A generic method for expression and use of "tagged" soluble versions of cell surface receptors.

A general method for expression, purification, immobilization, detection and radiolabeling of extracellular domains (ECD) of type I membrane proteins. The type I interleukin-1 receptor (IL-1RtI), the alpha-subunit of interleukin-2 receptor (IL-2R alpha) and E-selectin are used as illustrative examples of cell surface receptors. DNA encoding the ECD of the proteins are fused at their 3' end to a chimeric DNA which serves to generically "tag" the recombinant ECD. The resulting fusion protein contains a substrate sequence for protein kinase-A (PKA) adjacent to the signal sequence from human placental alkaline phosphatase (HPAP), The HPAP signal sequence directs the formation of the phosphatidylinositol-glycan (PI-G) anchorage of the protein at the cell surface. When these chimeric genes are expressed in CHO cells, the ECDs are detected on the cell surface and can be released by treatment with phosphatidylinositol-specific phospholipase-C (PI-PLC). Based on protein processing known to occur for native HPAP, twenty amino acids from the HPAP signal sequence remain at the C-terminus of the ECD. A high affinity monoclonal antibody was generated against this common epitope. This antibody can be used to detect, purify and immobilize the ECDs. In addition, the ECDs can be radiolabeled with 32P by treatment with PKA and maintain the ability to bind their natural ligands. This "tagging" method has been successfully applied to many other type I proteins which serve as cell surface receptors.

The use of light-directed combinatorial peptide synthesis in epitope mapping.

The application of light-directed combinatorial peptide synthesis to epitope mapping is described. Photolithography and solid phase peptide synthesis were combined in an automated fashion to assemble arrays containing 1024 peptide sequences on a glass support in ten steps with the precise location of each peptide known. The simultaneous synthesis of two slides containing three arrays of peptides each allowed for the independent screening of both a monoclonal antibody (mAb) and its Fab fragment at two different concentrations. A binary synthesis strategy was used to assemble the arrays, resulting in all deletions and truncations possible within the FLRRQFKVVT sequence being present and available for screening. The relative binding interactions of each peptide was determined by incubating the arrays with either mAb D32.39 and goat antimouse immunoglobulin G-FITC or mAb D32.39 Fab-FITC conjugate, followed by scanning the surface for fluorescence with an epifluorescence microscope. The fragment RQFKVVT was found to bind tightly to both the mAb and Fab fragment while tethered to the surface, and was measured to have 0.49 nM affinity in solution. The frame-shifted RRQFKVV sequence was found to have lower affinity both in solution (1.3 mM) and on the surface. The fragment RQFKVV was determined to be responsible for antibody recognition and was found to bind tightly when tethered to the surface, yet exhibited no binding in solution as the free acid, suggesting the requirement of an amidated C-terminus or an additional flanking residue. A deletion analysis revealed that the novel RQFKVT sequence exhibited higher affinity than the RQFKVV sequence while tethered to the surface.

Generation and screening of an oligonucleotide-encoded synthetic peptide library.

We have prepared a library of approximately 10(6) different peptide sequences on small, spherical (10-microns diameter) beads by the combinatorial chemical coupling of both L- and D-amino acid building blocks. To each bead is covalently attached many copies of a single peptide sequence and, additionally, copies of a unique single-stranded oligonucleotide that codes for that peptide sequence. The oligonucleotide tags are synthesized through a parallel combinatorial procedure that effectively records the process by which the encoded peptide sequence is assembled. The collection of beads was screened for binding to a fluorescently labeled anti-peptide antibody using a fluorescence-activated cell sorting instrument. Those beads to which the antibody bound tightly were isolated by fluorescence-activated sorting, and the oligonucleotide identifiers attached to individual sorted beads were amplified by the PCR. Sequences of the amplified DNAs were determined to reveal the identity of peptide sequences that bound to the antibody with high affinity. By combining the capacity for information storage in an oligonucleotide code with the tremendous level of amplification possible through the PCR, we have devised a means for specifying the identity of each member of a vast library of molecules synthesized from both natural and unnatural chemical building blocks. In addition, we have shown that the use of flow cytometry instrumentation permits facile isolation of individual beads that bear high-affinity ligands for biological receptors.

Controlling chemical reactivity with antibodies.

The remarkable specificity of an antibody molecule has been used to accomplish highly selective functional group transformations not attainable by current chemical methods. An antibody raised against an amine-oxide hapten catalyzes the reduction of a diketone to a hydroxyketone with greater than 75:1 regioselectivity for one of two nearly equivalent ketone moieties. The antibody-catalyzed reaction is highly stereoselective, affording the hydroxyketone in high enantiomeric excess. Similarly, the reduction of ketones containing branched and aryl substituents, including the highly symmetrical 1-nitrophenyl-3-phenyl-2-propanone, was enantioselective. The simple strategy presented herein may find general applicability to the regio- and stereoselective reduction of a broad range of compounds.

An efficient antibody-catalyzed aminoacylation reaction.

An antibody generated against a neutral phosphonate diester transition-state analog was found to catalyze the aminoacylation of the 3'-hydroxyl group of thymidine with an alanyl ester. A comparison of the apparent second-order rate constant of the antibody-catalyzed reaction [5.4 x 10(4) molar-1 minute-1 (M-1 min-1)] with that of the uncatalyzed reaction (2.6 x 10(-4) M-1 min-1) revealed this to be a remarkably efficient catalyst. Moreover, although the concentration of water (55 M) greatly exceeds that of the secondary alcohol, the antibody selectively catalyzes acyl transfer to thymidine. The antibody exhibits sequential binding, with Michaelis constants of 770 microM and 260 microM for acyl acceptor and donor, respectively, and a dissociation constant of 240 pM for hapten. This antibody-catalyzed reaction provides increased insight into the requirements for efficient aminoacylation catalysts and may represent a first step toward the generation of "aminoacyl transfer RNA synthetases" with novel specificities.

