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I Pecht

Publications and source records attributed to I Pecht.

At least 19 recordsLinked to original sources
PubMed1998

Purification and preliminary characterization of an Fc epsilon-receptor-activated protein-tyrosine phosphatase from mast cells.

Immunological stimulation of rat, mucosal type, mast cells (line RBL-2H3) by clustering the type I Fc(epsilon) receptor (Fc[epsilon]RI) causes a fast yet transient tyrosyl phosphorylation of several proteins. We report here the characterization of a protein-tyrosine phosphatase (PTP) involved in the very early steps coupling the Fc(epsilon)RI stimulus to the cell secretory response. We have observed earlier that a PTP activity present in one of the cells' solubilized particulate fractions is enhanced 2-3-fold upon cell stimulation by Fc(epsilon)RI clustering [Hampe, C. S. & Pecht, I. (1994) FEBS Lett. 346, 194-198]. This PTP (MpII PTP) was now isolated and purified to homogeneity and appears as a 45-kDa protein. Sequence comparison of two peptide stretches of MpII PTP with those of proteins present in the Swiss-Prot and EMBL data banks revealed no significant similarity to a known protein. Hence, we assume that the MpII PTP is a novel protein. The possible involvement of MpII PTP in the stimulus-secretion coupling cascade was investigated. Abrogation of the antigen-induced Fc(epsilon)RI clustering by an excess of monovalent hapten suppressed the Fc(epsilon)RI-mediated MpII PTP activity enhancement to the levels observed in resting cells. The activity enhancement required the presence of extracellular Ca2+ ions and was also induced by an artificial increase of the free intracellular concentration of these ions. It is apparently mediated by protein kinase C (PKC) since phorbol myristate induced an additive increase in the MpII PTP activity to that induced by antigen. Also, treatment with the PKC-specific inhibitor bisindolylmaleimide suppressed the antigen-induced MpII PTP activity enhancement. PKC involvement was further supported by the finding that the MpII PTP 45-kDa protein underwent seryl phosphorylation following antigen stimulation. This modification was found to be further enhanced by pretreatment with phorbol myristate prior to antigen stimulation.

Animals↗
PubMed1998

A hemagglutinin-based multipeptide construct elicits enhanced protective immune response in mice against influenza A virus infection.

Multipeptide constructs, comprising adjacent sequences of the 317-341 intersubunit region of immature influenza A hemagglutinin (H1N1), were designed and the functional properties of these branched peptides were compared to that of the corresponding linear peptides. In vivo studies revealed that the immunogenicity of the peptides was dependent on the presence of the hydrophobic fusion peptide (comprised in FP3), encompassing the N-terminal 1-13 sequence of the HA2 subunit. Antibody and T cell recognition, however, was directed against the 317-329 HA1 sequence, comprised in the P4 peptide. Multiple copies of P4, covalently linked by branched lysine residues, significantly enhanced the efficiency of antibody binding and the capacity of peptides to elicit B- and T-cell responses. A fraction of peptide induced antibodies reacted with immature or with proteolitically cleaved hemagglutinin (HA) molecules pretreated at low pH. Immunization with a multipeptide construct, (P4)4-FP3, not only resulted in elevated antibody and T cell responses but conferred enhanced protection against lethal A/PR/8/34 (H1N1) infection as compared to its subunit peptides. The beneficial functional properties of this artificial peptide antigen may be acquired by multiple properties including: (i) stabilized peptide conformation which promotes strong, polyvalent binding to both antibodies and MHC class II molecules; (ii) appropriate P4 conformation for antibody recognition stabilized by the covalently coupled fusion peptide, resulting in the production of virus cross reactive antibodies which inhibit the fusion activity of the virus; (iii) activation of peptide specific B cells which potentiate antigen presentation and peptide specific T cell responses; and (iv) generation of helper T cells which secrete lymphokines active in the resolution of infection.

Animals↗
PubMed1997

Mercuric chloride-induced vasculitis in the Brown Norway rat: alpha beta T cell-dependent and -independent phases: role of the mast cell.

