Epidermal Growth Factors (EGF) and Analogs
Definition
Epidermal Growth Factor (EGF) is part of a complex network of growth factors and receptors that together help to modulate the growth of cells. EGF is released by cells, and then is picked up either by the cell itself, stimulating its own growth, or by neighboring cells, stimulating their ability to divide 1.
Related Peptides
Depending on their binding specificities, EGF-related peptides can be subdivided into two classes. The first group of ligands binds to the EGFR (EGF receptors) and includes EGF itself, transforming growth factor (TGF) a, amphiregulin (AR), heparin-binding EGF-like growth factor (HB-EGF), and betacellulin (BTC). Each of these peptides competes with EGF for receptor binding and therefore this family of growth factors is referred to as the EGF agonists. Another family of EGF-related peptides is composed of the neu differentiation factors (NDFs)/heregulins ligands for ErbB-3 and ErbB-4. There are at least 12 different isoforms arising from a single gene by alternative splicing and depending on the sequence of their EGF-like repeat they are classified as either a or ß isoforms. However, despite the large number of NDFs no differences in receptor binding specificities appear to exist: ErbB-3 functions as a low affinity receptor for all NDF isoforms while ErbB-4 serves as a high affinity receptor 2.
Discovery
The discovery of EGF won Stanley Cohen a Nobel Prize in Physiology and Medicine in 1986 and was patented for cosmetic use by Greg Brown in 1989 .
Structural Characteristics EGF is a small mitogenic protein that is thought to be involved in mechanisms such as normal cell growth, oncogenesis, and wound healing. This protein shows both strong sequential and functional homology with human type-alpha transforming growth factor (Htgf-a), which is a competitor for EGF receptor sites. EGF is a small 53 amino acid residue long protein that contains three disulfide briges. The side chains of residues 13 (Tyr), 41 (Arg), and 47 (Leu) are all thought to play an important role in EGF's functionality 3.
Analogs
Three site-directed mutants of human epidermal growth factor, Leu-26-Gly, Leu-47-Ala, and Ile-23-Thr, were examined for their ability to stimulate the protein-tyrosine kinase activity of the epidermal growth factor receptor. The receptor binding affinities of the mutant growth factors were 20- to 50-fold lower, as compared to wild-type growth factor. At saturating concentrations of growth factor, the velocities of the phosphorylation of exogenously added substrate and receptor autophosphorylation were significantly lower with the mutant analogs, suggesting a partial 'uncoupling' of signal transduction. The mutant analogs were shown to compete directly with the binding of wild-type, resulting in a decrease in growth factor-stimulated kinase activity 4. Six mutants of human EGF, which carry single point substitutions within a surface patch proposed to juxtapose the bound receptor, were prepared and characterized for receptor affinity and mitogenicity. Receptor affinities relative to EGF are G12Q > H16D > Y13W > Q43A ˜ H16A ˜ EGF >> L15A. Notably, the reduced receptor affinity of mutant L15A indicates that Leu15 probably contributes substantially to receptor binding whereas unaltered receptor affinities observed for analogs H16A and Q43A indicate that neither His16 nor Gln43 contributes significantly to this interaction. On the other hand, the observed enhanced receptor affinities of analogs G12Q, Y13W and H16D highlight surface loci where additional productive receptor-binding contacts can be introduced. Interestingly, at acidic pH analog H16A reveals substantially greater receptor affinity than that of EGF, a property which may offer enhanced therapeutic utility in acidic environments in vivo 5.
Mode of Action
Receptors on the surface of the cell bind to EGF and relay the signal inside. When the receptor binds to EGF, it is activated by forming a dimer with other receptors. Four similar receptors have been discovered: the EGF receptor and three variants. These may dimerize with themselves, or mix-and-match to form heterodimers with the other types. The set of growth factors that interacts with these receptors is even more varied, with a dozen or so known examples, including EGF, transforming growth factor-a, and a number of neuregulins. The receptor is composed of a single chain with many functional parts. It is found in the cell membrane, with one portion facing out to receive the message and one portion facing inward to relay the message to the cell machinery. The outer portion forms an EGF-binding domain. It is composed of four articulated parts: two globular parts that grip EGF and two rod-shaped linkers that are rigidified by dozens of cysteine amino acids. When this multi-part domain binds to EGF, it changes shape, releasing one of the long, cysteine-rich sections. This allows the receptor to dimerize with other receptors. This brings the two kinase domains close to one another, allowing them to add phosphates to each other and activating the signaling process. Many hormone receptors act by binding to either side of a hormone, with the hormone in the center. The EGF receptor, surprisingly, forms dimers with the growth factors on opposite sides of the dimer, far from the point of contact between the two receptors6.
