[Show abstract][Hide abstract] ABSTRACT: Human ribonucleases A (hRNaseA) superfamily consists of thirteen members with high-structure similarities but exhibits divergent physiological functions other than RNase activity. Evolution of hRNaseA superfamily has gained novel functions which may be preserved in a unique region or domain to account for additional molecular interactions. hRNase3 has multiple functions including ribonucleolytic, heparan sulfate (HS) binding, cellular binding, endocytic, lipid destabilization, cytotoxic, and antimicrobial activities. In this study, three putative multifunctional regions, 34RWRCK38 (HBR1), 75RSRFR79 (HBR2), and 101RPGRR105 (HBR3), of hRNase3 have been identified employing in silico sequence analysis and validated employing in vitro activity assays. A heparin binding peptide containing HBR1 is characterized to act as a key element associated with HS binding, cellular binding, and lipid binding activities. In this study, we provide novel insights to identify functional regions of hRNase3 that may have implications for all hRNaseA superfamily members.
BioMed research international. 01/2013; 2013:170398.
[Show abstract][Hide abstract] ABSTRACT: Cell-penetrating peptides (CPPs) are short peptides which can carry various types of molecules into cells; however, although most CPPs rapidly penetrate cells , their tissue-targeting specificities are low. Herein, we describe cell-binding, internalization, and targeting characteristics of a newly identified 10-residue CPP, denoted ECP, derived from the core heparin-binding motif of human eosinophil cationic protein (ECP). Besides traditional emphasis on positively charged residues, the presence of cysteine and tryptophan residues was demonstrated to be essential for internalization. ECP entered Beas-2B and wild-type CHO-K1 cells, but not CHO cells lacking of cell-surface glycosaminoglycans (GAGs), indicating that binding of ECP to cell-surface GAGs was required for internalization. When cells were cultured with GAGs or pre-treated with GAG-digesting enzymes, significant decreases in ECP internalization were observed, suggesting that cell-surface GAGs, especially heparan sulfate proteoglycans were necessary for ECP attachment and penetration. Furthermore, treatment with pharmacological agents identified two forms of energy-dependent endocytosis, lipid-raft endocytosis and macropinocytosis, as the major ECP internalization routes. ECP was demonstrated to transport various cargoes including fluorescent chemical, fluorescent protein, and peptidomimetic drug into cultured Beas-2B cells , and targeted broncho-epithelial and intestinal villi tissues . Hence this CPP has the potential to serve as a novel vehicle for intracellular delivery of biomolecules or medicines, especially for the treatment of pulmonary or gastrointestinal diseases.
PLoS ONE 01/2013; 8(3):e57318. · 3.53 Impact Factor
[Show abstract][Hide abstract] 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.
International Journal of Molecular Sciences 01/2013; 14(9):19067-85. · 2.46 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Heparan sulfate (HS), one kind of sulfated glycosaminoglycans (GAGs), plays an important role in growth factor-receptor interaction, thereby involving in growth, angiogenesis and innate immune signaling. It is also highly addressed that GAGs and their anchoring core proteins are abnormally expressed in various cancers including lung, liver, and colon cancers. Human ribonuclease A (RNaseA) family members, hRNase2, hRNase3 and hRNase5 are reported to bind to cell surface by recognition of sulfated GAGs. We have recently identified three functionally important HS and heparin binding regions in hRNase3, also named as human eosinophil cationic protein (ECP), and characterized a dual-functional peptide (CPPecp) with GAG-binding and cell-penetrating activities. Screening of in vitro binding activity on various gastrointestinal cell lines demonstrated that CPPecp bound to cells rich in HS on the cell surface. However, cellular binding activity of FITC-CPPecp was abolished while cell surface HS was removed by haparinase treatment. In order to characterize the specific binding of CPPecp, quantitative assay was carried out to precisely decipher binding affinity between CPPecp and a variety of GAGs. In addition, magnetic nanoparticle (Fe3O4)-conjugated CPPecp (MNP-CPPecp) was synthesized as a novel bioprobe to evaluate its feasibility in in vivo magnetic resonance imaging (MRI) analysis in colon tumor mouse model. Taken together, our CPPecp in vitro preferred to bind to sulfated GAGs and selectively attach to HS on cell surface. Moreover, CPPecp demonstrated in vivo tumor targeting activity in colon tumor mouse model, suggesting that CPPecp possesses high potential to be developed as a novel molecular imaging agent and cancer targeting delivery vector.
Complex, Intelligent, and Software Intensive Systems (CISIS), 2013 Seventh International Conference on; 01/2013
[Show abstract][Hide abstract] ABSTRACT: Human ribonuclease A (hRNaseA) super family members have similar biological functions such as catalytic activities against specific RNA substrates. However, these enzymes with high sequence similarity may exhibit divergent physiological functions other than RNase activity, for example, angio genesis and innate immunity. In our investigation, a novel heparin-binding motif (HBM), RWRCK, identified from hRNase3 contributed to specific protein-heparin/heparan sulfate (HS) interaction. Based on this core HBM sequence, a 10-amino acid heparin binding peptide (HBPRNase3), NYRWRCKNQN, has been designed and characterized. Employing Clustal W2 and Uniprot Blastn program, such HBP pattern is found to be conserved in human and higher primates. Multiple sequence alignment of 13 members of human RNase A family reveals that HBP regions in hRNase2 and hRNase8 share 80% and 50% sequence identity to HBPRNase3, but the corresponding sequences of Gorilla and Pan troglodytes RNase3 are 100% identical, strongly suggesting that HBPRNase3 is conserved in higher primates along with species evolution. Interestingly, the putative HBPRNase2, NYQRRCKNQN, shows much lower heparin binding activity than HBPRNase3. In summary, HBPRNase3 is not a conserved motif in RNaseA superfamily, but it is a unique motif presenting in higher primates to play a crucial role in molecular interaction to heparin and HS.
Complex, Intelligent and Software Intensive Systems (CISIS), 2012 Sixth International Conference on; 01/2012
[Show abstract][Hide abstract] ABSTRACT: Multiple sequence alignment is widely applied to discover core conserved regions among query sequences. However, the major deficiency is that alignment accuracy is extremely sensitive to primary sequence identity, which causes alignment of low identity sequences difficult. We propose a feature-integrated model called feature-incorporated alignment (FIA) which integrates relevant biological characteristics including aromatic amino acids, hydrophilicity, beta-stranded structure, and BLOSUM62 matrix to locate ligand-binding residue in carbohydrate binding modules (CBMs), a protein family with fairly low sequence identify but highly functional correlation. The results indicated that FIA can not only detect aromatic residues on the outer surface of structure, but also achieve better accuracy than ClustalW2 and DIALIGN-TX on entropy criterion in all three test datasets from CBMs. Computational analysis in CBMs can facilitate the discovery of crucial ligand-binding residues of carbohydrate-active enzymes.
2009 IEEE International Conference on Bioinformatics and Biomedicine, BIBM 2009, Washington, DC, USA, 1-4 November 2009, Proceedings; 01/2009