Crystallization and preliminary X-ray diffraction data of an anti-angiotensin II Fab and of the peptide-Fab complex

mAb-131 is a monoclonal antibody that binds with high affinity (K alpha = 7.4 x 10(9) M-1) to the 8-residue peptide hormone angiotensin II, the major effector of the renin/angiotensin system. mAb-131 is a member of a well characterized idiotypic antibody network since it was raised as an anti-anti-idiotype of an antibody raised against angiotensin II. mAb-131 Fabs prepared with papain contain four major charge isoforms that can be separated by pH gradient elution from an anion-exchange column. Diffraction quality isomorphous crystals of two of the isoforms and of the Fab.peptide complexes have been grown. The crystals diffract to 3.5 A resolution, are tetragonal, space group P4(1) (or P4(3] with cell dimensions a = b = 78.6 A, c = 125.2 A, and have two Fab molecules per asymmetric unit. By using a different buffer, a second crystal form has been grown which diffracts to 3.3 A. It also belongs to space group P4(1) (or P4(3] but has cell dimensions of a = b = 109.6 A and c = 125.2 A. Knowledge of the three-dimensional structure of this Fab and of the peptide.Fab complex will give insight into two problems: 1) the recognition of small peptide hormones (which exist as random coils in solution) with high affinity by proteins, and 2) the nature of conservation of antibody combining sites in idiotypic networks.     

Phosphorylation of calf uterine progesterone receptor by cAMP-dependent protein kinase

We have examined the potential for using calf uterine progesterone receptor (PR) as a substrate for phosphorylation by cAMP-dependent protein kinase (cAMP-PK), PR was found to interact with anti-PR monoclonal antibody alpha PR6 (Sullivan et al., 1986), which was to immunopurify the receptor. Protein staining of the purified preparation revealed the presence of two major bands corresponding to 114 kDa and 90 kDa peptides; only 114 kDa peptide could be photoaffinity-labeled with R5020. The 90 kDa peptide co-migrated with 90 kDa heat shock protein (hsp-90) precipitated by anti-hsp-90 monoclonal antibody AC88 (Riehl et al., 1985). Incubation of the immunopurified protein-A-Sepharose-adsorbed PR with the catalytic subunit of cAMP-PK in the presence of gamma-[32P]ATP and divalent cations resulted in a Mg++-dependent incorporation of 32P-radioactivity into both the 114 kDa and the hsp-90 peptides. Small 32P-incorporation was also seen in the 114 kDa peptide in the presence of Mn++. A 60 degrees C preincubation of immunopurified PR increased the extent of phosphorylation of the hsp-90 peptide. A pretreatment with alkaline phosphatase reduced the ability of PR to act as a substrate while the steroid occupancy of PR appeared to enhance the phosphorylation of the 114 kDa peptide. The differential cation requirement for the phosphorylation of 114 kDa and hsp-90 peptides and a selective hormone-dependent increase in the phosphorylation of the 114 kDa peptide suggest a possible role of phosphorylation in mediating progesterone action in the calf uterus.     

Localization of a highly immunogenic region on the acetylcholine receptor alpha-subunit

Antibodies to synthetic peptides were employed in order to map domains on the alpha-subunit of the acetylcholine receptor to which several monoclonal antibodies are directed. Five peptides corresponding to residues 1-20, 126-143, 169-181, 330-340 and 351-368 of the receptor alpha-subunit were synthesized and antibodies against them were elicited. The anti-peptide antibodies were employed along with the monoclonal antibodies to identify fragments of S. aureus V8 protease digested- alpha-subunit in immunoblotting experiments. Our results demonstrate that a highly immunogenic region of the alpha-subunit is located on a carboxy-terminal 14 kDa portion of the alpha-subunit. This region also seems to undergo antigenic changes during muscle development. A monoclonal antibody directed against the cholinergic binding site of the acetylcholine receptor reacted with an 18 kDa segment of the alpha-subunit which bound alpha-bungarotoxin as well as antibodies directed against peptide 169-181.     

