Sequence Of Epidermal Growth Factor Research Articles

Versatile Mitogenic and Differentiation-Inducible Layer Formation by Underwater Adhesive Polypeptides.

Artificial materials have no biological functions, but they are important for medical devices such as artificial organs and matrices for regenerative medicine. In this study, mitogenic and differentiation‐inducible materials are devised via the simple coating of polypeptides, which contain the sequence of epidermal growth factor or insulin‐like growth factor with a key amino acid (3,4‐dihydroxyphenylalanine) of underwater adhesive proteins. The adhesive polypeptides prepared via solid‐phase synthesis form layers on various substrates involving organic and inorganic materials to provide biological surfaces. Through the direct activation of cognate receptors on interactive surfaces, the materials enable increased cell growth and differentiation compared to that achieved by soluble growth factors. This superior growth and differentiation are attributed to the long‐lasting signal transduction (triggered by the bound growth factors), which do not cause receptor internalization and subsequent downregulation.

Mussel-inspired adhesive epidermal growth factor for biological surface modification

Event Abstract Back to Event Mussel-inspired adhesive epidermal growth factor for biological surface modification Hongli Mao1, Shinhe Park1, 2*, Seiichi Tada1 and Yoshihiro Ito1, 2 1 RIKEN, Nano Medical Engineering Laboratory, Japan 2 Waseda University, Department of Life Science and Medical Bio-Science, Japan Introduction: Growth factor, as a fundamental requirement in tissue engineering, plays important roles in the regulation of a variety of cellular processes. It has been demonstrated that the immobilized growth factors on the material surfaces can stimulate the cognate receptors in the cell membrane and trigger the signal transduction. Although types of growth factors with binding ability to specific substrates were developed, simple and versatile strategies to modify growth factors for surface modification of multiple classes of materials are essential. Here inspired by underwater adhesive protein secreted by mussel, we incorporated the binding peptide sequence carrying 3,4-dihydroxyphenylalanine (DOPA) to the active sequence of epidermal growth factor (EGF). The binding affinity of the constructed growth factor (EGF-DOPA) to typical organic and inorganic materials and its biological activities were investigated. Materials and Methods: A peptide synthesizer ABI 433A was employed to synthesize the active sequence of EGF and the adhesive peptide carrying DOPAs and lysines (DOPA-Lys-DOPA-Lys-DOPA) by a solid-phase peptide synthesis method. Then the active sequence of EGF and the adhesive peptide was conjugated using three glycine residues as a spacer. After purification with a high performance liquid chromatography (HPLC), formulations of the synthesized peptide were confirmed by matrix-assisted laser desorption/ionization time-of-flight mass analysis (MALDI-TOF-MS). The binding affinity of EGF-DOPA to polystyrene (PSt) and titanium (Ti) surfaces was confirmed by using a quartz crystal microbalance with dissipation monitoring (QCM-D). The surface morphology and thickness of the substrates treated with EGF-DOPA was studied by atomic force microscopy (AFM), scanning electron microscopy (SEM) and ellipsometry. Rat kidney adherent fibroblast cell line, NRK49F, was cultured on the surfaces treated with EGF-DOPA. Cell growth and time course activation of cellular signal transduction was measured through WST-8 assay and phosphorylation analysis, respectively. Results and Discussion: MALDI-TOF-MS result showed that the expected 54-residue peptide (CPLSH-DGYCL-HDGVC-MYIEA-LDKYA-CNCVV-GYIGE-RCQYR-DLKWW-E-GGG-DOPA-K-DOPA-K-DOPA, EGF-DOPA) was successfully synthesized. QCM measurement showed that the binding affinity of EGF-DOPA on both PSt and Ti surfaces was significantly higher than that of EGF. Concentration and pH dependent binding affinity of EGF-DOPA was confirmed. This result was also proved by thickness measurement using SEM and ellipsometry. The measured thicknesses of EGF-DOPA bound on the substrates displayed the same trend as those calculated from the QCM measurement. AFM results showed that aggregation of the EGF-DOPA happened at high pH condition. Substrates treated with EGF-DOPA significantly improved cell growth and this result can attributed to the long activation period of bound EGF-DOPA on the substrates. Conclusions: Mussel-inspired adhesive EGF derivative (EGF-DOPA) was constructed. The binding affinity of EGF-DOPA to both PSt and Ti surfaces was dramatically higher than that of EGF. The bound EGF-DOPA significantly promoted cell growth. This mussel-inspired dopamine modification provides an effective method for preparation of adhesive materials that may be useful for surface modification and the development of medical devices.