Immunocytochemical localization of monoamine oxidases A and B in human peripheral tissues and brain.

Monoamine oxidases (MAO; EC 1.4.3.4.) A and B occur in the outer mitochondrial membrane and oxidize a number of important biogenic and xenobiotic amines. Monoclonal antibodies specific for human MAO A or B and immunocytochemical techniques were used to visualize the respective enzymes in human placenta, platelets, lymphocytes, liver, brain, and a human hepatoma cell line. MAO A was observed in the syncytiotrophoblast layer of term placenta, liver, and a subset of neurons in brain, but was not observed in platelets or lymphocytes, which are known to lack type A enzyme. MAO B was observed in platelets, lymphocytes, and liver, but not in placenta, which contains little or no MAO B. MAO B was also observed in a subset of neurons in the brain that was distinct from that which contained MAO A. MAO A and MAO B were also observed in some glia. Unlike most tissues examined, liver cells appeared to contain both forms of the enzyme. These studies show that MAO A and MAO B can be specifically visualized by immunocytochemical means in a variety of human cells and tissues and can provide a graphic demonstration of the high degree of cell specificity of expression of the two forms of the enzyme.

Assignment of genes for human monoamine oxidases A and B to the X chromosome.

Monoclonal antibodies that immunoprecipitate human monoamine oxidase (MAO) A or human MAO B, but not the corresponding mouse enzymes, were used to assay for the presence of immunoprecipitable MAO A or MAO B (presumably coded by the respective human genes) in mouse-human hybrid somatic cell lines containing small numbers of human chromosomes. The results were as follow: Extracts of a human lymphoblastoid x mouse hepatoma hybrid line that retained the human X chromosome contained immunoprecipitable MAO B, while a similar hybrid line that contained the same human chromosomes, except for the human X, did not. Extracts of a human fibroblast x mouse neuroblastoma hybrid cell line, whose human chromosomal material consisted solely of the X, contained both immunoprecipitable MAO A and MAO B. Extracts of a related hybrid line, whose human chromosomal material consisted solely of an autonomous fragment and a fragment translocated to a mouse chromosome, contained immunoprecipitable MAO A. However, the level of immunoprecipitable MAO B activity in extracts of this hybrid was low or undetectable. Among extracts of 33 human fibroblast x mouse hepatoma hybrids that had been selected for expression of the X-linked human enzyme HPRT, 60% contained immunoprecipitable MAO B. This figure was comparable to the 58% that expressed the X-linked human isozyme for glucose-6-phosphate dehydrogenase (G6PD). When 11 of these hybrid lines, which contained immunoprecipitable MAO B and human HPRT, were selected for loss of HPRT, all lost immunoprecipitable MAO B in addition to HPRT. These data demonstrate that genes controlling the expression of MAO A and MAO B, which can be immunoprecipitated with the human-specific monoclonal antibodies, are located on the human X chromosome. Properties of the immunological epitopes recognized by the monoclonal antibodies suggest that the X-linked genes detected in this study are probably structural genes for the enzymes.

Immunological uniqueness of human monoamine oxidases A and B: new evidence from studies with monoclonal antibodies to human monoamine oxidase A.

Monoamine oxidase (EC 1.4.3.4; MAO) is the primary enzyme responsible for the intraneuronal degradation of biogenic amines in the central nervous system. An understanding of the physiological significance of the functional and regulatory differences between the two forms of the enzyme, MAOs A and B, would be facilitated by the availability of antibodies specific for the two forms of the enzyme. We previously isolated and characterized a monoclonal antibody (MAO B-1C2, previously designated MAO-1C2) which binds human MAO B but not A. We describe here four new monoclonal antibodies (designated MAO A-3C9, A-4F10, A-7B10, and A-7E10) which were elicited to highly purified MAO A from human placenta and which, in the presence of antimouse IgG and Staphylococcus aureus, immunoprecipitate greater than 90% of the catalytically active purified MAO A. MAO A-3C9 appears to have a lower affinity for purified MAO A than the other three antibodies and does not immunoprecipitate either MAO A or MAO B from human platelets or from Triton X-100 extracts of human placental and liver mitochondria. MAO A-4F10, A-7B10, and A-7E10 immunoprecipitate catalytically active MAO A from Triton X-100 extracts of human placental and liver mitochondria, but not catalytically active MAO B from either pletelets or from Triton X-100 extracts of human liver mitochondria. Collectively, these anti-MAO monoclonal antibodies reveal unique epitopes on human MAO A not shared by MAO B, and at least one epitope on MAO B not shared by MAO A. These immunochemical differences support the hypothesis that MAO A and MAO B are different proteins, presumably isozymes.

Distinct monoamine oxidase A and B populations in primate brain.

Monoclonal antibodies specific for monoamine oxidase (MAO) A and MAO B, respectively, were used to localize these enzymes in primate brain. The reagents recognized different populations of neurons: those that recognized MAO A were located in cell groups containing catecholamines, including the substantia nigra, nucleus locus coeruleus, nucleus subcoeruleus, and the periventricular region of the hypothalamus, whereas those that recognized MAO B were observed in serotonin regions, including the nucleus raphe dorsalis and nucleus centralis superior. These data illustrate the physiological independence of MAO A and B and show that neurons may be specialized for their degradative as well as their synthetic functions.