SELF-ASSOCIATION OF N-SYNDECAN (SYNDECAN-3) CORE PROTEIN IS MEDIATED BY A NOVEL STRUCTURAL MOTIF IN THE TRANSMEMBRANE DOMAIN AND ECTODOMAIN PLANKING REGION
SELF-ASSOCIATION OF N-SYNDECAN (SYNDECAN-3) CORE PROTEIN IS MEDIATED BY A NOVEL STRUCTURAL MOTIF IN THE TRANSMEMBRANE DOMAIN AND ECTODOMAIN PLANKING REGION
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DOI：
10.1074/jbc.270.44.26404
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发表时间：
1995-11-03
影响因子：
4.8
通讯作者：
CAREY, DJ
中科院分区：
文献类型：
--
作者：
ASUNDI, VK;CAREY, DJ
我们单独表达了跨膜硫酸乙酰肝素蛋白聚糖 N-syndecan (syndecan-3) 的核心蛋白结构域，也可以将其作为麦芽糖结合蛋白融合蛋白表达。纯化蛋白质的生化表征表明，其中一些蛋白质能够自缔合并形成稳定的非共价多聚体复合物。 N-多聚糖核心蛋白复合物的形成也通过原位交联在哺乳动物细胞中得到证实。通过分析一系列包含蛋白质不同区域以及定点突变体的构建体，完成了 N-多配体核心蛋白中负责形成这些复合物的结构基序的鉴定。通过SDS-聚丙烯酰胺凝胶电泳、戊二醛交联和尺寸排阻高压液相色谱法测定自缔合。我们的结果表明，（i）N-syndecan 核心蛋白的跨膜结构域是形成稳定复合物所必需的，但不足以形成； (ii)赋予N-多聚糖核心蛋白形成多聚体复合物能力的最小氨基酸序列包括胞外结构域的最后四个氨基酸(ERKE)加上跨膜结构域； (iii)将该序列中的基本残基改变为丙氨酸残基的点突变部分或完全消除了N-syndecan核心蛋白形成复合物的能力； (iv)用亮氨酸替换跨膜结构域中的保守甘氨酸残基，消除了复合物的形成。这一特性与其他跨膜受体的寡聚活性相似，表明受调节的自缔合对于跨膜蛋白聚糖的生物活性可能很重要。
We expressed domains of the core protein of the transmembrane heparan sulfate proteoglycan N-syndecan (syndecan-3) either individually or as maltose-binding protein fusion proteins. Biochemical characterization of the purified proteins revealed that some of them were capable of self-association and formed stable, noncovalent multimeric complexes. The formation of N-syndecan core protein complexes was also demonstrated in mammalian cells by in situ cross-linking. Identification of structural motifs in the core protein of N-syndecan responsible for the formation of these complexes was accomplished by analyzing a series of constructs comprising different regions of the protein as well as site-directed mutants. Self-association was assayed by SDS-polyacrylamide gel electrophoresis, glutaraldehyde cross-linking, and size-exclusion high pressure liquid chromatography. Our results indicated that (i) the transmembrane domain of the N-syndecan core protein was required but not sufficient for the formation of stable complexes; (ii) the minimal amino acid sequence that conferred the ability of the N-syndecan core protein to form multimeric complexes included the last four amino acids (ERKE) of the extracellular domain plus the transmembrane domain; (iii) point mutations that changed the basic residues in this sequence to alanine residues either partially or completely abolished the ability of the N-syndecan core protein to form complexes; and (iv) replacement of conserved glycine residues in the transmembrane domain with leucines abolished complex formation. This property is similar to the oligomerization activity of other transmembrane receptors and suggests that regulated self-association may be important for the biological activity of transmembrane proteoglycans.