Laminin-1 is a novel carrier glycoprotein for the nonsulfated HNK-1 epitope in mouse kidney
Laminin-1 is a novel carrier glycoprotein for the nonsulfated HNK-1 epitope in mouse kidney
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DOI：
10.1093/glycob/cwn012
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发表时间：
2008-04-01
期刊：
影响因子：
4.3
通讯作者：
Oka, Shogo
中科院分区：
文献类型：
--
作者：
Kizuka, Yasuhiko;Kobayashi, Kyoko;Oka, Shogo
HNK-1表位具有独特的结构，包括硫酸化三糖（HSO 3 -3GlcA β 1-3Gal β 1-4GlcNAc），并且两种葡萄糖醛酸转移酶（GlcAT-P和GlcAT-S）是其生物合成的关键酶。然而，这些酶在其生物合成中的不同功能作用仍不清楚。最近，我们报道了这种表位的非硫酸化形式，它是由GlcAT-S生物合成的，但不是由GlcAT-P，在小鼠肾脏中的两种金属蛋白酶上表达。在这项研究中，我们发现一种新的糖蛋白携带非硫酸化HNK-1表位在小鼠肾脏中富集在核馏分。该蛋白经亲和纯化并鉴定为层粘连蛋白-1，我们还通过质谱法证实了N-连接的寡糖结构，包括来自层粘连蛋白-1的非硫酸化HNK-1表位。奇怪的是，肾切片的免疫荧光染色显示层粘连蛋白-1似乎不与非硫酸化HNK-1表位共定位。然而，蛋白酶处理加强了层粘连蛋白-1和非硫酸化HNK-1表位的信号，导致它们重叠。这些结果表明，层粘连蛋白-1上的非硫酸化HNK-1表位通常嵌入并掩蔽在坚固的基底膜中，与其他蛋白质紧密结合。为了阐明相关蛋白和碳水化合物表位的功能作用，我们研究了层粘连蛋白-1和α-肌营养不良蛋白聚糖之间的相互作用，通过它们的聚糖在小鼠肾脏中使用的覆盖测定技术。我们获得的证据表明，葡萄糖醛酸以及唾液酸抑制这种相互作用，这表明层粘连蛋白-1上的非硫酸化HNK-1表位可以调节其结合，并在维持肾脏基底层的适当结构中发挥作用。
The HNK-1 epitope has a unique structure comprising the sulfated trisaccharide (HSO3-3GlcA beta 1-3Gal beta 1-4GlcNAc), and two glucuronyltransferases (GlcAT-P and GlcAT-S) are key enzymes for its biosynthesis. However, the different functional roles of these enzymes in its biosynthesis remain unclear. Recently, we reported that a nonsulfated form of this epitope, which is biosynthesized by GlcAT-S but not by GlcAT-P, is expressed on two metalloproteases in mouse kidney. In this study, we found that a novel glycoprotein carrying the nonsulfated HNK-1 epitope in mouse kidney was enriched in the nuclear fraction. The protein was affinity-purified and identified as laminin-1, and we also confirmed the N-linked oligosaccharide structure including nonsulfated HNK-1 epitope derived from laminin-1 by mass spectrometry. Curiously, immunofluorescence staining of kidney sections revealed that laminin-1 appeared not to be colocalized with the nonsulfated HNK-1 epitope. However, proteinase treatment strengthened the signals of both laminin-1 and the nonsulfated HNK-1 epitope, resulting in overlapping of them. These results indicate that the nonsulfated HNK-1 epitope on laminin-1 is usually embedded and masked in the robust basement membrane in tight association with other proteins. To clarify the associated proteins and the functional role of the carbohydrate epitope, we investigated the interaction between laminin-1 and alpha-dystroglycan through their glycans in mouse kidney using the overlay assay technique. We obtained evidence that glucuronic acid as well as sialic acid inhibited this interaction, suggesting that the nonsulfated HNK-1 epitope on laminin-1 may regulate its binding and play a role in maintenance of the proper structure in the kidney basal lamina.