Nerve growth factor activity of several immunal related serine proteases

Rabbit was immuned by the previously purified protein with high nerve growth factor (NGF) bioactivity (NGF_like protease) from \%Agkistrodon halys Pallas\% and the antisera were collected. The polyclonal antibodies were tentatively purified and then used as ligands of an affinity column. The \%A.h.Pallas\% crude venom was fractionated by this affinity column and then by Mono Q on fast protein liquid chromatography (FPLC). As a result, fraction Ⅱ and fraction Ⅲ were purified respectively, whose N_terminal amino acid sequences show high homology with the serine proteases in snake venoms, as well as the previous NGF_like protease. However, they possessed different levels of NGF bioactivity. The NGF activity of the previous NGF_like protease is equivalent to that of NGF, while the activity of fraction Ⅱ seems relatively low in contrast to fraction Ⅲ which had no NGF activity.     

Similarity between the vaccinia virus 19K early protein and epidermal growth factor

An analysis of the 1,217-amino acid residue sequence of the precursor of mouse epidermal growth factor (mEGF) revealed regions of considerable similarity with bovine factor X, a blood coagulation factor. Similarities of mEGF itself with factor X, pancreatic secretory trypsin inhibitor and, most strikingly, transforming growth factor I (TGF-I) have been observed. On the basis of the comparisons described here, it seems that the presumptive 140-residue 19K early protein (relative molecular mass (Mr) 19,000) of vaccinia virus from residues 40-91 shows an overall identity of 36% (19/53 residues) with both mEGF and urogastrone (human epidermal growth factor, hEGF); a single deletion is assumed for vaccinia virus 19K protein which allows the six Cys residues (positions 45-80) to be aligned with those of mEGF or hEGF. This protein is encoded in the 10.3-kilobase (kb) inverted terminal repeat. Because it is an early protein with an EGF-like central portion, the 19K vaccinia virus protein may have an autocrine function and may be required for DNA synthesis.     

Tertiary structural differences between microbial serine proteases and pancreatic serine enzymes.

Although primary structural homology between bacterial serine proteases and those from the mammalian pancreas is slight, two-thirds of the residues in the bacterial enzyme SGPB as seen at 2.8-A resolution, adopt a similar polypeptide chain conformation to that of the chymotrypsin family. The three major regions of difference show how this family of proteolytic enzymes has developed from the more primitive bacterial to the relatively sophisticated pancreatic enzymes.     

ATP-stimulated interaction between epidermal growth factor receptor and supercoiled DNA

The receptor for epidermal growth factor (EGF) has been identified as a transmembrane glycoprotein that has tyrosine-specific kinase activity. The kinase activity of the receptor is enhanced in the presence of EGF (or related peptides), and the phosphorylation of a number of substrates, as well as autophosphorylation of the receptor, has been reported. Analogous findings have been described for the insulin receptor and the receptor for platelet-derived growth factor (PDGF). Thus, a number of hormone receptors and several viral transforming proteins appear to share the highly unusual property of tyrosine-specific kinase activity. Nevertheless, the specific relationship between tyrosine kinase activity and the control of cell growth and replication is unknown. It is known that after the initial binding of EGF to the plasma membrane, the hormone together with its receptor is rapidly internalized in endocytic vesicles and the hormone is eventually degraded in lysosomes. It is possible that the function of EGF is simply to stimulate internalization of its receptor, and that as a result of its altered location the receptor is able to phosphorylate a cytoplasmic component or even interact directly with a nuclear component. We now report that the purified receptor for EGF is able to interact with and nick supercoiled double-stranded DNA in an ATP-stimulated manner.     

The neu oncogene encodes an epidermal growth factor receptor-related protein

The neu oncogene is repeatedly activated in neuro- and glioblastomas derived by transplacental mutagenesis of the BDIX strain of rat with ethylnitrosourea. Foci induced by the DNAs from such tumours on NIH 3T3 cells contain the neu oncogene and an associated phosphoprotein of relative molecular mass 185,000 (p185). Previous work has shown that the neu gene is related to, but distinct from, the gene encoding the EGF receptor (c-erb-B). Here we describe a neu complementary DNA clone isolated from a cell line transformed by this oncogene; the clone has biological activity in a focus-forming assay. The nucleotide sequence of this clone predicts a 1,260-amino-acid transmembrane protein product similar in overall structure to the EGF receptor. We found that 50% of the predicted amino acids of neu and the EGF receptor are identical; greater than 80% of the amino acids in the tyrosine kinase domain are identical. Our results suggest strongly that the neu gene encodes the receptor for an as yet unidentified growth factor.     