Mercuric chloride induces a necrotizing vasculitis in the Brown Norway (BN) rat. This occurs in two phases, between 1 and 5 days (early) and between 12 and 20 days (late) after initiation of HgCl2. One outbred and four inbred rat strains were found to be susceptible to early vasculitis, but only the BN strain developed late vasculitis. In the BN strain, treatment with the mAb R73 (anti-alpha beta TCR) inhibited T cell function, completely prevented the late vasculitis, but had no effect against early vasculitis, indicating that early and late vasculitis is controlled by different genetic and cellular mechanisms. The role of the mast cell in the alpha beta T cell-independent early phase was studied. Serum concentrations of rat mast cell protease II rose following HgCl2 treatment, indicating mast cell degranulation. The reagents Doxantrazole and the mAb G63, which suppress mast cell secretory responses, also prevented the rise in rat mast cell protease II and significantly reduced the early vasculitis. The demonstration of an alpha beta T cell-dependent phase supports previous experimental data that T cells play an important role in the pathogenesis of vasculitis. The presence of an earlier alpha beta T cell-independent phase is a unique observation. The data support a role for the mast cell in the early vasculitis.

Animals↗
PubMed1997

Fc gamma receptor type IIb induced recruitment of inositol and protein phosphatases to the signal transductory complex of human B-cell.

Co-clustering of Fc gamma RIIb and B-cell receptor (BCR) inhibits cell activation by interrupting BCR stimulated signal transduction. The immunoreceptor tyrosine-based inhibitory motif (ITIM) of Fc gamma RIIb becomes tyrosyl phosphorylated (P-ITIM) upon co-clustering with BCR then P-ITIM interacts with several signalling molecules, some of which negatively regulate the cell activation process. The molecules recruited by the P-ITIM of human Fc gamma RIIb have not been characterised yet. In order to affinity isolate the potential functional partner molecules of human Fc gamma RIIb, synthetic peptides were designed to cover almost the entire intracellular Fc gamma RIIb domain, including Fc gamma RIIb2 specific sequences and stretches containing the phosphorylated and non-phosphorylated ITIM. We report here that several tyrosyl phosphorylated proteins bind to the P-ITIM peptide from both resting and activated B-cell lysates, the 53-56 kDa being the most prominent one. A fraction of the 53-56 kDa bands were identified as the protein tyrosine kinase (PTK), Lyn which also bound to ITIM peptide, pointing to its role in initiating Fc gamma RIIb-mediated negative regulation. Among the P-ITIM associated tyr phosphorylated components, the 145 kDa one was identified as the inositol polyphosphate 5-phosphatase, SHIP and the 72 kDa protein as the protein tyrosine phosphatase (PTP) SHP2, whereas SHP1 was not detected. Phosphatase activity assays showed that P-ITIM bound about five times higher SHIP and four times higher PTP activity than the ITIM containing peptide. Furthermore, we detected PKC and MAPK in both ITIM and P-ITIM peptides precipitated samples. Since human B-cells express both Fc gamma RIIb1 and Fc gamma RIIb2, differing in a 19 amino acid insert in the cytoplasmic tail of the former, we investigated the components binding to Fc gamma RIIb1 and Fc gamma RIIb2 specific sequences. Synthetic peptide representing Fc gamma RIIb1 and Fc gamma RIIb2 specific sequences weakly bound unidentified tyr phosphorylated proteins at 50-56 kDa, while the insert itself did not bind a detectable amount of protein. Neither of the ITIM or P-ITIM bound molecules were observed in samples precipitated with peptides corresponding to Fc gamma RIIb1 or Fc gamma RIIb2 specific sequences. These observations suggest that protein kinases associate with both ITIM and P-ITIM of human Fc gamma RIIb, Lyn being responsible for the tyrosyl phosphorylation of ITIM. SHIP and SHP2 phosphatases selectively bind to the phosphorylated ITIM. Based on these data we assume that SHIP and SHP2 recruited in vivo to the Fc gamma RIIb co-clustered BCR are responsible for the Fc gamma RIIb mediated negative regulation of human B-cell activation.

Amino Acid Sequence↗
PubMed1997

Analysis of the genes encoding the mast cell function-associated antigen and its alternatively spliced transcripts.

The mast cell function-associated Ag (MAFA) is a C-type lectin that, upon being clustered, inhibits the Fc epsilon RI stimulation-induced mast cell secretory response. We here report that MAFA is encoded by a single-copy gene that spans 13 kb in the rat genome and is composed of five exons. Three separate exons encode the carbohydrate recognition domain of the MAFA, defining its close homology to the genes of CD23, CD69, CD72, NKR-P1, and Ly49. Functional analysis of the 5' flanking region of the gene reveals that a cell type-specific promoter is located within the first 664 bp upstream of the transcription origin. The promoter lacks any obvious TATA box and drives gene transcription originating from multiple start sites. Examination for possible polymorphism of the MAFA transcripts revealed two novel transcripts, generated by alternative splicing. Deletion of the transmembranal exon in one of them does not result in a frameshift and would, upon translation, give rise to a soluble MAFA molecule. Splicing of two exons in a second transcript results in a new reading frame encoding a putative protein containing MAFA's cytoplasmic domain. The transcription of the MAFA gene was detected in normal rat lungs, where both transmembranal and soluble MAFA appear to be expressed. Lung immunohistochemical analysis further suggests that MAFA expression is restricted to mast cells.

