Human breast cancer cells synthesize and secrete an EGF-like immunoreactive factor in culture

A human breast cancer cell line, strain MCF-7, in culture synthesized and secreted a large amount of a polypeptide (designated as MCF-7 EGF) immunologically related to human epidermal growth factor (hEGF). The molecular weight of MCF-7 EGF estimated by gel filtration on Sephadex G-75 was similar to that of hEGF from human urine. On isoelectric focusing analysis, MCF-7 EGF gave a major peak at pH 4.6 and a minor peak at pH 5.0. In our enzyme immunoassay system, however, the dose-response curve of MCF-7 EGF did not show good parallelism with that of standard hEGF. From these results, it is suggested that MCF-7 cells synthesize and secrete a polypeptide immunologically related to hEGF into the culture medium.     

Synthesis and secretion of an hEGF-like immunoreactive factor by human gastric cancer cells (MKN-45)

A large amount of an immunoreactive factor was detected in the medium conditioned by human gastric cancer cells, strain MKN-45, by our enzyme immunoassay system for human epidermal growth factor (hEGF) based on hEGF isolated from urine. However, the dose-response curve of the immunoreactive factor (designated as MKN-45 EGF) was not parallel with the standard curve of hEGF. The molecular weight of MKN-45 EGF was slightly larger than that of hEGF and was estimated to be 7,000-8,000 by gel filtration on Sephadex G-50. On isoelectric focusing analysis, MKN-45 EGF gave a major peak at pH 5.0 and a minor one at pH 4.3. These results demonstrate that MKN-45 cells synthesize and secrete into the culture medium a polypeptide immunologically related to hEGF.     

Some properties of human epidermal growth factor (hEGF)-like immunoreactive material originating from platelets during blood coagulation

During blood coagulation, a considerable amount of human epidermal growth factor (hEGF)-like immunoreactive material (designated as platelet hEGF-LI) was liberated from platelets. The molecular nature of the platelet hEGF-LI was examined. The molecular weight of platelet hEGF-LI estimated by gel filtration was approximately 60,000-70,000. On chromatofocusing chromatography, platelet hEGF-LI was eluted mainly at pH 4.75 as a sharp peak with a minor peak at 4.30, like urine EGF. By treatment with 2-mercaptoethanol, the factor was converted into a material with molecular weight of 35,000-40,000. These results suggest that the majority of hEGF-LI in platelets may exist either in a covalently bound-form with some protein(s) in platelets or as a dimer intermolecularly cross-linked by an S-S linkage. Details of the biological properties and the physiological significance of the platelet hEGF-LI remain to be clarified.     

Human fibroblast cells synthesize and secrete nerve growth factor in culture.

Using our enzyme immunoassay system developed for recombinant hNGF, we examined the synthesis and secretion of human NGF (hNGF) by human fibroblast (WS-1) cells. The amount of the factor secreted by WS-1 cells increased linearly and a significant amount of NGF was detected in the conditioned medium of WS-1 cultures. WS-1 NGF showed properties identical to those of recombinant human NGF in immunoreactivity and molecular weight. An increase in cell density or the withdrawal of serum from the culture medium caused a drastic decrease in the rate of NGF secretion. These results suggest that WS-1 cells are able to synthesize and secrete hNGF in culture and that the synthesis/secretion is regulated in a growth phase-dependent manner.     

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.     

Production and properties of a monoclonal antibody against human epidermal growth factor

A monoclonal antibody against human epidermal growth factor (hEGF) was obtained from a mouse hybridoma cell line. The purified monoclonal antibody from the ascites fluid of a mouse injected with one of the cell lines was specific for hEGF and did not cross-react with mouse EGF (mEGF). Its Kd value for hEGF was 1.4 X 10(-9) M. This monoclonal antibody inhibited the biological activities of hEGF, including its binding to the receptor of BALB/3T3 cells and its stimulation of DNA synthesis in the cells, but did not affect the activities of mEGF. The monoclonal antibody completely inhibited DNA synthesis stimulated by human urine from a patient without a tumor, but only partially inhibited the stimulatory activity in urine from a tumor-bearing patient.     