Construction and characterization of a recombinant murine monoclonal antibody directed against human fibrin fragment-D dimer

cDNA libraries in lambda phage were generated from the murine hybridoma secreting mAb-15C5, a monoclonal antibody directed against fragment-D dimer of crosslinked human fibrin [Holvoet et al. (1989) Thromb. Haemostasis 61, 307-313], and clones encoding fragments of the heavy (gamma 1) and the light (kappa) chain were isolated. The kappa-chain cDNA was reconstructed from two overlapping clones encoding 20 amino acids of signal sequence and the 214 amino acids of the mature protein chain. The gamma 1-chain cDNA was reconstructed from the mAb-15C5 kappa-chain signal sequence, the mAb-15C5 gamma 1 variable-domain coding sequence and murine gamma 1-gene and gamma 1-chain cDNA fragments encoding the constant domains. These cDNAs were expressed in Chinese hamster ovary cells, selected cell lines were scaled up in roller bottle culture, and recombinant mAb-15C5 was purified from the conditioned medium by chromatography on Zn-chelate - Sepharose, protein-A - Sepharose and insolubilized fragment-D dimer, with a yield of 50 micrograms/l and a recovery of 20%. SDS-gel electrophoresis without reduction revealed a homogeneous band, and after reduction a light-chain band with identical and a heavy-chained band with a somewhat slower mobility than that of the natural mAb-15C5. Competitive binding revealed a comparable affinity of natural and recombinant mAb-15C5 for fibrin fragment-D dimer. Thus recombinant mAb-15C5, obtained by co-expression of the reconstructed cDNAs of the kappa and gamma 1 chain in Chinese hamster ovary cells, has very similar properties to natural mAb-15C5. These recombinant mAb-15C5 cDNAs may be useful for the construction of a humanized monoclonal antibody for thrombus imaging, and for targeting of thrombolytic agents to fibrin.     

A murine IgM monoclonal antibody binds an antigenic determinant in outer surface protein A, an immunodominant basic protein of the Lyme disease spirochete

A hybridoma cell line formed by the fusion of the P3x63-Ag8.653 myeloma cell line with splenocytes from BALB/c mice immunized with Borrelia burgdorferi produced an IgM monoclonal antibody (mAb-11G1) with kappa-light chains which detected an antigenic determinant in a major spirochetal protein of m.w. approximately 31,000, also known as outer surface protein A (OSP-A). Apparent saturation was reached in approximately 35 min with 34 ng of mAb-11G1 binding to 5 X 10(7) spirochetes giving an estimated 4.8 X 10(2) IgM molecules per spirochete and thus a minimum of 480 binding sites per organism. Enzymatic digestion studies suggest that the antigenic determinant to mAb-11G1 is contained within the peptide chain of OSP-A as binding could be eliminated by treatment of the spirochetes with proteinase K, Pronase and pepsin (100 to 200 micrograms/ml of enzyme) but not by trypsin or bromelain treatment. Periodate oxidation as well as mixed and endoglycosidase treatment of the spirochetes did not alter the binding of mAb-11G1. Two-dimensional gel electrophoresis of whole spirochetal cell lysates disclosed that OSP-A is a heterogeneously charged basic protein with an apparent isoelectric point range from 8.5 to 9.0. Amino acid analysis of OSP-A showed a 10% lysine component which could provide the basic nature to the protein. OSP-A with the intact antigenic determinant for mAb-11G1 can be found in the urine of hamsters experimentally infected with B. burgdorferi.     

