Advanced

C-terminal TFPI-1-derived peptides: their evolutionary host defense functions

Alyafei, Saud (2016) MOBN01 20161
Degree Projects in Molecular Biology
Abstract
Antimicrobial peptides (AMPs) are evolutionarily ancient molecules. They are essential innate immune response molecules that protect organisms from invading pathogens. Tissue factor pathway inhibitors (TFPI-1 and TFPI-2) C-terminal derived peptides exert host defense mechanisms against microbes. Studies showed that in human plasma, C-terminal TFPI-1 and -2 derived peptides mediate bacterial killing by boosting complement. In the present study, we showed the mechanism behind the classical complement activation by the TFPI-1 C-terminal GGL27 peptide. Protein-peptide interaction studies revealed that GGL27 peptide binds to IgM, IgG and to all IgG subunits. Importantly, IgG-GGL27 peptide interactions are facilitated through the Fc binding... (More)
Antimicrobial peptides (AMPs) are evolutionarily ancient molecules. They are essential innate immune response molecules that protect organisms from invading pathogens. Tissue factor pathway inhibitors (TFPI-1 and TFPI-2) C-terminal derived peptides exert host defense mechanisms against microbes. Studies showed that in human plasma, C-terminal TFPI-1 and -2 derived peptides mediate bacterial killing by boosting complement. In the present study, we showed the mechanism behind the classical complement activation by the TFPI-1 C-terminal GGL27 peptide. Protein-peptide interaction studies revealed that GGL27 peptide binds to IgM, IgG and to all IgG subunits. Importantly, IgG-GGL27 peptide interactions are facilitated through the Fc binding region. Furthermore, we showed that complement-mediated killing against E. coli by TFPI-1-derived peptides is evolutionarily conserved only in mammals except rodents, but not in reptiles, birds and fish due to absence of IgG. This complement-mediated killing is diminished when IgG is deactivated. Moreover, boosting of complement on bacterial surface by GGL27 shows an augmentation of phagocytosis activity. A previous study showed that GGL27 has an anti-coagulant activity in human plasma by blocking the intrinsic pathway, here we display the presence of anti-coagulant activity in mammalian and reptile peptides, while bird and fish peptides lack anti-coagulant activity. Finally, in vivo we showed that mouse TFPI-2-derived peptide (VKG24) is protective against bacterial infection. These findings conclude that vertebrate TFPI-derived peptides may exert a potential therapeutic approach against bacterial infections. (Less)
Popular Abstract
C-terminal TFPI-1 derived peptides: their evolutionary host defense functions

Antimicrobial peptides (AMPs) are evolutionarily ancient molecules. They function as innate host defense molecules by attacking pathogens by several mechanisms. These days AMPs are used as antibiotics to treat bacterial infections. The Tissue Factor Pathway Inhibitor (TFPI) is a serine protease inhibitor. Its carboxyl terminal end is cleaved by host derived enzymes such as; plasmin, thrombin and matrix metalloprotnaise-8 to produce C-terminal derived peptides. The C-terminal TFPI-1-derived peptide GGL27 is a multi-functional molecule. It exerts an antimicrobial activity by a direct killing as well as complement-mediated killing of gram-positive, gram-negative... (More)
C-terminal TFPI-1 derived peptides: their evolutionary host defense functions

Antimicrobial peptides (AMPs) are evolutionarily ancient molecules. They function as innate host defense molecules by attacking pathogens by several mechanisms. These days AMPs are used as antibiotics to treat bacterial infections. The Tissue Factor Pathway Inhibitor (TFPI) is a serine protease inhibitor. Its carboxyl terminal end is cleaved by host derived enzymes such as; plasmin, thrombin and matrix metalloprotnaise-8 to produce C-terminal derived peptides. The C-terminal TFPI-1-derived peptide GGL27 is a multi-functional molecule. It exerts an antimicrobial activity by a direct killing as well as complement-mediated killing of gram-positive, gram-negative and fungi. It also has an anti-coagulant effect by prolongation of coagulation time in human plasma.

