Trask B C, Broekelmann T, Ritty T M, Trask T M, Tisdale C, Mecham R P
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Biochemistry. 2001 Apr 10;40(14):4372-80. doi: 10.1021/bi002738z.
Microfibril-associated glycoprotein-1 (MAGP-1) is a small molecular weight protein associated with extracellular matrix microfibrils. Biochemical studies have shown that MAGP-1 undergoes several posttranslational modifications that may influence its associations with other microfibrillar components. To identify the sites in the molecule where posttranslational modifications occur, we expressed MAGP-1 constructs containing various point mutations as well as front and back half truncations in CHO cells. Characterization of transiently expressed protein showed that MAGP-1 undergoes O-linked glycosylation and tyrosine sulfation at sites in its amino-terminal half. This region of the protein also served as a major amine acceptor site for transglutaminase and mediated self-assembly into high molecular weight multimers through a glutamine-rich sequence. Fine mapping of the modification sites through mutational analysis demonstrated that Gln20 is a major amine acceptor site for the transglutaminase reaction and confirmed that a canonical tyrosine sulfation consensus sequence is the site of MAGP-1 sulfation. Our results also show that O-glycosylation occurs at more than one site in the molecule.
微原纤维相关糖蛋白-1（MAGP-1）是一种与细胞外基质微原纤维相关的小分子量蛋白质。生化研究表明，MAGP-1经历了几种可能影响其与其他微原纤维成分结合的翻译后修饰。为了确定分子中发生翻译后修饰的位点，我们在CHO细胞中表达了包含各种点突变以及前后半段截短的MAGP-1构建体。对瞬时表达蛋白的表征表明，MAGP-1在其氨基末端一半的位点进行O-连接糖基化和酪氨酸硫酸化。该蛋白区域也是转谷氨酰胺酶的主要胺受体位点，并通过富含谷氨酰胺的序列介导自组装成高分子量多聚体。通过突变分析对修饰位点进行精细定位表明，Gln20是转谷氨酰胺酶反应的主要胺受体位点，并证实了一个典型的酪氨酸硫酸化共有序列是MAGP-1硫酸化的位点。我们的结果还表明，O-糖基化发生在分子中的多个位点。