Manipulating Cell Migration and Proliferation with a Light‐Activated Polypeptide

Remote control of cells: A polypeptide has been made that stimulates proliferation and migration of cells upon photochemical activation. This light-activated polypeptide enables spatially defined control of cell populations at the scale of tissue organization; this is accomplished without physically contacting the cells or modifying their substrate. Polypeptide growth and differentiation factors modulate a wide variety of cell behaviors and can be used to manipulate cells in vitro for tissue engineering and basic studies of cell biology. To emulate in vitro the spatial aspect of growth factor function, new methods are needed to generate defined spatial gradients of activity. Polypeptide factors that are engineered to be activated with light provide a method for creating concentration gradients with the fine precision in space and time with which light can be directed. As a first test of this approach, we have chemically synthesized a polypeptide with the sequence of epidermal growth factor in which a critical glutamate is "caged" with a photoremovable group. Photolysis of this polypeptide afforded maximal mitogenic and chemokinetic activity at concentrations at which the caged factor was inactive. Spatially resolved photolysis of the factor resulted in spatial patterning of fibroblasts. This system will be useful for ex vivo tissue engineering and for investigating the interactions of cells with their matrix and the role of chemical gradients in biological pattern formation.

Peptide mimics of epidermal growth factor (EGF) with antagonistic activity

Epidermal growth factor is a potent growth-promoting factor for a variety of tissue cells in vivo and in vitro. Epidermal growth factor binds, phosphorylates, and activates epidermal growth factor receptors on the cell surface. In this study, we attempted to design functional peptide mimics by panning a phage display library on the anti-epidermal growth factor monoclonal antibody. By using anti-epidermal growth factor monoclonal antibody as a mold of the structure of the binding site of epidermal growth factor, high-efficiency probing was expected. From a random peptide phage display library, phage clones that bind to the anti-epidermal growth factor monoclonal antibody were isolated. One of the phage clones also exhibited binding activity to the epidermal growth factor receptor. The amino acid sequence of this phage clone showed slight similarity to the primary sequence of epidermal growth factor. We synthesized this motif to a 9-amino-acid intramolecularly disulfide-linked peptide. This synthetic peptide inhibited mitogenesis as well as epidermal growth factor receptor tyrosine phosphorylation, which is induced by epidermal growth factor. The present results suggest that the peptide synthesized in this study may mimic the epidermal growth factor receptor-binding region in epidermal growth factor.

Assessment of transcript polyadenylation by 3' RACE: the response of epidermal growth factor messenger ribonucleic acid to thyroid hormone in the thyroid and submaxillary glands.