Functions
The EGFR signaling pathway is one of the most important pathways that regulate growth, survival, proliferation, and differentiation in mammalian cells6. In animal models of acute renal injury, the administration of epidermal growth factor, insulin-like growth factor I (IGF-I), or hepatocyte growth factor accelerates the restoration of kidney function and the normalization of histology post-acute renal injury and reduces mortality. The mechanisms by which the growth factors act in acute renal failure include the stimulation of anabolism, the maintenance of glomerular filtration, and the enhancement of tubular regeneration7.
References
1. Goodsell DS (2003). The molecular perspective: epidermal growth factor. The Oncologist, 8(5):496–497.
2. Beerli RR, Hynes NE (1996). Epidermal growth factor-related peptides activate distinct subsets of erbb receptors and differ in their biological activities. J Biol Chem., 271:6071-6076.
3. Montelione GT, Wuthrich K, Burgess AW, Nice EC, Wagner G, Gibson KD, Scheraga HA (1992). Solution structure of murine epidermal growth factor determined by NMR spectroscopy and refined by energy minimization with restraints. Biochemistry, 31:236-242.
4. Matsunami RK, Campion SR, Niyogi SK, Stevens A (1990). Analogs of human epidermal growth factor which partially inhibit the growth factor-dependent protein-tyrosine kinase activity of the epidermal growth factor receptor. Febs letters, 264(1):105-108.
5. Mullenbach GT, Chiu CY, Gyenes A, Blaney J, Rosenberg S, Marlowe CK, Brown S, Stratton-Thomas J, Montelione GT, George-Nascimento C, Stauber G (1998). Modification of a receptor-binding surface of epidermal growth factor (EGF): analogs with enhanced receptor affinity at low pH or at neutrality. Protein Engineering, 11(6):473–480.
6. Oda K, Matsuoka Y, Funahashi A, Kitano H (2005). A comprehensive pathway map of epidermal growth factor receptor signaling. Molecular Systems Biology, 1:2005.
7. Hammerman MR, Miller SB (1994). Therapeutic use of growth factors in renal failure. Journal of the American Society of Nephrology, 5:1-11.
Eosinophilotactic Tetrapeptides
Definition
Eosinophilotactic tetrapeptides exhibit peak in vitro chemotactic activity for human eosinophils and rapidly deactivate eosinophils to homologous and other stimuli at concentrations as low as 10 -10 M 1.
Related Peptides
The ECF-A acidic tetrapeptides Val-Gly-Ser-Glu, Ala-Gly-Ser-Glu and the analogue Val-Gly-Asp-Glu are selectively chemotactic for human eosinophils over a narrow dose range. Histamine abrogates the chemotactic properties of the individual tetrapeptides 2.
Discovery
The eosinophil chemotactic factor of anaphylaxis, ECF-A, was discovered in 1971 by Kay et al., as a mediator released during immediate-type hypersensitivity reactions in guinea pig and human lung slices 1.
Structural Characteristics
Two eosinophilotactic tetrapeptides of amino acid sequence Val-Gly-Ser-Glu and Ala-Gly-Ser-Glu were recovered from the extracts in 4-12% overall yield of the low molecular weight peak from Sephadex G-25. Purified eosinophil chemotactic factor of anaphylaxis and the synthetic tetrapeptides were maximally active in a chemotactic chamber, and the activity was dependent on both the NH2 terminal and the COOH-terminal residues. Both natural and synthetic peptides were preferentially chemotactic for eosinophils and rendered them unresponsive to a subsequent stimulus1.
Mode of Action
The chemotactic activity of the tetrapeptide is dependent on both the hydrophobic NH2-terminal residue, which interacts with a hydrophobic domain in the chemotactic receptor, and the highly-charged COOH-terminal residue which is presumed to initiate eosinophil movement by perturbing a polar domain in the same receptor. The spatial requirement for effective interaction with both domains in the receptor is revealed by the lower potency and activity of the condensed tripeptides lacking glycine. The 10-fold greater potency of NH2-terminal tripeptide compared to the amide derivatives of NH2-terminal amino acids in reversibly inhibiting the intact tetrapeptides suggests a role for serine in binding to a portion of the receptor, possibly by hydrogen bonding. The COOH-terminal substituent tripeptide irreversibly suppresses eosinophil chemotaxis by a cell-directed action possibly reflecting its capacity to perturb the polar domain; this effect, resembling de-activation, requires higher concentrations than needed for deactivation by the tetrapeptide 3.
