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首页> 外文期刊>Biochemistry >The Structural Role of N-Linked Glycans on Human Glypican-1
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The Structural Role of N-Linked Glycans on Human Glypican-1

机译：N-连接聚糖对人Glypican-1的结构作用

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摘要

Glypicans are cell-surface heparan sulfate proteoglycans that regulate developmental signaling pathways by binding growth factors to their heparan sulfate chains. The primary structures of glypican core proteins contain potential N-glycosylation sites, but the importance of N-glycosylation in glypicans has never been investigated in detail. Here, we studied the role of the possible N-glycosylation sites at Asn-79 and Asn-116 in recombinant anchorless glypican-1 expressed in eukaryotic cells. Mutagenesis and enzymatic cleavage indicated that the potential N-glycosylation sites are invariably occupied. Experiments using the drug tunicamycin to inhibit the N-linked glycosylation of glypican-1 showed that secretion of anchorless glypican-1 was reduced and that the protein did not accumulate inside the cells. Heparan sulfate substitution of N-glycosylation mutant N116Q was similar to wild-type glypican-1 while the N79Q mutant and also the double mutant N79QN116Q were mostly secreted as high-molecular-weight heparan sulfate proteoglycan. N-Glycosylation mutants and N-deglycosylated glypican-1 had far-UV circular dichroism and fluorescence emission spectra that were highly similar to those of N-glycosylated glypican-1. A single unfolding transition at high concentrations of urea was found for both N-deglycosylated glypican-1 and glypican-1 in which the N-glycosylation sites had been removed by mutagenesis when chemical denaturation was monitored by circular dichroism and fluorescence emission spectroscopy. In summary, we have found that the potential N-glycosylation sites in glypican-1 are invariably occupied and that the N-linked glycans on glypican-1 affect protein expression and heparan sulfate substitution but that correct folding can be obtained in the absence of N-linked glycans.
机译：Glypicans是细胞表面硫酸乙酰肝素蛋白聚糖，通过将生长因子与其硫酸乙酰肝素链结合来调节发育信号通路。 Glypican核心蛋白的一级结构包含潜在的N-糖基化位点，但从未详细研究N-糖基化在Glypican中的重要性。在这里，我们研究了在真核细胞中表达的重组无锚glypican-1中Asn-79和Asn-116可能的N-糖基化位点的作用。诱变和酶促切割表明潜在的N-糖基化位点始终被占据。使用药物衣霉素抑制Glypican-1的N联糖基化的实验表明，无锚Glypican-1的分泌减少了，并且该蛋白没有在细胞内积聚。 N-糖基化突变体N116Q的硫酸乙酰肝素取代与野生型Glypican-1类似，而N79Q突变体和双重突变体N79QN116Q大多被分泌为高分子量硫酸乙酰肝素蛋白聚糖。 N-糖基化突变体和N-去糖基化的Glypican-1具有远紫外圆二色性和荧光发射光谱，与N-糖基化的Glypican-1高度相似。 N-去糖基化的Glypican-1和Glypican-1均在高浓度的尿素中出现了一个单一的展开转变，当通过圆二色性和荧光发射光谱法监测化学变性时，其中的N-糖基化位点已通过诱变去除。总之，我们发现glypican-1中潜在的N-糖基化位点总是被占据，并且glypican-1上的N-连接的聚糖影响蛋白质表达和硫酸乙酰肝素取代，但是在不存在N的情况下可以获得正确的折叠链聚糖。

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