title: Identification and Characterization of Roseltide, a Knottin-type Neutrophil Elastase Inhibitor Derived from Hibiscus sabdariffa.
abstract: Plant knottins are of therapeutic interest due to their high metabolic stability and inhibitory activity against proteinases involved in human diseases. The only knottin-type proteinase inhibitor against porcine pancreatic elastase was first identified from the squash family in 1989. Here, we report the identification and characterization of a knottin-type human neutrophil elastase inhibitor from Hibiscus sabdariffa of the Malvaceae family. Combining proteomic and transcriptomic methods, we identified a panel of novel cysteine-rich peptides, roseltides (rT1-rT8), which range from 27 to 39 residues with six conserved cysteine residues. The 27-residue roseltide rT1 contains a cysteine spacing and amino acid sequence that is different from the squash knottin-type elastase inhibitor. NMR analysis demonstrated that roseltide rT1 adopts a cystine-knot fold. Transcriptome analyses suggested that roseltides are bioprocessed by asparagine endopeptidases from a three-domain precursor. The cystine-knot structure of roseltide rT1 confers its high resistance against degradation by endopeptidases, 0.2 N HCl, and human serum. Roseltide rT1 was shown to inhibit human neutrophil elastase using enzymatic and pull-down assays. Additionally, roseltide rT1 ameliorates neutrophil elastase-stimulated cAMP accumulation in vitro. Taken together, our findings demonstrate that roseltide rT1 is a novel knottin-type neutrophil elastase inhibitor with therapeutic potential for neutrophil elastase associated diseases.
authors.affiliation: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, 637551, Singapore.
authors.forename: Shining
authors.initials: S
authors.lastname: Loo
authors.affiliation: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, 637551, Singapore.
authors.forename: Antony
authors.initials: A
authors.lastname: Kam
authors.affiliation: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, 637551, Singapore.
authors.forename: Tianshu
authors.initials: T
authors.lastname: Xiao
authors.affiliation: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, 637551, Singapore.
authors.forename: Giang K T
authors.initials: GK
authors.lastname: Nguyen
authors.affiliation: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, 637551, Singapore.
authors.forename: Chuan Fa
authors.initials: CF
authors.lastname: Liu
authors.affiliation: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, 637551, Singapore.
authors.forename: James P
authors.initials: JP
authors.lastname: Tam
mesh_terms: D000072283:A549 Cells; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D045744:Cell Line, Tumor; D003412:Cricetulus; D000242:Cyclic AMP / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000378:metabolism; D057771:Cystine-Knot Miniproteins / Q000037:antagonists & inhibitors*; D031584:Hibiscus / Q000737:chemistry*; D006801:Humans; D019272:Leukocyte Elastase / Q000037:antagonists & inhibitors*; D009504:Neutrophils / Q000187:drug effects*; D010447:Peptide Hydrolases / Q000378:metabolism; D053491:Proteinase Inhibitory Proteins, Secretory / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D015842:Serine Proteinase Inhibitors / Q000737:chemistry* / Q000494:pharmacology*; D059467:Transcriptome / Q000187:drug effects; D000099240:Asparaginyl Endopeptidase
chemical_list: D000242:Cyclic AMP; D003545:Cysteine; D003546:Cysteine Endopeptidases; D057771:Cystine-Knot Miniproteins; D019272:Leukocyte Elastase; D010447:Peptide Hydrolases; D053491:Proteinase Inhibitory Proteins, Secretory; D015842:Serine Proteinase Inhibitors; D000099240:Asparaginyl Endopeptidase