MOLECULAR-CLONING AND CHARACTERIZATION OF N-SYNDECAN, A NOVEL TRANSMEMBRANE HEPARAN-SULFATE PROTEOGLYCAN
MOLECULAR-CLONING AND CHARACTERIZATION OF N-SYNDECAN, A NOVEL TRANSMEMBRANE HEPARAN-SULFATE PROTEOGLYCAN
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DOI：
10.1083/jcb.117.1.191
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发表时间：
1992-04-01
影响因子：
7.8
通讯作者：
CIZMECISMITH, G
中科院分区：
文献类型：
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作者：
CAREY, DJ;EVANS, DM;CIZMECISMITH, G
通过用基于编码先前描述的蛋白聚糖核心蛋白的cDNA中的保守序列的寡核苷酸筛选，从新生大鼠雪旺细胞cDNA文库中分离编码膜蛋白聚糖核心蛋白的cDNA克隆。引物延伸和聚合酶链反应扩增用于获得另外的5'蛋白质编码序列。推导的氨基酸序列预测了一个353个氨基酸的多肽，具有一个单一的跨膜段和一个34个氨基酸的亲水性COOH-末端的胞质结构域。推定的胞外结构域包含三个潜在的糖胺聚糖附着位点，以及一个富含Thr和Pro残基的结构域。的cDNA和推导的氨基酸序列的分析显示了高度的同一性与以前描述的蛋白聚糖，但一个独特的胞外结构域序列的跨膜和胞质结构域。在北方印迹杂交的cDNA杂交到一个单一的5.6 kb的mRNA，是存在于雪旺细胞，新生大鼠脑，大鼠心脏，大鼠平滑肌细胞。将该cDNA编码的16 kD蛋白片段在细菌中表达并免疫家兔。所得抗体在免疫印迹上与去污剂提取的硫酸乙酰肝素蛋白聚糖的核心蛋白反应。核心蛋白的表观质量为120 kD。当使用抗核心蛋白抗体染色组织切片时，免疫反应性存在于外周神经、新生大鼠脑、心脏、主动脉和其他新生组织中。使用核糖核酸酶保护测定来定量核心蛋白mRNA的水平。在新生大鼠脑、心脏和雪旺细胞中发现高水平。mRNA在新生儿或成人肝脏或成人大脑中几乎检测不到。
A cDNA clone coding for a membrane proteoglycan core protein was isolated from a neonatal rat Schwann cell cDNA library by screening with an oligonucleotide based on a conserved sequence in cDNAs coding for previously described proteoglycan core proteins. Primer extension and polymerase chain reaction amplification were used to obtain additional 5' protein coding sequences. The deduced amino acid sequence predicted a 353 amino acid polypeptide with a single membrane spanning segment and a 34 amino acid hydrophilic COOH-terminal cytoplasmic domain. The putative extracellular domain contains three potential glycosaminoglycan attachment sites, as well as a domain rich in Thr and Pro residues. Analysis of the cDNA and deduced amino acid sequences revealed a high degree of identity with the transmembrane and cytoplasmic domains of previously described proteoglycans but a unique extracellular domain sequence. On Northern blots the cDNA hybridized to a single 5.6-kb mRNA that was present in Schwann cells, neonatal rat brain, rat heart, and rat smooth muscle cells. A 16-kD protein fragment encoded by the cDNA was expressed in bacteria and used to immunize rabbits. The resulting antibodies reacted on immunoblots with the core protein of a detergent extracted heparan sulfate proteoglycan. The core protein had an apparent mass of 120 kD. When the anti-core protein antibodies were used to stain tissue sections immunoreactivity was present in peripheral nerve, newborn rat brain, heart, aorta, and other neonatal tissues. A ribonuclease protection assay was used to quantitate levels of the core protein mRNA. High levels were found in neonatal rat brain, heart, and Schwann cells. The mRNA was barely detectable in neonatal or adult liver, or adult brain.