Hindawi Publishing Corporation BioMed Research International Volume 2013, Article ID 170398, 12 pages http://dx.doi.org/10.1155/2013/170398 Research Article In Silico Prediction and In Vitro Characterization of Multifunctional Human RNase3 Pei-Chun Lien, 1 Ping-Hsueh Kuo, 1 Chien-Jung Chen, 1 Hsiu-Hui Chang, 1 Shun-lung Fang, 1 Wei-Shuo Wu, 2 Yiu-Kay Lai, 2 Tun-Wen Pai, 3 and Margaret Dah-Tsyr Chang 1, 4 1 Institute of Molecular and Cellular Biology, National Tsing Hua University, No. 101, Section 2, Kuang Fu Road, Hsinchu 30013, Taiwan 2 Institute of Biotechnology, National Tsing Hua University, No. 101, Section 2, Kuang Fu Road, Hsinchu 30013, Taiwan 3 Department of Computer Science and Engineering, National Taiwan Ocean University, 2 Pei Ning Road, Keelung 20224, Taiwan 4 Department of Medical Science, National Tsing Hua University, No. 101, Section 2, Kuang Fu Road, Hsinchu 30013, Taiwan Correspondence should be addressed to Margaret Dah-Tsyr Chang; dtchang@life.nthu.edu.tw Received 31 October 2012; Accepted 2 December 2012 Academic Editor: Hao-Teng Chang Copyright © 2013 Pei-Chun Lien et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 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, 34 RWRCK 38 (HBR1), 75 RSRFR 79 (HBR2), and 101 RPGRR 105 (HBR3), of hRNase3 have been identi�ed 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. 1. Introduction Human ribonuclease A (hRNaseA) family members are encoded by unique genes located on human chromosome 14 [1]. e hRNaseA family is vertebrate cationic protein sharing conserved tertiary structure and speci�c enzymatic sites for RNase activity. It is in general considered to comprise eight members: RNase1 (pancreatic RNase), RNase2 (eosinophil derived neurotoxin/EDN), RNase3 (eosinophil cationic pro- tein/ECP), RNase4, RNase5 (angiogenin), RNase6, RNase7 (skin-derived RNase), and RNase8 (divergent paralog of RNase7) [2]. Analysis of human genome sequence has revealed the existence of �ve additional RNases named as RNases9–13, although they appear to lose enzymatic activity [3]. All hRNaseA family members encode relatively small polypeptides of 14 to 16 kDa containing signal peptides of 20 to 28 amino acids for protein secretion. Mature hRNaseA members contain 6 to 8 cysteine residues that are crucial to hold the overall tertiary structure [4]. ey possess an invariant catalytic triad including two histidines (one near the  terminus, and the other near the  terminus) and one lysine located within a conserved signature motif (CKXXNTF) [5]. ese RNaseAs are catalytically active to various degrees against standard polymeric RNA substrates [6]. Interestingly, their host defense functions including cytotoxic [7, 8], helminthotoxic [9, 10], antibacterial [11, 12], and antiviral [5, 13] activities have also been reported. However, the mechanisms of noncatalytic functions of some hRNaseA members, especially the ones with low RNase activities, are poorly understood. hRNase3 is found within the secondary granules of eosinophils and serves as a clinical asthma marker [14]. It is a multiple functional protein as the N-terminal domain 1−45