Abstract
Human eosinophil derived neurotoxin (EDN), a granule protein secreted by activated eosinophils, is a biomarker for asthma in children. EDN belongs to the human RNase A superfamily possessing both ribonucleolytic and antiviral activities. EDN interacts with heparin oligosaccharides and heparin sulfate proteoglycans on bronchial epithelial Beas-2B cells. In this study, we demonstrate that the binding of EDN to cells requires cell surface glycosaminoglycans (GAGs), and the binding strength between EDN and GAGs depends on the sulfation levels of GAGs. Furthermore, in silico computer modeling and in vitro binding assays suggest critical roles for the following basic amino acids located within heparin binding regions (HBRs) of EDN 34QRRCKN39 (HBR1), 65NKTRKN70 (HBR2), and 113NRDQRRD119 (HBR3) and in particular Arg35, Arg36, and Arg38 within HBR1, and Arg114 and Arg117 within HBR3. Our data suggest that sulfated GAGs play a major role in EDN binding, which in turn may be related to the cellular effects of EDN.
Highlights
Human eosinophil derived neurotoxin (EDN) is a basic protein normally stored in cytoplasmic granules and secreted by activated eosinophilic leukocytes [1]
To further investigate specificity of MBP-EDN binding, we measured the inhibitory activity of different types of GAGs including high molecular weight heparin (HMWH) which is highly sulfated, chondroitin sulfate C (CSC) and dermatan sulfate (DS) which are moderately sulfated compared to HMWH, and hyaluronic acid (HA)
Our model suggests that the bound heparin hexasaccharide was stabilized by ionic interactions with Arg36 and Lys38 in HBR1, and Lys1 and Arg132 which did not belong to heparin binding regions (HBRs), whereas Arg97 and basic residues in HBR2 and HBR3 were not located within van der Waals force/hydrogen bond distances to interact with the heparin hexasaccharide (Figure 5B)
Summary
Human eosinophil derived neurotoxin (EDN) is a basic protein (pI 8.9) normally stored in cytoplasmic granules and secreted by activated eosinophilic leukocytes [1]. Our previous study [19] showed that maltose-binding protein fused EDN (MBP-EDN) could interact with Beas-2B cells, a human bronchial epithelial cell line with limited expression of transcripts of TLR2 gene [16]. It suggested that MBP-EDN might interact with other components (other than TLR2) on cell surface of Beas-2B cells. The amino acid sequence of EDN contains 12 basic amino acids (8 Arg and 4 Lys residues), and nine of them are concentrated within three regions including 34QRRCKN39 in loop 3, 65NKTRKN70 in loop 4, and 113NRDQRRD119 in loop 7 [20]. The importance of sulfo groups of GAGs in interaction with EDN was characterized
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