SULFATE CONTRIBUTES TO THE NEGATIVE CHARGE OF PODOCALYXIN, THE MAJOR SIALOGLYCOPROTEIN OF THE GLOMERULAR-FILTRATION SLITS
SULFATE CONTRIBUTES TO THE NEGATIVE CHARGE OF PODOCALYXIN, THE MAJOR SIALOGLYCOPROTEIN OF THE GLOMERULAR-FILTRATION SLITS
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DOI：
10.1073/pnas.88.12.5398
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发表时间：
1991-06-01
影响因子：
11.1
通讯作者：
FARQUHAR, MG
中科院分区：
文献类型：
--
作者：
DEKAN, G;GABEL, C;FARQUHAR, MG
足糖萼蛋白是大鼠肾小球的主要唾液蛋白。其功能是凭借其高净负电荷维持肾小球上皮的过滤缝隙打开。我们已经使用生物合成标记和寡糖分析来表征该蛋白质上的阴离子电荷携带部分。用[S-35]CyS、[H-3]Man、[H-3]GlcN和（SO 4）-S-35生物合成标记来自2日龄大鼠的肾切片，之后免疫沉淀并通过SDS/PAGE纯化足糖萼蛋白。所有这些标记都被并入足糖萼蛋白中。免疫沉淀物用特异性糖苷酶消化或用链霉蛋白酶消化，然后对释放的糖肽进行色谱分析。足糖萼蛋白对N-聚糖酶和O-聚糖酶的消化敏感，表明存在N-和O-连接的寡糖。约30%的[H-3] GlcN-标记的糖肽结合到Con A，确认存在高甘露糖，杂合，或双触角N-连接的结构;碱性硼氢化物处理证实存在O-连接的寡糖。对（SO 4）-S-35标记的糖肽的分析表明，N-和O-连接结构均被硫酸化。我们的结论是，在新生大鼠肾脏中（i）足糖萼蛋白含有O-和N-连接的寡糖[高甘露糖或混合型，双触角，和复杂的（唾液酸化）型]，（ii）足糖萼蛋白是硫酸化的，和（iii）硫酸位于O-连接的寡糖和携带三或四个N-连接结构的糖肽。这些结果表明足糖萼蛋白的净负电荷最有可能来源于硫酸盐以及唾液酸残基。
Podocalyxin is the major sialoprotein of the rat glomerulus. Its function is to maintain the filtration slits of the glomerular epithelium open by virtue of its high net negative charge. We have used biosynthetic labeling and oligosaccharide analysis to characterize the anionic-charge-carrying moieties on this protein. Kidney slices from 2-day-old rats were biosynthetically labeled with [S-35]CyS, [H-3]Man, [H-3]GlcN, and (SO4)-S-35, after which podocalyxin was immunoprecipitated and purified bv SDS/PAGE. All these labels were incorporated into podocalyxin. Immunoprecipitates were subjected to digestion with specific glycosidases or digested with Pronase followed by chromatographic analysis of the released glycopeptides. Podocalyxin was susceptible to digestion with N-Glycanase and O-Glycanase, indicating the presence of both N- and O-linked oligosaccharides. Approximately 30% of the [H-3]GlcN-labeled glycopeptides bound to Con A, confirming the presence of high mannose, hybrid, or biantennary N-linked structures; alkaline borohydride treatment confirmed the presence of O-linked oligosaccharides. Analysis of the (SO4)-S-35-labeled glycopeptides indicated that both the N- and O-linked structures were sulfated. We conclude that in newborn rat kidney (i) podocalyxin contains both O- and N-linked oligosaccharides [high mannose or hybrid type, biantennary, and complex (sialylated) type], (ii) podocalyxin is sulfated, and (iii) sulfate is located on both O-linked oligosaccharides and on glycopeptides carrying tri- or tetrantennary N-linked structures. These results indicate that the net negative charge of podocalyxin is most likely derived from sulfate as well as from sialic acid residues.