Functions
ECF-A was discovered in 1971 as the mediator that is responsible for most of the eosinophil chemotactic activitv released during anaphylactic reactions. Of the other mediators of immediate hypersensitivity, only histamine stimulates directed migration of eosinophils in vitro; however, its action is transient and lacks apparent in vivo chemotactic activity 4.
References
1. Goetzl EJ, Austen KF (1975). Purification and synthesis of eosinophilotactic tetrapeptides of human lung tissue: Identification as eosinophil chemotactic factor of anaphylaxis (leukocyte chemotaxis/leukocyte deactivation/ anaphylactic mediators/acidic peptides of lung). PNAS., 72(10):4123-4127.
2. Turnbull LW, Evans DP, Kay AB (1977). Human eosinophils, acidic tetrapeptides (ECF-A) and histamine. Interactions in vitro and in vivo. Immunology, 32(1):57-63.
3. Goetzl EJ, Austen KF (1976). Structural determinants of the eosinophil chemotactic activity of the acidic tetrapeptides of Eosinophil Chemotactic Factor of Anaphylaxis. J Exp Med., 144:1424-1437.
4. Goetzl EJ (1976). Modulation of human eosinophil polymorphonuclear leukocyte migration and function. Am J Pathol., 85(2):419-436.
Enkephalins and Proenkephalins
Definition
Enkephalins are opioid peptides that are found at high levels in the brain and endocrine tissues. The major species of newly appearing enkephalin-containing peptide appears to be the intact precursor, proenkephalin 1.
Related Peptides
Opioid peptides constitute a large group of small proteins that interact with cell membrane receptors similarly to opiate alkaloids, morphine and heroin. Opiate alkaloid derivatives are extensively used for analgesia and anesthesia. The original opioid peptide families are enkephalins, dynorphins, and endorphins. Representative peptides from these three opioid peptide families have been found in the heart. Three different opiate receptors have been cloned and sequenced: mu (µ), delta (d), and kappa (?) 2.
Discovery
Kosterlitz and Hughes discovered enkephalins and endorphins in 1975 3.
Structural Characteristics
The amino acid sequence of methionine-enkephalin (Met-Enk) is tyrosine-glycine-glycine-phenylalanine-methionine. The proenkephalin sequence contains four copies of the pentapeptide Met-Enk, one of leu-enkephalin, and two extended forms of Met-enk (Met-enk-arg6-phe7 and met-enk-arg6-gly7-leu8). Pairs of basic amino acids mark these small peptides for cleavage from the precursor. Proenkephalin is processed by endoproteolytic enyzmes termed prohormone convertases, which recognize and cleave at dibasic amino acid sites. Initial proenkephalin processing starts before transport to the golgi network and are rapid. Later processing requires an acidic environment distal to the golgi network. Proenkephalin has a fast cleavage to peptide B, and slower cleavages yield other intermediate sized products that are cleaved ultimately to the penta to octapeptides. The different molecular-weight end products found in diverse tissues (muscle, neural, endocrine) may be due to variations in the cleavage sequence and local enzymatic conditions for processing 2.
Mode of Action
Pentapeptides Met-Enk and Leu-Enk, the endogenous ligands for the opiate receptor, function as neuromodulators or neurotransmitters. The most prominent action of enkephalins in the mammalian brain is depression of neuronal firing rate and it has been suggested that these peptides are inhibitory transmitters. The response of central neurones to several putative transmitter substances is depressed or enhanced by enkephalins, suggesting a postsynaptic action. It has also been shown that enkephalins suppress the K+-induced release of noradrenaline, dopamine and acetylcholine from rat brain slices, indicating a presynaptic effect. The firing of myenteric neurones in the guinea-pig ileum is inhibited by enkephalins. This inhibition is probably due to a direct postsynaptic action of the enkephalins resulting in a hyperpolarisation of the neuronal membrane 4. To achieve their biological function, enkephalins must be transported from an aqueous phase to the lipid-rich environment of their membrane bound receptor proteins. It is now known that Met-enk acts via three main subtypes of receptors referred to as µ, d and ? - receptors. While the first two receptor subtypes mediate the classic opioid effects of Met-enk, ?-receptors are reported to be involved in the non-opioid actions of the peptide, i.e. the inhibitory effect on the cell growth 5.
