Winzen Uwe, Cole Gregory J, Halfter Willi
Department of Neurobiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.
J Biol Chem. 2003 Aug 8;278(32):30106-14. doi: 10.1074/jbc.M212676200. Epub 2003 May 28.
Agrin is a large extracellular matrix protein that plays a key role in the formation and maintenance of the vertebrate neuromuscular junction. The amino acid sequence of agrin encodes a protein with a molecular size of 220 kDa, whereas SDS-PAGE shows a diffuse band around 400 kDa. Further studies showed that agrin is highly glycosylated and belongs to the family of heparan sulfate proteoglycans. By expressing different protein fragments, we localized the glycosaminoglycan (GAG) attachment sites to two locations within the agrin molecule. One site that is located between the seventh and eight follistatin-like domain includes 3 closely spaced serine-glycine (SG) consensus sequences and carries exclusively heparan sulfate side chains. The second site is located further downstream in the centrally located serine-threonine-rich domain and contains a cluster of 4 closely packed SG consensus sequences. This site predominantly carries chondroitin sulfate side chains. Investigating the contribution of individual serines in GAG priming by site-directed mutagenesis showed that each serine of the two SG clusters has the potential to carry GAGs. In accordance with the mixed GAG glycosylation of agrin peptide fragments, it was found that recombinant and in vivo-derived full-length agrin are not exclusively heparan sulfate proteoglycans but also carry chondroitin sulfate side chains.
集聚蛋白是一种大型细胞外基质蛋白，在脊椎动物神经肌肉接头的形成和维持中起关键作用。集聚蛋白的氨基酸序列编码一种分子大小为220 kDa的蛋白质，而SDS-PAGE显示在400 kDa左右有一条弥散带。进一步研究表明，集聚蛋白高度糖基化，属于硫酸乙酰肝素蛋白聚糖家族。通过表达不同的蛋白质片段，我们将糖胺聚糖（GAG）附着位点定位到集聚蛋白分子内的两个位置。一个位点位于第七和第八个卵泡抑素样结构域之间，包含3个紧密排列的丝氨酸-甘氨酸（SG）共有序列，且仅携带硫酸乙酰肝素侧链。第二个位点位于中央富含丝氨酸-苏氨酸结构域的更下游，包含一簇4个紧密排列的SG共有序列。该位点主要携带硫酸软骨素侧链。通过定点诱变研究单个丝氨酸在GAG引发中的作用表明，两个SG簇中的每个丝氨酸都有携带GAG的潜力。根据集聚蛋白肽片段的混合GAG糖基化情况，发现重组的和体内来源的全长集聚蛋白不仅是硫酸乙酰肝素蛋白聚糖，还携带硫酸软骨素侧链。