Close similarity of epidermal growth factor receptor and v-erb-B oncogene protein sequences

Each of six peptides derived from the human epidermal growth factor (EGF) receptor very closely matches a part of the deduced sequence of the v-erb-B transforming protein of avian erythroblastosis virus (AEV). In all, the peptides contain 83 amino acid residues, 74 of which are shared with v-erb-B. The AEV progenitor may have acquired the cellular gene sequences of a truncated EGF receptor (or closely related protein) lacking the external EGF-binding domain but retaining the transmembrane domain and a domain involved in stimulating cell proliferation. Transformation of cells by AEV may result, in part, from the inappropriate acquisition of a truncated EGF receptor from the c-erb-B gene.     

Vaccinia virus encodes a polypeptide homologous to epidermal growth factor and transforming growth factor

Epidermal growth factor (EGF) and transforming growth factor type I (TGF) are polypeptides of 53 and 50 amino acid residues, respectively. Both bind to EGF receptor, a 1,200-residue transmembranous glycoprotein, leading to phosphorylation of the receptor, enhancement of its tyrosine-specific kinase activity and ultimately to stimulation of cell growth. We report here that a 140-residue polypeptide encoded by one of the early genes of vaccinia virus (VV) is related closely to EGF and TGF. The presence of putative signal and transmembranous sequences further suggests that the viral protein might be an integral membrane protein, but that, as in the case of EGF itself, the membrane-associated form may be the precursor of a soluble growth factor. Production of EGF-like growth factors by virally infected cells could account for the proliferative diseases associated with members of the poxvirus family such as Shope fibroma virus, Yaba tumour virus, and molluscum contagiosum virus (MCV).     

Nucleotide sequence of epidermal growth factor cDNA predicts a 128,000-molecular weight protein precursor

Epidermal growth factor (EGF) has a profound effect on the differentiation of specific cells in vivo, and has been shown to be a potent mitogenic factor for a variety of cultured cells, of both ectodermal and mesodermal origin (see ref. 1 for review). This 53-amino acid polypeptide of known sequence contains six cysteine residues, which are thought to form three intrachain disulphide bonds. Urogastrone, a polypeptide bearing anti-gastric secretory activity isolated from human urine, which is presumably synthesized in submandibular and Brunner's glands, shares extensive sequence homology (70%) with EGF and may represent the human EGF equivalent. Here we present the sequence of a mouse EGF cDNA clone, which suggests that EGF is synthesized as a large protein precursor of 1,168 amino acids. Our data indicate that the discrepancy between EGF levels in male and female mouse submaxillary glands (MSGs) is due to different EGF mRNA levels in these tissues, and suggest that precursor EGF processing may differ from that described previously for other polypeptide hormones.     

New protein fold revealed by a 2.3-A resolution crystal structure of nerve growth factor

Nerve growth factor (NGF) is a member of an expanding family of neurotrophic factors (including brain-derived neurotrophic factor and the neurotrophins) that control the development and survival of certain neuronal populations both in the peripheral and in the central nervous systems. Its biological effects are mediated by a high-affinity ligand-receptor interaction and a tyrosine kinase signalling pathway. A potential use for NGF and its relatives in the treatment of neurological disorders such as Alzheimer's disease and Parkinson's disease requires an understanding of the structure-function relationships of NGF. NGF is a dimeric molecule, with 118 amino acids per protomer. We report the crystal structure of the murine NGF dimer at 2.3-A resolution, which reveals a novel protomer structure consisting of three antiparallel pairs of beta strands, together forming a flat surface. Two subunits associate through this surface, thus burying a total of 2,332 A. Four loop regions, which contain many of the variable residues observed between different NGF-related molecules, may determine the different receptor specificities. A clustering of positively charged side chains may provide a complementary interaction with the acidic low-affinity NGF receptor. The structure provides a model for rational design of analogues of NGF and its relatives and for testing the NGF-receptor recognition determinants critical for signal transduction.     

Dissection of the protein kinase cascade by which nerve growth factor activates MAP kinases.