Functions
Proenkephalin is a precursor for neuropeptides with a variety of functions in the neuroendocrine and nervous systems. Upon activation, T-helper lymphocytes were found to express high levels of proenkephalin mRNA and to secrete large amounts of the Met-Enk neuropeptide, perhaps indicating an axis by which the immune and nervous systems interact 6. Enkephalins cause antinociception and potentiated morphine analgesia but they also block the development of tolerance and physical dependence. In addition to their central and peripheral antinociceptive function, opioids can modulate immune activity and cell proliferation. Moreover it is known that they have significant role in different physiological processes like cell differentiation and regeneration, inflammation, cancer and angiogenesis and analgesia effects 5.
References
1. Fleminger G, Lahm HW, Udenfriend S (1984).Changes in rat adrenal catecholamines and proenkephalin metabolism after denervation. PNAS., 81(11):3587-3590.
2. Barbara A. Barron. 2000. Cardiac Opioids. Proceedings of the Society for Experimental Biology and Medicine, 224:1-7.
3. Fratta W, Yang HY, Hong J, Costa E (1977). Stability of Met-enkephalin content in brain structures of morphine-dependent or foot shock-stressed rats. Nature, 268(5619):452-453.
4. Wouters W, Den Bercken JV (1979). Hyperpolarisation and depression of slow synaptic inhibition by enkephalin in frog sympathetic ganglion. Nature, 277:53-54.
5. Tsanova A, Dacheva D, Penchev V, Georgiev G, Pajpanova T, Golovinski E, Lalchev Z (2009). Comparative study of the interaction between synthetic methionine-enkephalin and monolayers of zwitterionic and negatively charged phospholipids. Biotechnol & Biotechnol., 23:463-466.
6. Rattner A, Korner M, Rosen H, Baeuerle PA, Citri Y (1991). Nuclear factor Kappa B activates proenkephalin transcription in T lymphocytes. Molecular and Cellular Biology, 11(2):1017-1022.
Enkephalins and Proenkephalins
Definition
Enkephalins are opioid peptides that are found at high levels in the brain and endocrine tissues. The major species of newly appearing enkephalin-containing peptide appears to be the intact precursor, proenkephalin 1.
Related Peptides
Opioid peptides constitute a large group of small proteins that interact with cell membrane receptors similarly to opiate alkaloids, morphine and heroin. Opiate alkaloid derivatives are extensively used for analgesia and anesthesia. The original opioid peptide families are enkephalins, dynorphins, and endorphins. Representative peptides from these three opioid peptide families have been found in the heart. Three different opiate receptors have been cloned and sequenced: mu (µ), delta (d), and kappa (?) 2.
Discovery
Kosterlitz and Hughes discovered enkephalins and endorphins in 1975 3.
Structural Characteristics
The amino acid sequence of methionine-enkephalin (Met-Enk) is tyrosine-glycine-glycine-phenylalanine-methionine. The proenkephalin sequence contains four copies of the pentapeptide Met-Enk, one of leu-enkephalin, and two extended forms of Met-enk (Met-enk-arg6-phe7 and met-enk-arg6-gly7-leu8). Pairs of basic amino acids mark these small peptides for cleavage from the precursor. Proenkephalin is processed by endoproteolytic enyzmes termed prohormone convertases, which recognize and cleave at dibasic amino acid sites. Initial proenkephalin processing starts before transport to the golgi network and are rapid. Later processing requires an acidic environment distal to the golgi network. Proenkephalin has a fast cleavage to peptide B, and slower cleavages yield other intermediate sized products that are cleaved ultimately to the penta to octapeptides. The different molecular-weight end products found in diverse tissues (muscle, neural, endocrine) may be due to variations in the cleavage sequence and local enzymatic conditions for processing 2.
