Interaction of human macrophage C-type lectin with O-linked N-acetylgalactosamine residues on mucin glycopeptides
Interaction of human macrophage C-type lectin with O-linked N-acetylgalactosamine residues on mucin glycopeptides
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DOI：
10.1074/jbc.274.16.10697
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发表时间：
1999-04-16
影响因子：
4.8
通讯作者：
Irimura, T
中科院分区：
文献类型：
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作者：
Iida, SI;Yamamoto, K;Irimura, T
荧光素标记的合成肽PTTTPITTTT-TK，通过与UDP-Gal-NAc和LS 174 T人结肠癌细胞微粒体组分的体外糖基化，转化为具有不同数目的N-乙酰基-D-半乳糖胺（GalNAc）的O-糖基化糖肽，具有1、3、5和6个GalNAc残基的糖肽（G1、G3、G5和G6），并通过基质辅助激光解吸电离飞行时间质谱法确认它们的大小。通过肽测序仪确定它们的序列，对于G1为PTTTGalNAcPITTTTK，对于G3为PTGalNAcTTPITGalNAc TGalNAcTTK，G5的PTTGalNAcTGalNAcPITGalNAcTGalNAc- TGalNAcTK和PTGalNAcTGalNAcTGalNAcPITGalNAc。制备了重组钙型人巨噬细胞凝集素（HML），并利用表面等离子体共振（SPR）和荧光偏振法研究了HML与这些糖肽的相互作用。重组HML（rHML）固定化糖肽的亲和力增加，所揭示的SPR，在平行的GalNAc的数量。当G6-肽以高密度固定时，获得最高的亲和力。荧光偏振平衡结合测定还显示，rHML对可溶性糖肽的亲和力增加，这取决于附着的GalNAc的数量。制备了HML的糖识别结构域（CRD）片段，测定了它们对这四种糖肽的亲和力，其亲和力明显低于rHML。rHML对G3-和G5-肽的亲和力常数分别是G1-肽的11倍和38倍，而CRD片段的亲和力常数仅分别是G1-肽的2倍和6倍。化学交联研究表明，rHML而不是重组CRD在水溶液中形成三聚体。因此，密集糖基化的O-连接的糖肽的优先结合应该是由于rHML的三聚体形成。
A fluorescein-labeled synthetic peptide, PTTTPITTTT-TK, was converted into O-glycosylated glycopeptides with various numbers of attached N-acetyl-D-galactosamines (GalNAcs) by in vitro glycosylation with UDP-Gal-NAc and a microsomal fraction of LS174T human colon carcinoma cells, Glycopeptides with 1, 3, 5, and 6 GalNAc residues (G1, G3, G5, and G6) were obtained, and their sizes were confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, Their sequences were determined by a peptide sequencer to be PTTTGalNAcPITTTTK for G1, PTGalNAcTTPITGalNAc TGalNAcTTK for G3, PTTGalNAcTGalNAcPITGalNAcTGalNAc- TGalNAcTK for G5, and PTGalNAcTGalNAcTGalNAcPITGalNAc. TGalNAcTGalNAcTK for G6, A calcium-type human macrophage lectin (HML) was prepared in a recombinant form, and its interaction with these glycopeptides was investigated by surface plasmon resonance (SPR) spectroscopy and fluorescence polarization. The affinity of recombinant HML (rHML) for immobilized glycopeptides increased, as revealed by SPR, in parallel with the number of GalNAc. The highest affinity was obtained when the G6-peptide was immobilized at high density, Fluorescence polarization equilibrium-binding assays also revealed that the affinity of rHML for soluble glycopeptides increased, depending on the number of attached GalNAcs. Carbohydrate recognition domain (CRD) fragments of HML were prepared, and their affinity for these four glycopeptides was also determined, this affinity was apparently lower than that of rHML, Affinity constants of rHML for the G3- and G5-peptides were 11- and 38-fold higher, respectively, than for the G1-peptide, whereas those of CRD fragments were only 2- and 6-fold higher, respectively. A chemical cross-linking study revealed that rHML but not recombinant CRD forms trimers in an aqueous solution. Thus, preferential binding of densely glycosylated O-linked glycopeptides should be due to the trimer formation of rHML.