Quantitative immunofluorescence, anti-ras p21 antibody specificity, and cellular oncoprotein levels

A general approach to investigating specificity and saturation of antibodies by quantitative immunofluorescence is applied to monoclonal antibodies generated against p21 or ras oligopeptides to quantify ras p21 oncoprotein in cultured cells. Ras 10, a panreactive mouse monoclonal antibody, appears to be a superior probe for detection of p21 in cell extracts or fixed cells because it binds a 21 kD protein on SDS-PAGE/western blots and labels the cytoplasmic membrane in a saturable and competitive manner. RAP-5, a widely used mouse monoclonal antibody generated against an oligopeptide of ras p21, does not recognize p21 in denaturing immunoblots or in immunofluorescence of cultured cells.     

VAMP-7 mediates vesicular transport from endosomes to lysosomes.

A more complete picture of the molecules that are critical for the organization of membrane compartments is beginning to emerge through the characterization of proteins in the vesicle-associated membrane protein (also called synaptobrevin) family of membrane trafficking proteins. To better understand the mechanisms of membrane trafficking within the endocytic pathway, we generated a series of monoclonal and polyclonal antibodies against the cytoplasmic domain of vesicle-associated membrane protein 7 (VAMP-7). The antibodies recognize a 25-kD membrane-associated protein in multiple tissues and cell lines. Immunohistochemical analysis reveals colocalization with a marker of late endosomes and lysosomes, lysosome-associated membrane protein 1 (LAMP-1), but not with other membrane markers, including p115 and transferrin receptor. Treatment with nocodozole or brefeldin A does not disrupt the colocalization of VAMP-7 and LAMP-1. Immunoelectron microscopy analysis shows that VAMP-7 is most concentrated in the trans-Golgi network region of the cell as well as late endosomes and transport vesicles that do not contain the mannose-6 phosphate receptor. In streptolysin- O-permeabilized cells, antibodies against VAMP-7 inhibit the breakdown of epidermal growth factor but not the recycling of transferrin. These data are consistent with a role for VAMP-7 in the vesicular transport of proteins from the early endosome to the lysosome.     

p180, a novel recycling transmembrane glycoprotein with restricted cell type expression.

A 180-kilodalton (kDa) protein (p180) was identified among the antigens for a panel of monoclonal antibodies raised against human fibroblast cell surface proteins. Binding studies with 125I-Fab' fragments of an anti-p180 monoclonal antibody demonstrated that 10 to 30% of p180 was located on the plasma membrane and that the remaining 70 to 90% was on intracellular membranes. p180 was rapidly internalized from the cell surface at 37 degrees C, and kinetic analyses indicated that this was a constitutive process followed by the recycling of p180 back to the plasma membrane. Morphological studies demonstrated that on the cell surface p180 was concentrated in coated pits, whereas inside the cell it was found in endosomes as suggested by its colocalization with the transferrin receptor. Immunoblot analysis with a polyclonal antiserum raised against purified human protein showed that p180 has a restricted distribution with expression at high levels in fibroblast cultures and in tissues containing cells of mesodermal origin. A biochemical characterization of p180 showed it to be a transmembrane glycoprotein with an extracellular domain, which consists of approximately 30 kDa of complex oligosaccharides attached to at least 45 kDa of the protein core. The cytoplasmic domain of p180 was found to contain a serine residue(s) that was phosphorylated both in vivo and in vitro by activated protein kinase C. p180 was purified by subjecting solubilized membrane proteins from a human osteosarcoma cell line to immunoaffinity chromatography and gel filtration. The N-terminal sequence information obtained from the purified protein showed no homology to other known proteins. It was concluded that p180 may be a novel recycling receptor which is highly restricted in its expression to fibroblastlike cells.     

Plasma cell antigen PC-1 and the transferrin receptor in mouse, rat, and hamster: serologic and biochemical analysis

The plasma cell membrane antigen PC-1 and the receptor for the iron transport protein transferrin are high m.w., developmentally regulated proteins consisting of two similar or identical disulfide-bonded subunits. In this paper, we report the results of a serologic and biochemical analysis of these proteins in various strains of inbred mice, and in rats and hamsters. A monoclonal antibody against the PC-1a allelic product is shown to detect an antigenic determinant on the PC-1 molecule that has the same strain distribution as the antigen previously detected with polyclonal alloantisera. The mouse PC-1 protein was purified from plasma cells of the PC-1a genotype and was used to generate polyclonal rabbit anti-PC-1 antibodies. These antibodies precipitated a homologous protein from plasmacytoma cells derived from PC-1- congenic mice, demonstrating that PC-1b is not a "null" allele. The PC-1b allelic product had a slightly lower apparent m.w. than the PC-1a product, and had a slightly more basic isoelectric point. Rabbit anti-mouse PC-1 antibodies also precipitated a homologous protein from immunoglobulin-secreting cells of rat and hamster origin, but did not show detectable cross-reaction with the transferrin receptor. Disulfide bonding between chains was conserved in both PC-1 and the transferrin receptor in all species examined, but transferrin receptors from mouse cells had a significantly higher apparent m.w. than those of rat, hamster, or human cells.     