Mode of Action
Pentapeptides Met-Enk and Leu-Enk, the endogenous ligands for the opiate receptor, function as neuromodulators or neurotransmitters. The most prominent action of enkephalins in the mammalian brain is depression of neuronal firing rate and it has been suggested that these peptides are inhibitory transmitters. The response of central neurones to several putative transmitter substances is depressed or enhanced by enkephalins, suggesting a postsynaptic action. It has also been shown that enkephalins suppress the K+-induced release of noradrenaline, dopamine and acetylcholine from rat brain slices, indicating a presynaptic effect. The firing of myenteric neurones in the guinea-pig ileum is inhibited by enkephalins. This inhibition is probably due to a direct postsynaptic action of the enkephalins resulting in a hyperpolarisation of the neuronal membrane 4. To achieve their biological function, enkephalins must be transported from an aqueous phase to the lipid-rich environment of their membrane bound receptor proteins. It is now known that Met-enk acts via three main subtypes of receptors referred to as µ, d and ? - receptors. While the first two receptor subtypes mediate the classic opioid effects of Met-enk, ?-receptors are reported to be involved in the non-opioid actions of the peptide, i.e. the inhibitory effect on the cell growth 5.
Functions
Proenkephalin is a precursor for neuropeptides with a variety of functions in the neuroendocrine and nervous systems. Upon activation, T-helper lymphocytes were found to express high levels of proenkephalin mRNA and to secrete large amounts of the Met-Enk neuropeptide, perhaps indicating an axis by which the immune and nervous systems interact 6. Enkephalins cause antinociception and potentiated morphine analgesia but they also block the development of tolerance and physical dependence. In addition to their central and peripheral antinociceptive function, opioids can modulate immune activity and cell proliferation. Moreover it is known that they have significant role in different physiological processes like cell differentiation and regeneration, inflammation, cancer and angiogenesis and analgesia effects 5.
References
1. Fleminger G, Lahm HW, Udenfriend S (1984).Changes in rat adrenal catecholamines and proenkephalin metabolism after denervation. PNAS., 81(11):3587-3590.
2. Barbara A. Barron. 2000. Cardiac Opioids. Proceedings of the Society for Experimental Biology and Medicine, 224:1-7.
3. Fratta W, Yang HY, Hong J, Costa E (1977). Stability of Met-enkephalin content in brain structures of morphine-dependent or foot shock-stressed rats. Nature, 268(5619):452-453.
4. Wouters W, Den Bercken JV (1979). Hyperpolarisation and depression of slow synaptic inhibition by enkephalin in frog sympathetic ganglion. Nature, 277:53-54.
5. Tsanova A, Dacheva D, Penchev V, Georgiev G, Pajpanova T, Golovinski E, Lalchev Z (2009). Comparative study of the interaction between synthetic methionine-enkephalin and monolayers of zwitterionic and negatively charged phospholipids. Biotechnol & Biotechnol., 23:463-466.
6. Rattner A, Korner M, Rosen H, Baeuerle PA, Citri Y (1991). Nuclear factor Kappa B activates proenkephalin transcription in T lymphocytes. Molecular and Cellular Biology, 11(2):1017-1022.
Endothelin receptor antagonists
Definition
Endothelin receptor antagonists, by blocking the vasoconstrictor and cardiotonic effects of ET-1, produce vasodilation and cardiac inhibition. Endothelin receptor antagonists have been shown to decrease mortality and improve themodynamics in experimental models of heart failure 1.
Related Peptides
Novel cyclic pentapeptides, WS7338A, B, C and D [cyclo-(AAl -AA2-D-Trp-D-Glu-Ala)] were isolated as ET-receptor antagonists from the culture broth of Sfreptomyces sp. No. 7338 2.
Discovery
Based on conformational analysis, the minimum structural requirements for binding affinity was defined by Neya et al., and a series of acylated tripeptides with ET-A receptor binding affinity were synthesized. Through extensive chemical modification of the lead tripeptide, a potent and selective ETA receptor antagonist FR139317 was discovered. Further modification of this series of tripeptide ET antagonists has led to the discovery of a highly potent and selective ET-B receptor antagonist FR164343 2.
Structural Characteristics
Cyclic pentapeptide endothelin receptor antagonists:
WS7338A: 1 cyclo-(D-Val-Leu-D-Trp-D-Glu-Ala)
WS7338B: 2 cyclo-(D-allolle-Leu-D-Trp-D-Glu-Ma)
WS7338C: 3 cyclo-(D-Val-Val-D-Trp-D-Glu-Ala)
WS7338D: 4 cyclo-(D-Leu-Val-D-Trp-D-Glu-Ala)
Through extensive modification of the cyclic pentapeptide 2 on the basis of its conformation analysis a series of tripeptide ET receptor antagonists with ETA and ETB subtype-selectivity were discovered. Further in vivo characterization demonstrated that FR139317 is a highly potent and selective ET-A receptor antagonist, whereas FR164343 is a highly potent and selective ET-B receptor antagonist. It may be concluded that FR139317 and FR164343 are useful pharma- cological agents for investigating the pathophysiological roles of the ET system 2.