Localization of wheat germ agglutinin and antibody binding sites on the plasma membranes of sea urchin sperm heads as revealed by label-fracture and fracture-flip

Freeze-fracture electron microscopy reveals that intramembrane particles are concentrated in a band encircling the posterior portion of the acrosome of Strongylocentrotus purpuratus sperm. Two colloidal gold labeling methods, label-fracture and replica-staining fracture-flip, were employed to show that the plant lectin wheat germ agglutinin, which recognizes a 210 kDa sperm surface glycoprotein, binds to this localized band of intramembrane particles. Monoclonal antibody J18/2, which also recognizes the 210 kDa surface glycoprotein, shows this localized binding in approximately 20% of the sperm observed in this study. The majority of sperm displayed a uniform distribution of receptor sites for monoclonal antibody J18/2. Since wheat germ agglutinin and monoclonal antibody J18/2 are known to agglutinate Strongylocentrotus purpuratus sperm but not sperm of another sea urchin, Lytechinus pictus, similar determinations were made for the latter species. Lytechinus pictus sperm are not labeled with wheat germ agglutinin and are only sparsely labeled with monoclonal antibody J18/2. The acrosomal localizations of wheat germ agglutinin and monoclonal antibody J18/2 receptors in Strongylocentrotus purpuratus sperm are consistent with the involvement of the 210 kDa surface glycoprotein in an egg jelly-induced sperm acrosome reaction. Low-temperature post-embed labeling of thin sections with wheat germ agglutinin and monoclonal antibody J18/2 show concentrations of label within the acrosomal vesicle of Strongylocentrotus purpuratus sperm, suggesting the presence of an intracellular storage site for the 210 kDa glycoprotein.     

Characterization of the two heavy chains of the T3 complex on the surface of human T lymphocytes

The T3 complex has been defined by a group of monoclonal antibodies which react with all human peripheral blood T lymphocytes and a subpopulation of thymocytes. This membrane structure includes glycoproteins of 44 (alpha), 37 (beta), 25 (gamma), and 20 kDa (delta) as well as a nonglycosylated polypeptide of 20 kDa (epsilon). The characterization of the alpha and beta chains has been of particular interest because they may constitute the T cell receptor for antigen. Here we show that the T3 complex prepared by immunoprecipitation from T lymphocytes of a leukemic patient (Sezary syndrome) displays an unusually strong association of the alpha and beta chains with the 20/25-kDa T3 proteins. The alpha and beta chains (48 and 44 kDa) were co-precipitated by anti-20-kDa T3 monoclonal antibodies as a disulfide-linked 90-kDa heterodimer. A minor 220-kDa multimer composed of proteins similar to the alpha and beta chains was also present in these immunoprecipitates. This multimer could be independently precipitated with a new monoclonal antibody WT-31, which detects the larger polypeptide chains of the T3 complex on all human T lymphocytes. After removal of N-linked oligosaccharides, both the alpha and beta chain were found to have 33-kDa peptide backbones with distinct isoelectric points. Using a monoclonal reagent T40/25, a 90-kDa heterodimer, consisting of 40- and 49-kDa chains with peptide backbones of 34 kDa was found to be T3-associated on the T leukemic cell line HPB-ALL. When the alpha and beta chains from the Sezary patient were compared with the corresponding chains from HPB-ALL by peptide mapping, large differences were observed. Taken together, the data presented here provide strong evidence that the T cell receptor for antigen is part of the T3 complex on the surface of human T lymphocytes.     

Immunoprecipitation of the in vitro translation product of rabbit muscle protein phosphatase C-I mRNA

Rabbit muscle polyA+ mRNA was translated in vitro using a rabbit reticulocyte lysate system in the presence of [35S]methionine. A mouse monoclonal antibody to the catalytic subunit of rabbit muscle phosphorylase phosphatase ("phosphatase C-I") was used to immunoprecipitate the products which were then analyzed by SDS-PAGE and autoradiography. These studies showed that the major product of the phosphatase mRNA is a single ca. 36 kDa polypeptide. These findings are significant in view of suggestions that the catalytic subunit is derived from a larger precursor, and in view of the molecular cloning of two cDNAs for the phosphatase, which encode polypeptides of 35.4 kDa and 37.5 kDa, respectively.     

