Protein mapping in rat basophilic leukaemia cells

This review discusses some of our recent studies on rat basophilic leukaemia (RBL) cells, a model cell line for mast cell function. Our interest in these cells is a consequence of the role played by mast cells in the allergic response. Thus far we have described the identification of several spots, and their pI/M(r) co-ordinates. Here we describe how we can further decipher the mast cell proteome using a variety of staining/immuno-blotting procedures. We demonstrate the sensitivity of staining procedures and immuno-blotting using an anti-phosphotyrosine antibody. Our aim is to contribute to the ever-expanding two-dimensional gel and phosphoprotein databases currently available

Two-dimensional electrophoresis reveals differential protein expression in high- and low-secreting variants of the rat basophilic leukaemia cell line

The aim of this investigation was the identification of cellular proteins that confer a high secretory phenotype on subclones of the rat basophilic leukaemia (RBL) cell line as a model of mast cell regulated degranulation. Following protein separation by two-dimensional (2-D) electrophoresis and silver staining, more than 2000 polypeptide "spots" were resolved reproducibly. Higher sample loads and Coomassie blue staining facilitated the identification by delayed extraction-matrix-assisted laser desorption/ionization (DE-MALDI) mass spectrometry of several polypeptides that were differentially expressed in the high- and low-secreting clones. Several proteins were identified whose expression could contribute to the difference in secretory phenotype. Furthermore, silver-stained 2-D gel patterns suggested differential expression of proteins in the 20-25 kDa and the pI 4.5-7.5 range, characteristic of small guanosine 5'-triphosphate (GTP)-binding proteins. By a combination of "GTP overlay" and immunoblotting, we were able to demonstrate differential expression of small GTP binding-proteins, including Rab3 proteins, in high-and low-secreting clones. The sensitivity of this complementary approach facilitated the detection of some GTP binding and Rab3 proteins, whose expression was not evident in silver-stained 2-D gels

Differential expression of Rab3 isoforms in high- and low-secreting mast cell lines

The expression of several isoforms of the small-molecular-weight Rab3 GTP-binding proteins is a characteristic feature of all cell types undergoing regulated exocytosis, in which Rab3 proteins are considered to regulate the assembly/disassembly of a fusion complex between granule and plasma membrane in a positive and negative manner through interaction with effector proteins. The pattern of Rab3 protein expression may, therefore, provide a subtle means of regulating exocytosis. To investigate the relationship between Rab3 expression and secretory activity, we assessed the differential expression of individual Rab3 proteins in high- and low-secreting clones of the rat basophilic (RBL) cell line. mRNAs for Rab3 isoforms (a-d) were analyzed by constructing cDNA libraries of high- and low-secreting RBL clones. The relative abundance of mRNAs for Rab3 isoforms was initially determined from the clonal frequency of corresponding cDNA clones. RT-PCR using isoform-specific primers was successfully applied to the quantitation of Rab3a mRNA. The presence of individual Rab3 proteins was revealed by SDS-PAGE and immunoblotting, and also by in situ immunofluorescence confocal microscopy. We present evidence that Rab3a and Rab3c are expressed at high levels in the low-secreting variant, while Rab3d is predominant in the high secretor. Levels of the Rab3 effector proteins, Rabphilin and Noc2, are similar in both RBL cell lines. Subcellular fractionation of unstimulated high and low secretor RBL clones revealed that in both cell types Rab3a has a cytoplasmic location while Rab3d is present in a membrane/organelle fraction containing secretory vesicles. Differences in the pattern of expression of Rab3 isoforms in the two RBL cell lines and their localisation may influence the secretory potential. Furthermore, the presence of Rab3 and effector proteins indicates that the mechanism for regulated exocytosis in cells of mast cells/basophil lineage appears similar to that in pre-synaptic vesicles and pancreatic beta-cells

Assessing the role of Asp 194 in the transmembrane domains of the α-chain of the high-affinity receptor complex for immunoglobulin E in signal transduction

The high-affinity receptor complex for IgE plays a pivotal role in allergic responses since cross-linking of the high-affinity IgE receptor (FcɛRI) on target cells initiates a signaling cascade facilitating release of inflammatory mediators causing allergic responses. The transmembrane regions of the ligand binding domains of the high-affinity IgE and low-affinity IgG receptors share an invariant motif (LFAVDTGL) containing a polar aspartate within a predominantly non-polar setting. The functional importance of this aspartate residue (D194) in FcɛRI-mediated receptor signaling was assessed by site-directed mutagenesis. Rat basophilic leukemia cells (RBL-2H3) transfected with the human IgE binding subunit (FcɛRIα) incorporating polar substitutions like asparagine (D194N) or threonine (D194T) resulted in the formation of a functional rat/human chimeric receptor complex. When activated via huIgE and antigen, cells transfected with these variant receptor subunits supported mediator release, intracellular calcium mobilisation and tyrosine phosphorylation of γ-chain and Syk kinase while a non-polar substitution (D194L) gave rise to cell surface expression of the mutated receptor subunit but failed to initiate downstream signaling. No cell surface expression of huFcɛRIα gene constructs was observed when D194 was replaced with the non-polar Ile (D194I) residue of similar size, the larger positively charged Arg (D194R) or lysine (D194K) residues, or the negatively charged glutamate (D194E) and smaller polar Ser (D194S) non-polar Ala (D194A) and V (D194V). These observations highlight importance of the size and charge of amino acid residue at position 194 in determining IgE receptor subunit interactions, cell surface localization, and initiation of downstream signaling events

Synthesis and incorporation into cyclic peptides of tolan amino acids and their hydrogenated congeners: construction of an array of A–B-loop mimetics of the Cε3 domain of human IgE

The disruption of the human immunolobulin E–high affinity receptor I (IgE–FcεRI) protein–protein interaction (PPI) is a validated strategy for the development of anti asthma therapeutics. Here, we describe the synthesis of an array of conformationally constrained cyclic peptides based on an epitope of the A–B loop within the Cε3 domain of IgE. The peptides contain various tolan (i.e., 1,2-biarylethyne) amino acids and their fully and partially hydrogenated congeners as conformational constraints. Modest antagonist activity (IC50 660 μM) is displayed by the peptide containing a 2,2′-tolan, which is the one predicted by molecular modeling to best mimic the conformation of the native A–B loop epitope in IgE