Mode of Action
ET-receptor antagonists are designed such that they have very high binding affinities to endothelin receptors (ET-A and ET-B). Therefore these antagonists mediate their action by blocking the ET receptor binding sites 2.
Functions
ET-receptor antagonists have recently been proposed as an alternative to traditional therapies for pulmonary arterial hypertension 3. ET-receptor antagonists might be useful to treat myocardial ischemia. It is possible that during chronic therapy, inhibition of the proliferative effects of ET may be beneficial for structural changes. Hence, long-term studies with ET-receptor antagonists in coronary artery disease are necessary to determine their clinical potential 4. Significant hemodynamic and neurohumoral benefits were observed in patients with severe heart failure receiving the selective endothelin antagonist 5.
References
1. Book: Cardiovascular Physiology Concepts by Klabunde RE.
2. Neya M (1997). Discovery of endothelin antagonists. Pure & Appl Chem., 69(3):441-446.
3. Liu C, Chen J, Gao Y, Deng B, Liu K (2008). Endothelin receptor antagonists for pulmonary arterial hypertension. Database of Systematic Reviews, 4:1858-1865.
4. Wenzel RR, Fleisch M, Shaw S, Noll G, Kaufmann U, Schmitt R, Jones CR, Clozel M, Meier B, Lüscher TF (1998). Hemodynamic and coronary effects of the endothelin antagonist bosentan in patients with coronary artery disease. Circulation, 98(21):2235-2240.
5. Bergler-Klein J, Pacher R, Berger R, Bojic, A, Stanek B (2004). Neurohumoral and hemodynamic effects of the selective endothelin anEndothelin Antagoniststagonist darusentan in advanced chronic heart failure. J Heart Lung Transplant., 23(1):20-27.
Eglin c and Fragments
Definition
Eglin c is an 8.1 kDa protein proteinase inhibitor first isolated from the leech Hirudo medicinalis and now produced by genetic engineering as an N-acetyl derivative 1.
Related Peptides
Eglin c belongs to the potato inhibitor I family of serineproteinase inhibitors 1.
Discovery
Eglins are small protein inhibitors isolated from the leech Hirudo medicinalis by Seemueller et al.,in 1977 2.
Structural Characteristics
This inhibitor is composed of a single polypeptide chain of 70 amino acid residues. It is extremely stable despite the lack of disulphide bridges. This enzyme is a 25 kDa serine proteinase synthesized and stored in the pancreas as a proenzyme which is a single polypeptide chain of 241 amino acid residues 1. Eglin consists of a four-stranded beta-sheet with an alpha-helical segment and the protease-binding loop fixed on opposite sides. This loop, which contains the reactive site Leu45I--Asp46I, is mainly held in its conformation by unique electrostatic/hydrogen bond interactions of Thr44I and Asp46I with the side chains of Arg53I and Arg51I which protrude from the hydrophobic core of the molecule. The conformation around the reactive site is similar to that found in other proteinase inhibitors. The nine residues of the binding loop Gly40I--Arg48I are involved in direct contacts with subtilisin. In this interaction, eglin segment Pro42I--Thr44I forms a three-stranded anti-parallel beta-sheet with subtilisin segments Gly100--Gly102 and Ser125--Gly127. The reactive site peptide bond of eglin is intact, and Ser221 OG of the enzyme is 2.81 A apart from the carbonyl carbon 2.
Eglin C fragments:
In a study, various peptide fragments related to eglin c, which consists of 70 amino acid residues, were synthesized by a conventional solution method and their inhibitory effects on leukocyte elastase, cathepsin G and alpha-chymotrypsin were examined. Among them, H-Arg-Glu-Tyr-Phe-OMe (eglin c 22-25) and H-Ser-Pro-Val-Thr-Leu-Asp-Leu-Arg-Tyr-OMe (Eglin c 41-49) inhibited cathepsin G and alpha-chymotrypsin but not leukocyte elastase, while H-Thr-Asn-Val-Val-OMe (Eglin c 60-63) inhibited leukocyte elastase but not cathepsin G or alpha-chymotrypsin, although eglin c potently inhibited leukocyte elastase, cathepsin G and alpha-chymotrypsin. These results indicated that the interaction sites of eglin c with leukocyte elastase, cathepsin G and alpha-chymotrypsin might be different 3. In another study, a protected C-terminal triacontapeptide of eglin c, eglin c (31–70), eglin c (22–30) and eglin c (8–70) and finally eglin c were synthesized by a conventional solution method in order to study the relationship between their structure and the inhibitory activity against human leukocyte elastase, cathepsin G and a-chymotrypsin. Although the inhibitory activity of eglin c (31–70) and eglin c (22–70) against the aforementioned enzymes did not increase dramatically, eglin c (8–70) exhibited inhibitory activity against the above enzymes with similar or rather lower Ki-values than that of N a-acetyleglin c.
