In Silico Prediction and In Vitro Characterization of Unique Function of Human RNase3

Lien, Pei-Chun; Chen, Chien-Jung; Kuo, Ping-Hsueh; Pai, Tun-Wen; Chang, Hsiu-Hui; Lai, Yiu‐Kay; Wu, Wei-Shuo; Chang, Margaret Dah‐Tsyr

doi:10.1109/cisis.2012.84

Cited by 3 publications

(7 citation statements)

References 39 publications

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“…We identified a key antimicrobial region (residues 24–45) that is essential for membrane leakage, depolarization and LPS binding. Recent work on ECP‐binding domains also highlighted the involvement of the 34–38 stretch for both heterosaccharide and lipid binding . A second region, close to the N–terminus of the protein (residues 8–16) was found to be essential for bacteria agglutination .…”

Section: Discussionmentioning

confidence: 99%

Towards the rational design of antimicrobial proteins

et al. 2013

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The ribonuclease (RNase) A superfamily lineage includes distant members with antimicrobial properties, suggesting a common ancestral host‐defense role. In an effort to identify the minimal requirements for the eosinophil cationic protein (ECP or RNase 3) antimicrobial properties we applied site‐directed mutagenesis on its closest family homolog, the eosinophil‐derived neurotoxin (EDN or RNase 2). Both eosinophil secretion proteins are involved in human immune defense, and are reported as being among the most rapidly evolving coding sequences in primates. Previous studies in our laboratory defined two regions at the N–terminus involved in the protein antimicrobial action, encompassing residues 8–16 and 34–36. Here, we demonstrate that switching two single residues is enough to provide EDN with ECP antipathogen properties. That is, the EDN double‐mutant Q34R/R35W displays enhanced bactericidal activity, particularly towards Gram‐negative bacteria, and a significant increase in its affinity towards the bacterial outer membrane lipopolysaccharides. Moreover, we confirmed the direct contribution of residue W35 in lipopolysaccharide binding, membrane interaction and permeabilization processes. Furthermore, additional T13 to I substitution provides EDN with an exposed hydrophobic patch required for protein self‐aggregation and triggers bacterial agglutination, thereby increasing the final antimicrobial activity by up to 20–fold. Our results highlight how single selected mutations can reshape the entire protein function. This study provides an example of how structure‐guided protein engineering can successfully reproduce an evolution selection process towards the emergence of new physiological roles.

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Section: Discussionmentioning

confidence: 99%

Towards the rational design of antimicrobial proteins

et al. 2013

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“…Cys 37 also hydrogen bonded to SGN1. Regarding other putative HBRs in ECP [ 22 ], only Arg 105 located at HBR3 ecp 101 ,RPGRR 105 , interacted with 2- O -sulfated iduronic acid residue 6 (IDS6) through hydrogen bonding, whereas HBR2 ecp 73 ,RSRFR 77 , had no interaction with Hep6 in this model. The heparin molecule also interacted with residues including His 15 and Lys 38 which were expected to serve as proton donors in the case of RNase activity, suggesting that Hep6 shared the same surface area with RNA for binding ( Figure 3B , yellow spheres) [ 42 ].…”

Section: Resultsmentioning

confidence: 98%

“…Both ECP and EDN have three putative HBRs each of which contains three basic residues within contiguous five amino acids [ 22 ]. Among these HBRs only HBR1 on ECP (HBR1 ecp , 34 RWRCK 38 ), and HBR1 on EDN (HBR1 edn , 34 QRRCK 38 ), were located at a comparable position in primary sequence ( Figure 1 ).…”

Section: Resultsmentioning

confidence: 99%

“…Residues from sugars 7 to 12 were likely to interact with ECP at an extended part of the cavity on ECP surface by making additional interactions with Asn 19 , Arg 45 , Arg 101 and Arg 104 ( Figure 7B ). Among these residues Arg 101 and Arg 104 were located at HBR3 ecp [ 22 ], and Asn 19 and Arg 45 were located at a position close to non-reducing end of Hep12, suggesting that HBR3 ecp might participate in the interaction between wtECP and long-chain heparin.…”