Overproduction of human epidermal growth factor/urogastrone in Escherichia coli and demonstration of its full biological activities

A man-made gene coding for [21-Leu] human epidermal growth factor (hEGF)/β-urogastrone was constructed, inserted into a lacZ fusion-gene expression vector, and cloned into Escherichia coli. In the cloned cells the β-galactosidase/hEGF fusion gene was efficiently expressed and the yield of the hybrid protein reached 10% of the total cellular protein. The [21-Leu] hEGF synthesized in the bacterial cells was proved to be as functional as the natural hEGF or urogastrone and mouse EGF by the following criteria: (1) stimulation of cell proliferation, (2) stimulation of thymidine incorporation into cultured cells, (3) competition with mouse EGF for binding sites on the plasma membrane of human KB cells, and (4) stimulation of phosphorylation of a membrane-bound protein of human KB cell, which has an apparent molecular weight of 150 000 to 170 000 and is indistinguishable from the protein phosphorylated in the presence of mouse EGF in the sodium dodecyl sulfate—polyacrylamide electrophoretic gel. The hEGF produced in the bacterial cells also resulted in precocious eyelid-opening by the daily subcutaneous injection into newborn mice and in accelerated incisor eruption in the animals as mouse EGF did, indicating that the hEGF is also fully active in vivo or physiologically.     

Assessment by a two-site enzyme immunoassay of human epidermal growth factor (urogastrone) in the urine of patients with various gastrointestinal diseases including malignant tumors

By using our two-site enzyme immunoassay (EIA) system, the levels of human epidermal growth factor (hEGF) in the urine of patients with various gastrointestinal diseases including malignant tumors were measured. Urinary excretion of hEGF in patients having undergone gastric resection, expressed as a function of creatinine, was found to be somewhat decreased. While the levels of hEGF in patients with gastric cancer were significantly increased. Then, the molecular features of hEGF in the urine of patients with gastric cancer were examined by gel filtration. The elution profile demonstrated that high molecular weight components, which immunologically cross-reacted with hEGF, were considerably increased. On the other hand, the level of normal EGF with a molecular weight of 6500 was decreased to some extent. These results suggest that processing of the EGF precursor into an active EGF molecule is partially suppressed in patients with gastric cancer.     

人工合成表皮生长因子基因在甲基营养型酵母中的高效表达

选用酵母菌偏爱密码子人工合成了编码５１个氨基酸的人表皮生长因子（ｈＥＧＦ）基因．将合成基因与编码酵母α因子前导肽８５个氨基酸的ＤＮＡ片段融合后克隆到醇氧化酶基因启动子下游,并构建出多拷贝表达载体．此载体转化甲基营养型酵母株ＧＳ１１５后筛选出整合型ＭｕｔＳＨｉｓ＋基因型菌株．高密度培养及诱导表达后该株可分泌具完好生物活性和正确物理化学性质的人表皮生长因子,产量达１００ｍｇ／Ｌ,经３次柱层析纯度达９５％以上,为观察其生物学作用打下了良好基础     

Human epidermal growth factor and the proliferation of human fibroblasts

The effect of human epidermal growth factor (hEGF), a 5,400 molecular weight polypeptide isolated from human urine, on the growth of human foreskin fibroblasts (HF cells) was studied by measuring cell numbers and the incorporation of labeled thymidine. The addition of hEGF to HF cells growing in a medium containing 10% calf serum resulted in a 4-fold increase in the final density. The presence of hEGF also promoted the growth of HF cells in media containing either 1% calf serum or 10% gamma globulin-free serum. The addition of hEGF to quiescent confluent monolayers of HF cells, maintained in a medium with 1% calf serum for 48 hours, resulted in a 10- to 20-fold increase in the amount of ³H-thymidine incorporation after 20–24 hours. The stimulation of thymidine incorporation was maximal at an hEGF concentration of 2 ng/ml, was dependent on the presence of serum, and was enhanced by the addition of ascorbic acid. In confluent cultures of HF cells, subject to density dependent inhibition of growth, hEGF was able to stimulate DNA synthesis more effectively than fresh calf serum. Human EGF stimulated DNA synthesis in quiescent cultures, however, regardless of cell density. The addition of rabbit anti-hEGF inhibited all effects of this growth factor on HF cells.     

