Purification and characterization of two recombinant human glucuronyltransferases involved in the biosynthesis of HNK-1 carbohydrate in Escherichia coli

Shinako Kakuda, Shogo Oka, Toshisuke Kawasaki

Research output: Contribution to journalArticlepeer-review

14 Citations (Scopus)

Abstract

Two glucuronyltransferases (GlcAT-P and GlcAT-S) are involved in the biosynthesis of HNK-1 carbohydrate, which is spatially and temporally regulated in the nervous system. To clarify the enzymatic properties of the respective glucuronyltransferases, we established an expression system for producing large amounts of soluble forms of flag-tagged human GlcAT-P and GlcAT-S in Escherichia coli. Approximately 15 and 6 mg of enzymatically active flag-GlcAT-P and flag-GlcAT-S were purified from E. coli cells in 5 liters of culture medium, respectively. These recombinant enzymes transferred GlcA to a glycoprotein acceptor, asialo-orosomucoid (ASOR), as well as a glycolipid acceptor, paragloboside. The specific activity of the recombinant GlcAT-P (1100 nmol/ min/mg) toward a glycoprotein acceptor, ASOR, was comparable to that of the enzyme (4300 nmol/min/mg) purified from rat brain. Phosphatidylinositol (PI) is specifically required for expression of the activity of the recombinant enzymes toward a glycolipid acceptor, paragloboside. The recombinant GlcAT-P was highly specific for the terminal type II structure, Galβ1-4GlcNAc, while the recombinant GlcAT-S recognized not only the type II structure, Galβ1-4GlcNAc, but also the type I structure, Galβ1-3GlcNAc. These acceptor specificities were similar to those of the native enzymes.

Original languageEnglish
Pages (from-to)111-119
Number of pages9
JournalProtein Expression and Purification
Volume35
Issue number1
DOIs
Publication statusPublished - 05-2004
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biotechnology

Fingerprint

Dive into the research topics of 'Purification and characterization of two recombinant human glucuronyltransferases involved in the biosynthesis of HNK-1 carbohydrate in Escherichia coli'. Together they form a unique fingerprint.

Cite this