Chemical synthesis and characterization of the epidermal growth factor-like module of human complement protease Clr

Clr is one of the two serine proteases of Cl, the first component of complement, in which it is associated in a calcium-dependent manner to the homologous serine protease Cls. This interaction is mediated by the N-terminal region of Clr, which comprises a single epidermal growth factor (EGF)-like module containing the consensus sequence required for calcium binding, surrounded by two CUB modules. With a view to determine the structure of the EGF-like module of Clr and evaluate its contribution to calcium binding, this module [Clr(123–175)] was synthesized by automated solid-phase methodology using the Boc strategy. A first synthesis using the Boc-His(Z) derivative gave very low yield, due to partial deprotection of His residues leading to chain termination by acetylation, and to insertion of glycine residues. This could be circumvented by using the Boc-His(DNP) derivative and by condensation of appropriate glycine-containing segments. The synthetic peptide was efficiently folded under redox conditions to the species with three correct disulfide bridges, as determined by mass spectrometry and N-terminal sequence analyses of thermolytic fragments. The homogeneity of the synthetic peptide was assessed by reversed-phase HPLC and electrospray mass spectrometry. One-dimensional ¹H NMR spectroscopic analysis provided evidence that the EGF-like module had a well defined structure, and was able to bind calcium with an apparent K_d of 10 mM. This value, comparable to that found for the isolated EGF-like modules of coagulation factors IX and X, is much higher than that measured for native Clr. As already proposed for factors IX and X, it is suggested that neighbouring module(s), most probably the N-terminal CUB module, contribute(s) to the calcium binding site. © Munksgaard 1997.     

Chemical synthesis and biological activity of the EGF-like domain of heparin-binding epidermal growth factor-like growth factor (HB-EGF)

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a recently discovered member of the epidermal growth factor (EGF) family. This novel growth factor possesses the EGF-like domain in the carboxyl portion. In order to evaluate the biological function of the EGF-like domain in HB-EGF, human HB-EGF(44–86) corresponding to the EGF-like domain was synthesized by the solid-phase procedure using the Fmoc strategy. It was confirmed by amino acid microsequencing of cystine-containing fragments derived from thermolytic digestion that the pattern of three disulfide bond pairings in synthetic HB-EGF(44–86) was consistent with that of EGF and transforming growth factor-α (TGF-α). The homogeneity of the synthetic peptide was confirmed by analytical RP-HPLC, amino acid analysis and fast atom bombardment mass spectrometer (FAB-MS). Compared with h-EGF, the EGF-like domain of human HB-EGF showed a comparable mitogenic activity in the proliferation of NIH/3T3 fibroblast cells. These results suggest that the EGF-like domain of human HB-EGF may play an important role in mitogenic activity.     

Peptides corresponding to the second epidermal growth factor-like domain of human blood coagulation factor VII: synthesis,folding and biological activity

Factor VIIa (FVIIa) is the enzymatically active constituent of the FVIIa/tissue factor (TF) complex, the initiator of the extrinsic pathway of blood coagulation. The zymogen FVII and FVIIa are composed of discrete domains, two of which are homologous to the epidermal growth factor (EGF). This investigation examined the significance of the FVII EGF-2 domain in the processes leading to activation of factor X (FX). Peptides 47 residues in length and corresponding to the amino acid sequence of the EGF-2 domain of human FVII were prepared by solid-phase synthesis methods. Peptide variants with all six Cys residues replaced by l -2-aminobutyryl residues (1), or containing one (2a-c), two (3a,b) or three (4) disulfide bonds, were obtained by application of various S-protecting groups and oxidation methods. Peptide 4, containing the cystine bridge arrangement corresponding to that found in the native protein, was prepared by a two-step regioselective disulfide bond formation method. An evaluation of the anti-coagulant properties of peptides 1-4 revealed that all peptides, with the exception of the two-cystine isomer containing non-native disulfide pairings (3b), were potent inhibitors of TF/FVIIa-mediated activation of FX. The fully constrained peptide 4 was found to be twice as active as its completely non-constrained counterpart 1, the two peptides showing IC₅₀ values of 1.6 ± 0.5μm (1) and 0.8 ± 0.2 μm (4) with respect to TF/FVIIa-dependent FX activation. The results of this study demonstrate the functional importance of the EGF-2 domain of FVII in the induction of coagulation by the extrinsic pathway.     

Modulation of the activity and assessment of the receptor selectivity in a series of new RGD-containing peptides

We have investigated the structure-activity relationship of a series of new synthetic RGD analogs and their potential use as specific platelet aggregation inhibitors. Twelve short linear peptides showed high potency to inhibit aggregation in ADP-stimulated dog platelets. In order to assess the selectivity of these analogs towards platelet integrin GPIIb-IIIa, a new cell adhesion inhibition system was devised which was able to discriminate between the two closely related β3-integrins of the vasculature, GPIIb-IIIa (α_IIbβ₃), present in platelets, and the vitronectin receptor (α_vβ₃), expressed in endothelial cells and platelets. As reported for other peptides by Scarborough et. al. (1993, J. Biol. Chem. 2 68 , 1066), the analogs containing lysine instead of arginine in position 1 showed increased selectivity towards GPIIb-IIIa. One of them, in which the piperidine carboxylic group was attached to the ^N-terminus of KGDW, not only strongly inhibited platelet aggregation, but also selectively abolished cell adhesion mediated by GPIIb-IIIa without effect on the vitronectin receptor.     

