Identification of the target of monoclonal antibody A6H as dipeptidyl peptidase IV/CD26 by LC MS\MS.

The monoclonal antibody (MAb) A6H, originally developed to fetal renal tissues, was found to be highly reactive to renal cell carcinoma and was subsequently demonstrated to co-stimulate a subpopulation of T cells. The A6H antigen had not been identified heretofore. Antigen from detergent extracts of renal cell carcinoma cells (7860) was immunoabsorbed with A6H-agarose, and the resin-bound proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The antigen had a molecular weight of approximately 120 kDa as determined by Western blots. The 120-kDa protein band was excised and subjected to in-gel tryptic digestion, and the resulting peptides were separated and analyzed by liquid chromatography tandem mass spectrometry (LC MS\MS). The tandem mass spectra of the eluting peptides were used in combination with the SEQUEST computer program to search a human National Cancer Institute (NCI) protein database for the identity of the protein. The target antigen was shown to be dipeptidyl peptidase IV (DPP IV), which is also known as the cluster differentiation antigen CD26. Flow analysis of the expression of the A6H antigen and of CD26 on 7860 cells and on peripheral blood lymphocytes supported the identification of the A6H antigen as DPP IV. Recognition that the A6H antigen is DPP IV/CD26 afforded the opportunity to compare previous studies on A6H with those on other anti-CD26 antibodies in terms of expression in cancer cell lines and various tissues and as co-stimulators of T-cell activation.

Differential stable isotope labeling of peptides for quantitation and de novo sequence derivation.

We have demonstrated the use of per-methyl esterification of peptides for relative quantification of proteins between two mixtures of proteins and automated de novo sequence derivation on the same dataset. Protein mixtures for comparison were digested to peptides and resultant peptides methylated using either d0- or d3-methanol. Methyl esterification of peptides converted carboxylic acids, such as are present on the side chains of aspartic and glutamic acid as well as the carboxyl terminus, to their corresponding methyl esters. The separate d0- and d3-methylated peptide mixtures were combined and the mixture subjected to microcapillary high performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS). Parent proteins of methylated peptides were identified by correlative database searching of peptide tandem mass spectra. Ratios of proteins in the two original mixtures could be calculated by normalization of the area under the curve for identical charge states of d0- to d3-methylated peptides. An algorithm was developed that derived, without intervention, peptide sequence de novo by comparison of tandem mass spectra of d0- and d3-peptide methyl esters.

Cloning of an ecdysone receptor homolog from Manduca sexta and the developmental profile of its mRNA in wings.

Using the Drosophila melanogaster ecdysone receptor (DmEcR) B1 cDNA clone, we isolated three genomic clones for EcR from the tobacco hornworm, Manduca sexta. Subsequent isolation and sequencing of several cDNAs yielded a homolog of the B1 isoform with 50, 95 and 70% amino acid identities with DmEcR in the N-terminal A/B, the DNA binding and the ligand binding domains respectively. Unlike Drosophila, an intron occurs between the exons encoding the two zinc fingers of Manduca EcR (MsEcR). A 6.0 kb mRNA encoding MsEcR was found in both larval wing discs and prothoracic glands and in pupal wings. During the final larval instar, the mRNA was maximal in the wing discs at one day after wandering (W1), whereas in the prothoracic gland EcR mRNA increased rapidly to high levels on day 2 and remained high thereafter. During the onset of adult development, two peaks of EcR mRNA were observed in wings from day 3 to 5 and on day 8 after pupal ecdysis. These two peaks correlated with the time of increasing titers of ecdysone (E) and 20-hydroxyecdysone (20E), respectively. The EcR mRNA peaks always preceded the large ecdysteroid peak, suggesting that the transcription of the EcR gene is induced by a low concentration of ecdysteroid in vivo.

Potassium channels and epilepsy: evidence that the epileptogenic toxin, dendrotoxin, binds to potassium channel proteins.

Dendrotoxin-I, a component of the venom of the black mamba snake, Dendroaspis polylepsis, was used to affinity purify a potassium channel from bovine brain. This dendrotoxin-I binding protein was composed of several subunits with molecular weights of 35,000, 38,000, 42,000 and 74,000. Partial sequence resulting from Edman degradation of the N-terminus of the 74 kDa subunit was identical to the predicted amino acid sequence of the N-terminus of a protein encoded by a mouse/rat homologue of the Shaker gene family of potassium channels, MK2/RBK2 (RCK5). Polyclonal antibodies raised against synthetic peptides derived from the predicted amino acid sequence of another member of this family, MK1, recognized this 74 kDa subunit. Due to extensive amino acid sequence identity between MK2 and MK1, it is likely that antibodies recognized epitopes common to both. Thus, from an immunological standpoint, either MK1, MK2, or both channel proteins could have been present in this 74 kDa band on protein blots. Closely related K+ channels in bovine brain could have copurified based on their affinity for dendrotoxin-I (DTX-I). DTX-I was shown to inhibit MK1 currents in a time and voltage independent fashion. Physiological and molecular evidence indicates the existence of many types of DTX sensitive potassium channels in the mammalian brain, however, our protein sequencing of the 74 kDa subunit has detected the presence of only one unique N-terminal sequence, identical to MK2. The possible reason for the appearance of this discrepancy is discussed. This paper represents the first report identifying one dendrotoxin binding protein in bovine brain tissue (BK2) as a delayed rectifier type of potassium channel.

Immunological evidence for a relationship between the dendrotoxin-binding protein and the mammalian homologue of the Drosophila Shaker K+ channel.

Polyclonal antibodies were raised against two synthetic peptides from different parts of the predicted amino acid sequence of the mouse homologue (MBK1) of the Drosophila Shaker K+ channel. The antibodies recognized the toxin-binding subunit of the dendrotoxin-binding proteins from rat and bovine brain. The results suggest that the dendrotoxin-binding protein is related to the expression products of the mammalian homologue of the Shaker gene.