Anti-synthetic Peptide Antibodies Research Articles

Immunoprotection against lethal effects of Crotalus durissus snake venom elicited by synthetic epitopes trapped in liposomes

Snakebites caused by Crotalus genus are the second most frequent in Brazil. Crotoxin is a beta-neurotoxin responsible for the main envenomation effects of Crotalus biting, while crotamine immobilizes the animal hind limbs, contributing to prey immobilization and to envenoming symptoms. As crotoxin and crotamine represent about 90% of Crotalus venom dry weight, these toxins are of great importance for antivenom therapy. In this sense, knowledge regarding the antigenicity/immunogenicity at the molecular level of these toxins can provide valuable information for the improvement of specific antivenoms. Therefore, the aims of this study are the identification of the B-cell epitopes from crotoxin and crotamine; and the characterization of the neutralizing potency of antibodies directed against the corresponding synthetic epitopes defined in the current study. Linear B-cell epitopes were identified using the Spot Synthesis technique probed with specific anti-C. d. terrificus venom horse IgG. One epitope of crotamine (F12PKEKICLPPSSDFGKMDCRW32) and three of crotoxin (L10LVGVEGHLLQFNKMIKFETR30; Y43CGWGGRGRPKDATDRCCFVH63 and T118YKYGYMFYPDSRCRGPSETC138) were identified. After synthesis in their soluble form, the peptides mixture correspondent to the mapped epitopes was entrapped in liposomes and used as immunogens for antibody production in rabbits. Anti-synthetic peptide antibodies were able to protect mice from the lethal activity of C. d. terrificus venom.

Localization of the bioadhesive precursors of the sandcastle worm, Phragmatopoma californica (Fewkes)

The marine sandcastle worm bonds mineral particles together into underwater composite dwellings with a proteinaceous glue. The products of at least four distinct secretory cell types are co-secreted from the building organ to form the glue. Prominent hetereogeneous granules contain dense sub-granules of Mg and the (polyphospho)proteins Pc3A and B, as well as at least two polybasic proteins, Pc1 and Pc4, as revealed by immunolabeling with specific antibodies against synthetic peptides. Equally prominent homogeneous granules comprise at least two polybasic proteins, Pc2 and Pc5, localized by immunolabeling with anti-synthetic peptide antibodies. The components of the sub-micrometer granule types are unknown, though positive staining with a redox-sensitive dye suggests the contents include o-dihydroxy-phenylalanine (dopa). Quantitative PCR and in situ hybridization demonstrated that a tyrosinase-like enzyme with a signal peptide was highly expressed in both the heterogeneous and homogeneous granules. The contents of the granules are poorly mixed in the secreted mixture that forms the glue. Subsequent covalent cross-linking of the glue may be catalyzed by the co-secreted tyrosinase. The first three parapodia of the sandcastle worm also contain at least two distinct secretory tissues. The Pc4 protein was immunolocalized to the anterior secretory cells and the tryosinase-like gene was expressed in the posterior secretory cells, which suggests these proteins may have multiple roles.

Detection of the Sm31 antigen in sera of Schistosoma mansoni– infected patients from a low endemic area

Schistosoma mansoni cathepsin B (Sm31) is a major antigen from adult worms that circulates in the blood of infected patients (Li et al., Parasitol Res 1996; 82: 14-18). An analysis of the Sm31 sequence (Klinkert et al., Mol Biochem Parasitol 1989; 33: 113-122) allowed the prediction of seven hydrophilic regions that were confirmed to be exposed on the surface of a 3D model of Sm31; the species specificity of these regions was checked using BLAST analysis. The corresponding peptides were chemically synthesized in polymerazible forms using the t-Boc technique. Rabbits developed a high humoral response against these peptides as tested by a multiple antigen blot assay; it recognized native Sm31 in crude S. mansoni extracts and as circulating antigen in sera of S. mansoni-infected patients by western blot. Relevant antigenic determinants were located at the N- and C-terminus sequences. Antibodies against these regions recognized the native enzyme in an ELISA-like assay called cysteine protease immuno assay in which the immunocaptured enzyme was revealed by the intrinsic cathepsin B hydrolytic activity of Sm31. The method successfully and specifically detected Sm31 in sera of infected individuals, most of them (83.3%) with light infections, offering a rationale for the development of parasite enzyme capture assays using anti-synthetic peptide antibodies for possible use in the diagnosis of schistoso,iasis.

