Antimicrobial and Antitumor Activities of Novel Peptides Derived from the Lipopolysaccharide- and β-1,3-Glucan Binding Protein of the Pacific Abalone Haliotis discus hannai.

Bo-Hye Nam,Hee Kong,Dong-Gyun Kim,Chul An,Young-Ok Kim,Woo-Jin Kim,Jung-Kil Seo,Ji Moon,Eun Park

doi:10.3390/md14120227

Abstract

Antimicrobial peptides are a pivotal component of the invertebrate innate immune system. In this study, we identified a lipopolysaccharide- and β-1,3-glucan-binding protein (LGBP) gene from the pacific abalone Haliotis discus hannai (HDH), which is involved in the pattern recognition mechanism and plays avital role in the defense mechanism of invertebrates immune system. The HDH-LGBP cDNA consisted of a 1263-bp open reading frame (ORF) encoding a polypeptide of 420 amino acids, with a 20-amino-acid signal sequence. The molecular mass of the protein portion was 45.5 kDa, and the predicted isoelectric point of the mature protein was 4.93. Characteristic potential polysaccharide binding motif, glucanase motif, and β-glucan recognition motif were identified in the LGBP of HDH. We used its polysaccharide-binding motif sequence to design two novel antimicrobial peptide analogs (HDH-LGBP-A1 and HDH-LGBP-A2). By substituting a positively charged amino acid and amidation at the C-terminus, the pI and net charge of the HDH-LGBP increased, and the proteins formed an α-helical structure. The HDH-LGBP analogs exhibited antibacterial and antifungal activity, with minimal effective concentrations ranging from 0.008 to 2.2 μg/mL. Additionally, both were toxic against human cervix (HeLa), lung (A549), and colon (HCT 116) carcinoma cell lines but not much on human umbilical vein cell (HUVEC). Fluorescence-activated cell sorter (FACS) analysis showed that HDH-LGBP analogs disturb the cancer cell membrane and cause apoptotic cell death. These results suggest the use of HDH-LGBP analogs as multifunctional drugs.

Highlights

Invertebrates lack antibodies and an adaptive immune system; instead, they rely on innate immunity to defend themselves against invading pathogens
pattern recognition proteins (PRPs) bind to pathogen-associated molecule patterns (PAMPs) to form complexes that subsequently activate immune responses such as phagocytosis, nodule formation, encapsulation, activation of proteinase cascades, and synthesis of antimicrobial peptides
A 632-bp sequence was obtained from clone DGT-151, and the N-terminal coding sequence was obtained using the Rapid amplification of cDNA-end (RACE) method and gene-specific primers

Summary

Introduction

Invertebrates lack antibodies and an adaptive immune system; instead, they rely on innate immunity to defend themselves against invading pathogens. The innate immune system of marine invertebrates allows them to survive and grow in their microbe-rich benthic environment. The first stage of the immune response is the recognition of invasive pathogens. Microbial cell-wall components referred to as pathogen-associated molecule patterns (PAMPs), such as LPS, β-1,3-glucan, and peptidoglycans, are recognized by a specific pattern recognition receptors (PRRs) or. Mar. Drugs 2016, 14, 227 pattern recognition proteins (PRPs). PRPs bind to PAMPs to form complexes that subsequently activate immune responses such as phagocytosis, nodule formation, encapsulation, activation of proteinase cascades, and synthesis of antimicrobial peptides. Various types of invertebrate PRPs, such as peptidoglycan recognition proteins (PGRPs), C-type lectins, lipopolysaccharide (LPS)-binding proteins, and β-glucan binding proteins (βGBPs), have been reported

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