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首页> 外文学位 >Molecular cell biology of cell surface glycoproteins: Dipeptidyl peptidase IV as a model protein.
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Molecular cell biology of cell surface glycoproteins: Dipeptidyl peptidase IV as a model protein.

机译：细胞表面糖蛋白的分子细胞生物学：二肽基肽酶IV作为模型蛋白。

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摘要

Dipeptidyl peptidase IV (DPPIV) is a serine exopeptidase that cleaves N-terminal dipeptides from polypeptides if the second residue in the polypeptide is a proline or an alanine. As a membrane glycoprotein, DPPIV is expressed on the cell surface of many cell types as homodimers. DPPIV has been under extensive studies for more than two decades from different aspects since its original identification in rat liver and kidney. Recent studies have shown DPPIV may be involved in many biological processes, including interaction with extracellular matrix, metabolism and uptake of proline-containing peptides in the small intestine and kidney proximal tubules, regulation of lymphocyte activation and proliferation, and maturation and degradation of peptide hormone and neuropeptides (for example, substance P). In a process of identification and characterization of membrane glycoproteins that are expressed on distinct membrane domains in polarized cells (for example, liver hepatocytes and epithelial cells of intestine and kidney tubules), we successfully produced antibodies to a membrane glycoprotein with Mr. of 110,000 (originally denoted gp110) that is expressed in the bile canaliculus of hepatocytes and in the apical membrane domain of epithelial cells of small intestine and kidney proximal tubules. We have identified that this gp110 is DPPIV. Using antibodies to DPPIV, we isolated the full-length cDNA for DPPIV for the first time. After subcloning the cDNA into an in vitro expression vector pGEM-4Z, RNA was produced in vitro with Sp6 polymerase. RNA produced was used to study the membrane topology of DPPIV using rabbit reticulocyte lysate translation and translocation systems. These studies revealed that DPPIV has a type II membrane orientation (a single transmembrane domain located in the N-terminal region with most of the polypeptide in the extracellular side and only N-terminal 6 amino acids in the cytoplasmic side). Further studies have demonstrated that N-terminal 34 amino acid sequence (defined as N-terminal signal/anchor sequence) is sufficient for the signal/anchor function in translocating the polypeptide across microsomal membranes and anchoring the polypeptide in the membrane. Site-directed mutagenesis studies of this N-terminal signal/anchor sequence have revealed some important structural features of this type of targeting sequence for the rough endoplasmic reticulum (RER) (RER-targeting sequence) as compared to other types of RER-targeting sequences. After subcloning the DPPIV cDNA into the inducible eukaryotic expression vector pMSG, we have obtained stable transfected Chinese hamster ovary (CHO) cells that produce enzymatically active DPPIV. By Western and Northern blotting analysis, we were able to conclude that expression of DPPIV in rat tissues is mainly regulated at the mRNA levels.
机译：二肽基肽酶IV（DPPIV）是一种丝氨酸肽外切酶，如果多肽中的第二个残基是脯氨酸或丙氨酸，它可以从多肽上切割出N端二肽。 DPPIV作为膜糖蛋白，在许多细胞类型的细胞表面均以同二聚体的形式表达。自从DPPIV最初在大鼠肝脏和肾脏中鉴定以来，已经从不同方面进行了广泛的研究超过二十年。最近的研究表明，DPPIV可能参与许多生物学过程，包括与细胞外基质的相互作用，小肠和肾脏近端小管中含脯氨酸的肽的代谢和摄取，淋巴细胞活化和增殖的调节以及肽激素的成熟和降解和神经肽（例如，物质P）。在鉴定和表征在极化细胞（例如，肠和肾小管的肝肝细胞和上皮细胞）的不同膜结构域上表达的膜糖蛋白的过程中，我们成功生产了110,000先生的膜糖蛋白抗体。最初表示为gp110），在肝细胞的胆小管以及小肠和肾脏近端小管上皮细胞的顶膜结构域表达。我们已经确定此gp110是DPPIV。使用针对DPPIV的抗体，我们首次分离了DPPIV的全长cDNA。将cDNA亚克隆到体外表达载体pGEM-4Z中后，用Sp6聚合酶体外产生RNA。使用兔网织红细胞裂解物翻译和易位系统，将产生的RNA用于研究DPPIV的膜拓扑。这些研究表明DPPIV具有II型膜取向（位于N末端区域的单个跨膜结构域，其中大部分多肽位于细胞外侧，而仅N末端6个氨基酸位于细胞质侧）。进一步的研究表明，N末端的34个氨基酸序列（定义为N末端信号/锚序列）足以使信号/锚功能跨多肽膜转运多肽并将多肽锚定在膜中。对这种N末端信号/锚序列的定点诱变研究表明，与其他类型的RER靶向序列相比，这种针对内质网（RER）的靶向序列的重要结构特征（RER靶向序列） 。将DPPIV cDNA亚克隆到可诱导的真核表达载体pMSG中后，我们获得了稳定转染的中国仓鼠卵巢（CHO）细胞，可产生具有酶活性的DPPIV。通过Western和Northern印迹分析，我们可以得出结论，DPPIV在大鼠组织中的表达主要受mRNA水平的调节。

著录项

  • 作者

    Hong, Wanjin.;

  • 作者单位

    State University of New York at Buffalo.;

  • 授予单位 State University of New York at Buffalo.;
  • 学科 Biology Molecular.
  • 学位 Ph.D.
  • 年度 1990
  • 页码 124 p.
  • 总页数 124
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 分子遗传学;
  • 关键词

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