Characteristics of DNA-binding activity of human cytomegalovirus ppUL44

Human cytomegalovirus (HCMV)-specific monoclonal antibody, SCMVM34, recognizes the early antigen encoded by UL44 of HCMV. This antigen is confined to the nucleus of HCMV-infected cells. This study was performed to characterize the DNA-binding activity of the protein encoded by UL44 of HCMV. The nuclear and cytoskeletal fraction of HCMV-infected cells was subjected to 0.4 M NaCl extraction, DEAE-Sephacel ion exchange chromatography, DNA-cellulose chromatography and SDS-PAGE analysis with monitoring of the reactive protein using SCMVM34 monoclonal antibody. The molecular weights of the resultant proteins were found to be 34, 40 and 52 kDa. The internal peptide fragments were isolated by tryptic digestion and reverse-phase HPLC. The internal amino acid sequence analysis of the peptides from the HPLC profile revealed that the antigen recognized by SCMVM34 monoclonal antibody was ppUL44. The reactive antigen began to be eluted from 250 mM NaCl (Tris-HCl pH 7.4) in DNA cellulose. The 34 kDa protein seems to bind to DEAE more tightly than the 52 kDa protein. The surface charge of 34 kDa might be more basic. Conclusively, the antigen recognized by SCMVM34 was the protein encoded by HCMV UL44, which was localized in the nuclei after HCMV infection, and was the DNA-binding protein with the characteristic that the surface charge of the molecule was more basic, as the molecular weights of the protein were decreased.     

GD2 ganglioside-binding antibody 14G2a and specific aurora A kinase inhibitor MK-5108 induce autophagy in IMR-32 neuroblastoma cells

The process of autophagy and its role in survival of human neuroblastoma cell cultures was studied upon addition of an anti-GD2 ganglioside (GD2) 14G2a mouse monoclonal antibody (14G2a mAb) and an aurora A kinase specific inhibitor, MK-5108. It was recently shown that combination of these agents significantly potentiates cytotoxicity against IMR-32 and CHP-134 neuroblastoma cells in vitro, as compared to the inhibitor used alone. In this study we gained mechanistic insights on autophagy in the observed cytotoxic effects exerted by both agents using cytotoxicity assays, RT-qPCR, immunoblotting, and autophagy detection methods. Enhancement of the autophagy process in the 14G2a mAb- and MK-5108-treated IMR-32 cells was documented by assessing autophagic flux. Application of a lysosomotropic agent—chloroquine (CQ) affected the 14G2a mAb- and MK-5108-stimulated autophagic flux. It is our conclusion that the 14G2a mAb (40 μg/ml) and MK-5108 inhibitor (0.1 μM) induce autophagy in IMR-32 cells. Moreover, the combinatorial treatment of IMR-32 cells with the 14G2a mAb and CQ significantly potentiates cytotoxic effect, as compared to CQ used alone. Most importantly, we showed that interfering with autophagy at its early and late step augments the 14G2a mAb-induced apoptosis, therefore we can conclude that inhibition of autophagy is the primary mechanism of the CQ-mediated sensitization to the 14G2a mAb-induced apoptosis. Although, there was no virtual stimulation of autophagy in the 14G2a mAb-treated CHP-134 neuroblastoma cells, we were able to show that PHLDA1 protein positively regulates autophagy and this process exists in a mutually exclusive manner with apoptosis in PHLDA1-silenced CHP-134 cells.     

Partial purification and characterization of the Ca2(+)-pumping ATPase of the liver plasma membrane

A Ca2(+)-pumping ATPase has been characterized in rat hepatocyte plasma membranes. The enzyme has high Ca2+ affinity, and properties typical of a P-type ion pump. At variance with the Ca2+ pumps of other eukaryotic plasma membranes, it is not stimulated by calmodulin. The steady state concentration of the phosphoenzyme formed in the presence of ATP is increased by La3+. The enzyme cross-reacts with a monoclonal antibody (mAb-5F10) raised against the human erythrocyte Ca2+ pump. The enzyme has been purified using a mAb-5F10 antibody affinity column. CNBr digestion of the isolated protein has yielded two peptides which have been sequenced. One of them matches perfectly a sequence contained in the erythrocyte Ca2+ pump, the other is very homologous to another domain in the erythrocyte pump. In spite of the absence of calmodulin stimulation, 125I-calmodulin overlay experiments on the purified liver ATPase under denaturing conditions have revealed that the enzyme binds calmodulin even more strongly than the erythrocyte pump. Immunocytochemical experiments on liver slices using the mAb-5F10 antibody have shown that the enzyme is located predominantly in the blood sinusoidal domain of the hepatocyte plasma membrane.     

