Melittin-Hybrid 합성 폡타이드가 Fusarium oxysporum의 성장에 미치는 저해효과

이동건,신송엽,이성구,이명규,함경수 한국미생물생명공학회 ( 구 한국산업미생물학회 ) 1996 한국미생물·생명공학회지 Vol.24 No.5

꿀벌의 독액으로부터 분리된 ME은 강항 항균활성을 가지나, 진핵세포에 대하여 세포독성 활성을 포함하고 있다. 본 연구에서는 구조와 항진균활성과 상관관계를 검토하며, 세포독성을 가지지 않으며 보다 강한 항진균활성을 가진 펩타이드의 디자인하기 위하여, ME와 CA 또는 MA으로 이루어진 hybid 펩타이드인 MA(10-17)ME(1-12) 및 CA(1-8)ME(1-12)을 고상합성법의 의하여 합성 하였다. CA(1-8)ME(1-12) 및 MA(10-17)ME(1-12)는 인간의 적혈구에 대하여 용혈현상을 나타내지 않으며, Fusarium oxysporum에 대하여 ME 만큼의 강한 항진균활성을 나타내었다. 또한 이들 hybrid 펩타이드는 (1,3)-β-D-glucan synthase의 활성을 강하게 억제하였다. 이 결과는 Fusarium oxysporum에 대한 hybrid 펩타이드의 활성은 균의 세포벽의 합성의 억제에 의한 것과 관련성이 있는 것을 시사한다. 또한 본 연구의 결과는 세포독성을 가지며 강한 항진균활성을 가지는 펩타이드의 설계에 기초를 제공하였다고 생각된다. Melittin (ME) from honeybee venom has a broad range of strong antimicrobial activity, but it has hemolytic activity against eukaryotic cells. In order to design peptides with powerful antifungal activity without cytotoxic property of ME and understand structure-antifungal activity relationships, the hybrid peptides derived from the sequences of ME and cecropin A (CA) or magainin 2 (MA), MA(10-17) ME(1-12) and CA(1-8)ME(1-12), were synthesized by solid phase method. MA(10-17)ME(1-12) showed potent antifungal activity comparable to ME against Fusarium oxysporum with no hemolytic activity against human red blood cells. The hybrid peptides showed strong inhibition of (1,3)-β-D-glucan synthase. This result indicates that the antifungal activity of the hybrid peptides against Fusarium oxysporum is attributed to the inhibition of cell wall synthesis. The results therefore showed a successful design of a peptide having antifungal activity without hemolytic property.

Chemical Synthesis and Determination of Biological Activity of the Epidermal Growth Factor-Like Domain of Mouse Betacellulin

Shin, Song-Yub,Kang, Shin-Won,Ha, Jong-Myung Korean Society for Biochemistry and Molecular Biol 1995 Journal of biochemistry and molecular biology Vol.28 No.2

To investigate the biological functions of the EGF-like domain of mouse betacellulin (BTC), mouse BTC(33-80), a 48-residue peptide corresponding to the EGF-like domain, was synthesized by stepwise solidphase methods using a 9-fluorenylmethoxycarbonyl (Fmoc) strategy. The homogeneity of synthetic mouse BTC(33-80) was confirmed by analytical reversed phase (RP)-HPLC, amimo acid analysis, and fast atom bombardment mass spectrometer (FAB-MS). Three disulfide bond pairings of synthetic mouse BTC(33-80) were established by amino acid analysis of cysteine-containing fragments derived from thermolytic digestion. These were consistent with the pairings of EGF and transforming growth factor ($TGF-{\alpha}$). The EGF-Iike domain of mouse BTC showed equipotent activity in both EGF-receptor binding on A-431 epidermoid carcinoma cells, and mitogenesis on NIH-3T3 fibroblast cells, as compared with authentic h-EGF. Results suggest that the EGF-Iike domain of BTC plays a significant role in mitogenic activity with an EGF-receptor mediated system.

