Chromatofocusing fails to separate hFSH Isoforms on the basis of glycan structure
Chromatofocusing fails to separate hFSH Isoforms on the basis of glycan structure
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DOI：
10.1021/bi701764w
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发表时间：
2008-02-12
期刊：
影响因子：
2.9
通讯作者：
Desaire, Heather
中科院分区：
文献类型：
--
作者：
Bousfield, George R.;Butnev, Vladimir Y.;Desaire, Heather
促卵泡激素（FSH）糖基化受性腺反馈调节，产生一系列与垂体或尿液提取物池中FSH制剂相关的聚糖。FSH糖基化由于FSH β N-糖基化的抑制、成熟N-聚糖所具有的1-4个分支的加工以及末端唾液酸残基的数量和连接而变化。为了表征FSH糖基化，通常通过色谱聚焦分离垂体提取物和各种生理液体中的FSH同工型。所得FSH同种型制剂中免疫学和生物学活性比率的变化通常归因于糖基化的变化，糖基化最常根据唾液酸含量来定义。使用蛋白质印迹法评估人FSH β糖基化抑制，发现30-47%的非糖基化hFSH β与通过色谱聚焦衍生的6种hFSH亚型制剂中的4种相关。这些亚型中聚糖分支的糖肽质谱评估广泛表征了两个N-糖基化位点，一个位于FSH功能的关键聚糖α Asn（52），另一个位于β Asn（24）。每个FSH分子有2 - 4个N-聚糖，分布在这些位点上的电荷的许多组合可以提供相同的等电点。实际上，几种聚糖对于所分析的所有同种型级分是共同的。没有趋势显示主要是单触角聚糖与高pI组分相关，也没有主要是三触角和四触角聚糖与低pI组分相关。因此，受体结合活性的差异可能与激素中的任何特定聚糖类型或位置无关。FSH聚集与受体结合活性降低有关，但不影响免疫活性。然而，由于凝胶过滤表明每个亚型制备物中存在足够的异二聚体以生成完整的抑制曲线，因此几种制备物中受体结合活性的几乎完全丧失不能仅通过聚集来解释，并且机制仍然未知。
Follicle-stimulating hormone (FSH) glycosylation is regulated by feedback from the gonads, resulting in an array of glycans associated with FSH preparations derived from pools of pituitary or urine extracts. FSH glycosylation varies due to inhibition of FSH beta N-glycosylation, elaboration of 1-4 branches possessed by mature N-glycans, and the number and linkage of terminal sialic acid residues. To characterize FSH glycosylation, FSH isoforms in pituitary gland extracts and a variety of physiological fluids are commonly separated by chromatofocusing. Variations in the ratios of immunological and biological activities in the resulting FSH isoform preparations are generally attributed to changes in glycosylation, which are most often defined in terms of sialic acid content. Using Western blotting to assess human FSH beta glycosylation inhibition revealed 30-47% nonglycosylated hFSH beta associated with four of six hFSH isoform preparations derived by chromatofocusing. Glycopeptide mass spectrometry assessment of glycan branching in these isoforms extensively characterized two N-glycosylation sites, one at alpha Asn(52), the critical glycan for FSH function, and the other at beta Asn(24). With two to four N-glycans per FSH molecule, many combinations of charges distributed over these sites can provide the same isoelectric point. Indeed, several glycans were common to all isoform fractions that were analyzed. There was no trend showing predominantly monoantennary glycans associated with the high-pI fractions, nor were predominantly tri- and tetra-antennary glycans associated with low-pI fractions. Thus, differences in receptor binding activity could not be associated with any specific glycan type or location in the hormone. FSH aggregation was associated with reduced receptor binding activity but did not affect immunological activity. However, as gel filtration indicated sufficient heterodimer was present in each isoform preparation to generate complete inhibition curves, the near total loss of receptor binding activity in several preparations could not be explained by aggregation alone, and the mechanism remains unknown.