The production and characterization of monoclonal antibodies to a human colonic antigen associated with ulcerative colitis: cellular localization of the antigen by using the monoclonal antibody

We detected in human colon extracts a 40 kDa protein(s) that specifically reacts with tissue-bound IgG obtained from the colon of patients with ulcerative colitis or CCA-IgG. Using the hybridoma technology, we developed monoclonal antibodies to this 40 kDa protein. The specific immunoreactivity of one of the monoclonal antibodies (7E12H12, IgM isotype) against the 40 kDa protein was demonstrated both by ELISA and by immunotransblot. Competitive binding experiments showed that CCA-IgG inhibits the binding of 7E12H12 to the 40 kDa protein, suggesting the recognition of common epitope(s) on the 40 kDa protein by the monoclonal antibody and CCA-IgG. 7E12H12 was used to determine cellular localization of the 40 kDa protein. Biopsy tissue specimens from colon, esophagus, stomach, duodenum, jejunum, ileum, liver, pancreas, lungs, kidneys, salivary, and mammary glands were obtained. Tissue specimens were fixed in 4% paraformaldehyde or in 10% formalin. Sections were sequentially incubated with the hybridoma supernatant, biotinylated anti-mouse IgM, avidin-biotin-peroxidase complex, and 3,3'-diaminobenzidine. An unrelated hybridoma supernatant was used as control. The monoclonal antibody exclusively recognized colonic epithelial cells both in the crypt and on the luminal surface. Immunoreactivity was present on the plasma membrane chiefly along the basolateral areas of the cells. Plasma membrane localization of the 40 kDa protein was confirmed by immunoelectron microscopy. All colonic mucosal biopsy specimens from both adult and fetal colon reacted with the monoclonal antibody. None of the biopsy specimens from stomach, duodenum, jejunum, ileum, liver, pancreas, or non-gastrointestinal tissue reacted with the antibody, confirming the organ specificity of the 40 kDa protein. The interaction between this colonic epithelial membrane protein and the CCA-IgG may play an important role in the pathogenesis of ulcerative colitis.     

Phenylalanine hydroxylase-like antibody in human chorionic villi]

An antigen similar by electrophoretic mobility to liver phenylalanine hydroxylase (PH) and cross-reacting with monoclonal antibody PH8 against liver PH was detected in extracts of soluble proteins in 6 from 23 samples of chorionic villi. An antigen with electrophoretic mobility corresponding to 40-41 kDa was detected in extracts of membrane proteins from these 23 samples by immunoblotting with monoclonal antibody PH8. Its molecular weight was similar to that of major chymotryptic peptide of human liver PH. The content of the antigen varied with samples and was less than 20 ng/mg of the extracted protein. Two-dimensional gel electrophoresis revealed only 1 spot of the antigen. The antigen did not react with monoclonal antibodies PH7 and PH9 epitopes of which were located in N-terminal fragment of liver PH. These data suggest that the antigen of membrane fraction could be a PH protein without N-terminal domain.     

Characterization of anti-prostatic acid phosphatase monoclonal antibody and its medical significance