Binding properties of monoclonal antibody to the cytoplasmic domain of transferrin receptor

Hybridomas secreting monoclonal antibodies to transferrin receptor (TFR) were isolated. One of these antibodies, U-1, recognized the cytoplasmic domain of TFR and the others, N-2 and W-3, recognized its cell surface domains. Only antibody W-3 competed with transferrin (TF) for binding to TFR. Antibody U-1 bound to purified TFR but not to 35S- or 125I-TFR in cell extracts. 125I-Antibody U-1 bound to TFR alone in cell extracts when TFR was bound to antibody N-2-Sepharose 4B, but even in the presense of cell extracts it did not bind to TFR bound to antibody W-3-Sepharose 4B. Antibody W-3 co-precipitated TFR and a protein of about 30 kDa from cell extracts, and also reacted with the 30 kDa protein in cell extracts in the absence of TFR. Based on these results, the existence of two different states of the cytoplasmic domain of TFR is discussed.     

Both the SH2 and SH3 domains of human CRK protein are required for neuronal differentiation of PC12 cells.

Human CRK protein is a homolog of the chicken v-crk oncogene product and consists mostly of src homology region 2 (SH2) and SH3, which are shared by many proteins, in particular those involved in signal transduction. SH2 has been shown to bind specifically to phosphotyrosine-containing peptides. We report here that both SH2 and SH3 are required for signaling from CRK protein. Microinjection of the CRK protein induced neurite formation of rat pheochromocytoma cell line PC12. This activity was abolished by mutation of the CRK protein in either SH2 or SH3. The neuronal differentiation induced by the CRK protein was blocked by an excess amount of peptides containing CRK SH3. Moreover, we identified three proteins, of 118, 125, and 136 kDa, which bound specifically to CRK SH3. The CRK-induced neuronal differentiation was also suppressed by monoclonal antibodies against either CRK SH2 or p21ras. These results suggest that both SH2 and SH3 of the CRK protein mediate specific protein-protein binding and that the resulting multimolecular complex generates a signal for neurite differentiation through activation of p21ras.     

A 90,000-dalton binding protein common to both steroid receptors and the Rous sarcoma virus transforming protein, pp60v-src

Previous studies have shown that the avian progesterone receptor, when in the nontransformed 8 S state, is complexed to another cellular protein having a molecular weight of 90,000. In this report, we show that this receptor-binding protein is indistinguishable from the 90,000-dalton protein which associates in a complex with the Rous sarcoma virus transforming protein, pp60v-src. This identity was established by the following criteria. 1) Monoclonal antibodies directed against the pp60v-src-associated 90-kDa protein recognized the 90-kDa progesterone receptor binding protein in an immunoblot assay. Conversely, monoclonal antibodies that recognize the progesterone receptor binding protein bind to the 90-kDa protein which complexes with pp60v-src. 2) Peptide maps prepared from the 90-kDa proteins immunoprecipitated from chicken cells with monoclonal antibodies directed against either the 90-kDa receptor binding protein or the 90-kDa pp60v-src-associated protein were indistinguishable. 3) Preincubation of the progesterone receptor complex with monoclonal antibodies prepared against the pp60v-src-associated protein caused a shift in the sedimentation of the progesterone receptor. Previous studies have established that the pp60v-src-associated protein is indistinguishable from one of the major heat shock proteins which are induced under a variety of stress conditions in eukaryotic cells. These present studies implicate a new role for this 90-kDa protein in the action of steroid hormones.     

Antibodies to synthetic peptide from the residue 33 to 42 domain of c-Ha-ras p21 block reconstitution of the protein with different effectors.

