379f70675d8a6df013b82cc1b376047c IntJouMolSci_a2024v25n1p604.pdf 2d64070633a13468599739378dc982213e3b17cb IntJouMolSci_a2024v25n1p604.pdf 9f43079ab6f1b80bb0d4d829692d947cf4dafcaca74bfb8d87cc52562e93cf40 IntJouMolSci_a2024v25n1p604.pdf Title: A Common Polymorphism in RNASE6 Impacts Its Antimicrobial Activity toward Uropathogenic Escherichia coli Subject: Human Ribonuclease (RNase) 6 is a monocyte and macrophage-derived protein with potent antimicrobial activity toward uropathogenic bacteria. The RNASE6 gene is heterogeneous in humans due to the presence of single nucleotide polymorphisms (SNPs). RNASE6 rs1045922 is the most common non-synonymous SNP, resulting in a G to A substitution that determines an arginine (R) to glutamine (Q) transversion at position 66 in the protein sequence. By structural analysis we observed that R66Q substitution significantly reduces the positive electrostatic charge at the protein surface. Here, we generated both recombinant RNase 6-R66 and -Q66 protein variants and determined their antimicrobial activity toward uropathogenic Escherichia coli (UPEC), the most common cause of UTI. We found that the R66 variant, encoded by the major SNP rs1045922 allele, exhibited superior bactericidal activity in comparison to the Q66 variant. The higher bactericidal activity of R66 variant correlated with an increase in the protein lipopolysaccharide binding and bacterial agglutination abilities, while retaining the same enzymatic efficiency. These findings encourage further work to evaluate RNASE6 SNP distribution and its impact in UTI susceptibility. Keywords: RNase; RNase 6; RNase k6; single nucleotide polymorphisms; antimicrobial peptides; urinary tract infections; uropathogenic Escherichia coli Author: Raul Anguita, Guillem Prats-Ejarque, Mohammed Moussaoui, Brian Becknell and Ester Boix Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25 CreationDate: Wed Jan 3 05:29:22 2024 CET ModDate: Wed Jan 3 05:31:58 2024 CET Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 10 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 1566293 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- FJMEJE+VnURWPalladioL Type 1 Custom yes yes yes 10 0 FUWQRK+URWPalladioL-Bold Type 1 Custom yes yes yes 16 0 TBRRDK+URWPalladioL-Roma Type 1 Custom yes yes yes 22 0 SLZKKO+URWPalladioL-Ital Type 1 Custom yes yes yes 27 0 LOSPJF+URWPalladioL-BoldItal Type 1 Custom yes yes yes 32 0 IRWHZX+TimesNewRomanPSMT Type 1C WinAnsi yes yes no 48 0 CKUPDE+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 51 0 VYVZGZ+CMSY10 Type 1 Builtin yes yes yes 68 0 DCCJBN+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 85 0 DCCJBO+PalatinoLinotype CID TrueType Identity-H yes yes yes 88 0 DCCJBP+PalatinoLinotype TrueType WinAnsi yes yes no 94 0 DCCJCB+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 97 0 DCCJKA+PalatinoLinotype,BoldItalic TrueType WinAnsi yes yes no 100 0 DCCJLB+PalatinoLinotype TrueType MacRoman yes yes no 103 0 DCCJBN+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 118 0 DCCJBO+PalatinoLinotype CID TrueType Identity-H yes yes yes 121 0 DCCJBP+PalatinoLinotype TrueType WinAnsi yes yes no 127 0 DCCJCB+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 130 0 DCCJKA+PalatinoLinotype,BoldItalic TrueType WinAnsi yes yes no 133 0 DCCJLB+PalatinoLinotype TrueType MacRoman yes yes no 136 0 XIRVNA+EURB10 Type 1 Builtin yes yes yes 145 0 OVNEXW+EURM10 Type 1 Builtin yes yes yes 150 0 DCCJBN+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 165 0 DCCJBO+PalatinoLinotype CID TrueType Identity-H yes yes yes 168 0 DCCJBP+PalatinoLinotype TrueType WinAnsi yes yes no 174 0 DCCJCB+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 177 0 DCCJLB+PalatinoLinotype TrueType MacRoman yes yes no 180 0 SYVAVM+VnURWPalladioL-Italic Type 1 Custom yes yes yes 193 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2024-05-11 02:46:06 CEST RepresentationInformation: IntJouMolSci_a2024v25n1p604.