Effect of Double Replacement of L-Pro, D-Pro, D-Leu or Nleu in Hydrophobic Face of Amphipathic α-Helical Model Antimicrobial Peptide on Structure, Cell Selectivity and Mechanism of Action

Shin, Song Yub Korean Chemical Society 2014 Bulletin of the Korean Chemical Society Vol.35 No.11

In order to investigate the effects of the double replacement of $\small{L}$-Pro, $\small{D}$-Pro, $\small{D}$-Leu or Nleu (the peptoid residue for Leu) in the hydrophobic face (positions 9 and 13) of amphipathic ${\alpha}$-helical non-cell-selective antimicrobial peptide $L_8K_9W_1$ on the structure, cell selectivity and mechanism of action, we synthesized a series of $L_8K_9W_1$ analogs with double replacement of $\small{L}$-Pro, $\small{D}$-Pro, $\small{D}$-Leu or Nleu in the hydrophobic face of $L_8K_9W_1$. In this study, we have confirmed that the double replacement of $\small{L}$-Pro, $\small{D}$-Pro, or Nleu in the hydrophobic face of $L_8K_9W_1$ let to a great increase in the selectivity toward bacterial cells and a complete destruction of ${\alpha}$-helical structure. Interestingly, $L_8K_9W_1$-$\small{L}$-Pro, $L_8K_9W_1$-$\small{D}$-Pro and $L_8K_9W_1$-Nleu preferentially interacted with negatively charged phospholipids, but unlike $L_8K_9W_1$ and $L_8K_9W_1$-$\small{D}$-Leu, they did not disrupt the integrity of lipid bilayers and depolarize the bacterial cytoplasmic membrane. These results suggested that the mode of action of $L_8K_9W_1$-$\small{L}$-Pro, $L_8K_9W_1$-$\small{D}$-Pro and $L_8K_9W_1$-Nleu involves the intracellular target other than the bacterial membrane. In particular, $L_8K_9W_1$-$\small{L}$-Pro, $L_8K_9W_1$-$\small{D}$-Pro and $L_8K_9W_1$-Nleu had powerful antimicrobial activity (MIC range, 1 to $4{\mu}M$) against methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Pseudomonas aeruginosa (MDRPA). Taken together, our results suggested that $L_8K_9W_1$-$\small{L}$-Pro, $L_8K_9W_1$-$\small{D}$-Pro and $L_8K_9W_1$-Nleu with great cell selectivity may be promising candidates for novel therapeutic agents, complementing conventional antibiotic therapies to combat pathogenic microorganisms.

Structure and Antibiotic Activity of a Porcine Myeloid Antibacterial Peptide, PMAP-23 and its Analogues

Shin, Song-Yub,Kang, Joo-Hyun,Jang, So-Yun,Kim, Kil-Lyong,Hahm, Kyung-Soo Korean Society for Biochemistry and Molecular Biol 2000 Journal of biochemistry and molecular biology Vol.33 No.1

PMAP-23 is a 23-residue antimicrobial peptide derived from porcine myloid cells. In order to investigate the effects of two Pro residues at positions 12 and 15 of PMAP-23 on antibiotic activity, two analogues in which Ala was substituted for Pro residue at position 12 or 15 were synthesized. $Pro^{12}{\rightarrow}Ala$ (PMAPl) or $Pro^{15}{\rightarrow}Ala$(PMAP2) substitution in PMAP-23 caused a significant reduction on antitumor and phospholipid vesicle-disrupting activities, but did not cause a significant effect on antibacterial activity. PMAP-23 displayed the type I ${\beta}-turn$ structure with a negative ellipticity at near 205 om in SDS micelle, whereas PMAP1 and PMAP2 had a somewhat ${\alpha}-helical$ propensity in TFE solution, as compared to PMAP-23. These results suggest that two Pro residues of positions 12 and 15 in PMAP-23 play important roles in the formation of ${\beta}-turn$ structure on lipid membrane and its ${\beta}-turn$ structure may be essential for antibiotic activity including phospholipid vesicle-disrupting property.