Section: Resultsmentioning

confidence: 99%

“…Two more segments 73 RSRFR 77 and 101 RPGRR 105 were also predicted to be HBRs. Besides, in silico comparison of these three regions on ECP to corresponding regions in RNase1-13 demonstrated that the 34 RWRCK 38 was a unique peptide motif [ 22 ]. Notably, mutant recombinant ECP with Ala replacing all basic residues in 3 HBRs still possessed 19% binding activity to low molecular weight heparin (LMWH), suggesting that additional residues or factors are operative in the ECP-LMWH interaction [ 22 ].…”

Section: Introductionmentioning

confidence: 99%

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Functional Characterization of ECP-Heparin Interaction: A Novel Molecular Model

et al. 2013

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Human eosinophil cationic protein (ECP) and eosinophil derived neurotoxin (EDN) are two ribonuclease A (RNaseA) family members secreted by activated eosinophils. They share conserved catalytic triad and similar three dimensional structures. ECP and EDN are heparin binding proteins with diverse biological functions. We predicted a novel molecular model for ECP binding of heparin hexasaccharide (Hep6), [GlcNS(6S)-IdoA(2S)]3, and residues Gln40, His64 and Arg105 were indicated as major contributions for the interaction. Interestingly, Gln40 and His64 on ECP formed a clamp-like structure to stabilize Hep6 in our model, which was not observed in the corresponding residues on EDN. To validate our prediction, mutant ECPs including ECP Q40A, H64A, R105A, and double mutant ECP Q40A/H64A were generated, and their binding affinity for heparins were measured by isothermal titration calorimetry (ITC). Weaker binding of ECP Q40A/H64A of all heparin variants suggested that Gln40-His64 clamp contributed to ECP-heparin interaction significantly. Our in silico and in vitro data together demonstrate that ECP uses not only major heparin binding region but also use other surrounding residues to interact with heparin. Such correlation in sequence, structure, and function is a unique feature of only higher primate ECP, but not EDN.

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Heparan sulfate targeting strategy for enhancing liposomal drug accumulation and facilitating deep distribution in tumors

et al. 2020

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Nanoparticles (NPs), such as liposomes, effectively evade the severe toxicity of unexpected accumulation and passively shuttle drugs into tumor tissues by enhanced permeability and retention. In the case of non-small cell lung cancer and pancreatic ductal adenocarcinoma, cancer-associated fibroblasts promote the aggregation of a gel-like extracellular matrix that forms a physical barrier in the desmoplastic stroma of the tumor. These stroma are composed of protein networks and glycosaminoglycans (GAGs) that greatly compromise tumor-penetrating performance, leading to insufficient extravasation and tissue penetration of NPs. Moreover, the presence of heparan sulfate (HS) and related proteoglycans on the cell surface and tumor extracellular matrix may serve as molecular targets for NP-mediated drug delivery. Here, a GAG-binding peptide (GBP) with high affinity for HS and high cell-penetrating activity was used to develop an HS-targeting delivery system. Specifically, liposomal doxorubicin (L-DOX) was modified by post-insertion with the GBP. We show that the in vitro uptake of L-DOX in A549 lung adenocarcinoma cells increased by GBP modification. Cellular uptake of GBP-modified L-DOX (L-DOX-GBP) was diminished in the presence of extracellular HS but not in the presence of other GAGs, indicating that the interaction with HS is critical for the cell surface binding of L-DOX-GBP. The cytotoxicity of doxorubicin positively correlated with the molecular composition of GBP. Moreover, GBP modification improved the in vivo distribution and anticancer efficiency of L-DOX, with enhanced desmoplastic targeting and extensive distribution. Taken together, GBP modification may greatly improve the tissue distribution and delivery efficiency of NPs against HS-abundant desmoplastic stroma-associated neoplasm.

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In Silico Prediction and In Vitro Characterization of Unique Function of Human RNase3

Cited by 3 publications

References 39 publications

Towards the rational design of antimicrobial proteins

Towards the rational design of antimicrobial proteins

Functional Characterization of ECP-Heparin Interaction: A Novel Molecular Model

Heparan sulfate targeting strategy for enhancing liposomal drug accumulation and facilitating deep distribution in tumors

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