Proteomic analysis of β-1,3-glucanase in grape berry tissues

Grape berries are considered recalcitrant materials in proteomic analysis, because berry tissues contain large amounts of secondary metabolites, especially phenolic compounds, which severely interfere with protein extraction and electrophoresis separation. We report hereby a PVPP/TCA-based protein extraction protocol for grape berries. Phenolic compounds in berry extracts were removed with repeated PVPP cleanups, and proteins were recovered with TCA precipitation. Protein resolution in 2-D gels was gradually improved with the increase of PVPP cleanup steps. By the protocol, about 760 protein spots of berry tissues were clearly resolved in 2-D gels with CBB staining. This protocol was also used to analyze β-1,3-glucanase (EC 3.2.1.39) in berry tissues. An anti-synthetic peptide antibody was prepared against 15 amino acid sequence residing on the surface of β-1,3-glucanase molecule. It detected two major spots in 2-D blots of berry extracts. The spots were identified by MALDI-TOF analysis as β-1,3-glucanase. The present study validates that β-1,3-glucanase is present in higher abundance in berry skins than in pulps, and in red berries than in white berries. Therefore, β-1,3-glucanase displays a tissue-specific expression. The preferential accumulation of β-1,3-glucanase in skins may be relevant to berry ripening.

Immunochemical characterization and measurement of neuronal type nitric oxide synthase in human neuroblastoma NB-OK-1 cell using novel anti-synthetic peptide antibody and specific immunoassay system.

We developed a sensitive and specific immunoassay system for human neuronal nitric oxide synthase (hnNOS) using synthetic hnNOS(998-1024) peptide and anti-hnNOS(998-1024) antibody. The novel antibody and radioimmunoassay system revealed a typical nNOS protein in human neuroblastoma NB-OK-1 cell (160 kDa, 180 fmol/10(6) cells). The kinetic parameters of the enzyme were K(m)=4.88 microM and V(max)=4.34 pmol/min/mg protein for L-arginine. On incubation of NB-OK-1 cell for 24 h, betamethasone phosphate decreased both nNOS-immunoreactivity (nNOS-IR) and enzymatic activity in the cell dose-dependently. On the other hand, pituitary adenylate cyclase activating polypeptide(1-38) (PACAP38) increased both nNOS-IR and enzymatic activity at concentrations of 10(-10) and 10(-9) M, but inversely decreased both at 10(-7) M. These suggest the positive and negative implications of endogenous NO in proliferation and differentiation of the cell, which support mitogenic activity of NO generated by nNOS in the cell. The present findings also provided evidence that the quantitative change of nNOS protein controls the integrated activity of the enzyme in the cell and, in turn, substantiate the validity and reliability of the present immunoassay system for hnNOS and its practical usefulness.

The Conformation of Calreticulin Is Influenced by the Endoplasmic Reticulum Luminal Environment

The Conformation of Calreticulin Is Influenced by the Endoplasmic Reticulum Luminal Environment

Use of a chaotropic anion iodide in the purification of Z-line proteins: isolation of CapZ from fish white muscle.

In the present study, we have described an improved method allowing the isolation of proteins which form tightly associated complexes in organized structures such as Z line in skeletal muscle. This procedure is based on both extraction and chromatography in the presence of a chaotropic agent. KI at medium concentration (0.6 M) was selected, taking into account its dissociating activity and mild effect on the native state of proteins. This procedure was applied to purify and to characterize for the first time a CapZ from fish white muscle, a protein involved in the stabilization of the filaments in Z line. The alpha and beta CapZ subunits were identified using anti-synthetic peptide antibodies directed against conserved sequences derived from chicken CapZ. The protocol can be also used for the isolation of other muscular proteins such as alpha-actinin and actin. Finally this technique may be utilized to obtain a good amount of capping protein which could be employed in experiments of microfilament dynamics.

Application of region-specific immunoassay for human chromogranin A: Substantial clue for detection and measurement of chromogranin A in human plasma