Mode of Action
The interaction of Eglin with subtilisin looks quite similar to the interaction observed in the proteinase complexes of the other 'small' seine proteinase inhibitor proteins, obeying the 'standard mechanism' proposed by Laskowski and Kato. In Eglin, the binding loop is in a conformation which allows it to bind tightly to the cognate enzyme, under formation of a three-stranded (a new feature, not yet observed in other complexes) intermolecular ß-sheet. This complex is in a conformation similar to that expected for a pre-transition state complex. The relatively rigid and densely packed structure of the complex and the high association rates observed suggest that the loop structure in the free inhibitor will possess a similar conformation 2.
Functions
It potently inhibits chymotrypsin, subtilisin, neutrophil elastase and cathepsin G, forms loose complexes with bovine pancreatic trypsin and pig pancreatic elastase, and does not inhibit plasmin, thrombin and kallikrein 1. Eglin-c treatment prevents MCT-induced ventilatory dysfunction and suggest that endogenous elastase may play an important role in MCT-induced inflammation-mediated ventilatory abnormality 5.
References
1. Faller B, Dirrig S, Rabaud M, Bieth JG (1990). Kinetics of the inhibition of human pancreatic elastase by recombinant eglin c. Influence of elastin. Biochem. J., 270(3):639-644.
2. Bode W, Papamokos E, Musil D, Seemueller U, Fritz H(1986).. Refined 1.2 A crystal structure of the complex formed between subtilisin Carlsberg and the inhibitor eglin c. Molecular structure of eglin and its detailed interaction with subtilisin. EMBO J., 5(4):813-818.
3. Tsuboi S, Nakabayashi K, Matsumoto Y, Teno N, Tsuda Y, Okada Y, Nagamatsu Y, Yamamoto J (1990). Amino acids and peptides. XXVIII. Synthesis of peptide fragments related to eglin c and studies on the relationship between their structure and effects on human leukocyte elastase, cathepsin G and alpha-chymotrypsin. Chem Pharm Bull., 38(9):2369-2376.
4. Okada Y, Tsuboi S (1991). Amino acids and peptides. Part 32. Total synthesis of eglin c. Part 2. Synthesis of a heptacontapeptide corresponding to the entire amino acid sequence of eglin c and of related peptides, and studies on the relationship between the structure and inhibitory activity against human leukocyte elastase, cathepsin G and a-chymotrypsin. J. Chem. Soc., 1991:3321-3328.
5. Lai YL, Zhou KR (1997). Eglin-c prevents monocrotaline-induced ventilatory dysfunction. Appl Physiol., 82:324-328.
Eglin c and Fragments
Definition
Eglin c is an 8.1 kDa protein proteinase inhibitor first isolated from the leech Hirudo medicinalis and now produced by genetic engineering as an N-acetyl derivative 1.
Related Peptides
Eglin c belongs to the potato inhibitor I family of serineproteinase inhibitors 1.
Discovery
Eglins are small protein inhibitors isolated from the leech Hirudo medicinalis by Seemueller et al.,in 1977 2.
Structural Characteristics
This inhibitor is composed of a single polypeptide chain of 70 amino acid residues. It is extremely stable despite the lack of disulphide bridges. This enzyme is a 25 kDa serine proteinase synthesized and stored in the pancreas as a proenzyme which is a single polypeptide chain of 241 amino acid residues 1. Eglin consists of a four-stranded beta-sheet with an alpha-helical segment and the protease-binding loop fixed on opposite sides. This loop, which contains the reactive site Leu45I--Asp46I, is mainly held in its conformation by unique electrostatic/hydrogen bond interactions of Thr44I and Asp46I with the side chains of Arg53I and Arg51I which protrude from the hydrophobic core of the molecule. The conformation around the reactive site is similar to that found in other proteinase inhibitors. The nine residues of the binding loop Gly40I--Arg48I are involved in direct contacts with subtilisin. In this interaction, eglin segment Pro42I--Thr44I forms a three-stranded anti-parallel beta-sheet with subtilisin segments Gly100--Gly102 and Ser125--Gly127. The reactive site peptide bond of eglin is intact, and Ser221 OG of the enzyme is 2.81 A apart from the carbonyl carbon 2.
