CS Lyases: Structure, Activity, and Applications in Analysis and the Treatment of Diseases

We previously reported that the xyloside 2-(6-hydroxynaphthyl) ␤-D-xylopyranoside (XylNapOH), in contrast to 2-naphthyl ␤-D-xylopyranoside (XylNap), specifically reduces tumor growth both in vitro and in vivo. Although there are indications that this could be mediated by the xyloside-primed glycosaminoglycans (GAGs) and that these differ in composition depending on xyloside and cell type, detailed knowledge regarding a structure-function relationship is lacking. In this study we isolated XylNapOH-and XylNap-primed GAGs from a breast carcinoma cell line, HCC70, and a breast fibroblast cell line, CCD-1095Sk, and demonstrated that both XylNapOH-and XylNap-primed chondroitin sulfate/dermatan sulfate GAGs derived from HCC70 cells had a cytotoxic effect on HCC70 cells and CCD-1095Sk cells. The cytotoxic effect appeared to be mediated by induction of apoptosis and was inhibited in a concentration-dependent manner by the XylNap-primed heparan sulfate GAGs. In contrast, neither the chondroitin sulfate/ dermatan sulfate nor the heparan sulfate derived from CCD1095Sk cells primed on XylNapOH or XylNap had any effect on the growth of HCC70 cells or CCD-105Sk cells. These observations were related to the disaccharide composition of the XylNapOH-and XylNap-primed GAGs, which differed between the two cell lines but was similar when the GAGs were derived from the same cell line. To our knowledge this is the first report on cytotoxic effects mediated by chondroitin sulfate/dermatan sulfate.

Section: Structure Of Cytotoxic Xyloside-primed Glycosaminoglycansmentioning

confidence: 99%

Xyloside-primed Chondroitin Sulfate/Dermatan Sulfate from Breast Carcinoma Cells with a Defined Disaccharide Composition Has Cytotoxic Effects in Vitro

Persson

Tykesson

Westergren‐Thorsson

et al. 2016

“…Polysaccharide lyases depolymerize GAGs through ␤-elimination reactions and generally produce unsaturated disaccharides containing unsaturated uronic acid with C-C double bonds at nonreducing termini (Fig. 1B) (11). Carbon atoms C-3, C-4, C-5, and C-6 of unsaturated GlcUA (⌬GlcUA) and IdoUA (⌬IdoUA) are located in a single plane because of the formation of double bonds between C-4 and C-5 (12).…”

mentioning

confidence: 99%

Crystal Structure of a Bacterial Unsaturated Glucuronyl Hydrolase with Specificity for Heparin

Nakamichi

Mikami

Murata

et al. 2014

Background: Bacterial unsaturated glucuronyl hydrolase (UGL) is essential for complete degradation of host glycosaminoglycans. Results: Crystal structure of Pedobacter heparinus UGL, Phep_2830 specific for heparin degradation, was determined. Conclusion: The pocket-like structure and lid loop of Phep_2830 are involved in heparin disaccharide recognition. Significance: This work contributes to understanding the bacterial degradation of host extracellular matrix components.

“…These data also showed that the Tyr-242 residue acted as a base to abstract a proton from the C5 position of GlcA, while the Asn-183 and His-233 residues neutralized the charge on the acidic group of GlcA (16). ChnAC and several other CS lyases have been widely used in conjunction with modern separation and analytical methods for disaccharide analysis, polysaccharide sequencing, and biological evaluations (19). However, very little is known about the direction of the ChnAC-catalyzed degradation of polysaccharides.…”

mentioning

confidence: 92%

“…Although these CS lyases followed the same gen-eral mechanism, in that they catalyzed a ␤-elimination reaction at the C4 position of GlcA to yield ⌬4,5-unsaturated GlcA at the non-reducing end of the oligosaccharide products, these enzymes exhibited different substrate specificities (17)(18)(19) (16,20), Flavobacterium heparinum (21,22), Serratia marcescens (23) and Bacteroides stercoris (24), and these enzymes have been subjected to extensive biochemical and x-ray crystallographic analyses. Interestingly, the enzymatic characterization of the ChnAC from F. heparinum showed that it acted as an endolyase toward a polysaccharide substrate, producing a mixture of unsaturated oligosaccharides of different sizes (25).…”

mentioning

confidence: 99%

“…In this study, we decided to synthesize chimeric oligosaccharides concurrently containing repeating HS and CS disaccharide units. This selection was based on the fact that the CS backbone structure (-GalNAc-␤-1,4-GlcA-) can be cleaved by ChnAC, whereas the HS backbone structure (-GlcNAc-␣-1,4-GlcA-) remains unchanged (16,19). The site resistant to being cleaved by the ChnAC enzyme was positioned on the reducing or the non-reducing terminus, and the products resulting from the treatment of these substrates with ChnAC were identified by electrospray ionization mass spectrometry (ESI-MS).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Uncovering the Catalytic Direction of Chondroitin AC Exolyase

Yin

Wang

Sheng

2016

Glycosaminoglycans (GAGs) are polysaccharides that play vital functional roles in numerous biological processes, and compounds belonging to this class have been implicated in a wide variety of diseases. Chondroitin AC lyase (ChnAC) (EC 4.2.2.5) catalyzes the degradation of various GAGs, including chondroitin sulfate and hyaluronic acid, to give the corresponding disaccharides containing an ⌬ 4 -unsaturated uronic acid at their non-reducing terminus. ChnAC has been isolated from various bacteria and utilized as an enzymatic tool for study and evaluating the sequencing of GAGs. Despite its substrate specificity and the fact that its crystal structure has been determined to a high resolution, the direction in which ChnAC catalyzes the cleavage of oligosaccharides remain unclear. Herein, we have determined the structural cues of substrate depolymerization and the cleavage direction of ChnAC using model substrates and recombinant ChnAC protein. Several structurally defined oligosaccharides were synthesized using a chemoenzymatic approach and subsequently cleaved using ChnAC. The degradation products resulting from this process were determined by mass spectrometry. The results revealed that ChnAC cleaved the ␤1,4-glycosidic linkages between glucuronic acid and glucosamine units when these bonds were located on the reducing end of the oligosaccharide. In contrast, the presence of a GlcNAc-␣-1,4-GlcA unit at the reducing end of the oligosaccharide prevented ChnAC from cleaving the GalNAc-␤1,4-GlcA moiety located in the middle or at the non-reducing end of the chain. These interesting results therefore provide direct proof that ChnAC cleaves oligosaccharide substrates from their reducing end toward their non-reducing end. This conclusion will therefore enhance our collective understanding of the mode of action of ChnAC.