Chemical synthesis of per-selenocysteine human epidermal growth factor

Human seleno-epidermal growth factor (seleno-EGF), a 53-residue peptide where all six cysteine residues of the parent human EGF sequence were replaced by selenocysteines, was synthesized and the oxidative folding of a polypeptide containing three diselenide bonds was compared to that of the parent cysteine peptide. The crude high performance liquid chromatography (HPLC) profiles clearly showed that both the native EGF and its selenocysteine-analogue fold smoothly, yielding a single sharp peak, proving that even in the case of three disulfide-bonded polypeptides the disulfide-to-diselenide bond substitution is highly isomorphous, as confirmed by conformational circular dichroism measurements and particularly by the biological assays.     

Human epidermal growth factor. High resolution solution structure and comparison with human transforming growth factor alpha.

The solution structure of the 53 amino acid peptide hormone, human epidermal growth factor (hEGF), has been determined to high resolution from nuclear magnetic resonance (n.m.r.) data. A large number of internuclear distance and dihedral restraints was obtained, including data from uniformly 15N-labelled hEGF. Dynamical simulated annealing methods using the program XPLOR were used for structure calculation. An improved protocol was developed combining efficient conformational searching at a reduced computational cost. The general fold of the calculated structures compared well with that of a derivative of the carboxy-terminally truncated hEGF determined previously. A group of 44 structures were calculated with no violations greater than 0.3 A and 3 degrees for distance and dihedral restraints, respectively. The average pairwise root mean square (r.m.s.) deviation of all backbone atoms for these structures was 2.25 A for all 53 residues, 0.92 A for the bulk of the protein, and 0.23 A for the functionally important carboxy-terminal domain. Two new helical segments containing highly conserved amino acids have been identified; one between cysteines 6 and 14 and a second at the end of the carboxy-terminal domain. New insight into the molecular architecture of the site of putative receptor binding was provided by comparing the structure of hEGF with its biologically equipotent analogue, human transforming growth factor alpha. This comparison revealed a close structural relationship between the two growth factors and provides an improved understanding of the structure/function relationships in EGF.     

Chemical synthesis and cloning of the human epidermal growth factor gene

The solid-phase phosphotriester method was used to synthesise 24 oligodeoxyribonucleotides, which were enzymatically joined together to give the human epidermal growth factor gene and its analogue containing Leu codon in position 21. The primary structure of the cloned genes were confirmed by the Maxam-Gilbert technique. It is demonstrated that the high purity degree of oligonucleotides allows to synthesise and clone genes without purification of intermediate fragments.     

Rat Sertoli cells secrete a growth factor that blocks epidermal growth factor (EGF) binding to its receptor

The conditioned medium from Sertoli cells contains a potent mitogen(s) that can markedly stimulate the proliferation of 4 different cell lines of endoderm or mesoderm origin in the presence or absence of serum. With A431 cells, conditioned medium produced in a dose-dependent manner up to a 5.2-fold increase in cell number after 5 days in culture. Addition of follicle-stimulating hormone (FSH), testosterone, retinol, and insulin to the Sertoli cells increased the secretion of the mitogenic activity. The ability of Sertoli cell conditioned medium (SCCM) to displace 125I-labeled epidermal growth factor (125I-EGF) from formalin-fixed A431 cells was also examined. The SCCM from Sertoli cells incubated with insulin contained 1.42 ng eq of EGF/ml; testosterone, retinol, and FSH (in the presence of insulin) further increased the secretion of this EGF competing activity to 2.09, 2.56, and 3.22 ng eq/ml, respectively. The amount of EGF competing activity was positively correlated with mitogenic activity. Separation of SCCM by gel filtration on Bio-Gel P-10 produced three major peaks of EGF-competing activity at apparent Mr = 1800-2100, 3800-4200, and 8000-9500. Chromatographing SCCM (in the presence of protease inhibitors) on size exclusion high performance liquid chromatography revealed two peaks of EGF competing activity at Mr about 8000 and 2000 coincident with and proportional to peaks of mitogenic activity. This activity was heat-sensitive and resistant to reducing agents, and addition of an equivalent amount of EGF as that present in SCCM produced an inhibition in growth of the A431 cells compared to a 3-fold stimulation with SCCM. Thus, the Sertoli cells secrete a potent mitogen that is distinct from EGF and alpha TGF. This factor that we have termed Sertoli cell-secreted growth factor is hormonally regulated by FSH, testosterone, and retinol and may play an important role in controlling spermatogenesis.     

