Jain Renu K, Chang Wen Tzu, Geetha Chitta, Joyce Paul B M, Gorr Sven-Ulrik
Department of Periodontics, Endodontics and Dental Hygiene, Health Sciences Center, University of Louisville, Louisville, KY 40292, U.S.A.
Biochem J. 2002 Dec 1;368(Pt 2):605-10. doi: 10.1042/BJ20021195.
Aggregation chaperones, consisting of secretory proteins that contain a hexa-histidine epitope tag, enhance the calcium-induced aggregation of regulated secretory proteins and their sorting to secretory granules. The goal of this study was to gain a better understanding of this unusual aggregation mechanism. Hexa-histidine-epitope-tagged secreted alkaline phosphatase, an aggregation chaperone, enhanced the in vitro aggregation of chromogranin A in the presence of calcium, but not in the presence of magnesium or other divalent cations. As an exception, chromogranin was completely aggregated by zinc, even in the absence of the aggregation chaperone. In addition, fluorescence spectroscopy of the aggregation reaction mixture showed an increase in fluorescence intensity consistent with the formation of protein aggregates. The calcium-induced aggregation of chromogranin A was completely inhibited by 0.2% Triton X-100, suggesting that it involves hydrophobic interactions. In contrast, the detergent did not affect chaperone-enhanced aggregation, suggesting that this aggregation does not depend on hydrophobic interactions. EDTA-treated chaperone did not enhance chromogranin A aggregation, indicating that divalent cations are necessary for chaperone action. Although the structure of the aggregation chaperone was not important, the size of the chaperone was. Thus the free His-hexapeptide could not substitute for the aggregation chaperone. Based on these results, we propose that the hexa-histidine tag, in the context of a polypeptide, acts as a divalent cation-dependent nucleation site for chromogranin A aggregation.
聚集伴侣由含有六组氨酸表位标签的分泌蛋白组成，可增强钙诱导的调节性分泌蛋白聚集及其向分泌颗粒的分选。本研究的目的是更好地理解这种不同寻常的聚集机制。作为聚集伴侣的带有六组氨酸表位标签的分泌性碱性磷酸酶，在有钙存在的情况下增强了嗜铬粒蛋白A的体外聚集，但在有镁或其他二价阳离子存在时则不然。作为一个例外，即使在没有聚集伴侣的情况下，嗜铬粒蛋白也能被锌完全聚集。此外，聚集反应混合物的荧光光谱显示荧光强度增加，这与蛋白质聚集体的形成一致。0.2%的 Triton X-100完全抑制了钙诱导的嗜铬粒蛋白A聚集，这表明它涉及疏水相互作用。相比之下，去污剂并不影响伴侣增强的聚集，这表明这种聚集不依赖于疏水相互作用。经EDTA处理的伴侣不能增强嗜铬粒蛋白A的聚集，这表明二价阳离子是伴侣发挥作用所必需的。虽然聚集伴侣的结构并不重要，但伴侣的大小很重要。因此，游离的六组氨酸肽不能替代聚集伴侣。基于这些结果，我们提出，在多肽背景下的六组氨酸标签作为嗜铬粒蛋白A聚集的二价阳离子依赖性成核位点。
