Synthesis of sialoglycopolypeptide for potentially blocking influenza virus infection using a rat alpha2,6-sialyltransferase expressed in BmNPV bacmid-injected silkworm larvae.
Synthesis of sialoglycopolypeptide for potentially blocking influenza virus infection using a rat alpha2,6-sialyltransferase expressed in BmNPV bacmid-injected silkworm larvae.
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DOI：
10.1186/1472-6750-9-54
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发表时间：
2009-06-05
影响因子：
3.5
通讯作者：
Park EY
中科院分区：
文献类型：
--
作者：
Ogata M;Nakajima M;Kato T;Obara T;Yagi H;Kato K;Usui T;Park EY
唾液酸是一种具有九个碳骨架的脱氧糖醛酸，主要存在于动物细胞表面。细胞表面的唾液酸通过充当微生物、病毒、毒素和激素的受体来发挥各种生物学功能；通过掩蔽受体；并通过调节免疫系统。为了合成人工唾液酸糖蛋白，我们开发了大规模生产的大鼠α2,6-唾液酸转移酶（ST6Gal1）。使用半胱氨酸蛋白酶和几丁质酶缺陷的重组家蚕核多角体病毒 (BmNPV-CP--Chi-) 杆粒在五龄蚕幼虫血淋巴中表达 ST6Gal1。表达的 ST6Gal1 被纯化、表征并用于脱唾液酸糖多肽的唾液酸化。我们测试了合成的α2,6-唾液酸糖多肽对黑接骨木（SNA）凝集素的血凝抑制作用。 FLAG标签的重组ST6Gal1被高效表达，并通过硫酸铵沉淀、抗FLAG M2柱亲和层析纯化，仅从11只蚕幼虫中产生2.2 mg纯化的融合蛋白，回收率为64%。对纯化的ST6Gal1进行了表征，通过HPLC作图方法发现其N-聚糖模式近似为少甘露糖苷型。通过化学酶法合成含有丹酰基的荧光标记 N-乙酰基乳糖胺 (LacNAc) 糖苷，作为 ST6Gal1 的高灵敏度受体底物。该酶的受体底物特异性与大鼠肝脏 ST6Gal1 相似。荧光糖苷可用作 ST6Gal1 高灵敏度皮摩尔测定的底物。利用ST6Gal1，通过末端Gal残基的催化反应对去唾液酸糖多肽进行区域选择性和定量唾液酸化，得到α2,6-唾液酸糖多肽。 α2,6-唾液酸糖多肽选择性抑制黑接骨木 (SNA) 凝集素诱导的血凝，显示出比对照胎球蛋白高约 780 倍的亲和力。去唾液酸糖多肽和γ-聚谷氨酸不影响SNA凝集素介导的血凝。来自蚕表达系统的重组 ST6Gal1 可用于脱唾液酸糖肽的唾液酸化。唾液酸化糖蛋白是研究生物和生理事件分子机制的重要工具，例如感染期间的细胞间识别和病毒进入。
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. 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. 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.
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影响因子：
2.7
作者：
Sun, Mingchi;Li, Yanhong;Chen, Xi
通讯作者：
Chen, Xi
DOI：
10.1038/nbt0291-165
发表时间：
1991-02-01
期刊：
BIO-TECHNOLOGY
影响因子：
--
作者：
DAVIS, GT;BEDZYK, WD;JACOBS, TW
通讯作者：
JACOBS, TW
影响因子：
3.1
作者：
Hiyoshi, Masato;Kageshima, Ayano;Park, Enoch Y.
通讯作者：
Park, Enoch Y.
影响因子：
3.5
作者：
Ogata, Makoto;Murata, Takeomi;Usui, Taichi
通讯作者：
Usui, Taichi
影响因子：
4.1
作者：
Park, Enoch Y.;Ishikiriyama, Motoki;Ueda, Hiroshi
通讯作者：
Ueda, Hiroshi