In Silico Prediction and In Vitro Characterization of Multifunctional Human RNase3

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.


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 protein/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  possesses antipathogenic activities such as antibacterial, antihelminthic, and antiviral competencies [15][16][17].In terms of key amino acids involving speci�c functions, Trp 35 of hRNase3 interacts with cell membrane to form transmembrane pores in an arti�cial lipid bilayer, suggesting that lipid destruction is a crucial step in bactericidal activity [18].In addition, Arg 1 , Trp 10 , Gln 14 , Lys 38 , and Gln 40 located in antipathogenic domain are identi�ed to bind to lipopolysaccharide (LPS) and peptidoglycan with high affinity, which may also be important for its bactericidal activity [19,20].Moreover, hRNase3 possesses cytotoxic activity against various mammalian cell lines including those derived from blood and epidermis [17], and Arg 97 is the key residue for its cytotoxicity [21].It is also highly associated with host in�ammatory response and thus involved in tumor microenvironment to exercise its antitumor response [22][23][24].Interestingly, hRNase3 reduces the infectivity of human respiratory syncytial virus in an RNase activity-dependent manner [25].Treatment of bronchial epithelial cells with hRNase3 induces production of tumor necrosis factor alpha (TNF-) and triggers apoptosis via a caspase-8-dependent pathway [26].Furthermore, hRNase3 is reported to bind to a class of cell surface receptors termed as heparan sulfate proteoglycans [27] and thereby internalizes target cells through macropinocytosis [28].Taken together, basic and aromatic residues, especially Arg and Trp, are considered to play important roles in enzymatic and other biological functions of hRNase3.
In this study, in combination with in silico analyses employing Reinforced Merging for Unique Segments (ReMUS) system, we have identi�ed three heparin binding regions (HBRs) in hRNase3.We focused on their roles in heparin and cellular binding and endocytic and cytotoxic activities employing in vitro functional analyses.Our results showed that HBR1 ( 34 RWRCK 38 ) is crucial for enzymatic RNase function and serves as a major heparin binding site for endocytosis, HBR2 ( 73 RSRFR 77 ) contributes toward cell binding and endocytic activities, and HBR3 ( 101 RPGRR 105 ) plays a critical role in cytotoxicity.In addition, a noncytotoxic HBR1-derived peptide was characterized to bind to negatively charged molecules including glycosaminoglycans (GAGs) and lipids on cell surface.In summary, we have identi�ed multifunctional regions of hRNase3, which may provide novel insights to implicate for all hRNaseA superfamily members.

In Silico Analysis.
Unique peptides of query proteins, 13 hRNaseA family members, were identi�ed employing Reinforced Merging for Unique Segments ReMUS system (ReMUS) (http://140.121.196.30/remus.asp)[42].e system adopted a bottom-up strategy to extract unique patterns in each sequence at different unique levels.A fundamental unique peptide segment with previously de�ned pattern length, named as primary pattern was extracted at the �rst step.e rule of thumb for primary pattern lengths is that a shorter length setting for similar protein sequences and a longer length for dissimilar ones.e length of primary pattern in this study is set as 3 residues for hRNaseA protein family.Aer that Boyer Moore algorithm was performed to efficiently retrieve all primary patterns among all sequences.�ach veri�ed fundamental unique peptide segment was analyzed based on its frequencies of appearance, and its representation level of uniqueness was calculated for the merging processes in the next module.e last merging algorithm concatenated these extracted unique peptide segments through a bottom-up approach only if the primary unique peptide segments were overlapped within a sequence.e merged segments were guaranteed with unique features compared to all other protein sequences in the query dataset.

Fluorescence-Assisted Carbohydrate Electrophoresis (FACE).
Carbohydrates were labeled with 2-aminoacridone (AMAC) according to previous study [44].e AMAClabeled carbohydrate and peptide were mixed and incubated at 25 ∘ C for 15 min.e complex was then loaded onto 1% agarose gels and electrophoresed in the buffer containing 40 mM Tris-acetic acid, 1 mM EDTA, pH 8.0 for 20 to 30 min.is experiment was performed in dark or under red light to prevent from light exposure.e AMAC labeled probe was observed under UV light (424 nm) and scanned by transilluminator.

