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Role of Synthetic Proteins and Peptides in Animals

The document discusses the role of cationic antimicrobial peptides in animal defenses. It describes how such peptides are produced in animals and play an important role in innate host defenses against bacteria. They are able to kill bacteria rapidly, prevent endotoxin induction, and block sepsis in animal models. The document also discusses the potential of antimicrobial peptides as novel antibiotic therapeutics due to their broad spectrum of activity and ability to kill bacteria and neutralize endotoxins.

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124 views7 pages

Role of Synthetic Proteins and Peptides in Animals

The document discusses the role of cationic antimicrobial peptides in animal defenses. It describes how such peptides are produced in animals and play an important role in innate host defenses against bacteria. They are able to kill bacteria rapidly, prevent endotoxin induction, and block sepsis in animal models. The document also discusses the potential of antimicrobial peptides as novel antibiotic therapeutics due to their broad spectrum of activity and ability to kill bacteria and neutralize endotoxins.

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roni
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ROLE OF SYNTHETIC PROTEINS\PEPTIDES

IN ANIMALS

The role of antimicrobial peptides in animal defenses:


It is becoming clear that the cationic antimicrobial peptides are an important
component of the innate defenses of all species of life. Such peptides can
be constitutively expressed or induced by bacteria or their products. The
best peptides have good activities vs. a broad range of bacterial strains,
including antibiotic-resistant isolates. They kill very rapidly, do not easily
select resistant mutants, are synergistic with conventional antibiotics, other
peptides, and lysozyme, and are able to kill bacteria in animal models. It is
known that bacterial infections, especially when treated with antibiotics, can
lead to the release of bacterial products such as lipopolysaccharide (LPS)
and lipoteichoic acid, resulting in potentially lethal sepsis. In contrast to
antibiotics, the peptides actually prevent cytokine induction by bacterial
products in tissue culture and human blood, and they block the onset of
sepsis in mouse models of endotoxemia.

The Nature of Cationic Antimicrobial Peptides:

We use the term (cationic) antimicrobial peptides to describe gene-


encoded peptides comprising between 12 and 50 amino acids, with at least
two excess positive changes due to lysine and arginine residues and
around 50% hydrophobic amino acids. They are found in all species of life,
ranging from plants and insects to animals, including molluscs,
crustaceans, amphibians, birds, fish, mammals, and humans. More than
500 such peptides have been discovered. They fit into at least four
structural classes, namely β-sheet, comprising two to three β-strands
stabilized by disulphide bridges, amphipathic α-helices, extended
structures, and loop structures (Table 1).
Some representative mammalian peptides

Peptide Class Amino acid sequence *†

HNP-1
(α- β-
AC YC RIPAC IAGERRYGTC IYQGRLWAFC C
defensi sheet
1 2 3 3 2 1

n)

HBD-2
(β- β- MRVLYLLFSFLFIFLMPLPGVFGGIGDPVTC LKSGAIC HPVFC P 1 2 3

defensi sheet RRYKQIGTC GLPGTKC C KKP


2 1 3

n)

Protegr β-
RGGRLC YC RRRFC VC VGR
in sheet
1 2 1 2

Indolic Exten
ILPWKWPWWPWRR-NH
idin ded
2

Exten
Bac5 RFRPPIRRPPIRPPFYPPFRPPIRPPIFPPIRPPFRPPLGPFP
ded

Bacteni
Loop RLC RIVVIRVC R
cin
1 1

α-
LL-37 helic LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES
al

α-
Cecropi
helic SWLSKTAKKLENSAKKRISEGIAIAIQGGPR
n P1
al

In addition to these classes, there is a variety of animal cationic proteins


including bactericidal permeability increasing protein, lactoferrin, transferrin,
cathepsin G, cystatin, CAP18, pepsinogen C, ribosomal protein S30, etc.,
whose antibacterial activities can be traced to a cationic peptide sequence
within these basic proteins.
Mechanism of Production in Animals:

The mucous layer of animals covers and protects all epithelial tissues
against microbial, mechanical, and chemical insults and forms an initial line
of defense. The main constituents are the mucins and other glycoproteins,
proteinase inhibitors, and cationic peptides, like defensins. Healthy-tissue
epithelial cells have been shown to express β-defensin genes at a low
level. However, some defensin genes can be induced on treatment with
proinflammatory cytokines, LPS, or bacteria. For example, production of
human β-defensin (HBD)-2 in keratinocytes is strongly induced on contact
of keratinocytes with Gram-negative bacteria or proinflammatory cytokines
such as tumor necrosis factor (TNF)-α or IL-1β. HBD-2 mRNA expression
has been observed in the skin, foreskin, lungs, and trachea, but not in the
kidney, salivary glands, small intestine, and liver. This is in contrast to
HBD-1, which is constitutively expressed mainly in the urogenital tract and
kidney.

