Substrate specificity of Chondroitinase ABC I based on analyses of biochemical reactions and crystal structures in complex with disaccharides

Makoto Takashima,Tadashi Eguchi,Akimasa Miyanaga,Ippei Watanabe

doi:10.1093/glycob/cwab086

Makoto Takashima, Tadashi Eguchi + Show 2 more

Open Access

https://doi.org/10.1093/glycob/cwab086

Copy DOI

Abstract

Chondroitinase ABC I (cABC-I) is the enzyme which cleaves the β-1,4 glycosidic linkage of chondroitin sulfate (CS) by β-elimination. To elucidate more accurately the substrate specificity of cABC-I, we evaluated the kinetic parameters of cABC-I and its reactivity with CS isomers displaying less structural heterogeneity as substrates, e.g., approximately 90 percent of disaccharide units in Chondroitin sulfate A (CSA) or Chondroitin sulfate C (CSC) is D-glucuronic acid and 4-O-sulfated N-acetyl galactosamine (GalNAc) (A-unit) or D-glucuronic acid and 6-O-sulfated GalNAc (C-unit), respectively. cABC-I showed the highest reactivity to CSA and CSC among all CS isomers, and the kcat/Km of cABC-I was higher for CSA than for CSC. Next, we determined the crystal structures of cABC-I in complex with CS disaccharides, and analyzed the crystallographic data in combination with molecular docking data. Arg500 interacts with 4-O-sulfated and 6-O-sulfated GalNAc residues. The distance between Arg500 and the 4-O-sulfate group was 0.8 Å shorter than that between Arg500 and the 6-O-sulfated group. Moreover, it is likely that the 6-O-sulfated group is electrostatically repulsed by the nearby Asp490. Thus, we demonstrated that cABC-I has the highest affinity for the CSA richest in 4-O-sulfated GalNAc residues among all CS isomers. Recently, cABC-I was used to treat lumbar disc herniation. The results provide useful information to understand the mechanism of the pharmacological action of cABC-I.

Highlights

As linear polysaccharides composed of repeating disaccharide units of uronic acid and hexosamine, the glycosaminoglycans (GAGs) are classified into, e.g., chondroitin sulfate (CS), dermatan sulfate (DS), hyaluronan (HA), heparan sulfate (HS) and keratin sulfate (KS)
According to its substrate specificity, Chondroitin sulfate lyase (CSase) is mainly categorized into three types: Chondroitinase ABC, Chondroitinase AC and Chondroitinase B. cABC has been used as a GAG-degrading enzyme for more than 30 years (Murata and Yokoyama 1985a; Yoshida et al 1989)
We evaluated the reactivity of Chondroitinase ABC I (cABC-I) toward five CS isomers adjusted to an molecular weight (Mw) of almost 25 kDa, and non-sulfated GAGs as substrates: chondroitin 4-O-sulfate (CSA), DS, Chondroitin sulfate C (CSC), CSD, CSE, CH and HA (Figure 1B). cABCI had a high reactivity to CSA and CSC compared with other CS isomers, and barely recognized the non-sulfated GAGs CH and HA (Figure 1B and D)

Summary

Introduction

As linear polysaccharides composed of repeating disaccharide units of uronic acid and hexosamine, the glycosaminoglycans (GAGs) are classified into, e.g., chondroitin sulfate (CS), dermatan sulfate (DS), hyaluronan (HA), heparan sulfate (HS) and keratin sulfate (KS). CS is a sulfated polysaccharide composed of repeating of D-glucuronic acid (GlcA) and N-acetyl galactosamine (GalNAc) with a β1–3 linkage. It is widely distributed in human or animal tissues and associated with biological and pathological phenomena (Linhardt and Toida 2004; Raman et al 2005; Gama et al 2006; Mikami and Kitagawa 2013). By degradation of CS, cABCI directly injected into the NP causes reduction in disc pressure and herniation volume, and relieves the symptoms associated with LDH (Sugimura et al 1996; Takahashi et al 1996; Yamada et al 2001)

Methods

Results

Conclusion