Characterization of KI-24, a Novel Murine Monoclonal Antibody with Specific Reactivity for the Human Immunodeficiency Virus-1 p24 Protein

Shin, Song-Yub,Park, Jung-Hyun,Lee, Myung-Kyu,Jang, So-Youn,Hahm, Kyung-Soo Korean Society for Biochemistry and Molecular Biol 2000 Journal of biochemistry and molecular biology Vol.33 No.1

The HIV-1 p24(202-221) sequence ETINNEEEWDRVHPV HAGP contains a B-cell epitope with the earliest immune response and the highest antibody titer against anti-mouse sera obtained by immunization with p24 antigens. A novel mouse monoclonal antibody (mAb) was generated against the immunodominant B-cell epitope of the HIV-1 p24 capsid protein, p24(202-221). BALB/c mice were immunized with the four branched multiple antigenic peptide (MAP) containing the HIV-1p24(202-221) sequence, and antibody-secreting hybridoma were produced by fusion of mouse splenocytes with P3X63Ag8.653, mouse myeloma cells. One clone which produced the antigen-specific mAb named KI-24 (Isotype IgG1, light chain: ${\kappa}$) was identified. mAb KI-24 was highly specific for both the p24(202-221) and p24 proteins when analyzed by ELISA and Western blotting. Since p24(202-221) also contains a cytotoxic T-lymphocyte epitope, this specfic peptide epitope and the monoclonal antibody with specific reactivity against the p24 protein and p24(202-221) can be used in peptide vaccine development and p24 antigen detection from HIV patients.

Effects of the Hinge Region of Cecropin A(1-8)-Melittin 2(1-12), a Synthetic Antimicrobial Peptide on Antibacterial, Antitumor, and Vesicle-Disrupting Activity

Shin, Song-Yub,Kang, Joo-Hyun,Jang, So-Yun,Kim, KiI-Lyong,Hahm, Kyung-Soo Korean Society for Biochemistry and Molecular Biol 1999 Journal of biochemistry and molecular biology Vol.32 No.6

CA(1-8)-ME(1-12) [CA-ME], composed of cecropin A(1-8) and melittin(1-12), is a synthetic antimicrobial peptide having potent antibacterial and antitumor activities with minimal hemolytic activity. In order to investigate the effects of the flexible hinge sequence, Gly-Ile-Gly, of CA-ME on antibiotic activity, CA-ME and three analogues, CA-ME1, CA-ME2, and CA-ME3, were synthesized. The Gly-Ile-Gly sequence of Ca-ME was deleted in CA-ME1 and replaced with Pro and Gly-Pro-Gly in CA-ME2 and CA-ME3, respectively. CA-ME1 and CA-ME3 showed a significant decrease in antitumor activity and phospholipid vesicle-disrupting ability. However, CA-ME2 showed similar antitumor and vesicle-disrupting activities, as compared with CA-ME. These results suggest that the flexibility or ${\beta}$-turn induced by Gly-Ile-Gly or Pro in the central part of CA-ME may be important in the electrostatic interaction of the N-terminus cationic ${\alpha}$-helical region with the cell membrane surface and the hydrophobic interaction of the C-terminus amphipathic ${\alpha}$-helical region with the hydrophobic acyl chains in the cell membrane. CA-ME3 exhibited lower antitumor and vesicle-disrupting activities than CA-ME and CA-ME2. This result suggests that the excessive ${\beta}$-turn structure caused by the Gly-Pro-Gly sequence in CA-ME3 seems to interrupt ion channel/pore formation in the lipid bilayer. We concluded that the appropriate flexibility or bilayer. We concluded that the appropriate flexibility or ${\beta}$-turn structure provided by the central hinge is responsible for the effective antibiotic activity of the antimicrobial peptides with the helix-hinge-helix structure.