Evidence for Their Role in Host Defenses:

The evidence for a role of cationic antimicrobial peptides in innate host


defenses has become quite convincing. It includes data demonstrating that
some peptides are inducible by bacterial products and convincing animal
model and transgenic animal experiments indicating that the peptides
protect against infection in experimental animals. The inducibility of
peptides has been summarized above. The kinetics of induction are highly
suggestive of a role in early defenses against infection.

Role in Counteracting Sepsis:

More than a half million patients suffer from sepsis every year in North
America. Sepsis is associated with the presence of pathogenic
microorganisms or their toxins in the blood. It can result from infections with
either Gram-negative or Gram-positive bacteria. Gram-negative sepsis is
usually caused by the release of a bacterial outer membrane component,
endotoxin (LPS). The toxicity of LPS is contained within its lipid A portion.
Gram-positive sepsis is also presumed to be caused by the release of
bacterial cell wall components. A number of Gram-positive cell wall
constituents, including lipoteichoic acid (LTA), peptidoglycan
(PG), Streptococcus rhamnose-glucose polymer,
and Staphylococcus capsular polysaccharide, have been shown to
stimulate the production of inflammatory mediators in vitro.

The mechanism by which LPS activates macrophages is now understood


in some detail. LPS-binding protein (LBP), an acute-phase reactant that is
present in the blood, binds LPS and transfers it to CD14, a protein that
exists as a soluble form in blood and as a glycosyl phosphatidylinositol-
linked molecule on the surface of monocytes and macrophages. LPS⋅CD14
complexes are thought to initiate intracellular signaling reactions by binding
to Toll-like receptors (TLRs) on macrophages and other cells. TLR4
appears to be required for LPS to initiate signaling and to induce
inflammatory responses. LPS⋅CD14 complexes cause activation of the NF-
κB transcription factor as well as activation of the ERK, JNK, and p38
mitogen-activated protein kinases, all of which mediate the production of
inflammatory cytokines. Despite their structural differences, both LTA and
PG also activate macrophages and polymorphonuclear leukocytes in
association with CD14 and TLR4/TLR2.

Potential as Therapeutics:

There is no question that, with the increasing antibiotic resistance problem,


there is a need to develop new classes of antibiotics. Cationic antimicrobial
peptides have many of the desirable features of a novel antibiotic class. In
particular, they have a broad spectrum of activity, kill bacterial rapidly, are
unaffected by classical antibiotic resistance mutations, do not easily select
antibiotic resistant variants, show synergy with classical antibiotics,
neutralize endotoxin, and are active in animal models. Despite this, many
issues remain to be solved. For example, these peptides have relatively
high molecular weights compared with most antibiotics and will have to be
produced recombinantly to keep prices down.

ENERGY-PROTEIN INTERRELATIONSHIPS

The utilization of dietary proteins must be put in the context of the available
energy supply. Energy is the main driving force of metabolism. If energy is
limiting dietary protein will be used inefficiently as another source of energy
instead of being converted into body protein.

Figure 1a shows the response of growing pigs given diets in which the
amount of protein, with a constant amino acid profile, was varied while
maintaining a constant energy supply by replacing starch with protein. In
addition, the diets were given at three levels of feeding which increased both
the protein and energy supply in a fixed ratio. Increasing protein from low
and limiting levels at constant energy increased protein deposition in the
carcase until energy limited the response. Giving more feed increased the
energy supply and allowed the response to dietary protein to continue until
the new energy level again became limiting. This will repeat until the genetic
potential of the animal or some other factor limits further protein accretion.

In vitro and in vivo toxicity studies

Determined the cytotoxic effects of peptides clavanin A and clavanin-


MO on mouse red blood cells (mRBCs), RAW264.7 macrophage, L929
mouse fibroblast cell lines and human embryonic kidney cells 293
(HEK-293). Cell viability, through detection of cellular oxidative
metabolism, was measured using the MTT assay after 24 hours of
treatment with different peptide concentrations. Similar to previous
studies, clavanin A showed no haemolytic activity and no significant
cytotoxic effects on cells in culture. Addition of the oligopeptide tag
did not result in increased toxicity, as clavanin-MO exhibited similar
levels of low toxicity as its parent peptide and did not cause
cytotoxicity towards RAW264.7, L929 cells and HEK-293 cells at the
concentrations required for antimicrobial and immunomodulatory
activity. Consistent with these in vitro results, neither clavanin A nor
clavanin-MO caused any apparent toxicity in mice treated
intraperitoneally with a dose as high as 50 mg.kg −1 of peptide (data
not shown), five times the maximum utilized here for successful anti-
infective therapy.

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