Novel poly-GalNAc β1-4GlcNAc (LacdiNAc) and fucosylated poly-LacdiNAc N-glycans from mammalian cells expressing β1,4-N-acetylgalactosaminyltransferase and α1,3-fucosyltransferase
Novel poly-GalNAc β1-4GlcNAc (LacdiNAc) and fucosylated poly-LacdiNAc N-glycans from mammalian cells expressing β1,4-N-acetylgalactosaminyltransferase and α1,3-fucosyltransferase
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DOI：
10.1074/jbc.m414273200
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发表时间：
2005-04-01
影响因子：
4.8
通讯作者：
Cummings, RD
中科院分区：
文献类型：
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作者：
Kawar, ZS;Haslam, SM;Cummings, RD
含有 GalNAc beta 1-4GlcNAc（LacdiNAc 或 LDN）基序的聚糖由许多无脊椎动物表达，但该基序也出现在脊椎动物中，并且在几种哺乳动物糖蛋白激素中发现。该基序与更常见的 Gal beta 1-4GlcNAc（LacNAc 或 LN）基序形成对比。为了更好地了解LDN的生物合成和调控，我们在中国仓鼠卵巢（CHO）Lec8细胞中稳定表达编码秀丽隐杆线虫β1,4-N-乙酰半乳糖胺基转移酶（GalNAcT）的cDNA，以建立L8-GalNAcT CHO细胞，该酶在体外产生LDN。通过质谱和连锁分析确定这些细胞的聚糖结构。 L8-GalNAcT 细胞系产生复合型 N-聚糖，其触角上定量地带有 LDN 结构。出乎意料的是，大多数这些复合型N-聚糖含有由重复LDN基序(-3GalNAc beta 1-4GlcNAc beta 1-)(n)组成的新型“poly-LDN”结构。这些新颖的结构与众所周知的由重复 LN 基序 (-3Gal beta 1-4GlcNAc beta 1-)(n) 组成的聚 LN 结构形成对比。我们还在L8-GalNAcT细胞中稳定表达人α1,3-岩藻糖基转移酶IX，建立了新的细胞系L8-GalNAcT-FucT。这些细胞产生具有 α 1,3-岩藻糖基化 LDN (LDNF) GalNAc beta 1-4(Fuc beta 1-3) GlcNAc beta 1-R 的复合型 N-聚糖以及新型“聚-LDNF”结构 (-3GalNAc beta 1-4(Fuc beta 1-3)GlcNAc beta 1-)(n)。通过在 L8-GalNAcT 细胞中表达重组形式的常见 α 亚基，研究了这些细胞系利用含有 LDN 的 N-聚糖产生糖蛋白激素的能力。 α亚基N-聚糖携带LDN结构，通过人GalNAc 4-磺基转移酶I的共表达进一步修饰，生成SO4-4GalNAc beta 1-4GlcNAc-R。因此，这些稳定的哺乳动物细胞的产生将有助于未来对糖蛋白中LDN相关结构的生物活性和特性的研究。
Glycans containing the GalNAc beta 1-4GlcNAc (LacdiNAc or LDN) motif are expressed by many invertebrates, but this motif also occurs in vertebrates and is found on several mammalian glycoprotein hormones. This motif contrasts with the more commonly occurring Gal beta 1-4GlcNAc (LacNAc or LN) motif. To better understand LDN biosynthesis and regulation, we stably expressed the cDNA encoding the Caenorhabditis elegans beta 1,4-N-acetylgalactosaminyltransferase (GalNAcT), which generates LDN in vitro, in Chinese hamster ovary (CHO) Lec8 cells, to establish L8-GalNAcT CHO cells. The glycan structures from these cells were determined by mass spectrometry and linkage analysis. The L8-GalNAcT cell line produces complex-type N-glycans quantitatively bearing LDN structures on their antennae. Unexpectedly, most of these complex-type N-glycans contain novel "poly-LDN" structures consisting of repeating LDN motifs (-3GalNAc beta 1-4GlcNAc beta 1-)(n). These novel structures are in contrast to the well known poly-LN structures consisting of repeating LN motifs (-3Gal beta 1-4GlcNAc beta 1-)(n). We also stably expressed human alpha 1,3-fucosyltransferase IX in the L8-GalNAcT cells to establish a new cell line, L8-GalNAcT-FucT. These cells produce complex-type N-glycans with alpha 1,3-fucosylated LDN (LDNF) GalNAc beta 1-4(Fuc beta 1-3) GlcNAc beta 1-R as well as novel "poly-LDNF" structures (-3GalNAc beta 1-4(Fuc beta 1-3)GlcNAc beta 1-)(n). The ability of these cell lines to generate glycoprotein hormones with LDN-containing N-glycans was studied by expressing a recombinant form of the common alpha-subunit in L8-GalNAcT cells. The alpha-subunit N-glycans carried LDN structures, which were further modified by co-expression of the human GalNAc 4-sulfotransferase I, which generates SO4-4GalNAc beta 1-4GlcNAc- R. Thus, the generation of these stable mammalian cells will facilitate future studies on the biological activities and properties of LDN-related structures in glycoproteins.