Characterization of a high-molecular-weight form of epidermal growth factor in an extract of human urine

We purified from a side fraction of the commercial preparation of urokinase from large volumes of human urine a high-molecular-weight (HMW) form of human epidermal growth factor (hEGF). Sequence analysis of the amino terminus of the intact molecule and of two tryptic fragments and carboxypeptidase Y analysis revealed the molecule to correspond to residues 828-1023 of the hEGF precursor predicted by the nucleotide sequence of human renal hEGF mRNA, with hEGF forming its carboxyl terminus. HMW hEGF bound poorly to concanavalin A-agarose, quite avidly to wheat germ lectin-agarose, and completely to phenyl boronate-agarose, suggesting that it was O-glycosylated. Sephacryl S-200 chromatography of freshly-voided urine revealed mostly hEGF, with smaller amounts of a much higher molecular weight hEGF, but little material that was the size of the HMW hEGF we characterized. The large fragment we characterized presumably is cleaved from the larger form by enzyme(s) present in urine during the collection, storage, and processing of urine. We have confirmed that hEGF is synthesized as a large precursor molecule, as predicted by the nucleotide sequence of hEGF mRNA.     

Partial purification and characterization of epidermal growth factor in human breast milk

Human epidermal growth factor (hEGF), a potent growth stimulator of many tissues in culture, has been isolated from human urine and subsequently identified in many human biological fluids including breast milk. In this study, partial purification and characterization of hEGF-like substance(s) in human milk were performed using homologous hEGF radioimmunoassay (RIA) and radioreceptor assay (RRA). hEGF-like material(s) was extracted from pooled human milk by ethanol precipitation, followed by adsorption to cation- and anion-exchange resin. DEAE-Sephadex G-25 ion-exchange chromatography of human milk extracts revealed three major components with hEGF activity (peak I, II, III) eluted with a linear gradient by ammonium acetate. The competitive binding curves for these components were parallel to those for standard hEGF in both RIA and RRA. The apparent molecular weight of peak I was approximately 6,500 and that of peak II and III was approximately 7,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The pI value for peak I was approximately 4.5 and that for peaks II and III was approximately 5.0 by isoelectric focusing. These data are comparable to the size and charge heterogeneity of hEGF in human urine extracts. In conclusion, the major components of hEGF in human milk appear to be physicochemically, immunologically and biologically (receptor binding activity) indistinguishable from hEGF of urinary origin.     

人表皮生长因子的研究进展

人表皮生长因子是激活表皮生长因子受体的生长因子家族的典型成员,由人体的多个组织器官合成与分泌,通过结合受体激活一系列信号途径,调控细胞的增殖、分化和迁移等。近年来,有关人表皮生长因子的研究已扩展到其在人类生理和病理作用的领域,尤其在组织再生和伤口愈合方面成为研究热点。文中综述了人表皮生长因子的研究进展,简要描述了其基因和蛋白的结构与特点、作用机制与生物学效应,重点介绍该生长因子在胃肠溃疡愈合、皮肤伤口修复和肿瘤病理过程中的作用与影响,从而为相关研究提供辅助信息。     

Monoclonal antibodies specific for high molecular weight form of human epidermal growth factor

Hybridomas that secrete monoclonal antibodies specific for the high molecular weight (HMW) form of human epidermal growth factor (hEGF) were established by fusing spleen cells obtained from mice immunized with purified urinary HMW-hEGF with myeloma P3 x 63Ag8.653. The resulting monoclonal antibodies were characterized basically into two groups. One group recognized both EGF and HMW-hEGF, while the other recognized HMW-hEGF specifically on radio immunoprecipitation. Surprisingly, the majority of the isolates was positive by western blotting. Utilizing these monoclonal antibodies for affinity chromatography, we purified HMW-hEGF successfully from urine. These antibodies may be an extraordinarily powerful tool for histological study related to both forms of EGF.     

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.     

Expression of a fusion protein containing human epidermal growth factor and the collagen-binding domain of <Emphasis Type="Italic">Vibrio mimicus</Emphasis> metalloprotease

Human epidermal growth factor (hEGF) is a polypeptide of 53 amino acids, is an important autocrine/paracrine factor in the human body, and is used in the pharmaceutical and cosmetics industries. We constructed a fusion hEGF protein with a collagen-binding domain (CBD) composed of 33 amino acids from Vibrio mimicus metalloprotease (VMCBD). The CBD segment of the metalloprotease was fused at the C terminus of the hEGF protein. The recombinant fusion protein was expressed in Escherichia coli and purified. The purified hEGF protein promoted greater growth of human/A-431 cells than did the control hEGF. The fusion EGF protein also showed collagen-binding activity with type I collagen. In contrast, hEGF did not bind to type I collagen. These results suggest that recombinant hEGF protein fused to VMCBD may be able to remain for a long period at injured epidermal tissue acting as a healing agent.     