RNase Activity Assay.
RNase activity assay of recombinant wild-type and mutant hRNase3 were performed using yeast tRNA (Invitrogen), and bovine RNaseA (USB) was typically RNase and used as positive control.ree hundred microliters of 100 mM sodium phosphate (NaPO 4 ) buffer, pH 7.4, and 500 L diethylpyrocarbonate-(DEPC-) ddH 2 O were mixed including 50 L of 0.05 M RNaseA and 5 M of wild-type and mutant hRNase3, separately.Ten microliters of 5 mg/mL yeast tRNA was added and incubated at 37 ∘ C for 0, 5, 10, and 15 min, respectively.Ice-cold 500 L stop solution (1 : 1 (v/v) 40 mM lanthanum nitrate and 6% perchloric acid) was added and mixed for 10 min to stop reaction.Entire yeast tRNA was suspended by centrifugation at 16,100 ×g at 25 ∘ C for 5 min.One hundred microliters of supernatant in each tube was placed on to a 96-well plate.e amount of soluble tRNA in supernatant was determined by UV absorbance at 260 nm.

In Silico Analysis of Unique Peptide Regions in hRNaseA
Superfamily.To predict unique peptide regions possibly involved in multifunctions of hRNase3, ReMUS system was employed to analyze sequences of hRNase3 and the other 12 members of hRNaseA family.Eleven unique peptide motifs including HISLNPPR, RCTIAMRA, NYR-WRC, SIRCPHNRTLNNC, RSRFRVP, PLLHCD, DLINP, PGAQN, NCTYADRPGRRFYV, DPRDSPRY, and LDTTI in hRNase3 were identi�ed as shown in blue and light blue in Figure 1.Among which three unique segments rich in positively charged amino acids were denoted as putative HBRs including 34 RWRCK 38 (HBR1), 73 RSRFR 77 (HBR2), and 101 RPGRRR 105 (HBR3).Since heparin binding activity of HBR1 has been previously reported [27], the presence of 3 HBRs might possibly correlate with stronger heparin binding features of hRNase3 than other hRNaseA family members.Subsequently, Clustal W2 was applied to compare primary sequence of hRNase3 with the other hRNaseAs and alignment of putative HBRs of hRNase3 with correspondent segments of the other 12 hRNaseAs.Figure 2 revealed that HBR1 in hRNase3 was 60% identical to the corresponding segments of hRNase1, hRNase2, hRNase7, and hRNase8, but these HBRs were not conserved with any of the other hRNase family members, suggesting that these three HBRs might account for unique functions of hRNase3.erefore, seven mutant hRNase3 constructs were generated by site-directed mutagenesis with selective alanine replacement in each HBR in order to investigate unique functions of HBR1, HBR2 and HBR3 in hRNase3 (Supplementary Figure 1 available online at http://dx.doi.org/10.1155/2013/170398).

RNase Activity of Wild-Type and Mutant
RNase3.Aer 0.05 M bovine RNaseA, 5 M wild-type or mutant hRNase3 was incubated with 50 g tRNA at 37 ∘ C for 0, 5, 10, and 15 min, the amount of digested ribonucleotides was examined by UV absorbance at 260 nm. Figure 3(a) showed that RNase activity of HBR1-mt RNase3 was signi�cantly reduced and that of HBR3-mt RNase3 was 15% less than wild type hRNase3.Besides, the RNase activity of HBR2-mt RNase3 was comparable to that of wild-type hRNase3.Moreover, double and triple mutation abolished hRNase activities of HBR12-, HBR13-and HBR123-mtRNase3 expect HBR23-mtRNase3 (Figure 3(b)).ese results suggested that HBR1 of hRNase3 played a critical role in ribonucleolytic activity, mainly due to the presence of a catalytic residue Lys in the sequence.

Endocytosis Activity of Wild-Type and Mutant hRNase3 to
Beas-2B Cells.To determine the in�uence of different HBRs on endocytosis activity of hRNase3, intracellular uptake assay was performed.Beas-2B cells were treated with wild type or mutant hRNase3 at 37 ∘ C for 1 h, followed by trypsin digestion for 15 min to remove surface-bound recombinant proteins before being analyzed by western blotting.When 40 g of total cell lysates were examined with an exposure time of 1 min, only HBR2-mtRNase3 and HBR3-mtRNase3 could be detected in cytosol of Beas-2B cells (Figure 4, lanes 3 and 4).In addition, none of the double and triple HBR mutants of hRNase3 was able to enter Beas-2B cells (Figure 4, lanes 5, 6, 7, and 8).ese results indicated that the importance of HBRs associated with endocytosisactivity of hRNase3 to Beas-2B cells in increasing order was HBR3, HBR2, and HBR1.