The aim of the study is to highlight the importance of evolution on the activity of TFPI-1-derived peptides and the molecular mechanism behind complement activation. Viable count assay was performed to test the presence of complement-mediated killing of E. coli by TFPI-1-derived peptides in different type of species. Furthermore, western blot analysis was conducted to investigate the binding of GGL27 to IgM, IgG subclasses and its specific binding site on IgG, which might be the factor in classical complement pathway activation. Again, viable count assay was used to test the relation between GGL27 and IgG binding to mediate complement killing of bacteria. To examine the effect of GGL27 on complement-related responses, phagocytosis assay was performed. In addition, an activated partial thromboplastin time (aPPT) assay was performed to investigate the presence of anti-coagulation function by TFPI-1-derived peptides of different type of species. Finally, in a therapeutic context, we used a mouse TFPI-2-derived peptide (VKG24) in E. coli infection mouse model.

We found out that only mammalian TFPI-1-derived peptides have a complement-mediated killing against bacteria, but not birds, reptiles and fish peptides. This function is dependent on the binding of TFPI-1-derived peptides to IgG, which in turn leads to the augmentation of classical complement pathway, resulting in bacterial killing. We showed that GGL27 clearly binds to IgM and all IgG subclasses via the Fc region by western blot analysis. These results indicate that this binding might occur in vivo and contribute in boosting the classical complement pathway. When IgG was deactivated (by IdeS enzyme), GGL27 did not show any killing of bacteria, this experiment supports that the complement-mediated killing of bacteria is actually dependent on the binding of GGL27 to IgG. Since, GGL27 enhances classical complement pathway, we showed that it also has an effect on phagocytosis. GGL27 significantly increased the phagocytosis activity of murine macrophages to E. coli particles when E. coli particles were incubated with GGL27. In addition to the antimicrobial activity, mammalian and reptiles TFPI-1-derived peptides showed an anti-coagulation function, where they inhibited the intrinsic pathway which led to prolongation of coagulation time. In vivo, TFPI-2-derived peptide VKG24 decreased inflammatory responses as indicated by the decline of ROS production in treated mice. Also, a systemic bacterial killing was observed in organs such as; liver, kidney, spleen, lung and blood of treated mice, providing a promising future therapeutic application.

Advisor: Praveen Papareddy
Master’s Degree Project in Molecular Biology, 45 credits
Department of Biology, Lund University (Less)
Please use this url to cite or link to this publication:
author
Alyafei, Saud
supervisor
organization
course
MOBN01 20161
year
type
H2 - Master's Degree (Two Years)
subject
language
English
id
8893108
date added to LUP
2016-10-06 12:08:15
date last changed
2016-10-06 12:08:15
@misc{8893108,
  abstract     = {Antimicrobial peptides (AMPs) are evolutionarily ancient molecules. They are essential innate immune response molecules that protect organisms from invading pathogens. Tissue factor pathway inhibitors (TFPI-1 and TFPI-2) C-terminal derived peptides exert host defense mechanisms against microbes. Studies showed that in human plasma, C-terminal TFPI-1 and -2 derived peptides mediate bacterial killing by boosting complement. In the present study, we showed the mechanism behind the classical complement activation by the TFPI-1 C-terminal GGL27 peptide. Protein-peptide interaction studies revealed that GGL27 peptide binds to IgM, IgG and to all IgG subunits. Importantly, IgG-GGL27 peptide interactions are facilitated through the Fc binding region. Furthermore, we showed that complement-mediated killing against E. coli by TFPI-1-derived peptides is evolutionarily conserved only in mammals except rodents, but not in reptiles, birds and fish due to absence of IgG. This complement-mediated killing is diminished when IgG is deactivated. Moreover, boosting of complement on bacterial surface by GGL27 shows an augmentation of phagocytosis activity. A previous study showed that GGL27 has an anti-coagulant activity in human plasma by blocking the intrinsic pathway, here we display the presence of anti-coagulant activity in mammalian and reptile peptides, while bird and fish peptides lack anti-coagulant activity. Finally, in vivo we showed that mouse TFPI-2-derived peptide (VKG24) is protective against bacterial infection. These findings conclude that vertebrate TFPI-derived peptides may exert a potential therapeutic approach against bacterial infections.},
  author       = {Alyafei, Saud},
  language     = {eng},
  note         = {Student Paper},
  title        = {C-terminal TFPI-1-derived peptides: their evolutionary host defense functions},
  year         = {2016},
}