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.

Figure 1. MS profiles of different plant parts of Hibiscus sabdariffa.

(A) calyces, (B) capsule, (C) seed, (D) leaves, and (E) flower of Hibiscus sabdariffa were collected and profiled using Maldi-TOF MS.

Figure 2. De novo sequencing of roseltide rT1.

(A) S-alkylated roseltide rT1 was digested with trypsin, resulting in two tryptic fragments with the m/z of 545 and 2442; (B) MS/MS spectra of 545 Da fragment; (C) MS/MS spectra of 2442 Da fragment; (D) S-alkylated roseltide rT1 was digested with chymotrypsin at two sites, resulting in fragments with the m/z of 1045 and 1328. The third peptide fragment of m/z 1640 could not be detected.; (E) MS/MS spectra of 1045 Da fragment; (F) MS/MS spectra of 1328 Da fragment.

Figure 3. Roseltide transcripts from Hibiscus sabdariffa.

The histogram depicts conservation among the putative amino acid sequences of roseltides by amino acid property grouping as determined by Jalview software. AEP: asparagine endopeptidase; PDI: protein disulfide isomerase; SPase: signal peptidase.

Figure 4

(A) Sequence alignment of CKAI with roseltide rT1; allotide Ac2 from Allamanda cathartica; wrightide Wr-Al1 from Wrightia religiosa; alstotides As1, As3 and As4 from Alstonia scholaris (B) Solution structure of roseltide rT1; (C) Superimposition of roseltide rT1 (green) on wrightide Wr-AI1 (PDB entry 2MAU) (purple) and alstotides As1 (PDB entry 2MM6) (blue).

Figure 5

(A) Acid, (B) human serum, (C) trypsin, and (D) pepsin stability of roseltide rT1. All results were expressed as mean ± S.E.M. (n = 3).

Figure 6

Roseltide rT1 does not show cytotoxic activities. Effects of roseltide rT1 on (A) Huh7 and (B) A549 cells. All results were expressed as mean ± S.E.M. (n = 3). *P < 0.05 compared to control group.

Figure 7. The effects of roseltide rT1 on human neutrophil elastase.

(A) The effects of different concentrations of roseltide rT1 or synthetic elastase inhibitor, N-methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone, on human neutrophil elastase (HNE) activity was measured at 405 nm at 37 °C using N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide as a substrate. All results were expressed as mean ± S.E.M. (n = 3); (B) MS spectra showed biotinylation of roseltide rT1; (C) The effects of biotin-rT1 on HNE activity. All results were expressed as mean ± S.E.M. (n = 3). *P < 0.05 compared to control; (D) Silver-stained SDS-PAGE of the pull-down complex between HNE and biotin-rT1. The left-most lane shows a protein marker (Bio-rad, US); HNE-only lane: purified HNE only; biotin lane: purified HNE incubated with biotin and NeutrAvidin resin (control), biotin-rT1 lane: purified HNE incubated with biotin-rT1 and NeutrAvidin resin. The full-length gel image is provided in Supplementary data S11.

Figure 8. Roseltide rT1 ameliorates human neutrophil elastase (HNE)-stimulated cAMP accumulation in CHO-K1 cells co-expressed with PAR2 receptor and Glosensor cAMP biosensor constructs.

Comparison of PAR2 receptor expressions in cAMP-CHO cells using (A) confocal microscopy and (B) flow cytometry; (C) Effects of cAMP activator Forskolin (10 μM) on cAMP accumulation in PAR2-cAMP-CHO cells; (D) Effects of HNE with or without roseltide rT1 on cAMP accumulation in PAR2-cAMP-CHO cells. All results were expressed as mean ± S.E.M. (n = 3). *P < 0.05 compared to HNE-treated group.

Figure 9

(A) Modeling the interaction between roseltide rT1 and the human neutrophil elastase (PDB entry: 1HNE) using the ClusPro Version 2.0 server. Blue: Negatively-charged, Red: Positively-charged and White: Neutral.; (B) Interactions between roseltide rT1 and peptide chloromethyl ketone inhibitor (AAPA-CMK) (PDB entry: 1HNE) with the catalytic triad of human neutrophil elastase formed by His57, Asp102, and Ser195.