Chromogranin A (CgA), a secretory protein, is co-released with catecholamines from storage vesicles. It is known to be elevated in the circulation of patients with neuroendocrine and endocrine tumors. For further investigation of the protein, especially in humans, it is essential to facilitate quantitative analysis of the protein in human biological materials. In order to introduce novel immunological methodology for this purpose, we purposely selected human CgA(344–374) for the synthetic immunogen to produce region-specific CgA antibodies. The anti-synthetic peptide antibody thus obtained made it possible to develop an immunological method for measurement and characterization of CgA in human plasma. The plasma CgA-immunoreactivity (LI) level measured by the method was 0.31±0.01 pmol/ml (mean±SEM) in normal subjects and 1.55±0.29 pmol/ml in pheochromocytoma. On gel chromatography and HPLC analysis of the plasma of patients with pheochromocytoma, the region-specific assay system enabled us to show the presence of N-terminal truncated CgA, besides CgA itself. By following up changes of plasma CgA-LI in a pheochromocytoma patient using samples that were collected consecutively over a two-year period, the present assay system using the region-specific antibody, anti-human CgA (344–374) serum, was confirmed to be extremely valuable for the measurement of CgA-LI in human plasma. The characteristic features and high sensitivity of the present assay system will give us a substantial clue to the detection and measurement of CgA to develop further investigation of the protein in humans.

Synthesis, secretion, degradation, and fate of ameloblastin during the matrix formation stage of the rat incisor as shown by immunocytochemistry and immunochemistry using region-specific antibodies.

Rat ameloblastin is a recently cloned tooth-specific enamel matrix protein containing 422 amino acid residues. We investigated the expression of this protein during the matrix formation stage of the rat incisor immunohistochemically and immunochemically, using anti-synthetic peptide antibodies that recognize residues 27-47 (Nt), 98-107 (M-1), 224-232 (M-2), 386-399 (M-3), and 406-419 (Ct) of ameloblastin. Immunohistochemical preparations using antibodies Nt and M-1 stained the Golgi apparatus and secretory granules of the secretory ameloblast and the entire thickness of the enamel matrix. Only M-1 intensely stained the peripheral region of the enamel rods. Immunostained protein bands were observed near 65, 55, and below 22 kD. Immunohistochemical preparations using antibodies M-2 and Ct stained the Golgi apparatus and secretory granules of the ameloblast and the immature enamel adjacent to the secretion sites, but not deeper enamel layers. Immunostaining using M-2 and Ct revealed protein bands near 65 and 40-56 kD, and 65, 55, 48, 36, and 25 kD, respectively. M-3 stained the cis side of the Golgi apparatus but not the enamel matrix. This antibody recognized a protein band near 55 kD, but none larger. After brefeldin A treatment, immunoreaction of the 55-kD protein band intensified, and dilated cisternae of rER of the secretory ameloblast contained immunoreactive material irrespective of the antibodies used. These data indicate that ameloblastin is synthesized as a 55-kD core protein and then is post-translationally modified with O-linked oligosaccharides to become the 65-kD secretory form. Initial cleavages of the 65-kD protein generate N-terminal polypeptides, some of which concentrate in the prism sheath, and C-terminal polypeptides, which are rapidly degraded and lost from the enamel matrix soon after secretion.

Expression of the Retinal Pigmented Epithelial Cell-Specific pP344 Gene during Development of the Chicken Eye and Identification of Its Product: (eye development/retinal pigmented epithelium/gene expression/in situ hybridization).

In order to understand the transdifferentiation of the retinal pigmented epithelial cells (PECs) into the lens cells at the level of gene expression, a gene, tentatively called pP344 gene, was studied, because its expression appeared to be closely related with the differentiated state of PECs. We analyzed pP344 gene expression during chicken eye development by RT-PCR and in situ hybridization and also characterized the pP344 protein using antipeptide antibodies. In addition to the previous observation that the transcript of pP344 gene is limited to the pigmented epithelium and not detected in the melanocytes, we show here that the transcript is limited to retinal PECs and is never observed in iris or ciliary PECs. The time course of expression level showed two peaks; the first peak occurred around the 10th day similarly to the expression of melanosome-related genes, while the second peak occurred just after hatching when PECs had completely differentiated, suggesting that pP344 gene may be related to the function of fully differentiated PECs. Antisynthetic peptide antibodies detected pP344 protein in the culture medium of the PECs but not within the cells. Thus, we concluded that pP344 gene is specifically expressed by the retinal PEC and its product is a secreted protein.

