\u03b2-elimination of hyaluronate by red king crab hyaluronidase

Dmitrii Sliadovskii,Victor Marchenkov,Oleg Gusev,Evgeny Sogorin,Irina Pozdnyakova-Filatova,Maxim Molchanov,Maria Timchenko,Tatyana Ponomareva

doi:10.1038/s41598-021-01890-3

Abstract

Crustacean hyaluronidases are poorly understood both in terms of their enzymatic properties and in terms of their structural features. In this work, we show that the hepatopancreas homogenate of the red king crab has a hyaluronidase activity that is an order of magnitude higher than its commercial counterpart. Zymography revealed that the molecular weight of a protein with hyalorunidase activity is 40–50 kDa. Analysis of the hepatopancreas transcriptome and results of cloning and sequencing of cDNA revealed a hyaluronidase sequence with an expected molecular weight of 42.5 kDa. Further analysis showed that hyaluronat enzymatic cleavage follows the beta -elimination mechanism, which is well known for bacterial hyaluronidases. The results of ion-exchange chromatography showed that the final product of hyaluronate degradation is unsaturated tetrasaccharide. Thus, we identified a new hyaluronidase of higher eukaryotes, which is not integrated into the modern classification of hyaluronidases.

Highlights

Crustacean hyaluronidases are poorly understood both in terms of their enzymatic properties and in terms of their structural features
We shown that the red king crab hyaluronidase cleaves β-(1→4)-glycosidic bonds by the mechanism of β-elimination
Transcriptome analysis of red king crab hepatopancreas, cloning and sequencing of cDNA encoding the mature form of hyaluronidase

Summary

Introduction

Crustacean hyaluronidases are poorly understood both in terms of their enzymatic properties and in terms of their structural features. Zymography revealed that the molecular weight of a protein with hyalorunidase activity is 40–50 kDa. Analysis of the hepatopancreas transcriptome and results of cloning and sequencing of cDNA revealed a hyaluronidase sequence with an expected molecular weight of 42.5 kDa. Further analysis showed that hyaluronat enzymatic cleavage follows the β-elimination mechanism, which is well known for bacterial hyaluronidases. The reaction products contain N-acetyl-D-glucosamine at the non-reduced end and D-glucuronic acid at the reduced end of the oligosaccharides. A significant part of the preparations are of animal origin, namely homogenates of testicular tissues (for example, Lydazum, Liporaza, Vitrase, Amphadase and others). The popularity of medical preparations obtained on the basis of testicular tissues of farm animals can be explained by the low cost of raw materials and the simplicity of obtaining the final drug. The resulting homogenate contains a heterogeneous mixture of proteins (Supplementary Fig. 1)

Methods

Results

Conclusion