A comparison of folding techniques in the chemical synthesis of the epidermal growth factor‐like domain in neu differentiation factor α/β

Abstract: The 52‐residue α/β chimera of the epidermal growth factor‐like domain in neu differentiation factor (NDF_eα/β) has been synthesized and folded to form a three disulfide bridge (Cys¹⁸²–Cys¹⁹⁶, Cys¹⁹⁰–Cys²¹⁰, Cys²¹²–Cys²²¹) containing peptide. We investigated two general strategies for the formation of the intramolecular disulfide bridges including, the single‐step approach, which used fully deprotected and reduced peptide, and a sequential approach that relied on orthogonal cysteine protection in which specific pairs are excluded from the first oxidation step. Because there are 15 possible disulfide bridge arrangements in a peptide with six cysteines, the one‐step approach may not always provide the desired disulfide pairing. Here, we compare the single‐step approach with a systematic evaluation of the sequential approach. We employed the acetamidomethyl group to protect each pair of cysteines involved in disulfide bridges, i.e. Cys¹⁸² to Cys¹⁹⁶, Cys¹⁹⁰ to Cys²¹⁰ and Cys²¹² to Cys^221. This reduced the number of possible disulfide patterns from 15 to three in the first folding step. We compared the efficiencies of folding for each protected pair using RP‐HPLC, mapped the disulfide connectivity of the predominant product and then formed the final disulfide from the partially folded intermediate via I₂ oxidation. Only the peptide having the Cys¹⁸²–Cys¹⁹⁶ pair blocked with acetamidomethyl forms the desired disulfide isomer (Cys¹⁹⁰–Cys²¹⁰/Cys²¹²–Cys²²¹) as a single homogeneous product. By optimizing both approaches, as well as other steps in the synthesis, we can now rapidly provide large‐scale syntheses of NDF_eα/β and other novel EGF‐like peptides.     

From pro defensins to defensins: synthesis and characterization of human neutrophil pro α‐defensin‐1 and its mature domain

Abstract: Human neutrophil α‐defensins (HNPs) are small, cationic, Cys‐rich antimicrobial proteins that play important roles in innate immunity against infectious microbes such as bacteria, fungi and enveloped viruses. Synthesized as inactive precursors in vivo (pre‐proHNPs), HNPs are activated through proteolytic removal of the inhibitory pro‐peptide required for subcellular sorting and correct folding. We seek to understand the molecular basis for the recognition between the 45‐residue pro‐peptide and the C‐terminal functional domain. Here we described, total chemical synthesis of the 75‐residue human neutrophil pro α‐defensin‐1 (proHNP1) via native chemical ligation. After oxidative folding, proHNP1 is cleaved by cyanogen bromide at the Met⁴⁵–Ala⁴⁶ peptide bond to release the mature form. The native disulfide connectivity in HNP1, i.e. Cys¹–Cys⁶, Cys²–Cys⁴ and Cys³–Cys⁵, is verified by mass mapping of peptide fragments generated by proteolytic digestion and Edman degradation. Fluorescence spectroscopy studies and antimicrobial activity assays further support that synthetic proHNP1 and HNP1 are correctly folded. While largely unstructured in aqueous solution, the pro‐peptide binds to HNP1 intermolecularly with an apparent K_d value of 6.2 μm at pH 7.4, confirming the mode of intramolecular inactivation of human α‐defensin precursors.     

Transinactivation of the epidermal growth factor receptor tyrosine kinase and focal adhesion kinase phosphorylation by dietary flavonoids: effect on invasive potential of human carcinoma cells

Focal adhesion kinase (FAK), a member of a growing family of structurally distinct protein tyrosine kinases (PTK), has been linked to specific phosphorylation events, and the elevation of FAK activity in human carcinoma cells correlated with increased invasive potential. Transactivation of the epidermal growth factor receptor (EGFR) tyrosine kinase activity is proposed to stimulate cell migration and the subsequent activation of downstream signaling pathways. Quercetin (Qu) and luteolin (Lu), are potent PTK inhibitors as well as putative chemopreventive agents. The present work, we demonstrate that Qu and Lu at concentration of 20 microM transinactivated EGFR tyrosine kinase activity with marked reduction in phosphotyrosyl level of 170, 125, 65, 60 and 42 kDa cellular proteins, and induced apoptosis in MiaPaCa-2 cells. The 125 kDa protein was further identified as a FAK by immunoprecipitation and immunoblotting analyses. Tumor cells treated with Lu or Qu dampened the phosphorylation of FAK. In addition, our data clearly demonstrated that tumor cells responded to Qu and Lu by parallel reductions in the levels of phosphorylated FAK and the secreted matrix metalloproteinase (MMP) that may lead to the suppression of invasive potential and cell migration in vitro. While the molecular mechanism of FAK regulation of MMP secretion in tumor cells remains unclear, our results suggested that blockade of the EGFR-signaling pathway may contributed to the net effect. As suggested in the current study, targeting EGFR and FAK with the objective of modulating their regulatory pathways could offer prospects for the treatment of EGFR-responsive cancers in the future.