Koles Kate, van Berkel Patrick H C, Pieper Frank R, Nuijens Jan H, Mannesse Maurice L M, Vliegenthart Johannes F G, Kamerling Johannis P
Bijvoet Center for Biomolecular Research, Department of Bio-Organic Chemistry, Section of Glycoscience and Biocatalysis, Utrecht University, Padualaan 8, NL-3584 CH Utrecht, The Netherlands.
Glycobiology. 2004 Jan;14(1):51-64. doi: 10.1093/glycob/cwh010. Epub 2003 Sep 26.
Human C1 inhibitor (hC1INH) is a therapeutic N, O-glycoprotein with a growing number of clinical applications, but the current natural supplies are not likely to meet the clinical demands. Therefore, recombinant approaches are of interest, whereby specific attention has to be paid to the generated glycosylation patterns. Here, the N,O-glycoprotein was expressed in the mammary gland of transgenic rabbits and subjected to glycan analysis. After release of the N-glycans of recombinant-rabbit human C1 inhibitor (rhC1INH) by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F, the oligosaccharides were separated from the O-glycoprotein by centrifugal filtration, then fractionated by a combination of anion-exchange, normal-phase, and high-pH anion-exchange liquid chromatography. The O-glycans, released from the O-glycoprotein by alkaline borohydride treatment, were fractionated by anion-exchange high-performance liquid chromatography (HPLC). The structures of individual components were analysed by 500 MHz 1H NMR spectroscopy, in most cases combined with MALDI-TOF MS. In contrast to the structural data reported for native serum hC1INH, rhC1INH contained a broad array of different N-glycans, made up of oligomannose-, hybrid-, and complex-type structures. In the case of complex-type N-glycans (partially) (alpha2-6)-sialylated (N-acetylneuraminic acid only), mono- and diantennary chains were found; part of the diantennary structures were (alpha1-6)-core-fucosylated or (alpha1-3)-fucosylated in the lower or upper antenna (Lewis x). The manno-oligosaccharide pattern of part of the hybrid- and oligomannose-type structures indicates that besides the usual N-glycan processing route, also the alternative endo-mannosidase pathway is followed. The small core 1-type O-glycans showed the usual (alpha2-3)- and (alpha2-6)-sialylation pattern of O-glycoproteins of nonmucinous origin.
人C1抑制剂（hC1INH）是一种治疗性N、O-糖蛋白，临床应用日益增多，但目前的天然供应不太可能满足临床需求。因此，重组方法备受关注，其中必须特别注意产生的糖基化模式。在此，该N、O-糖蛋白在转基因兔的乳腺中表达并进行聚糖分析。用肽-N4-（N-乙酰-β-氨基葡萄糖基）天冬酰胺酶F释放重组兔人C1抑制剂（rhC1INH）的N-聚糖后，通过离心过滤将寡糖与O-糖蛋白分离，然后通过阴离子交换、正相和高pH阴离子交换液相色谱联用进行分级分离。通过碱性硼氢化物处理从O-糖蛋白释放的O-聚糖，通过阴离子交换高效液相色谱（HPLC）进行分级分离。大多数情况下，结合基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS），通过500 MHz 1H核磁共振光谱分析各个组分的结构。与天然血清hC1INH报道的结构数据相比，rhC1INH含有多种不同的N-聚糖，由低聚甘露糖型、杂合型和复合型结构组成。在复合型N-聚糖（部分）（α2-6）-唾液酸化（仅N-乙酰神经氨酸）的情况下，发现了单天线和双天线链；部分双天线结构在下部或上部天线（Lewis x）中（α1-6）-核心岩藻糖基化或（α1-3）-岩藻糖基化。部分杂合型和低聚甘露糖型结构的甘露寡糖模式表明，除了通常的N-聚糖加工途径外，还遵循了替代的内切甘露糖苷酶途径。小核心1型O-聚糖显示了非黏液来源O-糖蛋白通常的（α2-3）-和（α2-6）-唾液酸化模式。