The complementary DNA (cDNA) sequence of epidermal growth factor (EGF) indicates that its 3' untranslated region (3' UTR) is 745 bases long, with polyadenylation occurring at residue 4749. However, when we used reverse transcriptase-polymerase chain reaction (RT-PCR) with an anchored 3' primer [3'rapid amplification of cDNA ends (RACE)] to amplify the 3' ends of cDNA, we actually detected two major products [800 and 600 base pairs (bp)] and a minor product (400 bp) in the thyroid or submaxillary glands (SMGs) of male mice. Analysis of genomic DNA with a battery of primer pairs gave only the predicted PCR products from the 3' UTR, demonstrating the lack of introns in this region of genomic DNA and eliminating alternate splicing as the explanation of the transcript diversity we detected. We confirmed that two potential polyadenylation sites proximal to residue 4749 are used in vivo by hybridizing the same 3' RACE products with probes specific for the 5' end of the 3' UTR, and also for poly-A tails. To assess the distribution of poly-A tail lengths on transcripts using the terminal polyadenylation site (4749), we used several different approaches to analyze 3' RACE products. Solution hybridization with 3' UTR probes revealed a striking difference between transcripts in SMG and thyroid: SMG contained two large 3' RACE populations (approximately 770 and 870 bp), whereas thyroid only contained one (approximately 770 bp). EGF transcript heterogeneity due to different poly-A tail lengths was confirmed using an upstream primer 400 bases closer to the 3' end of the 3' UTR, and TaqI digestion. Again we found two major populations in SMG (approximately 380 and 480 bp), but only one (380 bp) in thyroid, which upon TaqI digestion showed tissue-specific heterogeneity only in the 3' fragment. T4 treatment of male mice (0.25 microgram T4/gm ip) increased the intensity of both populations in SMG and the smaller population in thyroid within 24 h. However, after a week of T4 injections, only the intensity of the population with the longer poly-A tails in the SMG remained elevated, a finding consistent with tissue-specific enhanced stability of transcripts due to polyadenylation. Finally, to resolve poly-A tail lengths more precisely, we used an upstream primer that was specific for the 3' end of murine 3' UTR. This approach revealed that the thyroid contains three major populations of EGF transcripts, with poly-A tail lengths of approximately 20, 50, and 70 A's. After T3 treatment for 24 h, the intensity of transcripts containing 20 A's increased 52% (P < 0.02) and those with 50 A's increased 130% (P < 0.01), whereas there was no change in transcripts with tails > or = 70 A's. On the other hand, there were no distinct bands in SMG samples, but rather a heterogeneous distribution of poly-A tail lengths from approximately 20-120 A's that showed an overall increase of approximately 60% in response to T3.

Studies on peptides. CXXVII. Synthesis of a tripentacontapeptide with epidermal growth factor activity.

The tripentacontapeptide corresponding to the entire linear sequence of epidermal growth factor was synthesized by assembling 15 peptide fragments and one His residue (position 22), followed by deprotection with trifluoromethanesulfonic acid-thioanisole in trifluoroacetic acid. The deprotected peptide was subjected to air-oxidation. After purification by ion-exchange chromatography on diethyl aminoethyl cellulose followed by high performance liquid chromatography, a peptide with powerful anti-gastric activity was obtained.

Computer-based characterization of epidermal growth factor precursor

The cDNA sequence of the precursor of mouse epidermal growth factor (EGFP) has recently been reported by two groups, both of whom noted the presence of repeated similar segments, each about 40 residues long. One of these repeat units overlaps with the sequence of epidermal growth factor itself. The sequence of epidermal growth factor has been reported to be similar to that of pancreatic secretory trypsin inhibitor (PSTI) and a somewhat better match has been found with part of the sequence of bovine factor X, one of the blood coagulating factors. We report here that there is an even stronger similarity between the sequences of some of the repeat units of epidermal growth factor precursor and certain segments in factor X. This sequence similarity is also apparent in comparisons with other blood coagulation factors. On the basis of these sequence comparisons we suggest a scheme for the evolution of the epidermal growth factor precursor. We have also identified certain structural features in the precursor sequence that bear on the way in which the mature epidermal growth factor is generated.

Epidermal Growth Factor: LOCATION OF DISULFIDE BONDS

The three disulfide bridges in epidermal growth factor have been identified by the isolation of three unique cystinyl peptides obtained by thermolytic digestion. The cysteic acid-containing peptide pairs derived from each of the cystinyl peptides were isolated. Their amino acid compositions were in agreement with those predicted from the known amino acid sequence of epidermal growth factor. The cysteinyl residues in positions 6 and 20, 14 and 31, and 33 and 42 were linked as disulfides in this 53 amino acid residue polypeptide.

The Primary Structure of Epidermal Growth Factor

The complete amino acid sequence of epidermal growth factor (EGF) has been established through the use of automated Edman degradation and standard enzymatic and chemical techniques. The location of the half-cystinyl residues was facilitated by the use of the S-[14C]aminoethyl derivative of EGF. The sequence is: [see PDF for sequence]. The calculated molecular weight of the 53-residue polypeptide is 6045, a value that is in agreement with the molecular weight of 6400 established by physical measurements. The peptide is acidic and the 6 half-cystines exist in disulfide linkage. Four arginine residues are located in the COOH-terminal portion of the molecule. The six COOH-terminal amino acids are not necessary for biological activity and EGF1–47 has activity identical with that of the 53-residue native molecule.