Neu5Acα2-3Galβ1-3GalNAc 三糖结构特异性猪 α2,3-Gal-β1,3-GalNAc-α2,6-唾液酸转移酶 (pST6GalNAc IV) 基因的分子表征,Glycoconjugate Journal

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Neu5Acα2-3Galβ1-3GalNAc 三糖结构特异性猪 α2,3-Gal-β1,3-GalNAc-α2,6-唾液酸转移酶 (pST6GalNAc IV) 基因的分子表征
Glycoconjugate Journal ( IF 2.7 ) Pub Date : 2010-03-22 , DOI: 10.1007/s10719-010-9284-3
Hyun-Kwon Ko ₁ , Kwon-Ho Song , Un-Ho Jin , Hwan-Hoo Seong , Young-Chae Chang , Nam-Hyung Kim , Dong-Soo Kim , Young-Choon Lee , Cheorl-Ho Kim

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糖缀合物的唾液酸在各种生物过程中起着至关重要的作用，例如细胞-细胞通讯和细胞-底物相互作用。一种水杨酸转移酶 ST6GalNAc IV（Neu5Ac-α2,3-Gal-β1,3-GalNAc-α2,6-唾液酸转移酶）催化 α2-6-连接到蛋白质的 O-糖苷连接的 Ser/Thr 的 GalNAc 残基上的形成。在这项研究中，我们克隆了猪 ST6GalNAc IV (pST6GalNAc IV) 并研究了其功能表征。pST6GalNAc IV cDNA 已从猪肝组织中分离出来，它包含一个完整的开放阅读框（ORF，906 bp），编码 302 个氨基酸残基。pST6GalNAc IV 的整个 ORF 包含唾液酸基序 'L'-（大）、'S'-（小）和 '-VS'（非常小）与智人(90%)、Pan troglodytes具有高度的序列相似性(91%) 和Mus musculus (87%)。pST6GalNAc IV mRNA 在各种猪组织中的表达通过逆转录聚合酶链反应 (RT-PCR) 分析进行鉴定。pST6GalNAc IV mRNA 在舌、肌肉和心脏中高度表达，而在胰腺中不表达。为了对猪肾 PK15 细胞中 pST6GalNAc IV 基因的功能表征，我们还建立了 pST6GalNAc IV 转染的 PK15 细胞，该细胞稳定表达 pST6GalNAc IV 基因。通过流式细胞术并用免疫荧光分析检测pST6GalNAc IV转染的PK15细胞的糖基化模式山槐凝集素（MAA），山槐血凝素（MAL II），西洋接骨木凝集素（SNA）和花生凝集素（PNA）凝集素。被MAL-II和SNA识别的Neu5Acα2-3Galβ1-3(Neu5Acα2-6)GalNAc四糖或Neu5Acα2-6GalNAc二糖的特定碳水化合物结构被揭示是由pST6GalNAc IV新合成的。结果表明，猪pST6GalNAc IV基因能够在O-糖蛋白上合成Neu5Acα2-3Galβ1-3(Neu5Acα2-6)GalNAc四糖结构。

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Molecular characterization of pig α2,3-Gal-β1,3-GalNAc-α2,6-sialyltransferase (pST6GalNAc IV) gene specific for Neu5Acα2-3Galβ1-3GalNAc trisaccharide structure

Sialic acids of glycoconjugates play crucial roles in various biological processes, such as cell-cell communication and cell-substrate interaction. A sisalyltransferase, ST6GalNAc IV (Neu5Ac-α2,3-Gal-β1,3-GalNAc-α2,6-sialyltransferase), catalyzes the formation of α2-6-linkages onto GalNAc residues of O-glycosidically linked Ser/Thr of proteins. In this study, we cloned the pig ST6GalNAc IV (pST6GalNAc IV) and investigated its functional characterization. pST6GalNAc IV cDNA has been isolated from pig liver tissues and it contains an entire open reading frame (ORF, 906 bp) coding for 302 amino acid residues. Entire ORF of pST6GalNAc IV containing sialylmotif ‘L’—(Large), ‘S’—(Small) and ‘—VS’ (Very small) has a high degree of sequence similarity with Homo sapiens (90%), Pan troglodytes (91%) and Mus musculus (87%). Expression of pST6GalNAc IV mRNA in various pig tissues was identified by reverse transcription polymerase chain reaction (RT-PCR) analysis. pST6GalNAc IV mRNA was highly expressed in tongue, muscle and heart, whereas it was not expressed in pancreas. For functional characterization of pST6GalNAc IV gene in pig kidney PK15 cells, we have also established pST6GalNAc IV-transfected PK15 cells, which are stably expressing the pST6GalNAc IV gene. The glycosylation pattern of pST6GalNAc IV-transfected PK15 cells was detected by flow cytometry and immunofluorescence analysis with Maackia amurensis agglutinin (MAA), Maackia amurensis hemagglutinin (MAL II), Sambucus nigra agglutinin (SNA) and peanut agglutinin (PNA) lectins. The specific carbohydrate structures of Neu5Acα2-3Galβ1-3(Neu5Acα2-6)GalNAc tetrasaccharide or Neu5Acα2-6GalNAc disaccharide recognized by MAL-II and SNA were revealed to be newly synthesized by pST6GalNAc IV. From the results, it was suggested that the pig pST6GalNAc IV gene is capable of synthesizing Neu5Acα2-3Galβ1-3(Neu5Acα2-6)GalNAc tetrasaccharide structures on O-glycoproteins.

更新日期：2010-03-22

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