Residues 32 to 40, which are conserved among ras proteins from different species, are likely to participate in interactions with the p21 effector system. With the goal of understanding the structural basis of the regulatory functions of c-Ha-ras p21, we produced rabbit antisera against a synthetic peptide corresponding to amino acids 33 to 42 of the protein. The affinity-purified antibodies interacted specifically with p21 and with the antigenic peptide. The epitope recognized by the antibodies appeared to be centered on threonine 35. The antibodies inhibited both in vitro p21-induced production of cyclic AMP in detergent extracts of RAS-defective yeast membranes and GAP-stimulated GTPase activity. However, monoclonal anti-ras antibodies Y13-259 and Y13-238 were not capable of specifically inhibiting interactions of p21 with these two putative effector proteins. The apparent inhibitory effect of Y13-259 on stimulation of p21 by GAP was due to a greatly reduced rate of exchange of nucleotides in the binding pocket of the protein. These findings provide additional support for the essential role of the residue 32 to 40 domain as the true effector site and further evidence of the involvement of GAP as a cellular effector of ras proteins.     

Abundant expression of ras proteins in Aplysia neurons

We have cloned a DNA fragment from the marine mollusc Aplysia californica, which contains sequences homologous to mammalian ras genes, by screening a genomic library with a viral Ha-ras oncogene probe under conditions of low stringency hybridization. Nucleotide sequencing revealed a putative exon that encodes amino acids sharing 68% homology with residues 5 to 54 of mammalian p21ras polypeptides, and which therefore is likely to encode a ras-like Aplysia protein. The cloned locus, designated Apl-ras, is distinct from the Aplysia rho (ras-homologue) gene and appears to be more closely related to mammalian ras. We used a panel of monoclonal antibodies raised against v-Ha-ras p21 to precipitate an Mr 21,000 protein from extracts of Aplysia nervous tissue, ovotestis, and, to a much lesser degree, buccal muscle. Fluorescence immunocytochemistry revealed that ras-like protein is most abundant in neuronal cell bodies and axon processes, with staining most prominent at plasma membranes. Much less was present in other tissues. The prominence of ras protein in neurons, which are terminally differentiated and non-proliferating, indicates that the control of cell division is not the sole function of this proto-oncogene. The large identified neurons of Aplysia offer the opportunity to examine how ras protein might function in mature nerve cells.     

Monoclonal antibodies provide specific intramolecular markers for the study of epithelial tonofilament organization

The tonofilament-associated protein antigens recognized in epithelial cells by a group of six monoclonal antibodies have been studied by immunofluorescence and gel immunoautoradiography. The monoclonal antibodies were generated against detergent insoluble cytoskeleton extracts from a cultured simple epithelium derived cell line, Ptk1 cells. They show various tissue specificities, and while they all recognize components at the low end of the molecular weight range for intermediate filament proteins, they confirm that single antibody species can react with multiple polypeptides of different molecular weights in the tonofilament complex. The monoclonal antibodies described here demonstrate the presence of a simple epithelium antigenic determinant associated with intermediate filaments that is not detectable in the specialized cells of squamous and keratinizing epithelia but can reappear in such cells after transformation.     

RAS enzyme-linked immunoblot assay discriminates p21 species: a technique to dissect gene family expression

The members of the RAS gene family of protooncogenes are of implied biological significance in oncogenesis. The precise role of these genes is unclear. One difficulty has been the inability to discriminate the individual p21 protein products of various ras genes in cell lines, de novo human tumors, and related normal tissues. In this report, specific proteins of the human c-Ha-ras-1, c-Ki-ras-2, and c-N-ras genes have been detected and discriminated by the differential use of various antisera recognizing these p21s. This enzyme-linked immunoblot assay utilizes a double antibody system in which monoclonal antibodies are initially used to immunoprecipitate the p21ras proteins. Immunoprecipitates are then subjected to one-dimensional Western blot analysis utilizing other antibodies raised against p21s, coupled with nonradiolabeled enzyme-linked colorimetric detection. By direct detection, the specific products of the three human ras genes can be discriminated. In addition, we describe the generation and characterization of a new anti-p21c-N-ras-specific antibody. The simultaneous expression into protein of multiple ras genes is unequivocally demonstrated in both homogeneous cell lines and heterogeneous human tissues. This new technique is also applicable for discrimination of the protein products of other gene families.     

Analysis and isolation of human transferrin receptor using the OKT-9 monoclonal antibody covalently crosslinked to magnetic beads.

A method is described for the use of magnetic beads as a solid phase for the immunoprecipitation of labeled proteins. The anti-human transferrin receptor monoclonal antibody OKT-9 has been coupled to sheep anti-mouse IgG1-coated magnetic beads using the crosslinking agent dimethyl pimelimidate. The transferrin receptor is readily detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography following immunoprecipitation from 35S-labeled cell lysates. When compared with precipitations using OKT-9 coupled to protein G Sepharose the magnetic beads result in fewer nonspecific bands. The protocol described is generally applicable to the identification of labeled proteins. In addition, because magnetic beads are amenable to covalent crosslinking procedures they can be used for the purification of proteins from complex mixtures. Covalently crosslinked OKT-9 sheep anti-mouse IgG1-coated magnetic beads have been used to affinity purify unlabeled transferrin receptor from cell lysates giving comparable purity and yield to transferrin Sepharose isolated transferrin receptor. The major advantages offered by magnetic beads compared to conventional affinity matrices are low nonspecific binding and the rapidity with which the purification can be performed.     