Physiological Characterization of Putative High-Affinity Glucose Transport Protein Hxt2 of Saccharomyces cerevisiae by Use of Anti-Synthetic Peptide Antibodies

Physiological Characterization of Putative High-Affinity Glucose Transport Protein Hxt2 of <i>Saccharomyces cerevisiae</i> by Use of Anti-Synthetic Peptide Antibodies

Physiological characterization of putative high-affinity glucose transport protein Hxt2 of Saccharomyces cerevisiae by use of anti-synthetic peptide antibodies

Characterization and quantification of the Hxt2 (hexose transport) protein of Saccharomyces cerevisiae indicate that it is one of a set of differentially expressed high-affinity glucose transporters. The protein product of the HXT2 gene was specifically detected by antibodies raised against a synthetic peptide encompassing the 13 carboxyl-terminal amino acids predicted by the HXT2 gene sequence. Hxt2 migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a broad band or closely spaced doublet with an average M(r) of 47,000. Hxt2 cofractionated with the plasma membrane ATPase, Pma1, indicating that it is a plasma membrane protein. Hxt2 was not solubilized by high pH or urea but was solublized by detergents, which is characteristic of an integral membrane protein. Expression of the Hxt2 protein was measured under two different conditions that produce expression of high-affinity glucose transport: a medium shift from a high (2.0%) to a low (0.05%) glucose concentration (referred to below as high and low glucose) and growth from high to low glucose. Hxt2 as measured by immunoblotting increased 20-fold upon a shift from high-glucose to low-glucose medium, and the high-affinity glucose transport expressed had a strong HXT2-dependent component. Surprisingly, Hxt2 was not detectable when S. cerevisiae growing in high glucose approached glucose exhaustion, and the high-affinity glucose transport expressed under these conditions did not have an HXT2-dependent component. The role of Hxt2 in growth during aerobic batch culture in low-glucose medium was examined. An hxt2 null mutant grew and consumed glucose significantly more slowly than the wild type, and this phenotype correlated directly with appearance of the Hxt2 protein.

30-kDa heparin-binding protein of brain (amphoterin) involved in neurite outgrowth. Amino acid sequence and localization in the filopodia of the advancing plasma membrane.

A cDNA library constructed from mRNA of rat brain was used to clone the cDNA that encodes the 30-kDa heparin-binding protein (amphoterin) that is developmentally regulated in brain and enhances neurite outgrowth in cerebral neurons. cDNA and peptide sequencing identified a dipolar sequence that has been previously found in studies of high mobility group 1 protein: the 184-amino acid cationic region is followed by a cluster of 30 anionic residues. The mRNA encoding amphoterin is also developmentally regulated; it is strongly reduced in quantity after the rapid perinatal growth phase of the rat brain. Anti-synthetic peptide antibodies raised according to the sequence of amphoterin were shown to bind specifically to the protein isolated from brain, and were used to detect amphoterin in subcellular fractions and in immunostaining of cells. Amphoterin was found in the cytoplasm of the cell soma, in the cell processes, and the substrate-attached material. In cells that are at an active stage of spreading and extending their cytoplasmic processes amphoterin was especially associated with plasma membrane filopodia. The distinct localization to the filopodia of the advancing plasma membrane suggests that endogenous amphoterin has a role in the extension of neurite-type cytoplasmic processes in developing cells. This inference is further supported by the finding that both anti-amphoterin and the anti-synthetic peptide antibodies in the culture media strongly inhibit the outgrowth of cytoplasmic processes.

Anti-synthetic peptide antibody reacting at the fusion junction of deletion-mutant epidermal growth factor receptors in human glioblastoma.

We have investigated human gliomas that amplify and rearrange the epidermal growth factor receptor gene, with generation of an in-frame deletion mutation of 802 nucleotides in the external domain. This in-frame deletion mutation generates a local amino acid sequence at the fusion junction of what normally were distant polypeptide sequences in the intact epidermal growth factor receptor. This 14-amino acid peptide was chemically synthesized, coupled to keyhole limpet hemocyanin, and used as an immunogen in rabbits. The elicited antibody reacted specifically with the fusion peptide in ELISA. The anti-fusion junction peptide antibody was purified by passage of the antiserum over a peptide affinity column with acidic elution. The purified antibody selectively bound the glioma deletion mutant as compared to the intact epidermal growth factor receptor as assessed by immunocytochemistry, immunofluorescence, immunoprecipitation with gel electrophoresis, and binding experiments using radioiodinated antibody. These data indicate that it is feasible to generate site-specific anti-peptide antibodies that are highly selective for mutant proteins in human tumors. The anti-peptide antibody described here, and other mutation site-specific antibodies, should be ideal candidates for tumor immunoimaging and immunotherapy.

Thyroid peroxidase and thyroid microsomal autoantibodies.

