Weyer Kathrin, Overgaard Michael T, Laursen Lisbeth S, Nielsen Claus G, Schmitz Alexander, Christiansen Michael, Sottrup-Jensen Lars, Giudice Linda C, Oxvig Claus
Department of Molecular Biology, Science Park, University of Aarhus, Gustav Wieds Vej 10C, DK-8000 Aarhus C, Denmark.
Eur J Biochem. 2004 Apr;271(8):1525-35. doi: 10.1111/j.1432-1033.2004.04061.x.
The metalloproteinase pregnancy-associated plasma protein-A (PAPP-A) cleaves a subset of insulin-like growth factor binding proteins (IGFBP), which inhibit the activities of insulin-like growth factor (IGF). Through this proteolytic activity, PAPP-A is believed to regulate IGF bioavailability in several biological systems, including the human reproductive system and the cardiovascular system. PAPP-A adheres to mammalian cells by interactions with glycosaminoglycan (GAG), thus targeting the proteolytic activity of PAPP-A to the cell surface. Based on site-directed mutagenesis, we here delineate the PAPP-A GAG-binding site in the C-terminal modules CCP3 and CCP4. Using heparin affinity chromatography, commonly employed in such studies, we define three clusters of arginines and lysines of CCP3, which are important for the interaction of PAPP-A with heparin. In a model of PAPP-A CCP3-CCP4, basic residues of these sequence clusters form a contiguous patch located on one side of the structure. Binding to the unknown, natural cell surface receptor of PAPP-A, assessed by flow cytometry, also depends on residues of these three basic clusters. However, single or double residue substitutions generally have a modest effect on PAPP-A heparin binding assessed by chromatography, but cell surface adhesion was critically reduced by several of these substitutions, emphasizing the relevance of analysis by flow cytometry. The contributions of positively charged residues located in CCP4 were all minor when analyzed by heparin affinity chromatography. However, the mutation of CCP4 residues Arg1459 and Lys1460 to Ala almost abrogated cell surface adhesion. Furthermore, when acidic residues of the homologous proteinase PAPP-A2 (Asp1547, Glu1555 and Glu1567) were introduced into the corresponding positions in the sequence of PAPP-A, located in each of the three basic clusters of CCP3, binding to heparin was strongly impaired and cell surface binding was abrogated. This explains, at least in part, why PAPP-A2 lacks the ability of cell surface adhesion, and further emphasizes the role of the basic clusters defined in PAPP-A.
金属蛋白酶妊娠相关血浆蛋白-A（PAPP-A）可裂解一部分胰岛素样生长因子结合蛋白（IGFBP），这些蛋白会抑制胰岛素样生长因子（IGF）的活性。通过这种蛋白水解活性，PAPP-A被认为在包括人类生殖系统和心血管系统在内的多个生物系统中调节IGF的生物利用度。PAPP-A通过与糖胺聚糖（GAG）相互作用而黏附于哺乳动物细胞，从而将PAPP-A的蛋白水解活性靶向到细胞表面。基于定点诱变，我们在此确定了C端模块CCP3和CCP4中的PAPP-A GAG结合位点。使用此类研究中常用的肝素亲和色谱法，我们确定了CCP3中对PAPP-A与肝素相互作用很重要的三个精氨酸和赖氨酸簇。在PAPP-A CCP3-CCP4模型中，这些序列簇的碱性残基形成了位于结构一侧的连续区域。通过流式细胞术评估，与PAPP-A未知的天然细胞表面受体的结合也取决于这三个碱性簇的残基。然而，单残基或双残基取代通常对通过色谱法评估的PAPP-A肝素结合影响不大，但其中一些取代会严重降低细胞表面黏附，这突出了流式细胞术分析的相关性。通过肝素亲和色谱法分析时，位于CCP4中的带正电荷残基的贡献都很小。然而，将CCP4残基Arg1459和Lys1460突变为丙氨酸几乎消除了细胞表面黏附。此外，当同源蛋白酶PAPP-A2的酸性残基（Asp1547、Glu1555和Glu1567）被引入位于CCP3的三个碱性簇中的PAPP-A序列的相应位置时，与肝素的结合受到强烈损害，细胞表面结合被消除。这至少部分解释了为什么PAPP-A2缺乏细胞表面黏附能力，并进一步强调了PAPP-A中定义的碱性簇的作用。
