Wong S Y, Guile G R, Dwek R A, Arsequell G
Department of Biochemistry, University of Oxford, U.K.
Biochem J. 1994 Jun 15;300 ( Pt 3)(Pt 3):843-50. doi: 10.1042/bj3000843.
A simple and efficient synthetic glycosylation method suitable for use in solid-phase enzymic oligosaccharide synthesis and site-specific glycosylation of recombinant proteins to produce defined glycoforms is described. This strategy utilizes N-(beta-saccharide) haloacetamides for attaching oligosaccharides specifically to cysteine residues of proteins in solution to form neoglycoproteins. The alkylation reaction was tested using N-(beta-chitotriose) bromoacetamide and an unprotected synthetic hexapeptide containing a single cysteine residue. The glycosylated product was confirmed by amino acid and hexosamine analyses as well as laser desorption mass spectrometry. Similarly N-(beta-chitotriose) iodoacetamide was covalently linked to non-reduced BSA to produce a defined glycoform of this protein. The specific attachment of chitotriose at the single cysteine residue in non-reduced serum albumin was suggested by Ellman's assay for free thiols. This was verified by amino acid sequencing of tryptic glycopeptide derived from this neoglycoprotein. Multiple sugar attachment was accomplished using fully reduced serum albumin as demonstrated by the formation of two neoglycoproteins using iodoacetamide derivatives of galactose beta 1-3-N-acetylgalactosamine (Gal beta 1-3GalNAc) and the major xylose/fucose-class plant-type oligosaccharide of horseradish peroxidase. These two neoglycoproteins with an average of 18-21 sugar residues attached were assayed positively for binding to peanut agglutinin and a sugar-specific anti-(horseradish peroxidase) monoclonal antibody YZ1/2.23 respectively. Sialylation of the neoglycoprotein containing Gal beta 1-3GalNAc was accomplished using alpha-2,3-sialyltransferase and radiolabelled CMP-N-acetylneuraminic acid. Significantly, glycan attachment using this conjugation method is reversible as demonstrated by the release of oligosaccharides from these two neoglycoproteins using hydrazinolysis. Therefore this method could provide invaluable reagents for many glycobiological studies.
本文描述了一种简单高效的合成糖基化方法，适用于固相酶促寡糖合成以及重组蛋白的位点特异性糖基化，以产生特定的糖型。该策略利用N-（β-糖基）卤代乙酰胺将寡糖特异性连接到溶液中蛋白质的半胱氨酸残基上，形成新糖蛋白。使用N-（β-壳三糖）溴乙酰胺和含有单个半胱氨酸残基的未保护合成六肽对烷基化反应进行了测试。通过氨基酸和己糖胺分析以及激光解吸质谱法确认了糖基化产物。同样，N-（β-壳三糖）碘乙酰胺与未还原的牛血清白蛋白共价连接，产生了该蛋白的特定糖型。通过埃尔曼法检测游离巯基表明，壳三糖特异性连接到未还原血清白蛋白中的单个半胱氨酸残基上。通过对源自该新糖蛋白的胰蛋白酶糖肽进行氨基酸测序验证了这一点。使用完全还原的血清白蛋白实现了多个糖基的连接，这通过使用半乳糖β1-3-N-乙酰半乳糖胺（Galβ1-3GalNAc）的碘乙酰胺衍生物和辣根过氧化物酶的主要木糖/岩藻糖类型的植物型寡糖形成两种新糖蛋白得到证明。这两种平均连接有18-21个糖残基的新糖蛋白分别被检测到能与花生凝集素和糖特异性抗（辣根过氧化物酶）单克隆抗体YZ1/2.23结合。使用α-2,3-唾液酸转移酶和放射性标记的CMP-N-乙酰神经氨酸实现了含有Galβ1-3GalNAc的新糖蛋白的唾液酸化。重要的是，通过肼解从这两种新糖蛋白中释放寡糖证明，使用这种共轭方法进行的聚糖连接是可逆的。因此，该方法可为许多糖生物学研究提供宝贵的试剂。