Cytotoxicity of Wild-Type and Mutant hRNase3 to
Beas-2B Cells.Beas-2B cells were incubated individually with 15 M of wild-type or mutant hRNase3 in serumfree medium at 37 ∘ C for 48 h followed by MTT assay.e cell viability of PBS treatment to Beas-2B cells was set as 100% to normalize that of different protein treatment.Figure 5 revealed that the viability of wild-type hRNase3-treated cells decreased to 50%, while that of HBR1-, HBR2-, HBR3-, HBR12-, HBR13-, HBR23-, and HBR123-mtRNase3-treated cells increased to 69%, 60%, 101%, 73%, 96%, 88%, and 97%, respectively.e cytotoxicity of HBR3-mtRNase3, HBR13-mtRNase3, HBR23-mtRNase3, and HBR123-mtRNase3 apparently diminished as compared to that of wild-type hRNase3, suggesting that HBR3 mutation played a major role in loss of cytotoxicity of hRNase3.It should be noted that HBR3 was located on  sheet 6 of hRNase3, hence mutation of HBR3 to alanine stretch might possibly lead to conformational change and subsequent of functional variation.

Discussion
hRNaseA superfamily members share diverse protein identities to hRNase3 even though they contain conserved 3dimensional structures and enzymatic functions.In addition to RNase activity, a variety of biological features including immune-regulatory, cytotoxic, antimicrobial, antitumor, and heparin/HS binding activities have been reported.In terms of RNase activities, hRNases1-to-8 have obvious catalytic activities� however, such function is unidenti�ed in hRNases9-to-13 and remains to be elucidated.As for cytotoxicity, hRNases1-to-5 are harmful to various cells or organisms, but hRNases6-to-13 are indistinct.In terms of lipid binding activity, hRNase2, hRNase3, and hRNase7 have been reported to interact with lipids [30][31][32], while that of the other hRNaseA family members are not well studied.As for antimicrobial activity, hRNase2, hRNase3, hRNase5, hRNase7, and hRNase8 are harmful to microorganisms (Table 1).In addition, hRNase2 and hRNase3 have been elucidated to mediate immune responses [31,32].Taken together, comparison of hRNaseA family members indicates that only hRNase2 has similar sequences and functions to hRNase3 (Table 1).
Structural analysis reveals that all hRNaseA family members share very similar secondary structures in three putative HBRs (Table 2).Since hRNase7 shares high primary sequence identity to hRNase6 and hRNase8, 58% and 75%, respectively, its structure was used as a template for structure simulation of hRNase6 and hRNase8 which have no resolved 3D structures yet.e HBR1 and HBR3 in all hRNaseA family members are present, respectively, in loop and -strand conformation.e secondary structure for HBR2 of hRNase4 is -strand, and that of the others is loop conformation.Hence, these results suggested that sequence composition, rather than secondary structure contents of each HBR-like segment in hRNaseA family members, is crucial for differential heparin binding activities.Among all hRNaseA superfamily members, hRNase2, hRNase3, and hRNase7 possess conventional heparin binding motifs in several HBRs.However, only hRNase2, hRNase3, and hRNase5 have been reported to demonstrate heparin binding activities [48,49].Here, three unique functional peptides encoded HBRs have been predicted in hRNase3 by ReMUS and demonstrated heparin binding properties at both molecular and cellular levels, and the correspondent HBR1 of hRNase2 has been identi�ed with heparin binding features too.Interestingly, in the primary sequence hRNase5 positively charged residue-rich regions, that is, 31 RRR 33 and R 70 have been reported to involve heparin binding by site-directed mutagenesis [49], and they are located pretty close to putative HBR1 and HBR2 of hRNase5 in this study.Finally, hRNase7 has been reported to be puri�ed through a heparin a�nity column despite unclear heparin binding mechanism, indicating that hRNase7 also possesses heparin binding potency.is �nding will contribute to further understanding of protein-ligand interaction in hRNaseA members.

Conclusion
In this study, we identify three functionally important HBRs in hRNase3, including 34

F 3 :
RNase activity of wild-type and mutant hRNase3.Five mg of yeast tRNA was added to 0.05 M bovine RNaseA and 5 M of (a) HBR1-mtRNase3, HBR2-mtRNase3, and HBR3-mtRNase3 and (b) HBR12-mtRNase, HBR13-mtRNase3, HBR23-mtRNase3, and HBR123-mtRNase3 separately to examine the RNase activity.e amount of digested ribonucleotides in supernatant was detected by monitoring OD 260 and RNase activity of bovine RNaseA was set as a positive control.
Sequences of HBR1, HBR2, and HBR3 in hRNase3 and corresponding regions of other 12 hRNaseA members.Sequences of hRNase3 and other hRNaseA superfamily members are aligned using ClustalW2 soware.Putative HBR1, HBR2, and HBR3 separately located on residues 34-38, 73-77, and 101-105 are predicted from hRNase3.Residues in black, red, and green boxes indicate corresponding sequence motifs aligned with, respectively, HBR1, HBR2, and HBR3 in all hRNaseA superfamily members.