Purification of human epidermal growth factor (urogastrone) from urine

Human epidermal growth factor has been isolated from a concentrated chromatographic eluate of human urine. The purification method utilizes six chromatographic steps including adsorption to aminoethylcellulose (AE-11), gel filtration on Sephadex G-50, carboxymethylcellulose (CM-52) chromatography, ion-exchange HPLC and reverse-phase HPLC. The final product appears homogeneous and identical to pure gamma-urogastrone when analyzed by polyacrylamide gel electrophoresis and reverse-phase HPLC using two eluent systems. The yield of the method described above allowed the development of a sensitive radioimmunoassay system for this growth factor.     

Regulation of epidermal growth factor (EGF) receptor activity during the modulation of protein synthesis.

The capacity of cultured human fibroblasts to bind 125I-labeled epidermal growth factor (EGF) was measured during protein synthesis inhibition and reinitiation. Protein synthesis was inhibited by incubation of human fibroblasts in histidine-free medium supplemented with L-histidinol to produce a stringent amino acid starvation. Under these conditions 125 I-EGF binding activity decreased with a half-life of 14.5 hours. Protein synthesis could be rapidly reinitiated by the addition of L-histidine to human fibroblasts which had been preincubated in histidinol containing media for 36 to 48 hours. 125I-EGF binding activity rapidly increased upon the reinitiation of protein synthesis. In the presence of serum 100% of the original binding capacity was recovered ten hours after the reinitiation or protein synthesis, while 70% of the binding capacity was recovered in 12 hours in serum-free media. The recovery of 125I-EGF binding activity after the reinitiation of protein synthesis, was not blocked by the presence of Actinomycin D, indicating that the messenger RNA for the EGF receptor may accumulate during the period of histidinol-mediated inhibition of protein synthesis. The time course of recovery of 125I-EGF binding activity after the reinitiation of protein synthesis is very similar to that observed during the recovery of receptor activity following "down regulation" of EGF receptor activity. Recovery from down regulation, however, was markedly sensitive to Actinomycin D.     

Localization of the epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) in the bovine testis

In the last few decades, several growth factors were identified in the testis of various mammalian species. Growth factors are shown to promote cell proliferation, regulate tissue differentiation, and modulate organogenesis. In the present investigation we have studied the localization of EGF and EGFR in the adult bovine testis by means of immunohistochemical method. Our results demonstrated that EGF and EGFR were localized solely to the bovine testicular germ cells (spermatogonia, spermatocytes, and round spermatids). In contrast, the somatic testicular cells (i.e., Sertoli, Leydig, and myofibroblast cells) exhibited no staining affinity. EGF and EGFR were additionally detected in the epithelial lining of straight tubules and rete testis. Interestingly, the distribution of EGF and EGFR in the germ cells was mainly dependent upon the cycle of the seminiferous epithelium since their localization appeared to be preponderant during the spermatogonia proliferation and during the meiotic and spermiogenic processes. In conclusion, such findings may suggest that EGF and EGFR are important paracrine and/or autocrine regulators of spermatogenesis in bovine.     

Purification and characterization of epidermal growth factor (beta-urogastrone) and epidermal growth factor fragments from large volumes of human urine

We purified human epidermal growth factor (hEGF) and hEGF fragments from a benzoic acid precipitate of the materials not adsorbed to silica gel in 330 liters of human urine by a relatively brief, simple, and efficient method employing sequential batch absorption to and stepwise elution from CM-cellulose and DEAE-cellulose, Bio-Gel P-10 chromatography in 50 mM HCl, and three reverse-phase HPLC steps for final resolution and purification of hEGF components. Recovery of hEGF was 29%. Eight apparently homogeneous hEGF components were recovered, each of which had similar activities in a homologous hEGF radioimmunoassay and an EGF radioreceptor assay using human placental membranes. Amino acid composition analysis indicated that there were four pairs of components that represented intact 53-amino acid hEGF, hEGF-(1-52), hEGF-(1-51), and hEGF-(1-50); intact hEGF accounted for one-third of the total materials recovered. Automated Edman degradation of each component for at least 10 cycles revealed a single amino acid sequence identical to that proposed for human beta-urogastrone. Similar immunoreactive hEGF components were observed in similar proportions in freshly voided urine, indicating that they were not artifacts of the purification process. Thus, multiple forms of fully biologically active hEGF (i.e., beta-urogastrone) can be relatively easily and efficiently purified from large volumes of human urine.     

The factor IXa second epidermal growth factor (EGF2) domain mediates platelet binding and assembly of the factor X activating complex.