Monoclonal antibody to purified prostatic acid phosphatase from seminal plasma was produced by fusion of spleen cells from immunized mice with the Sp2/O-Ag 14 cell line. This hybridoma-derived antibody, designated MAb-14, was classified as IgG1 immunoglobulin. The apparent affinity constant of phosphatase.MAb-14 complex formation calculated by using the Langmuir isotherm is 2.4 x 10(-8) M. The molecular weight of the complex formed under the condition of antibody excess was found to be 350K, which suggests that 2 molecules of prostatic phosphatase bind to 1 molecule of the antibody. The MAb-14 antibody bound to phosphatase had a negligible effect on the catalytic activity of the enzyme. All isoenzymatic forms of catalytically active prostatic phosphatase, resolved by isoelectric focusing or by chromatofocusing in different pH gradients, reacted with the monoclonal antibody. Several peptides of Mr 25K to 76K and of 13K to 76K were adsorbed from the prostatic tissue extract and from seminal fluid, respectively, on an MAb-14-Sepharose column. The MAb-14 monoclonal antibody was applied to the immunohistochemical investigation of prostatic phosphatase distribution in normal human prostate gland, in nodular hyperplasia, and in adenocarcinoma of the prostate. Immunostaining was observed in prostatic secretory epithelium, within the luminal content of prostatic glands, and in the neighborhood of prostatic cancer cells. Metastatic prostatic carcinoma was also strongly immunoreactive with the antibody. There was no cross-reactivity with leukocytes, kidney, liver, pancreas, spleen, breast, stomach mucosal, and colon tissues.     

Effect of removal of the cytolytic factor of Bacillus thuringiensis subsp. israelensis on mosquito toxicity

Solubilized crystal protein of Bacillus thuringiensis subsp. israelensis was fractionated by affinity chromatography using a monoclonal antibody directed against the crystal's 28 kDa peptide. The 28 kDa peptide was found to be relatively nontoxic to mosquito larvae although it does contain the hemolytic activity of the crystals. The crystal protein fraction depleted of the 28 kDa peptide was found to be nonhemolytic and to retain nearly full toxicity to mosquito larvae. These results suggest that the 28 kDa peptide is not required for the toxicity of solubilized crystal protein.     

Purification and functional characterization of bovine RP-A in an<Emphasis Type="Italic">in vitro</Emphasis> SV40 DNA replication system

The single-stranded DNA binding protein RP-A is required in SV40 DNAin vitro replication. The RP-A purified from calf thymus contains 4 polypeptides with molecular weights 70kDa, 53kDa, 32kDa, and 14kDa. The p70 subunit and its proteolysed form p53 are recognized by the monoclonal antibody 70C (Kenny et al. (1990)) and bind to ssDNA. The p70 and p32 subunits of bovine RP-A are phosphorylated by CDC2-cyclin B kinase. Bovine RP-A supports the origin dependent unwinding of SV40 DNA by T antigen. Furthermore, bovine RP-A can efficiently substitute for human RP-A in SV40 DNA replicationin vitro. A modified blotting technique revealed that RP-A interacts specifically and directly with the p48 subunit of DNA polymerase α-primase complex.     

Construction and characterization of a functional chimeric murine-human antibody directed against human fibrin fragment-D dimer

Fibrin-directed monoclonal antibodies may be clinically useful for in vitro thrombus imaging and for the targeting of fibrinolytic agents to blood clots. One such murine monoclonal antibody, (mAb-15C5), raised against the fragment-D dimer epitope of cross-linked human fibrin, was previously characterized [Holvoet, P., Stassen, J. M., Hashimoto, Y., Spriggs, D., Devos, P. & Collen, D. (1989) Thromb. Haemostasis 61, 307-313] has recently been cloned and expressed [Vandamme, A.-M., Bulens, F., Bernar, H., Nelles, L., Lijnen, H. R. & Collen, D. (1990) Eur. J. Biochem. 192, 767-775]. In order to reduce the immunogenicity of the murine mAb-15C5 in man, we have now constructed a murine--human chimera of mAb-15C5, by substituting the cDNA sequences encoding the constant regions of the murine kappa light chain and gamma 1 heavy chain by the corresponding human genomic sequences. Both chimeric murine--human Ig chains were cloned into two separately selectable expression vectors, which were contransfected into Chinese hamster ovary (CHO) cells. Murine--human chimeric mAb-15C5 (mAb-15C5Hu) was purified from the conditioned medium of selected cell lines by chromatography on Zn-chelating Sepharose, protein-A-Sepharose and on insolubilized antigen (fragment-D dimer), with a final yield of 29 micrograms/l and a recovery of 33%. SDS/PAGE without reduction revealed a homogeneous band with a mobility similar to that of natural mAb-15C5, whereas after reduction, both the heavy and the light chains had slightly slower mobilities than their natural counterparts. Expression in the presence of tunicamycin suggested that the differences in gamma 1-chain mobility were due to different N-glycosylation patterns. Immunoblotting of proteins from SDS gels showed immunological reactivity of recombinant mAb-15C5Hu with goat anti-(human IgG) IgG and of recombinant and natural murine mAb-15C5 with goat anti-(mouse IgG) IgG. Competitive binding revealed a comparable affinity of recombinant murine mAb-15C5, recombinant mAb-15C5Hu and natural mAb-15C5, for fragment-D dimer, indicating that recombinant mAb-15C5Hu was obtained in a functionally intact form. Thus, mAb-15C5Hu may constitute a useful alternative to mAb-15C5 for in vivo use in man.     