The antithyroid microsomal antibodies found in the serum of patients with autoimmune thyroid disease are directed largely, if not entirely, against thyroid peroxidase (TPO). In this study we used a highly purified, well characterized, large tryptic fragment of porcine TPO (hereafter referred to as purified porcine TPO) to examine possible differences among microsomal antibodies in patients with autoimmune thyroid disease. Antibodies against this TPO preparation and also against a synthetic peptide corresponding to residues 780-793 of the deduced sequence of the native enzyme were compared with microsomal antibodies from patients in immunoblot experiments. The antiporcine TPO and antisynthetic peptide antibodies reacted with crude preparations of human TPO. Binding of serum microsomal antibodies to purified porcine TPO was also found. Purified porcine TPO shows two fragments after gel electrophoresis under reducing conditions: a 59K fragment corresponding to the amino end of the molecule, and two approximately 30K fragments corresponding to the carboxyl end. Using an immunoblot procedure with purified porcine TPO as the antigen, we found that at least two epitopes were involved in microsomal antibody production: one associated with the 59K fragment and the other with the approximately 30K fragment(s). The distribution of serum antibodies against these epitopes differed among the patients, indicating that these antibodies comprise a heterogeneous group. Serum from patients with autoimmune thyroid disease significantly inhibited human TPO activity, raising the possibility that microsomal antibodies may contribute to the impaired thyroid function that occurs in some patients with autoimmune thyroid disease.

Demonstration of two distinct insulin-binding components in solubilized human placental membranes by radioimmunoassay using synthetic peptide and anti-synthetic peptide antibody

A peptide corresponding to the 957–980 sequence of human placental insulin receptor precursor (HIRP) was synthesized and antisera were produced against the synthetic peptide. Anti-synthetic HIRP(957–980) serum HIR-27 was proved to cross-react with HIRP-related proteins in solubilized human placental membranes. A radioimmunoassay developed with the antiserum and synthetic peptide HIRP(957–980) enabled us to separate, in combination with gel filtration, two insulin-binding components in solubilized human placental membranes which conceivably correspond to the α 2 β 2 and gaβstructures of the placental insulin receptor. The two components were shown to be distinct in insulin-binding behavior depending on conditions of pH and ionic strength in the binding assay.

The adhesive and neurite-promoting molecule p30: analysis of the amino-terminal sequence and production of antipeptide antibodies that detect p30 at the surface of neuroblastoma cells and of brain neurons.

A membrane-bound adhesive protein that promotes neurite outgrowth in brain neurons has been isolated from rat brain (Rauvala, H., and R. Pihlaskari. 1987. J. Biol. Chem. 262:16625-16635). The protein is an immunochemically distinct molecule with a subunit size of approximately 30 kD (p30). p30 is an abundant protein in perinatal rat brain, but its content decreases rapidly after birth. In the present study the amino-terminal sequence of p30 was determined by automated Edman degradations. A single amino-terminal sequence was found, which is not present in previously studied adhesive molecules. This unique sequence has a cluster of five positive charges within the first 11 amino acid residues: Gly-Lys-Gly-Asp-Pro-Lys-Lys-Pro-Arg-Gly-Lys. Antisynthetic peptide antibodies that recognize this sequence were produced in a rabbit, purified with a peptide affinity column, and shown to bind specifically to p30. The antipeptide antibodies were used, together with anti-p30 antibodies, to study the localization of p30 in brain cells and in neuroblastoma cells as follows. (a) Immunofluorescence and immunoelectron microscopy indicated that p30 is a component of neurons in mixed cultures of brain cells. The neurons and the neuroblastoma cells expressed p30 at their surface in the cell bodies and the neurites. In the neurites p30 was found especially in the adhesive distal tips of the processes. In addition the protein was detected in ribosomal particles and in intracellular membranes in a proportion of cells. (b) The antibodies immobilized on microtiter wells enhanced adhesion and neurite growth indicating that p30 is surface exposed in adhering neural cells. (c) Immunoblotting showed that p30 is extracted from suspended cells by heparin suggesting that a heparin-like structure is required for the binding of p30 to the neuronal cell surface. A model summarizing the suggested interactions of p30 in cell adhesion and neurite growth is presented.

Immunological cross-reactivity among α-zeins of maize ( Zea mays L.)