Previously we have determined that residues 88-109 (but not Arg(94)) in the second epidermal growth factor (EGF2)-like domain of factor IXa (FIXa) are important for assembly of the factor X (FX) activating complex on phospholipid vesicles (Wilkinson, F. H., London, F. S., and Walsh, P. N. (2002) J. Biol. Chem. 277, 5725-5733). Here we report that these residues are important for platelet binding affinity, stoichiometry, and assembly of the FX activating complex. We prepared several chimeric FIXa proteins using homologous sequences from factor VII (FVII): FIXa(FVIIEGF2) (FIX Delta 88-124,inverted Delta FVII91-127), FIXa(loop1) (FIX Delta 88-99,inverted Delta FVII91-102), FIXa(loop2) (FIX Delta 95-109,inverted Delta FVII98-112), and FIXa(loop3) (FIX Delta 111-124,inverted Delta FVII114-127) and tested their ability to bind to thrombin-activated platelets. Binding affinities (K(d) values in 10(-9) m) for the proteins were as follows in the presence and absence of FVIIIa, respectively: FIXa(N) (0.55 +/- 0.06, 2.9 +/- 0.45), FIXa(WT) (0.80 +/- 0.08, 3.5 +/- 0.5), FIXa(loop1) (19 +/- 4.0, 27 +/- 5.0), FIXa(loop2) (35 +/- 9.0, 65 +/- 12.0), and FIXa(loop3) (1.1 +/- 0.09, 5.0 +/- 0.90). These K(d) values are in good agreement with K((d)(app)) values (in 10(-9) m) determined from the activation of FX (in the presence and absence of FVIIIa, respectively): FIXa(N) (0.46 +/- 0.05, 1.40 +/- 0.14), FIXa(WT) (0.72 +/- 0.08, 3.8 +/- 0.08), FIXa(loop1) (3.2 +/- 0.72, 14.0 +/- 1.60), FIXa(loop2) (18.4 +/- 1.60, 26.3 +/- 3.40), and FIXa(loop3) (0.7 +/- 0.05, 3.0 +/- 0.15). Moreover, the stoichiometry of binding (sites/platelet) showed an agreement with V(max) of FX activation and was reduced in those proteins that also showed a decreased platelet binding affinity. A peptide corresponding to the FIX EGF2 domain (Leu(84)-Val(128)) was an effective inhibitor of FIXa binding to platelets in both the presence (K(i) = 0.7 x 10(-6) m) and the absence (K(i) = 1.5 x 10(-6) m) of FVIIIa and FX. We conclude that residues 88-109 of the FIXa EGF2 domain mediate binding to platelets and assembly of the FX activating complex.ut not Ar     

Binding of epidermal growth factor by human colon carcinoma cell (Caco-2) monolayers

The objective of this study was to investigate whether Caco-2 cells bind and internalize epidermal growth factor (EGF). [125I]EGF was presented to the apical (AP) or basolateral (BL) side of Caco-2 monolayers, grown on microporous membranes, at different times in culture. At day 10, [125I]EGF binding (at 37 degrees C) to the BL membrane was 2-3 times greater than binding to the AP membrane. Of that [125I]EGF bound to the AP membrane 76% was internalized within 3 h while internalization from the BL membrane was 90%. At lower temperatures membrane-bound [125I]EGF increased while internalization decreased. At day 16, AP and BL binding decreased and then remained constant through day 25. [125I]EGF was bound to the BL membrane of 10 days old monolayers with a Kd of 0.67 nM. There was a single binding site whose numbers in the BL membrane was about 5500/cell.     

Roles of epidermal growth factor (EGF) in the growth and differentiation of human endometrium

Endometrial tissues undergo drastic changes during menstrual cycle. After menstruation, they proliferate and differentiate into cells with secretory activity in the preparation for egg implantation. Although sex steroids play an important role in the development of endometrial tissues, sequential events occurring in the endometrium can not be fully explained by the direct actions of sex steroids. In this study, we offer evidences that EGF is released from endometrial cells and they possess the receptor for EGF. These findings prompted us to explore the biological roles of EGF in endometrial tissues. Here we clearly demonstrate that EGF is involved in the proliferation of endometrial cells. Moreover, EGF is found to enhance both glycogenesis and glycogenolysis, thus increasing the supply of glucose for blastocysts. We further set forth that EGF augments the capacity of progestin receptor and release of prostaglandins in endometrial cells. In summary, this study emphasizes that EGF may participate in the development of human endometrial tissues in concert with sex steroids, thus contributing to the acquisition of receptivity of eggs in the endometrium.     

Renal synthesis of epidermal growth factor is unchanged by vitamin D deficiency.

