Real time analysis of the enzymatic digestion of chondroitin sulfate: role of the sulfation pattern

Carla Silva¹, Ramon Novoa-Carballal¹, Rui Reis¹ and Iva Pashkuleva^*1

 ^1*3B´s Research Group – Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal; ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal.

E-mail: carla.silva@dep.uminho.pt

INTRODUCTION

Chondroitin sulfate (CS) consists of repeating glucoronic acid (GlcA)/N-acetyl-galactosamine (GalNAc) disaccharide units joined by β 1,4 and 1,3 linkages respectively.¹ Multiple sulfotransferases involved in 4-O and 6-O sulfation of GalNAc units are responsible for the obtaining of CS with different degree of sulfation (0.1 – 1.3 per disaccharide unit) and patterns; CS may contain sulfate groups in both 4- and 6-positions the GalNAc unit (CS-E) but may also be exclusively 4-sulfated (CS-A) or 6-sulfated (CS-C).^1,2 These different sulfation patterns influence the interactions between CS and proteins in the extracellular matrix or/and in the basement membrane and thus, can be critical in important physiological processes.² Herein, we demonstrate how the activity of two important enzymes, namely hyaluronidase and chondroitinase, are affected by the sulfation pattern of CS. We have established a protocol including GPC for the real time determination of the mechanism of enzyme action and ultrafiltration to obtain well defined oligosaccharides.

EXPERIMENTAL METHODS

The enzymatic digestion of CS-A, CS-C and CS-E by hyaluronidase and chondroitinase ABC was performed using 0.02Uenzyme/mgin phosphate buffer (0.1M,  pH= 7.4) at 37ºC for 48h. Aliquots of the reaction were taken at 1, 4, 8, 24 and 48 h. The enzyme was deactivated by heating and removed by centrifugation. The digested samples were then fractionated by ultrafiltration using 10kDa and 1kDa cut off membranes. All the products were lyophilized and analysed by gel permeation chromatography (GPC) and nuclear magnetic resonance (NMR).

RESULTS AND DISCUSSION

Digestion with Hyaluronidase

Table 1: Molecular weight and Infra-red (IR) area of digested species of CS by hyaluronidase for 24h

CS-A (84kDa)

CS-C (54kDa)

CS-E(146kDa)

1h

42kDa (71.0%)

37kDa (73.6%)

72.6kDa (82.0%)

1.2kDa (0.6%)

-

1.75kDa (1.1%)

914Da (23.9%)

774Da (21.3%)

914Da (15.5%)

558Da (4.6%)

495Da (5.0%)

690Da (1.0%)

24h

24.1kDa (72.4%)

11.4kDa (77.3%)

54.6kDa (76.4%)

1.9kDa (1.6%)

-

1.9kDa (2.7%)

940Da (22.0%)

369Da (17.7%)

936Da (18%)

581Da (3.9%)

254Da (5.1%)

573Da (2.9%)

Hyaluronidase showed enhanced activity toward a 6-sulfation-rich substrate (CS-C, Table 1). In addition, the substantial distributions toward medium sized-polysaccharides support an endolytic mechanism of action (Fig. 1).

Digestion with Chondroitinase ABC

Similarly to the hyaluronidase, the CS-C was digested at higher degree (higher amount of low molecular weight species) as compared with CS-A and CS-E (Table 2). However, in this case low molecular weight species (disaccharides) were the major products of digestion, suggesting exolytic cleavage activity for chondroitinase (Fig. 2).

Table 2: Molecular weight and Infra-red (IR) area of digested species of CS by chondroitinase for 24h

CS-A (84kDa)

CS-C (54kDa)

CS-E (146kDa)

1h

88kDa (83.0%)

-

130kDa (84.0%)

1.8kDa (1.2%)

4.3kDa (14.4%)

2.2kDa (3.0%)

948Da (13.1%)

1.7kDa (68.4%)

1.7kDa (68.4%)

558Da (2.3%)

861Da (17.2%)

633Da (1.6%)

24h

78.7kDa (80.8%)

-

118kDa(82.0%)

4.7kDa (1.3%)

5.5kDa (6.9%)

2.1kDa (3.9%)

1.71kDa (1.0%)

1.9kDa (75.3%)

991Da (12.2%)

934Da (14.3%)

990Da (17.7%)

-

574Da (2.6%)

-

585Da (1.8%)

CONCLUSION

The catalytic profiles of the studied enzymes were determined: hyaluronidase is mainly endolytic whereas chondroitinase acts via both endolytic and exolytic mechanisms. Both enzymes showed preference for CS-C, however in a different extent. Medium sized polysaccharides are obtained with hyaluronidase whereas higher amount of low molecular species are obtained with chondroitinase. This protocol can be also applied for obtaining of well defined oligosaccharides in a large scale.

REFERENCES

1.Yamada S. and Sugahara K., Curr Drug Discov Technol. 5: 289-301, 2008

2.Mikami, T. and Kitagawa H., Biochim. Biophys. Acta.: General subjects 1830(10): 4719-4733, 2013

ACKNOWLEDGMENTS

The FP7 project Polaris (316331) and the Programme NORTE-07-0124 FEDER-000016 are acknowledged.