In vitro Impact of Yeast Expressed Hybrid Peptide CATH-2TP5 as a Prophylactic Measure Toward Sepsis and Inflammation

Abstract

CATH-2TP5 is a linear cationic hybrid peptide, consequent from naturally occurring antimicrobial peptide (AMPs) Cathelicidin-2 (CATH-2) and Immunomodulatory peptide Thymopentin (TP5) having dynamic and potent anti-inflammatory activities without hemolytic effect. The biocompatible mechanism of CATH-2TP5 is favored to explore new methodologies in the direction of biomedical applications. In this retrospectively study, an antiendotoxin and anti-inflammatory hybrid peptide CATH-2TP5 was emulated into pPICZα-A and successfully expressed in Pichia pastoris (P. pastoris). The recombinant CATH-2TP5 was purified through the Ni-affinity column and reversed-phase HPLC. The purified CATH-2TP5 peptide exhibited robust anti-endotoxin activity and significantly (p < 0.05) neutralized the effect of lipopolysaccharide (LPS). Furthermore, the down-regulated effect of CATH-2TP was more pronounced (p < 0.05) on LPS-induced cytotoxic effects, nitric oxide secretion and pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) in murine RAW264.7 macrophages. As associated to control and parental peptide the number of apoptotic cells was also contracted with the treatment of CATH-2TP5. Thus, we concluded that CATH-2TP5 peptide may be used in various biomedical applications as a therapeutic drug.

Keywords: apoptosis; cathelicidin; endotoxin neutralization; hybrid peptide; inflammation.

FIGURE 1

Effects of pH value, methanol concentration, and inductive time and inductive on the peptide expression. (A) Recombinant peptide expression was conducted at different pH values (pH 5-6.5), induced by 1% final methanol concentration at 28°C for 120 h. (B) Recombinant peptide expression was conducted at pH 6, inducted by various methanol concentration (0.5–2.5%) at 28°C for 120 h. (C) Recombinant peptide expression was conducted at pH 6.0, inducted by 1% methanol concentration at 28°C for 24–120 h. Data are presented as mean ± SD, n = 3. While **p < 0.01, ***p < 0.001 vs. Controls.

FIGURE 2

Recombinant hybrid peptide CATH-2TP5 analysis, (A) Tricine-SDS-PAGE of yeast expressing CATH-2TP5 medium. Molecular mass markers (Lane M); Control PpICZαA-blank (Lane C); Supernatant PpICZαA-CATH-2TP5, separated by 15% Tricine-SDS-PAGE with sliver stained and arrow showed peptide (Lane 1). (B) Tricine-SDS-PAGE of the purified peptide. Molecular weight markers (Lane M); Control PpICZαA-blank (Lane C). A sample of the purified peptide and arrow specified CATH-2TP5 2.6 kDa (Lane 1). (C) RP-HPLC of hybrid peptide and high peak exhibited fraction that contains CATH-2TP5. (D) ESI-MS of purified hybrid recombinant CATH-2TP5 peptide.

FIGURE 3

Endotoxin neutralization, cytotoxicity, and hemolytic activity test of parental and recombinant CATH-2TP5 peptide. (A) LPS neutralization by CATH-2 and CATH-2TP5 indomitable by using an endotoxin quantitation kit. Mean values accessible; n = 3 ± SD (^#p < 0.05 and ^##p < 0.01 revealed contrast of CATH-2 vs. CATH-2TP5). (B) CATH-2TP5 diminished cytotoxicity in the supernatant of endotoxin-infected murine RAW264.7 macrophages. Data signified as mean ± standard deviation (SDs) of independent experiments. **p < 0.01 vs. LPS. While, ^#p < 0.05 and ^##p < 0.01 specifies significant variance related with parental CATH-2 peptide. (C) Hemolytic activities of CATH-2TP5 in contradiction of mouse RBCs. The data resemble to the mean values of three independent experiments and are expressed as a % age of hemolysis ± standard deviation (**p < 0.01, ***p < 0.001 vs. Triton X-100). Whereas, ^#p < 0.05 and ^##p < 0.01 indicates significant difference compared with parental CATH-2 peptide.

FIGURE 4

Anti-inflammatory effects of parental and novel CATH-2TP5 peptides on LPS-influenced murine RAW264.7 macrophages. Production of (A) NO, (B) TNF-α, (C) IL-6, and (D) IL-1β. Cells were cured with various doses of CATH-2 and CATH-2TP5 peptides for 1 h before the treatment of cells with LPS to triggered infection. After a 24 h incubation, the supernatant was collected, and inflammatory cytokines levels were detected. Values are means ± SDs of three determinations. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. LPS. While, ^#p < 0.05, ^##p < 0.01 and ^###p < 0.001 designates significant variance compared with parental CATH-2 peptide.

FIGURE 5

Apoptosis of LPS-induced murine RAW264.7 macrophage at 4, 12, and 24 h with FITC-conjugated annexin-V (Green) and PI (Red). (A) The marked RAW264.7 macrophage was observed and analyzed by flow cytometry, the control signifies normal cells, the middle panel LPS discloses demonstrative RAW264.7 macrophage treated with LPS only, and the lower panel reveals RAW264.7 macrophages treated with LPS plus parental and hybrid peptides. (B) Observe the effects of LPS with CATH-2TP5 in RAW264.7 macrophages apoptosis. and **p < 0.01, ***p < 0.001 indicate the significance and highly significance difference vs. LPS. While ^#p < 0.05 indicated significance divulges between parental and hybrid peptide.

FIGURE 6

A schematic diagram of the possible mechanism by which CATH-2TP5 counteract endotoxin replies in mouse RAW264.7 macrophages. Left, LPS only; right LPS plus CATH-2TP5. The CATH-2TP5 binds with LPS and neutralizing it. ↑, Activation; ⊣, no activation; ↑, upper regulate responses; ↓, lower regulate responses.