| Summary: | 碩士 === 國立清華大學 === 分子與細胞生物研究所 === 94 === The eosinophil appears to be the primary leukocyte responsible for tissue damage in bronchial asthma, which occurs when the granule proteins, including eosinophil cationic protein (ECP) and eosinophil derived neurotoxin (EDN), are released into the extracellular space. ECP and EDN belong to the ribonuclease A superfamily. Both have been suggested as factors in allergic respiratory diseases, and thus used as clinical biomarkers for detecting the severity of asthma. Interestingly, ECP and EDN showed cytotoxicity toward several cell lines, including HL60 and A431 cells. The cytotoxicity has been correlated with the internalization of the RNases, but the cellular receptor for ECP or EDN has not been identified yet.
Cell surface glycosaminoglycan (GAG) proteoglycans act as docking sites for the binding of several different proteins to surface of eukaryotic cells. GAGs are presented almost ubiquitously on cell surface but vary with respect to their composition and quantity among different group of species, cell types, tissues, and cellular development stages. Heparan sulfate (HS), a ubiquitously expressed GAG, bind to a wide variety of extracellular ligands, including growth factors, virus, and bacteria. ECP and EDN known as heparin binding proteins may interact with the HS on the cell surface. Here, we demonstrate that HS is essential in the cellular uptake of ECP and EDN by immunofluorescence study. In addition, surface plasmon resonance instrument (SPR) was used to investigate the binding affinities and kinetics between ECP/EDN and heparan sulfate. The interaction between ECP and HS was obviously reduced while ECP was denatured with urea or boiling, indicating that ECP binding to HS in a 3 D structure dependent manner. The ECP/EDN-HS interactions are decreased at acidic pH, suggesting that ECP and EDN may dissociate from HS under acidic pH within endosomes. Taken together, these findings suggest that heparan sulfate proteoglycan serves as the cellular endocytic receptor for ECP and EDN.
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