), H2O2 decomposes to produce active radical dotOH, which can quickly attack the glycan chain and capture the H connected to the carbon to form a hydroxyalkyl radical. The radical dotC-O can also be oxidized by Cu2+ or Fe3+ to +C-O, and finally, the +C-O will be converted into ketone, aldehyde, or acid based on the reaction conditions or the reaction medium. Free-radical degradation of various GAGs can maintain the original structural information, does not cause branch breakage or loss of sulfate groups, is highly reproducible, is inexpensive, and, therefore, is widely used. However, free-radical degradation is a complex process that results in byproducts. Thus, the development of suitable analytical methods is challenging.

Structural analysis of the various domains of GAGs is necessary in order to determine their biological activities, but these experiments have been limited by the extreme heterogeneity of GAGs. Mass spectrometry (MS) is now emerging as the method of choice for GAG oligosaccharide analysis (Galeotti & Volpi, 2016; Saad et al., 2005; Sanderson et al., 2018; Shi & Zaia, 2009). Linhardt and coworkers (Ly et al., 2011) first determined the structural sequence of GAGs in bikunin based on a top-down method using FTMS and FT-ICR-MS. Capillary amide hydrophilic interaction chromatography (HILIC) with online electrospray ionization (ESI) MS and high-performance liquid chromatography (HPLC) has been used for the analysis of released glycans (Wuhrer, Koeleman, Deelder, & Hokke, 2004), glycopeptides (Merry et al., 2002; van Schaick, Pirok, Haselberg, Somsen, & Gargano, 2019), glycosphingolipids (Wing et al., 2001), and GAGs (Staples et al., 2010). Li, Zhang, Zaia, and Linhardt, (2012)) used HILIC-FTMS to analyze enoxaparin sodium and obtained more than 300 pieces of glycan-related data, including non-reducing end components, glycans with or without unsaturated bonds, and glycans containing 1, 6-anhydro derivatives at their reducing ends. Although the combination of online high-performance chromatography and mass spectrometry provides an efficient technical basis for structural and compositional analysis of GAGs, specialized analytical techniques and bioinformatics are also necessary. Therefore, we utilized the publicly available open source GlycReSoft software developed by Zaia and coworkers (Maxwell et al., 2012) to calculate bioinformatics search space sets for GAG samples and to assign oligosaccharides.

In this study, HILIC in combination with high-resolution FTMS were applied for the identification and quantification of the GAG-derived oligosaccharides generated by free-radical degradation. By analyzing the structural characteristics of the products, the structural quantification of GAG-derived oligosaccharides was established, which provides a critical advantage for revealing the reaction mechanism of different GAGs in free-radical degradation. This finding was also further used to study the structure-activity relationships and potential pharmaceutical values of GAGs.

Section snippets

Materials

Heparan sulfate (CID 53477715 - National Center for Biotechnology Information, PubChem Compound Database) and heparin (CID 772 - National Center for Biotechnology Information, PubChem Compound Database) were purchased from Celsus (Cincinnati, Ohio, USA); NS-heparosan (Bhaskar et al., 2015) and OSCS (B. Li et al., 2009) were prepared in our laboratory. CSA (CID 4368136 - National Center for Biotechnology Information, PubChem Compound Database), H2O2, cupric acetate monohydrate, acetonitrile

Free-radical depolymerization of different types of GAGs

Compared to enzymatic degradation, the chemical degradation of polysaccharides has a wide range of applications due to its low cost. Chemical degradation methods include acid or alkali degradation, oxidant degradation, and free-radical degradation. In acid or alkali degradation, the reaction conditions are difficult to control, often leading to structural damage to the GAGs. Free-radical degradation based on the Fenton reaction is the most common chemical degradation method employed in the

Conclusions

Free-radical degradation of polysaccharides may result in nonspecific and/or specific scission of carbohydrate chains, an action that is related to their structural units and special substituent groups. In this study, various GAGs were degraded utilizing a micro-reaction Fenton depolymerization process through copper ions catalysis, and then characterized using HILIC-FTMS. Comparing different types of GAG-derived oligosaccharides eluting on a HILIC column, the resolution characteristics of the

Acknowledgments

This work was supported by the National Natural Science Foundation of China (31971210, 81991522), the National Science and Technology Major Project for Significant New Drug Development (2018ZX09735004), the Marine S&T Fund of Shandong Province for the Pilot National Laboratory for Marine Science and Technology (Qingdao) (2018SDKJ0401, 2018SDKJ0404), the Fundamental Research Funds for the Central Universities (201762002), and the Taishan Scholar Project Special Funds (TS201511011).

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