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2024-05-10 16:34:17 CEST Size: 1566293 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 395 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Results Destination: section.2 Children: Item: Title: RNASE6 rs1045922 Is a Common, Non-Synonymous SNP That Alters RNase 6Antimicrobial Activity Destination: subsection.2.1 Item: Title: Both SNP RNase6-R66 and -Q66 Display the Same Catalytic Activity Destination: subsection.2.2 Item: Title: The RNase6 R66Q Substitution Significantly Reduces the Positive Electrostatic Charge at the Protein Surface Destination: subsection.2.3 Item: Title: Discussion Destination: section.3 Item: Title: Materials and Methods Destination: section.4 Children: Item: Title: Materials Destination: subsection.4.1 Item: Title: Expression of RNase6-R66 and -Q66 Variants Destination: subsection.4.2 Item: Title: Minimum Bactericidal Concentration (MBC) Determination Destination: subsection.4.3 Item: Title: Lipopolysaccharide Binding Assay Destination: subsection.4.4 Item: Title: Bacterial Agglutination Assay Destination: subsection.4.5 Item: Title: Spectrophotometric Kinetic Assay Destination: subsection.4.6 Item: Title: 3D Structure Modelling Destination: subsection.4.7 Item: Title: References Destination: appendix.A. Info: Title: A Common Polymorphism in RNASE6 Impacts Its Antimicrobial Activity toward Uropathogenic Escherichia coli Author: Raul Anguita, Guillem Prats-Ejarque, Mohammed Moussaoui, Brian Becknell and Ester Boix Subject: Human Ribonuclease (RNase) 6 is a monocyte and macrophage-derived protein with potent antimicrobial activity toward uropathogenic bacteria. The RNASE6 gene is heterogeneous in humans due to the presence of single nucleotide polymorphisms (SNPs). RNASE6 rs1045922 is the most common non-synonymous SNP, resulting in a G to A substitution that determines an arginine (R) to glutamine (Q) transversion at position 66 in the protein sequence. By structural analysis we observed that R66Q substitution significantly reduces the positive electrostatic charge at the protein surface. Here, we generated both recombinant RNase 6-R66 and -Q66 protein variants and determined their antimicrobial activity toward uropathogenic Escherichia coli (UPEC), the most common cause of UTI. We found that the R66 variant, encoded by the major SNP rs1045922 allele, exhibited superior bactericidal activity in comparison to the Q66 variant. The higher bactericidal activity of R66 variant correlated with an increase in the protein lipopolysaccharide binding and bacterial agglutination abilities, while retaining the same enzymatic efficiency. These findings encourage further work to evaluate RNASE6 SNP distribution and its impact in UTI susceptibility. Keywords: RNase; RNase 6; RNase k6; single nucleotide polymorphisms; antimicrobial peptides; urinary tract infections; uropathogenic Escherichia coli Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25 CreationDate: Wed Jan 03 05:29:22 CET 2024 ModDate: Wed Jan 03 05:31:58 CET 2024 ID: 0xff28e0cfbe78c653b280b5634597ac5d, 0xff28e0cfbe78c653b280b5634597ac5d Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: FUWQRK+URWPalladioL-Bold FontSubset: true FirstChar: 3 LastChar: 134 FontDescriptor: FontName: FUWQRK+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: LOSPJF+URWPalladioL-BoldItal FontSubset: true FirstChar: 46 LastChar: 115 FontDescriptor: FontName: LOSPJF+URWPalladioL-BoldItal Flags: Symbolic FontBBox: -170, -300, 1073, 935 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: XIRVNA+EURB10 FontSubset: true FirstChar: 22 LastChar: 22 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