N-GLYCOSYLATION SITE MAPPING OF HUMAN SEROTRANSFERRIN BY SERIAL LECTIN AFFINITY-CHROMATOGRAPHY, FAST-ATOM-BOMBARDMENT MASS-SPECTROMETRY, AND H-1 NUCLEAR-MAGNETIC-RESONANCE SPECTROSCOPY
N-GLYCOSYLATION SITE MAPPING OF HUMAN SEROTRANSFERRIN BY SERIAL LECTIN AFFINITY-CHROMATOGRAPHY, FAST-ATOM-BOMBARDMENT MASS-SPECTROMETRY, AND H-1 NUCLEAR-MAGNETIC-RESONANCE SPECTROSCOPY
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DOI：
10.1016/s0003-2697(05)80010-7
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发表时间：
1992-10-01
影响因子：
2.9
通讯作者：
VANHALBEEK, H
中科院分区：
文献类型：
--
作者：
FU, DT;VANHALBEEK, H
本文报道了人血清转铁蛋白（h-STF）的N-糖基化位点图谱。用胰蛋白酶或胰凝乳蛋白酶消化还原的和S-羧甲基化的h-STF。用刀豆球蛋白A（ConA）、接骨木凝集素（SNA）和菜豆白细胞凝集素（LPHA）亲和层析分离蛋白水解酶中的糖肽，并用1HNMR谱进行初步分析。然后用N-聚糖酶单独消化糖肽级分。通过快速原子轰击-质谱（FAB-MS）分析每个级分的一部分消化物，以鉴定糖基化位点的肽序列。另一部分用凝集素亲和层析法分离寡糖，用1H NMR和FAB-MS对分离的寡糖进行结构表征。结果表明，与Con A结合的寡糖具有双-α（2→6）-唾液酸双触角结构。SNA结合级分被证明含有三唾液酸，三触角结构。在h-STF的两个N-糖基化位点（Asn 413和Asn 611）上发现二触角和三触角寡糖以约85：15的比例存在。SNA结合的糖肽通过LPHA亲和层析进一步分级分离。对两种不同的寡糖进行了表征，即三唾液酸2，4-三触角聚糖和三唾液酸2，6-三触角聚糖。发现连接到糖基化位点Asn 413的2，4-三触角寡糖与2，6-三触角寡糖的比例为10.5：1，而发现两种异构的三触角寡糖以10.1：1的比例连接到糖基化位点Asnell。
This report describes the N-glycosylation site mappingof human serotransferrin (h-STF). Reduced and S-carboxymethylated h-STF was digested with trypsin or chymotrypsin. Glycopeptides in the proteolytic digests were isolated by serial concanavalin A (Con A), Sambucus nigra agglutinin (SNA), and Phaseolus vulgaris leukoagglutinin (LPHA) affinity chromatography and subjected to preliminary analysis by1H NMR spectroscopy. The glycopeptide fractions were then individually digested with N-glycanase. One part of the digest of each fraction was analyzed by fast atom bombardment-mass spectrometry (FAB-MS) to identify the peptide sequences of the glycosylation sites. The other part was used to isolate the oligosaccharide by the corresponding lectin affinity chromatography and to characterize the structures of the isolated oligosaccharides by1H NMR spectroscopy and FAB-MS. The oligosaccharides in the Con A-bound fraction were shown to have bi-α(2→6)-sialyl, diantennary structures. The SNA-bound fraction was shown to contain trisialyl, triantennary structures. Di- and triantennary oligosaccharides were found to occur on each of the two N-glycosylation sites of h-STF (Asn413and Asn611) in the ratio of ∼85:15. The SNA-bound glycopeptides were further fractionated by LPHA affinity chromatography. Two different oligosaccharides were characterized, namely, a trisialyl2,4-triantennary and a trisialyl2,6-triantennary glycan. The ratio of 2,4-triantennary vs 2,6-triantennary oligosaccharides attached to glycosylation site Asn413was found to be ∼5:1, whereas the two isomeric triantennary oligosaccharides were found to be attached to glycosylation site Asnell in the ratio ∼1:1.