Polyclonal antisera were produced against four different α-zein polypeptides representing M r -23800, -25000 and -26700 size classes, respectively, as well as a truncated M r -26700 polypeptide whose size was similar to M r -23800 polypeptides. Both SDS-denatured and ‘native’ forms of polypeptides were used for immunization. Seven different antisera to intact α-zein polypeptides and one to a synthetic peptide were reacted with each of the different α-zein polypeptides used for immunization and whole α-zein fraction from a maize inbred (K55) in ELISA. The same antisera were used to immunostain whole α-zein blots after SDS-PAGE of inbreds K55 and W64A and also whole zein blots after IEF. The results indicated extensive immunological cross-reactivity among α-zein polypeptides when antisera to denatured immunogens were used in assays, whereas antisera to native immunogens yielded varying degrees of reduced cross-reactivity. Antisera to native immunogens showed little or no reactivity towards some α-zein charge components in IEF blots belonging presumably to a size class different from that of the homologous antigen. The results indicated also that the M r -25000 α-zein size class was immunologically related more to M r -26700 than M r -23800 polypeptides. Likewise, a M r -23800 α-zein polypeptide from inbred K55 was shown to be a tr M r -26700 polypeptide by polyclonal, monoclonal and anti-synthetic peptide antibodies. It is concluded that polyclonal antisera to intact α-zein polypeptides could be used to distinguish polypeptides from different homology classes, but they would be of limited use, if any, in discriminating individual polypeptides within a homology class. Thus, monoclonal antibodies to intact α-zein polypeptides and polyclonal and monoclonal antibodies to synthetic peptides corresponding to sites unique to individual α-zein polypeptides show a greater promise of studying structural relationships among different α-zeins by immunological probes.

Localization of neutralizing regions of the envelope gene of feline leukemia virus by using anti-synthetic peptide antibodies

We synthesized 27 synthetic peptides corresponding to approximately 80% of the sequences encoding gp70 and p15E of Gardner-Arnstein feline leukemia virus (FeLV) subtype B. The peptides were conjugated to keyhole limpet hemocyanin and injected into rabbits for preparation of antipeptide antisera. These sera were then tested for their ability to neutralize a broad range of FeLV isolates in vitro. Eight peptides elicited neutralizing responses against subtype B isolates. Five of these peptides corresponded to sequences of gp70 and three to p15E. The ability of these antipeptide antisera to neutralize FeLV subtypes A and C varied. In certain circumstances, failure to neutralize a particular isolate corresponded to sequence changes within the corresponding peptide region. However, four antibodies which preferentially neutralized the subtype B viruses were directed to epitopes in common with Sarma subtype C virus. These results suggest that distal changes in certain subtypes (possibly glycosylation differences) alter the availability of certain epitopes in one virus isolate relative to another. We prepared a "nest" of overlapping peptides corresponding to one of the neutralizing regions of gp70 and performed slot blot analyses with both antipeptide antibodies and a monoclonal antibody which recognized this epitope. We were able to define a five-amino-acid sequence required for reactivity. Comparisons were made between an anti-synthetic peptide antibody and a monoclonal antibody reactive to this epitope for the ability to bind both peptide and virus, as well as to neutralize virus in vitro. Both the anti-synthetic peptide and the monoclonal antibodies bound peptide and virus to high titers. However, the monoclonal antibody had a 4-fold-higher titer against virus and a 10-fold-higher neutralizing titer than did the anti-synthetic peptide antibody. Competition assays were performed with these two antibodies adjusted to equivalent antivirus titers against intact virions affixed to tissue culture plates. The monoclonal antibody had a greater ability to compete for virus binding, which suggested that differences in neutralizing titers may relate to the relative affinities of these antisera for the peptide conformation in the native structure.

The structure and function of the epidermal growth factor receptor studied by using antisynthetic peptide antibodies.

The human epidermal growth factor receptor has been purified and partial amino acid sequence obtained. A synthetic oligonucleotide was used to select complementary DNA clones from placental and A431 clone banks. The nucleotide sequence of a 5.8 kilobase transcript was determined and used to predict the total amino acid sequence of the receptor. We have predicted a model for the receptor which has an external ligand binding domain of 621 amino acids, a transmembrane region of 23 amino acids, and a cytoplasmic domain of 542 amino acids having protein tyrosine kinase activity. The kinase autophosphorylation sites have been mapped onto the primary amino acid sequence. Analysis of protein sequence databases have shown that the erb-B oncogene of avian erythroblastosis virus has acquired part of the avian EGF receptor gene. The hypothesis has been proposed that transformation by this virus is the result of expression of a truncated EGF receptor which lacks the majority of the EGF binding domain and delivers a continuous proliferation signal to transformed cells. We describe here the production of polyclonal and monoclonal antibodies to selected synthetic peptides from the EGF receptor and v-erb B sequences. Antisera to sequences encompassing the three major sites of autophosphorylation and the putative ATP binding site all recognize the native EGF receptor molecule. We have used these reagents to test our model of EGF receptor structure and v-erb B function.