In mammalian kidney, epidermal growth factor (EGF) is produced as a small internal domain of an abundant high molecular weight peptide associated with the luminal membrane of the thick ascending limb of Henle's loop and distal convoluted tubule. At present, there is no evidence to indicate a mitogenic function for the EGF-containing molecule in kidney; consideration of its molecular structure suggests the possibility of a membrane-associated physiologic role. In this study, we examine regulation of renal EGF synthesis during induction of vitamin D deficiency in mice. Despite evidence of marked hyperparathyroidism, urinary excretion of EGF was equivalent in control (2.54 +/- 0.72 micrograms/mg creatinine) and vitamin D deficient (2.13 +/- 0.97 micrograms/mg creatinine) animals. Similarly, EGF mRNA levels in kidney were comparable in the two groups. These data indicate that parathyroid hormone has no effect on renal EGF regulation, although it is known to stimulate calcium reabsorption in distal nephron segments.     

The solution structures of epidermal growth factor and transforming growth factor alpha

The structures of human epidermal growth factor (EGF) and human transforming growth factor alpha (TGFα) have been determined in solution using nuclear magnetic resonance techniques. The features of each structure are described and similarities and differences between them are discussed. The structures are combined with information from sequence homologies to produce a model of the receptor-recognition sites of EGF and TGFα, which can be tested in a site-directed mutagenesis programme. The model assists in explaining previous observations of sequence-activity relationships. The TGFα and EGF structures also serve as models for homologous modules in other extracellular proteins.     

Secretion of human epidermal growth factor by Corynebacterium glutamicum

AIMS: To examine the secretion of human epidermal growth factor (hEGF) by Corynebacterium glutamicum. METHODS AND RESULTS: We recently showed that a novel protein-secretion system in C. glutamicum could produce Streptomyces mobaraensis transglutaminase. In the present study, the industrially important protein hEGF was secreted into the culture medium in a fully active form by C. glutamicum and accumulated at a rate of up to 156 mg l(-1) day(-1). CONCLUSIONS: These results demonstrated that the hEGF protein could be secreted in an active form by C. glutamicum. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data confirmed that the pharmaceutically important human protein hEGF could be efficiently secreted in an active form by the C. glutamicum protein-expression system. Moreover, we demonstrated that this bacterium has potential as a host for the industrial-scale production of human proteins.     

Cytoskeleton-dependent release of human platelet epidermal growth factor.

To study the source of immunoreactive epidermal growth factor (ir-EGF) released by thrombin formation we removed 99.9% of the leukocytes normally present in platelet-rich plasma and induced coagulation with 30 mM of Ca2+. The absence of leukocytes did not reduce the amount of ir-EGF released; thus platelets are most likely the only source of the ir-EGF released during aggregation. To identify the site of ir-EGF in platelets we exposed washed platelets to collagen or thrombin and compared the kinetics of releases of ir-EGF, beta-thromboglobulin (bTG, an alfa-granule marker), ATP (dense granule marker), N-acetyl-beta-D-glucosaminidase (NAGA, a lysosome marker) and lactate dehydrogenase (LDH, a cytoplasmic marker). Release of ir-EGF started immediately and continued linearly. The process differed clearly from the releases of the granule markers, which occurred readily, and were completed in a few minutes. The release of ir-EGF also differed from the leakage of LDH, the start of which was delayed greater than 5 min, but then proceeded linearly. Cytochalasin B inhibited the release of hEGF, but demecolcine had no effect. We conclude that the ir-EGF released from platelets during aggregation derives neither from the granules nor the cytoplasma. The assembly of cytoskeleton is needed for its release.     

Total synthesis of biotinylated N domain of human hepatocyte growth factor

Hepatocyte growth factor/scatter factor (HGF/SF) is the high affinity ligand of MET tyrosine kinase receptor. We report here the total synthesis of a biotinylated analogue of human HGF/SF N domain. Functionally, N domain is part of the HGF/SF high affinity binding site for MET and also the main HGF/SF binding site for heparin. The 97 Aa linear chain featuring a C-terminal biotin group was assembled in high yield using an N-to-C one-pot three segments assembly strategy relying on a sequential Native Chemical Ligation (NCL)/bis(2-sulfanylethyl)amido (SEA) native peptide ligation process. The folded protein displayed the native disulfide bond pattern and showed the ability to bind heparin.     

Age-related decrease of urinary excretion of human epidermal growth factor (hEGF)

Epidermal growth factor (EGF) has been first isolated from the submandibular glands of the male mouse and recently from human urine. Despite its potent mitogenic effect in a variety of tissues, the physiological functions of EGF in human still remain undetermined. In order to study the effect of age on urinary human EGF (hEGF), we have evaluated urinary excretion of hEGF in normal subjects over a wide range of age (20–79 yr.) using homologous radioimmunoassay (RIA) for hEGF. Urinary excretion of hEGF expressed as a function of creatinine significantly decreased with increasing age, age, while females excreted significantly more hEGF than males. These data suggest that urinary excretion of hEGF decreases with age in normal subjects which may be due to reduced synthesis and/or secretion of hEGF.