In:
BMC Biotechnology, Springer Science and Business Media LLC, Vol. 9, No. 1 ( 2009-12)
Abstract:
Sialic acid is a deoxy uronic acid with a skeleton of nine carbons which is mostly found on cell surface in animals. This sialic acid on cell surface performs various biological functions by acting as a receptor for microorganisms, viruses, toxins, and hormones; by masking receptors; and by regulating the immune system. In order to synthesize an artificial sialoglycoprotein, we developed a large-scale production of rat α2,6-sialyltransferase (ST6Gal1). The ST6Gal1 was expressed in fifth instar silkworm larval hemolymph using recombinant both cysteine protease- and chitinase-deficient Bombyx mori nucleopolyhedrovirus (BmNPV- CP - - Chi - ) bacmid. The expressed ST6Gal1 was purified, characterized and used for sialylation of asialoglycopolypeptide. We tested the inhibitory effect of the synthesized α2,6-sialoglycopolypeptide on hemagglutination by Sambucus nigra (SNA) lectin. Results FLAG-tagged recombinant ST6Gal1 was expressed efficiently and purified by precipitation with ammonium sulphate followed by affinity chromatography on an anti-FLAG M2 column, generating 2.2 mg purified fusion protein from only 11 silkworm larvae, with a recovery yield of 64%. The purified ST6Gal1 was characterized and its N -glycan patterns were found to be approximately paucimannosidic type by HPLC mapping method. Fluorescently-labelled N -acetyllactosamine (LacNAc) glycoside containing dansyl group was synthesized chemo-enzymatically as high-sensitivity acceptor substrate for ST6Gal1. The acceptor substrate specificity of the enzyme was similar to that of rat liver ST6Gal1. The fluorescent glycoside is useful as a substrate for a highly sensitive picomole assay of ST6Gal1. Asialoglycopolypeptide was regioselectively and quantitatively sialylated by catalytic reaction at the terminal Gal residue to obtain α2,6-sialoglycopolypeptide using ST6Gal1. The α2,6-sialoglycopolypeptide selectively inhibited hemagglutination induced by Sambucus nigra (SNA) lectin, showing about 780-fold higher affinity than the control fetuin. Asialoglycopolypeptide and γ-polyglutamic acid did not affect SNA lectin-mediated hemagglutination. Conclusion The recombinant ST6Gal1 from a silkworm expression system is useful for the sialylation of asialoglycopeptide. The sialylated glycoprotein is a valuable tool for investigating the molecular mechanisms of biological and physiological events, such as cell-cell recognition and viral entry during infection.
Type of Medium:
Online Resource
ISSN:
1472-6750
DOI:
10.1186/1472-6750-9-54
Language:
English
Publisher:
Springer Science and Business Media LLC
Publication Date:
2009
detail.hit.zdb_id:
2052746-9
SSG:
12