Multispecificity of a recombinant anti‐ras monoclonal antibody

Recombinant monoclonal antibodies (Ab's) have widespread application as research tools, diagnostic reagents and as biotherapeutics. Whilst studying the cellular molecular switch protein m‐ras, a recombinant monoclonal antibody to m‐ras was generated for use as a research tool. Antibody genes from a single rabbit B cell secreting IgG to an m‐ras specific peptide sequence were expressed in mammalian cells, and monoclonal rabbit IgG binding was characterized by ELISA and peptide array blotting. Although the monoclonal Ab was selected for specificity to m‐ras peptide, it also bound to both recombinant full‐length m‐ras and h‐ras proteins. The cross‐reactive binding of the monoclonal Ab to h‐ras was defined by peptide array blot revealing that the Ab showed preference for peptide sequences containing multiple positively charged amino acid residues. These data reinforce the concept of antibody multispecificity through multiple interactions of the Ab paratope with diverse polypeptides. They also emphasize the importance of immunogen and Ab selection processes when generating recombinant monoclonal Ab's.     

Isolation and characterization of a transferrin binding protein from rat plasma

A transferrin binding protein was isolated from normal rat placenta and from iron-deficient rat plasma using a human transferrin affinity column. The yield of the isolated pure protein from iron-deficient rat plasma was about 0.5 micrograms/ml plasma. The major protein had a molecular mass of 85 kDa and contained carbohydrate. Reduction with mercaptoethanol did not change the molecular mass of the plasma transferrin binding protein whereas the native placental transferrin receptor of 180 kDa was reduced to 90 kDa. The transferrin binding protein reacted with both monoclonal and polyclonal antibodies raised against rat transferrin receptor. Immunoblotting of both normal and iron deficient rat plasma showed that the transferrin binding protein had a molecular mass of 85 kDa. In vitro digestion of purified rat placental transferrin receptor and red blood cells with trypsin provided an identical peptide profile, suggesting that the transferrin binding protein in rat plasma is derived from proteolysis of the extracellular portion of the transferrin receptor of the erythroid tissues.     

Characterization of proteins that associate with an unglycosylated form of the transferrin receptor in A431 cells

A protein doublet (Mr = 135,000/130,000) was found to coprecipitate with an unglycosylated form of the transferrin receptor in tunicamycin-treated A431 cells. This doublet is not detected with either a monoclonal or polyclonal antibody to the transferrin receptor on Western blots indicating that these proteins do not interact directly with transferrin receptor antibody. Proteolytic digestion patterns of the individual proteins of the Mr = 135,000/130,000 doublet suggest that they are related to one another and are distinct from the transferrin receptor. Further characterization of these proteins indicates that they form a high molecular weight complex with the unglycosylated but not the glycosylated form of the transferrin receptor. Pulse-chase experiments demonstrate that the proteins post-translationally associate with the receptor.     

Entamoeba histolytica: transferrin binding proteins

Entamoeba histolytica trophozoites depend on iron for their growth; thus, they must use some host iron-containing molecules to fulfill this requirement. In this work we report that amoebas are able to utilize human holo-Tf as iron source and to recognize it through transferrin binding proteins. By use of an anti-human transferrin antiserum in an immunoblotting assay, two main polypeptides with apparent molecular masses of 70 and 140 kDa were found in total extract of trophozoites cultured in vitro. However, when a monoclonal anti-human transferrin receptor antibody was used, only one band with molecular mass of 140 kDa was observed. Both the human transferrin and the monoclonal antibody recognized a protein on the amoebic surface, demonstrated by confocal microscopy. Furthermore, the complex transferrin-transferrin binding protein was internalized by an endocytic process and probably dissociated inside the cell. This mechanism could be one manner in which E. histolytica acquires iron from the human host transferrin.     