Albumin-binding proteins of endothelial cells: immunocytochemical detection of the 18 kDa peptide.

Extracts of isolated microvascular endothelial cells (MEC) and cultured bovine aortic endothelial cells (BAEC) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), electrotransfer and incubation with albumin either radioiodinated or adsorbed to 5-nm gold particles. Both ligands reacted exclusively with two peptides of 18 and 31 kDa. To the 18 kDa peptide (excised from preparative SDS-PAGE), an antibody was raised in rabbits and purified by affinity on 18 kDa obtained from two-dimensional gel electrophoresis and immobilized on nitrocellulose paper. The specificity of the anti-18 kDa was assessed by immunoblotting and immunoprecipitation of endothelial cell extracts. To check whether the 18 kDa peptide is exposed on the endothelial cell surface and/or its components (uncoated pits, open plasmalemmal vesicles), the apical membrane of BAEC was radioiodinated, the solubilized proteins incubated with the anti-18 kDa, and the immune complexes formed were precipitated with protein A-Sepharose CL-4B. The ensuing SDS-PAGE and autoradiography revealed that from all radioiodinatable surface proteins, the 18 kDa was the only polypeptide immunoprecipitated by the anti-18 kDa antibody. To localize the 18 kDa peptide, we applied indirect immunofluorescence technique on cultured MEC and BAEC and immunoelectron microscopy (EM) on ultrathin cryosections of mouse heart. Nonpermeabilized whole MEC and BAEC incubated with anti-18 kDa followed by rhodamine-conjugated second antibody showed a relatively intense surface fluorescence often appearing as small dots. At the EM level, heart ultrathin cryosections exposed anti-18 kDa followed by gold-conjugated second antibody revealed that 18 kDa was primarily associated with the membrane of plasmalemmal vesicles of capillary endothelia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular cloning of the 31 kDa cytosolic phospholipase A2, as an antigen recognized by the lung cancer-specific human monoclonal antibody, AE6F4

The human monoclonal antibody AE6F4 specifically reacts with human lung cancer tissues but does not with normal tissues. This monoclonal antibody recognizes a cytosolic 31 kDa antigen in the cancer cells. In a previous study, we elucidated that the 31 kDa antigen belonged to a family of proteins collectively designated as 14-3-3 proteins, which were known as protein kinase-dependent activators of tyrosine/trytophan hydroxylases, or protein kinase C inhibitor proteins. Here we report molecular cloning of the 31 kDa antigen from the human lung adenocarcinoma cell line, A549. Sequencing analysis indicates that the cloned cDNA is identical to that of previously reported human placental cytosolic phospholipase A₂ (cPLA₂), which is also a member of the 14-3-3 protein family. Western analysis demonstrated that a 31 kDa recombinant cPLA₂ expressed in monkey COS cells was recognized by the AE6F4 monoclonal antibody. Binding of the monoclonal antibody to the recombinant cPLA₂ was abolished when treated with sodium periodate, suggesting that not only are carbohydrate chains associated with the cPLA₂, but they also play a crucial role in antigen recognition by the monoclonal antibody.