School of Life Sciences, University of Hyderabad, Hyderabad 500 046, India.
J Biosci. 2011 Aug;36(3):545-53. doi: 10.1007/s12038-011-9082-5.
The process of uptake of hexamerins during metamorphosis from insect haemolymph by fat body cells is reminiscent of receptor-mediated endocytosis. Previously, we had identified a hexamerin-binding protein (HBP) and reported for the first time that uptake of hexamerins is dependent on the phosphorylation of HBP partly by a tyrosine kinase, which is, in turn, activated by 20-hydroxyecdysone (20E). However, the exact nature of HBP and the mechanism of interaction are still unknown. Here we report the possibility of HBP being a GPI-anchored protein in the fat body of Achaea janata and its role in the tyrosine-kinase-mediated phosphorylation signalling. Digestion of fat body membrane preparation with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC), and the subsequent recognition by antibodies specific for the cross-reacting determinant (CRD), revealed that HBP is glycosylphosphatidylinositol (GPI)-anchored protein and, further, that the hexamerin binding to HBP was inhibited after digestion. Hexamerin overlay assay (HOA) of co-immunoprecipitated in vitro phosphorylated HBP showed exclusive binding to ~120 kDa protein. Lectin-binding analysis of hexamerins revealed the presence of N-acetylgalactosamine (GalNAc) and N-acetylglucosamine (GluNAc), whereas HBP showed the presence of GalNac alone. Mild chemical deglycosylation studies and binding interaction in the presence of sugars revealed that glycan moieties are possibly not involved in the interaction between HBP and hexamerins. Taken together, these results suggest that HBP may be a GPI-anchored protein, and interaction and activation of HBP is through lipid-linked non-receptor src tyrosine kinases. However, additional studies are needed to prove that HBP is a GPI-anchored protein.
在昆虫血淋巴到脂肪体细胞的变态过程中六聚体的摄取过程让人想起受体介导的内吞作用。此前，我们已经鉴定出一种六聚体结合蛋白（HBP），并首次报道六聚体的摄取依赖于 HBP 的磷酸化，部分是由一种酪氨酸激酶介导的，而酪氨酸激酶又被 20-羟基蜕皮酮（20E）激活。然而，HBP 的确切性质和相互作用的机制仍然未知。在这里，我们报告了 HBP 作为 Achaea janata 脂肪体中糖基磷脂酰肌醇（GPI）锚定蛋白的可能性，以及它在酪氨酸激酶介导的磷酸化信号中的作用。用细菌磷脂酰肌醇特异性磷脂酶 C（PI-PLC）消化脂肪体细胞膜制剂，并用针对交叉反应决定簇（CRD）的特异性抗体进行后续识别，表明 HBP 是糖基磷脂酰肌醇（GPI）锚定蛋白，并且，在消化后，六聚体与 HBP 的结合被抑制。共免疫沉淀体外磷酸化 HBP 的六聚体覆盖测定（HOA）显示仅与~120 kDa 蛋白结合。六聚体的凝集素结合分析显示存在 N-乙酰半乳糖胺（GalNAc）和 N-乙酰葡萄糖胺（GluNAc），而 HBP 仅显示存在 GalNac。温和的化学去糖基化研究和存在糖的结合相互作用表明糖基部分可能不参与 HBP 和六聚体之间的相互作用。总之，这些结果表明 HBP 可能是一种 GPI 锚定蛋白，并且 HBP 的相互作用和激活是通过脂连接的非受体 src 酪氨酸激酶进行的。然而，还需要进一步的研究来证明 HBP 是一种 GPI 锚定蛋白。