Alternative Splicing↗
PubMed1997

The mast cell function-associated antigen exhibits saccharide binding capacity.

The mast cell function-associated antigen (MAFA) is a membrane glycoprotein first identified on rat mucosal type mast cells (line RBL-2H3) and known to inhibit the Fc epsilon RI-mediated secretory response. In its extracellular domain, an amino acid stretch homologous to the carbohydrate binding domain of calcium-dependent animal lectins has been found. To investigate its carbohydrate binding capacity, the MAFA has been expressed in the Spodoptera frugiperda insect cell line (Sf9) using the baculovirus expression system. Analysis by flow cytometry and surface labeling with 125I showed that the recombinant MAFA (rMAFA) was expressed as a monomeric and disulfide-linked homodimeric glycoprotein in the membrane of the insect cells, and both forms exhibited the same epitopes as the protein isolated from RBL-2H3 cells. Immunoaffinity-purified rMAFA was then employed for studies of its saccharide binding capacity by using different neoglycans and glycoproteins. The rMAFA was found to bind specifically terminal mannose residues in a Ca(2+)-dependent manner. These results support the notion that the extracellular domain of the MAFA is indeed able to bind ligands, which may be modulatory for the mast cell response.

Animals↗
PubMed1997

Characterizing immunodominant and protective influenza hemagglutinin epitopes by functional activity and relative binding to major histocompatibility complex class II sites.

In the present study the analysis of functional activity and major histocompatibility complex (MHC) binding of two adjacent MHC class II-restricted epitopes, located in the C-terminal 306-329 region of human influenza A virus hemagglutinin 1 subunit (HA1) conserved with subtype sequences and not affected by antigenic drift, was undertaken to explore the hierarchy of local immunodominance. The functional activity of two T cell hybridomas of the memory/effector Th1 phenotype in combination with in vivo immunization studies provided a good tool for investigating the functional characteristics of the T cell response. The in vitro binding assays performed with a series of overlapping, N-terminal biotinylated peptides covering the 306-341 sequence enabled us to compare the relative binding efficiency of peptides, comprising two distinct epitopes of this region, to I-Ed expressed on living antigen-presenting cells. Our studies revealed that (i) immunization of BALB/c mice with the 306-329 H1 or H2 peptides resulted in the activation and proliferation of T cells recognizing both the 306-318 and the 317-329 epitopes, while the 306-329 H3 peptide elicits predominantly 306-318-specific T cells, (ii) the 317-329 HA1 epitope of the H1 and H2 but not the H3 sequence is recognized by T cells and is available for recognition not only in the 317-329 peptide but also in the extended 306-329 or 306-341 peptides, (iii) the 306-318 and the 317-329 hemagglutinin peptides encompassing the H1, H2 but not the H3 sequence bind with an apparently similar affinity to and therefore compete for I-Ed binding sites, and (iv) the 317-341, the 317-329 peptides and their truncated analogs show subtype-dependent differences in MHC binding and those with lower binding capacity represent the H3 subtype sequences. These results demonstrate that differences in the binding capacity of peptides comprising two non-overlapping epitopes located in the C-terminal 306-329 region of HA1 of all three subtype-specific sequences to MHC class II provide a rationale for the local and also for the previously observed in vivo immunodominance of the 306-318 region over the 317-329 epitope in the H3 but not in the H1 or H2 sequences. In good correlation with the results of the binding and functional inhibition assays, these data demonstrate that in the H1 and H2 subtypes both regions are available for T cell recognition, they compete for the same restriction element with an apparently similar binding efficiency and, therefore, function as co-dominant epitopes. Due to the stabilizing effect of the fusion peptide, peptides comprising the 306-341 or 317-341 H1 sequences are highly immunogenic and elicit a protective immune response which involves the production of antibodies and interleukin-2 and tumor necrosis factor producing effector Th1 cells both directed against the 317-329 region. Based on the similarity of the I-Ed and HLA-DR1 peptide binding grooves and motifs, these results suggest that amino acid substitutions inserted to the H3 subtype sequence during viral evolution can modify the relative MHC binding capacity and invert the local hierarchy of immunodominance of two closely situated epitopes that are able to bind to the same MHC class II molecule.

Amino Acid Sequence↗
PubMed1997

Analysis of Fc(epsilon)RI-mediated mast cell stimulation by surface-carried antigens.

