Ram Narendra, Aroui Sonia, Jaumain Emilie, Bichraoui Hicham, Mabrouk Kamel, Ronjat Michel, Lortat-Jacob Hugues, De Waard Michel
INSERM U836, Grenoble Institute of Neurosciences, Research Group 3, Calcium Channels, Functions, and Pathologies Laboratory, Université Joseph Fourier, Grenoble Cedex 9, France.
J Biol Chem. 2008 Aug 29;283(35):24274-84. doi: 10.1074/jbc.M709971200. Epub 2008 Jul 3.
Maurocalcine (MCa), initially identified from a Tunisian scorpion venom, defines a new member of the family of cell penetrating peptides by its ability to efficiently cross the plasma membrane. The initiating mechanistic step required for the cell translocation of a cell penetrating peptide implicates its binding onto cell surface components such as membrane lipids and/or heparan sulfate proteoglycans. Here we characterized the interaction of wild-type MCa and MCa K20A, a mutant analogue with reduced cell-penetration efficiency, with heparin (HP) and heparan sulfates (HS) through surface plasma resonance. HP and HS bind both to MCa, indicating that heparan sulfate proteoglycans may represent an important entry route of the peptide. This is confirmed by the fact that (i) both compounds bind with reduced affinity to MCa K20A and (ii) the cell penetration of wild-type or mutant MCa coupled to fluorescent streptavidin is reduced by about 50% in mutant Chinese hamster ovary cell lines lacking either all glycosaminoglycans (GAGs) or just HS. Incubating MCa with soluble HS, HP, or chondroitin sulfates also inhibits the cell penetration of MCa-streptavidin complexes. Analyses of the cell distributions of MCa/streptavidin in several Chinese hamster ovary cell lines show that the distribution of the complex coincides with the endosomal marker Lyso-Tracker red and is not affected by the absence of GAGs. The distribution of MCa/streptavidin is not coincident with that of transferrin receptors nor affected by a dominant-negative dynamin 2 K44A mutant, an inhibitor of clathrin-mediated endocytosis. However, entry of the complex is greatly diminished by amiloride, indicating the importance of macropinocytosis in MCa/streptavidin entry. It is concluded that (i) interaction of MCa with GAGs quantitatively improves the cell penetration of MCa, and (ii) GAG-dependent and -independent MCa penetration rely similarly on the macropinocytosis pathway.
毛罗卡辛（MCa）最初是从突尼斯蝎子毒液中鉴定出来的，因其能够有效穿过质膜而成为细胞穿透肽家族的新成员。细胞穿透肽进行细胞转运所需的起始机制步骤涉及其与细胞表面成分（如膜脂和/或硫酸乙酰肝素蛋白聚糖）的结合。在这里，我们通过表面等离子体共振表征了野生型MCa和细胞穿透效率降低的突变类似物MCa K20A与肝素（HP）和硫酸乙酰肝素（HS）的相互作用。HP和HS均与MCa结合，表明硫酸乙酰肝素蛋白聚糖可能是该肽的重要进入途径。这一点得到以下事实的证实：（i）这两种化合物与MCa K20A的结合亲和力降低；（ii）在缺乏所有糖胺聚糖（GAGs）或仅缺乏HS的突变中国仓鼠卵巢细胞系中，与荧光链霉亲和素偶联的野生型或突变型MCa的细胞穿透率降低了约50%。用可溶性HS、HP或硫酸软骨素孵育MCa也会抑制MCa-链霉亲和素复合物的细胞穿透。对几种中国仓鼠卵巢细胞系中MCa/链霉亲和素的细胞分布分析表明，复合物的分布与内体标记物溶酶体追踪红一致，并且不受GAGs缺失影响。MCa/链霉亲和素的分布与转铁蛋白受体的分布不一致，也不受网格蛋白介导的内吞作用抑制剂显性负性发动蛋白2 K44A突变体的影响。然而，阿米洛利大大降低了复合物的进入，表明巨胞饮作用在MCa/链霉亲和素进入中的重要性。得出的结论是：（i）MCa与GAGs的相互作用在数量上提高了MCa的细胞穿透率；（ii）依赖GAG和不依赖GAG的MCa穿透同样依赖巨胞饮作用途径。
