Characterization of the N-linked oligosaccharides from human chorionic gonadotropin expressed in the methylotrophic yeast Pichia pastoris
Characterization of the N-linked oligosaccharides from human chorionic gonadotropin expressed in the methylotrophic yeast Pichia pastoris
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DOI：
10.1007/s10719-006-9010-3
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发表时间：
2007-01-01
影响因子：
3
通讯作者：
Kamerling, Johannis P.
中科院分区：
文献类型：
--
作者：
Blanchard, Veronique;Gadkari, Rupali A.;Kamerling, Johannis P.
人绒毛膜促性腺激素（hCG）是一种异源二聚体的胎盘糖蛋白激素，在妊娠早期参与维持黄体。具有生物活性的hCG已在毕赤酵母中成功表达。在phCG的结构研究和治疗应用的背景下，关于其糖基化模式的详细信息是先决条件。为此，用肽-N（4）-（N-乙酰基-β-葡糖胺基）天冬酰胺酰胺酶F释放N-聚糖，并通过阴离子交换色谱（Resource Q）分级分离，产生中性（80%）和带电的含磷酸盐（20%）的高甘露糖型结构。通过正相（Lichrosorb-NH 2）和高pH阴离子交换（CarboPac PA-1）色谱法进行亚分级。通过使用荧光2-氨基苯甲酰胺衍生物的HPLC分析、MALDI-TOF质谱和500-MHz H-1-NMR光谱对释放的N-聚糖进行结构分析。可以确定Man（8）GlcNAc（2）至Man（11）GlcNAc（2）（包括分子异构体）范围内的详细中性寡糖结构，并指出Man（15）GlcNAc（2）的结构。含磷酸盐寡糖的范围为Man（9）PGlcNAc（2）至Man（13）PGlcNAc（2）。未检出甘露糖基O-聚糖。对不同生产批次进行的分析研究表明，寡糖结构相似，但其相对量随培养基而变化。
Human chorionic gonadotropin (hCG) is a heterodimeric, placental glycoprotein hormone involved in the maintenance of the corpus luteum during the first trimester of pregnancy. Biologically active hCG has been successfully expressed in the yeast Pichia pastoris (phCG). In the context of structural studies and therapeutic applications of phCG, detailed information about its glycosylation pattern is a prerequisite. To this end N-glycans were released with peptide-N (4)-(N-acetyl-beta-glucosaminyl)asparagine amidase F and fractionated via anion-exchange chromatography (Resource Q) yielding both neutral (80%) and charged, phosphate-containing (20%) high-mannose-type structures. Subfractionations were carried out via normal phase (Lichrosorb-NH2) and high-pH anion-exchange (CarboPac PA-1) chromatography. Structural analyses of the released N-glycans were carried out by using HPLC profiling of fluorescent 2-aminobenzamide derivatives, MALDI-TOF mass spectrometry, and 500-MHz H-1-NMR spectroscopy. Detailed neutral oligosaccharide structures, in the range of Man(8)GlcNAc(2) to Man(11)GlcNAc(2) including molecular isomers, could be established, and structures up to Man(15)GlcNAc(2) were indicated. Phosphate-containing oligosaccharides ranged from Man(9) PGlcNAc(2) to Man(13) PGlcNAc(2). Mannosyl O-glycans were not detected. Profiling studies carried out on different production batches showed that the oligosaccharide structures are similar, but their relative amounts varied with the culturing media.