Mitogen activated protein (MAP) kinases (MAPKs) are a family of protein-serine/threonine kinases activated as an early intracellular response to a variety of hormones and growth factors. They are unique in requiring both serine/threonine and tyrosine phosphorylation to become active and are the only examples of protein-serine/threonine kinases activated by tyrosine phosphorylation. Nerve growth factor (NGF) promotes differentiation of phaeochromocytoma (PC12) cells, which respond by conversion within hours from a chromaffin-like to a sympathetic neuron-like phenotype. NGF stimulation of PC12 cells increases the activity of two protein kinases by greater than 20-fold within minutes, both strikingly similar to MAPKs. They are inactivated by either protein-tyrosine phosphatases or the protein-serine/threonine phosphatase termed protein phosphatase 2A (ref. 8), they activate protein S6 kinase-II (refs 9, 10), and they phosphorylate identical threonine residues on myelin basic protein (our unpublished results) to those phosphorylated by other MAPKs. Immunological data indicate that these protein kinases, termed peak-I and peak-II (Fig. 1a) are probably ERK2 and ERK1, respectively, two widely expressed MAPK isoforms. Here we identify the 'MAP kinase kinases' (MAPKKs) in PC12 cells which are activated by NGF and report that MAPKKs are dependent on serine/threonine phosphorylation for activity and promote phosphorylation of serine/threonine and tyrosine residues on MAPKs.     

重组人表皮生长因子在毕赤酵母X33中的构建、表达和纯化

在毕赤酵母X33中表达人表皮生长因子并纯化,对表皮生长因子(EGF)进行密码子优化,构建pPICZa A-EGF真核表达质粒,转化X33感受态细胞;利用抗性筛选以及PCR鉴定阳性菌株。经过甲醇诱导和镍柱纯化,聚丙烯酰胺凝胶电泳检测蛋白分泌表达情况。成功构建了表达pPICZa A-EGF真核表达质粒,转入X33中获得阳性菌株,成功诱导蛋白分泌表达并纯化。在毕赤酵母X33中成功表达人表皮生长因子,纯化后纯度为80%,收率为5.8 mg/L。     

Epidermal growth factor and the multiplication of cultured human epidermal keratinocytes.

The culture lifetime of epidermal cells of newborn humans is increased from 50 to 150 generations by adding to the medium epidermal growth factor, a polypeptide mitogen. EGF seems to delay senescence of the cells by maintaining them in a state further removed from terminal differentiation. This effect is revealed by a greater ability of the cells to survive subculture and initiate new colonies, but not necessarily by an increased growth rate.     

Human chromosomal mapping of genes for insulin-like growth factors I and II and epidermal growth factor

Many of the actions previously attributed to pituitary-derived growth hormone are mediated by polypeptide growth factors. These include the insulin-like growth factors I and II (IGF-I and IGF-II), which are members of the insulin family of proteins. We report here the chromosomal mapping of the human genes for IGF-I and IGF-II. IGF-II maps to the short arm of chromosome 11, which also contains the gene for insulin and the proto-oncogene c-Ha-ras1 (ref. 9). IGF-I maps to chromosome 12, which is evolutionarily related to chromosome 11 and carries the gene for the proto-oncogene c-Ki-ras2 (refs 10,44). We have also localized the human gene for an unrelated polypeptide hormone, epidermal growth factor, to chromosome 4q, in the same region as another specialized growth factor, T-cell growth factor. We speculate that these map assignments reflect the existence of gene families involved in growth control.     

Rapid induction of mRNAs forPC3 by partial hepatectomy and epidermal growth factor

The expression of immediate early gene plays a pivotal role in rat hepatocyte proliferation from G₀ to G₁ phases and the progression through G₁ phase of the cell cycle within several hours after 2/3 hepatectomy. We investigated the different gene expressions within 1 h after 2/3 hepatectomy by representational difference analysis. Sequence analysis indicated thatPC3 induced by NGF was a kind of immediate early gene and might be correlated with liver regeneration. Moreover, we found that 2/3 hepatectomy could induce the expressing ofPC3 mRNA by Northern blot with a peak 1–2 h after surgery. In primary cultures of rat hepatocytes, addition of EGF resulted in rapid and transient induction ofPC3 mRNA. It was first reported thatPC3 gene belongs to immediate early gene associated with liver regeneration.