Eglin C fragments:
In a study, various peptide fragments related to eglin c, which consists of 70 amino acid residues, were synthesized by a conventional solution method and their inhibitory effects on leukocyte elastase, cathepsin G and alpha-chymotrypsin were examined. Among them, H-Arg-Glu-Tyr-Phe-OMe (eglin c 22-25) and H-Ser-Pro-Val-Thr-Leu-Asp-Leu-Arg-Tyr-OMe (Eglin c 41-49) inhibited cathepsin G and alpha-chymotrypsin but not leukocyte elastase, while H-Thr-Asn-Val-Val-OMe (Eglin c 60-63) inhibited leukocyte elastase but not cathepsin G or alpha-chymotrypsin, although eglin c potently inhibited leukocyte elastase, cathepsin G and alpha-chymotrypsin. These results indicated that the interaction sites of eglin c with leukocyte elastase, cathepsin G and alpha-chymotrypsin might be different 3. In another study, a protected C-terminal triacontapeptide of eglin c, eglin c (31–70), eglin c (22–30) and eglin c (8–70) and finally eglin c were synthesized by a conventional solution method in order to study the relationship between their structure and the inhibitory activity against human leukocyte elastase, cathepsin G and a-chymotrypsin. Although the inhibitory activity of eglin c (31–70) and eglin c (22–70) against the aforementioned enzymes did not increase dramatically, eglin c (8–70) exhibited inhibitory activity against the above enzymes with similar or rather lower Ki-values than that of N a-acetyleglin c.
Mode of Action
The interaction of Eglin with subtilisin looks quite similar to the interaction observed in the proteinase complexes of the other 'small' seine proteinase inhibitor proteins, obeying the 'standard mechanism' proposed by Laskowski and Kato. In Eglin, the binding loop is in a conformation which allows it to bind tightly to the cognate enzyme, under formation of a three-stranded (a new feature, not yet observed in other complexes) intermolecular ß-sheet. This complex is in a conformation similar to that expected for a pre-transition state complex. The relatively rigid and densely packed structure of the complex and the high association rates observed suggest that the loop structure in the free inhibitor will possess a similar conformation 2.
Functions
It potently inhibits chymotrypsin, subtilisin, neutrophil elastase and cathepsin G, forms loose complexes with bovine pancreatic trypsin and pig pancreatic elastase, and does not inhibit plasmin, thrombin and kallikrein 1. Eglin-c treatment prevents MCT-induced ventilatory dysfunction and suggest that endogenous elastase may play an important role in MCT-induced inflammation-mediated ventilatory abnormality 5.
References
1. Faller B, Dirrig S, Rabaud M, Bieth JG (1990). Kinetics of the inhibition of human pancreatic elastase by recombinant eglin c. Influence of elastin. Biochem. J., 270(3):639-644.
2. Bode W, Papamokos E, Musil D, Seemueller U, Fritz H(1986).. Refined 1.2 A crystal structure of the complex formed between subtilisin Carlsberg and the inhibitor eglin c. Molecular structure of eglin and its detailed interaction with subtilisin. EMBO J., 5(4):813-818.
3. Tsuboi S, Nakabayashi K, Matsumoto Y, Teno N, Tsuda Y, Okada Y, Nagamatsu Y, Yamamoto J (1990). Amino acids and peptides. XXVIII. Synthesis of peptide fragments related to eglin c and studies on the relationship between their structure and effects on human leukocyte elastase, cathepsin G and alpha-chymotrypsin. Chem Pharm Bull., 38(9):2369-2376.
4. Okada Y, Tsuboi S (1991). Amino acids and peptides. Part 32. Total synthesis of eglin c. Part 2. Synthesis of a heptacontapeptide corresponding to the entire amino acid sequence of eglin c and of related peptides, and studies on the relationship between the structure and inhibitory activity against human leukocyte elastase, cathepsin G and a-chymotrypsin. J. Chem. Soc., 1991:3321-3328.
5. Lai YL, Zhou KR (1997). Eglin-c prevents monocrotaline-induced ventilatory dysfunction. J Appl Physiol., 82:324-328.