The EhADH112 recombinant polypeptide inhibits cell destruction and liver abscess formation by Entamoeba histolytica trophozoites

The Entamoeba histolytica EhCPADH complex, formed by a cysteine proteinase (EhCP112) and an adhesin (EhADH112), is involved in adherence, phagocytosis and cytolysis. This makes this complex an attractive candidate as a vaccine against amoebiasis. Here, we produced the recombinant polypeptide EhADH243, which includes the adherence epitope detected by a monoclonal antibody against the EhCPADH complex. EhADH243 was purified, and the effect of the polypeptide on in vitro and in vivo virulence was studied. Antibodies against EhADH243 reacted with the EhCPADH complex and with the recombinant polypeptide. EhADH243 and antibodies against this polypeptide inhibited adherence, phagocytosis and destruction of cell monolayers by live trophozoites, but had little effect on cell monolayer destruction by trophozoite extracts. EhADH243 recognized a 97 kDa protein in the MDCK membrane fraction that could be a putative receptor for E. histolytica trophozoites. Hamsters immunized with EhADH243 developed humoral response against EhCPADH, and animals were partially protected from amoebic liver abscess.     

Monoclonal antibody OKT-9 recognizes the receptor for transferrin on human acute lymphocytic leukemia cells

The monoclonal antibody OKT-9 recognizes a surface protein of human lymphocytes that consists of a disulfide bonded homodimer of m.w. 200,000 intact and 95,000 reduced. A similar protein is precipitated by transferrin-agarose, but not by agarose alone. Peptide mapping by limited proteolysis shows that the proteins precipitated by OKT-9 antibodies and transferrin-agarose are homologous. It is concluded that OKT-9 antibodies recognize the transferrin receptor. Expression of receptors for transferrin may be useful as a marker for activated or dividing cells.     

Point-mutated p21ras couples a muscarinic receptor to calcium channels and polyphosphoinositide hydrolysis

A high-affinity muscarinic receptor is detectable both in normal 3T3 mouse fibroblasts and in their transformed counterpart obtained by transfection with the oncogene EJ/T24-H-ras. However, only the transformed cell line is responsive to muscarinic agonist carbamylcholine in terms of Ca2+ influx and polyphosphoinositide hydrolysis, whereas the normal cell line is unresponsive. Using a point-mutated p21ras protein and monoclonal antibodies anti-p21ras, we provide evidences that p21ras couples to receptor-operating calcium channels and to polyphosphoinositide hydrolysis a muscarinic receptor which is uncoupled in normal mouse fibroblasts.     

Synthesis of the transferrin receptor by cultures of embryonic chicken spinal neurons

We have purified a glycoprotein from chicken sciatic nerves, sciatin, which has pronounced trophic effects on avian skeletal muscle cells in culture. Recent studies have shown that sciatin is identical to the iron-transport protein, transferrin, in terms of its physicochemical structure, immunological reactivity, and biological activity. To determine whether transferrin is synthesized and released by neuronal tissue, we incubated cultures of dissociated chicken spinal neurons in a medium free of L-leucine containing either L-3H-amino acids or L-[14C]leucine and immunoprecipitated transferrin with highly specific antibodies. The radiolabeled protein precipitated by rabbit heteroclonal, goat heteroclonal, or mouse monoclonal antitransferrin antibodies increased in specific activity in a linear manner for at least 30 min. Synthesis of this protein was abolished by the presence of puromycin (20 micrograms/ml) or cycloheximide (10(-5) M). The disappearance of the radiolabeled protein from cells was linear with a half-life (t 1/2) of 8-10 h. When immunoprecipitates were separated by SDS gel electrophoresis, a prominent band corresponding to transferrin (Mr 84,000) was visualized by staining with Coomassie Blue. However, when such gels were fluorographed, no radioactivity was apparent in the transferrin region of the gel although a prominent radioactive band was visualized at an Mr of 56,000. The protein of Mr 56,000 was not simply a degradation product of transferrin because this particular protein band was not generated by incubating radiolabeled transferrin with unlabeled neuronal homogenates. The protein of Mr 56,000 was purified from embryonic chicken brain and spinal cord by immunoabsorption chromatography on mouse monoclonal antitransferrin IgG conjugated to Sepharose 4B followed by affinity chromatography on immobilized transferrin. The purified protein bound radioiodinated transferrin and was precipitated by rabbit anti-chicken transferrin-receptor antibodies. Furthermore, this receptor protein was found to be localized on the plasma membrane of dorsal root ganglion neurons by immunocytochemistry using the peroxidase-antiperoxidase technique, and by blocking experiments, which showed that antitransferrin receptor IgG could inhibit the binding of fluorescein-conjugated transferrin at 4 degrees C to cultured neurons in vitro. From these data, we conclude that transferrin is not synthesized by cultures of chicken spinal cord neurons, but that the receptor for transferrin is synthesized by these cultures and is precipitated by antitransferrin antibodies as an antigen-receptor complex.