The Antimicrobial Activity Of Human High Mobility Group Protein 17 Isolated From Human LAK Cells

Human lymphokine-activated killer cells (LAK) have been proved useful to therapy of tumor and intracellular infections, in this study, our objective was to identify new antibacterial peptides from human LAK cells. Human LAK cells were obtained by culturing human peripheral blood mononuclear cells in the presnence of rIL-2 and PHA for one week. Acid soluble proteins of LAK cells were prepared by homogenizing the cells with 5% acetic acid. Using AU-PAGE and gel overlay assay, three main protein bands that exhibited antibacterial activity, named HLP-1, HLP-2, HLP-3 (Human Lymphocyte Peptides, HLPs) were isolated. The three main antibacterial components were further purified by RP-HPLC. Tricine-SDS-PAGE with silver staining showed that HPLC fraction 21 ( HLP-3 P21) that had potent antibacterial activity was of a purified single polypeptide with a apparent molecular weight of 16-17 kDa. N-terminal sequence of this polypeptide was as foloows: PKRKAGEDAK. The N-terminal amino acid sequences of HLPS-21 were analysed through NCBI BLAST to find out related proteins or mRNAs. In the data bank of BLAST, there were 9 related proteins with the same N-terminal amino acid sequences and 5'oligo nucleotide sequences. One is HMG-17 and the other four are similar protein of HMG-17, another four are hypotheticproteins deduced from human mRNAs. Race-PCR was used to clone the full length of HLP-3P21 cDNA. Three different clloones with 11 OObp, 750bp, and 500bp respectively were obteined. Nucleotide sequences analysis showed that only the clone with 1100bp was the coding sequence of HLP-3P21, and the other two (750bp, 500bp) were nonspecific polymerasing. Besides, the sequence of 1100bp was identical to HMG-17. Thus, the antibacterial peptide HLP-3P21 was HMG-17. To study HMG-17 the relationship of structure and antibacterial function, we constructed HMG-17 and its a-helic domain prokariotic expression vectors pGEX-1 A T-HMG17 and pGEX-1 X T-HMG17 a . HMG17 and HMG17 a-helic domain were isolated and purified from their constructs-transformed E.coli. The MIC and MBC antibacterial assays showed that HMG17 and HMG17 a-helic domain were both negative bacterial specific, and had no effects on positive bacterial and fungi at the same concentrations. At the same mole concentrations, no difference in MIC system occurred beteewn HMG-17 and HMG a-helic domain, however, the bactericidal activity of HMG17 was two-fold higher than that of HMG 17 a-helic domain. This results suggested that HMG-17 a-helic domain that is described as the DNA-binding domain would be essential in the antibacterial activity of HMG-17 via a nonlytic mechanisms as does the lenear proline/arginine-rich bactennecins whose antibacterial activity also centred on Gram-negative species by interrupting both DNA and protein synthesis.