Characterization of incilarin-like peptide with antibacterial activity from Pacific abalone, Haliotis discus hannai

Abstract

Incilarin A-like peptide containing C-type lectin domain was shown to be related to faster growth of Pacific abalones, Haliotis discus hannai. C-type lectin domain have several major characterizations that bind carbohydrate in a Ca2+-dependent manner and have conserved cysteine residues with double-loop structure. Marine invertebrates such as abalones, oysters, mussels have an innate immune system for protecting from pathogens in microbe-rich aquatic environment. Antimicrobial peptides (AMPs) act as the first line of the biological defense playing an important role in the innate immune system in marine invertebrates. In this study, the gene encoding Incilarin A of Haliotis discus hannai was identified an open reading frame that consisted of 157 amino acids. The gene encoding incilarin A consisted of 447 bp encoding 149 amino acids was cloned into pET 44a(+) expression vector and purified the recombinant incilarin A-like peptide (rHdhIncA) from Escherichia coli BL21. Recombinant incilarin A-like peptide (rHdhIncA) expressed from E. coli was purified by affinity chromatography. Antimicrobial activity of the recombinant peptides was analyzed against pathogens by using Ultrasensitive radial diffusion assay (URDA) with ampicillin as a positive control against Gram-positive bacteria (Bacillus subtilis and Streptococcus parauberis) and Gram-negative bacteria (Vibrio harveyi, Escherichia coli). Various activities of the recombinant proteins including bacterial binding and agglutination, DNA binding were also tested. NPN uptake assay was carried out to examine the mechanism of antimicrobial activity against pathogens. Antimicrobial activity of rHdhIncA was revealed from the minimum inhibitory concentration (MIC) indicating that antimicrobial activity of 2 uM purified rHdhIncA is similar to the activity of 1.4 uM ampicillin in B. subtilis, S. parauberis, V. harveyi. Also, bacterial binding in E. coli BL21-GFP, B. subtilis was detected and bacterial agglutination was detected dependent Ca2+. DNA binding of rHdhIncA was detected by gel retardation, but rHdhIncA have slight results in outer membrane permeabilization in E. coli.|선행연구에 따르면 북방전복의 종묘 단계에 있어서 작은 개체군에 비해 큰 개체군에서의 Incilarin A 유전자의 발현이 높다. 이는 전복의 성장에 있어서 항균 단백질인 Incilarin A의 면역작용이 영향이 있다고 여겨진다. 본 연구는 in vitro 상에서의 incilarin A의 항균활성을 분석했다. 북방전복의 incilarin A는 474 bp의 염기서열과 159개의 아미노산으로 구성되어 있었다. 그 중 signal peptide는 아미노 말단의 17개 아미노산으로 구성되어 있었고, signal peptide를 제외한 incilarin A를 his6-tag를 표지하여 pET44a vector에 삽입하여 E. coli(DE3)에 발현하였다. 그 결과 447 bp의 염기서열과 149 아미노산 서열의 재조합 Incilarin A-유사 단백질을 정제하였다. Incilarin A-유사 단백질이 C-type lectin domain을 가지는 것을 확인했고, C-type lectin의 특성인 항균활성에 대해 분석연구를 진행하였다. 그람 음성균(V. harveyi, E. coli)와 그람 양성균(B. subtilis, S. parauberis)에서의 ampicillin과 최소 저해농도를 비교한 결과, incilarin A-유사 단백질 2 uM과 ampicillin 1.4 uM 의 활성이 유사하였다. Incilarin A-유사 단백질과 그람 음성균, 양성균과의 결합을 확인하였고, Ca2+에 의존하여 응집함을 확인하였다. Incilarin A-유사 단백질과 DNA의 결합에 의해 DNA 저해가 일어나는 것을 확인했고, 세포 외막 투과 실험을 통해 세포외막 파괴활성은 없는 것으로 확인되었다.