Isolation and characterization of epidermal growth factor from human milk

Epidermal growth factor (EGF) has been purified from human milk. The purification was monitored with a human placental membrane radioreceptor assay using murine salivary epidermal growth factor I (mEGF I) as a competitive ligand and was achieved exclusively by the use of reverse-phase liquid chromatography (RPLC). The sequential use of preparative, semipreparative and analytical RPLC on an octylsilica support with solvent systems of different solute selectivity such as pyridine formate, triethylammonnium phosphate or perfluorocarbonic acids in the presence of n-propanol or acetonitrile allowed purification to homogeneity with 5 consecutive runs. The molecular mass, amino acid composition and NH2-terminal sequence of human EGF were determined. Gas-phase microsequencing of residues 1-17 revealed the following sequence: Asn-Ser-Asp-Ser-Glu-X-Pro-Leu-Ser-His-Asp-Gly-Tyr-X-Leu-X-Asp which is identical with the NH2-terminof urogastrone from human urine. The purified polypeptide competes with mEGF for the placental membrane receptor with a ki of 1 ng. Furthermore, it stimulates the anchorage-dependent as well as -independent proliferation of human and rat indicator cells with half-maximal stimulation at 1 and 2.5 ng/ml, respectively. Although human epidermal growth factor has been unequivocally identified in human milk and -for the first time-shown to be identical with urogastrone from human urine, the high-resolution techniques employed have also revealed the presence of EGF-related molecules which await further characterization. It is possible that EGF and the EGF-related growth factors possess important regulatory functions in normal growth of the human breast during pregnancy and lactation as well as in abnormal growth during mammary tumor formation and progression.     

Receptor recognition by histidine 16 of human epidermal growth factor via hydrogen-bond donor/acceptor interactions

Human epidermal growth factor (hEGF) and human transforming growth factor alpha (hTGFalpha) are prototypical of structurally related polypeptide mitogens which interact with the epidermal growth factor receptor (EGFR). Several determinants of receptor recognition that specify function have been proposed on the basis of structural criteria. This study evaluates the role of one such candidate, H16 of hEGF, by site-specific mutagenesis. When assayed for receptor tyrosine kinase stimulation using (Glu4,Tyr1)n as the exogenous substrate in vitro, the relative agonist activities of position 16 mutants range from 14-263% of wild-type hEGF. The rank order of potency was found to correlate with the relative receptor binding affinities of the mutants, which range from 7-272% of wild-type, as determined by radioreceptor competition assays. The mitogenic activity of the H16 mutants is similar to that of wild-type hEGF as determined by clonogenic assays using rat tracheal epithelial cells. While the colony forming efficiencies do not reflect significant differences in growth rate or survival characteristics in the presence of the hEGF variants, it is reduced to 1.6% in control cultures which lack EGF in the medium. The results show that H16 of hEGF, although not essential for mitogenic activity, optimizes receptor recognition by hydrogen-bond donor/acceptor interactions and may share this feature with H18 of hTGFalpha.     

转基因迷你番茄及其对酒精引起的胃伤害的保护作用

表皮生长因子(EGF)具有促进多种细胞增殖的作用, 尤其在维持消化道黏膜完整和促进消化道溃疡愈合方面作用巨大。以前的研究多集中在细菌和酵母中表达, 本研究试图以番茄作为生物反应器生产重组人 EGF, 使之成本降低, 应用方便。依据人 EGF 的基因序列, 设计合成了番茄密码子偏爱的人 EGF 基因, 并将其构建到植物表达载体 pCAMBIA2300 中, 通过农杆菌介导得到了含有人 EGF 基因的转基因番茄。放射免疫法检测到每克鲜重果实中的表达量达 3.48 ± 1.01 ng。将果汁灌喂小白鼠 15 天(相当于每鼠每天喂服 24 ng rhEGF)能显著抵抗酒精引起的胃溃疡形成, 溃疡指数由 42.20 ± 18.13 下降为 16.25 ± 9.57。     

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.