ABSTRACTS ; STRUCTURE - ACTIVITY RELATIONSHIPS OF HUMAN EPIDERMAL GROWTH FACTOR AND EGF - LIKE DOMAIN IN EGF FAMILY PROTEINS

Song Yub Shin,Eisuke Munekata 생화학분자생물학회(구 한국생화학분자생물학회) 1995 추계학술대회집 Vol.- No.-

Characterization of KI - 24 , a Novel Murine Monoclonal Antibody with Specific Reactivity for the Human Immunodeficiency Virus - 1 p24 Protein

Shin, Song Yub,Lee, Myung Kyu,Hahm, Kyung Soo,Park, Jung Hyun,Jang, So Youn 생화학분자생물학회 2001 BMB Reports Vol.33 No.1

The HIV-1 p24(202-221) sequence ETINNEEEWDRVHPV HAGP contains a B-cell epitope with the earliest immune response and the highest antibody titer against anti-mouse sera obtained by immunization with p24 antigens. A novel mouse monoclonal antibody (mAb) was generated against the immunodominant B-cell epitope of the HIV-1 p24 capsid protein, p24(202-221). BALBlc mice were immunized with the four branched multiple antigenic peptide (MAP) containing the HIV-1 p24(202-221) sequence, and antibodysecreting hybridoma were produced by fusion of mouse splenocytes with P3X63Ag8.653, mouse myeloma cells. One clone which produced the antigen-specific mAb named KI-24 (Isotype IgG1, light chain: k) was identified. mAb KI-24 was highly specific for both the p24(202-221) and p24 proteins when analyzed by ELISA and Western blotting. Since p24(202-221) also contains a cytotoxic T-lymphocyte epitope, this specfic peptide epitope and the monoclonal antibody with specific reactivity against the p24 protein and p24(202-221) can be used in peptide vaccine development and p24 antigen detection from HIV patients.

Structure and Antibiotic Activity of a Porcine Myeloid Antibacterial Peptide , PMAP - 23 and its Analogues

Shin, Song Yub,Kim, Kil Lyong,Hahm, Kyung Soo,Kang, Joo Hyun,Jang, So Yun 생화학분자생물학회 2001 BMB Reports Vol.33 No.1

PMAP-23 is a 23-residue antirnicrobial peptide derived from porcine myloid cells. In order to investigate the effects of two Pro residues at positions 12 and 15 of PMAP-23 on antibiotic activity, two analogues in which Ala was substituted for Pro residue at position 12 or 15 were synthesized. Pro^(12)→Ala (PMAP1) or Pro^(15)→Ala (PMAP2) substitution in PMAP-23 caused a significant reduction on antitumor and phospholipid vesicle-disrupting activities, but did not cause a significant effect on antibacterial activity. PMAP-23 displayed the type I β-turn structure with a negative ellipticity at near 205 nm in SDS micelle, whereas PMAP1 and PMAP2 had a somewhat a-helical propensity in TFE solution, as compared to PMAP-23. These results suggest that two Pro residues of positions 12 and 15 in PMAP-23 play important roles in the formation of β-turn structure on lipid membrane and its S-turn structure may be essential for antibiotic activity including phospholipid vesicle-disrupting property:

Characterization and Epitope Mapping of KI - 41 , a Murine Monoclonal Antibody Specific for the gp41 Envelope Protein Protein of the Human Immunodeficiency Virus - 1

Shin, Song Yub,Lee, Myung Kyu,Hahm, Kyung Soo,Park, Jung Hyun,Jang, So Youn 생화학분자생물학회 1999 BMB Reports Vol.31 No.1

In this study, a mouse monoclonal antibody (mAb) against gp41(584-618), the immunodominant epitope of HIV-1 gp41 envelope protein, was generated. For this purpose, BALB/c mice were immunized with double branched multiple antigenic peptides derived from the HIV-1 gp41(584-618) sequence, and antibody-secreting hybridoma were produced by fusion of mice splenocytes with SP2/0 myeloma cells. One clone producing an antigen specific mAb, termed KI-41 (isotype IgGl) was identified, whose specific reactivity against gp4l(584-618) could be confirmed by ELISA and Western blot analysis. Epitope mapping revealed the recognition site of the mAb KI-41 to be located around the sequence RILAVERYLKDQQLLG, which comprises the N-terminal region within the immunized gp41(584-618) peptide. Since this mAb recognizes this specific epitope within the HIV-1 gp41 without any cross-reactivity to other immunodominant regions in the HIV-2 gp36, KI-41 will provide some alternative possibilities in further applications such as the development of indirect or competitive ELISA for specific antibody detection in HIV-1 infection or for other basic researches regarding the role and function of HIV-1 gp41.