Clustering of the type I receptor for IgE (Fc[epsilon]RI) on mast cells initiates a cascade of biochemical processes that result in secretion of inflammatory mediators. To determine the Fc(epsilon)RI proximity, cluster size, and mobility requirements for initiating the Fc(epsilon)RI cascade, a novel experimental protocol has been developed in which mast cells are reacted with glass surfaces carrying different densities of both antigen and bound IgE, and the cell's secretory response to these stimuli is measured. The results have been analyzed in terms of a model based on the following assumptions: 1) the glass surface antigen distribution and consequently that of the bound IgE are random; 2) Fc(epsilon)RI binding to these surface-bound IgEs immobilizes the former and saturates the latter; 3) the cell surface is formally divided into small elements, which function as a secretory stimulus unit when occupied by two or more immobilized IgE-Fc(epsilon)RI complexes; 4) alternatively, similar stimulatory units can be formed by binding of surface-carried IgE dimers to two Fc(epsilon)RI. This model yielded a satisfactory and self-consistent fitting of all of the different experimental data sets. Hence the present results establish the essential role of Fc(epsilon)RI immobilization for initiating its signaling cascade. Moreover, it provides independent support for the notion that as few as two Fc(epsilon)RIs immobilized at van der Waals contact constitute an "elementary stimulatory unit" leading to mast cell (RBL-2H3 line) secretory response.

Animals↗
PubMed1997

Binding of copolymer 1 and myelin basic protein leads to clustering of class II MHC molecules on antigen-presenting cells.

Copolymer 1 (Cop 1), a synthetic copolymer of amino acids, effective in suppression of experimental allergic encephalomyelitis (EAE) and myelin basic protein (MBP), was shown to bind extensively and promiscuously to the class II MHC molecules on antigen-presenting cells (APC) without prior processing. In the case of human APC, binding has earlier been demonstrated to DR but not DQ or class I molecules. In the present study, we examined whether binding of Cop 1 and MBP affects MHC class II expression on the cell membrane. Biotinylated derivatives of these antigens were used to monitor their direct binding to MHC molecules on living APC by flow cytometry using phycoerythrin-streptavidin, while the levels of MHC surface expression were monitored by staining with FITC-conjugated anti-class I- and class II-specific antibodies. When Cop 1 or MBP were incubated with the APC, intensity of cell staining with anti-DR, but not with anti-DQ or anti-class I antibodies, was significantly increased, compared to the staining of control APC not reacted with these antigens. In contrast, staining intensity was unaffected when p84-102, a human immunodominant epitope of MBP, or ovalbumin (OVA), a protein which undergoes proteolytic degradation prior to binding, were incubated with the APC. Cycloheximide, a protein synthesis inhibitor, had no effect on the enhanced staining intensity with anti-DR antibody of cells treated with Cop 1 or MBP, whereas it inhibited the enhanced staining of both DR and DQ molecules caused by the respective antibodies, in the absence of these antigens. Brefeldin A, a protein transport inhibitor, lowered the levels of staining intensity with anti-DR and anti-DQ antibodies in both cases, with and without antigen added to the APC. Fluorescence microscopic analysis revealed that cells incubated with Cop 1 or MBP, but not with p84-102 or OVA, exhibit both bright staining of the cell membrane and clusters produced by the aggregation of DR molecules with these antigens. Taken together, these observations indicate that Cop 1 and MBP, due to their polyvalent character, lead to increased fluorescence intensity of their complexes with HLA-DR, possibly due to recruitment and clustering of previously synthesized DR molecules. This can explain the efficient binding of these antigens to the MHC class II molecules.

Adjuvants, Immunologic↗
PubMed1997

The effect of linomide, an immunoregulator in experimental autoimmune diseases, on humoral antibody responses in mice.

Linomide (quinoline-3-carboxamide), a well tolerated, orally administered compound was recently shown to be effective in the prevention and treatment of several autoimmune diseases in experimental animal models. We have investigated its effect on specific humoral immune responses directed to T-cell-dependent soluble or particulate antigens and to a T cell-independent antigen in several mouse strains. Linomide administered after antigen priming did not affect primary and secondary antibody responses directed to T-cell particulate antigens (SRBC) or soluble antigens given with or without complete Freund's Adjuvant (CFA). Linomide treatment given prior to antigen priming did not affect the antibody response to a soluble antigen (TNP-KLH) given with an adjuvant. In contrast, dose-dependent down regulation of primary antibody responses was observed when T cell-dependent (BSA-dextran) or T-cell-independent (TNP-Ficoll) antigens were administered in an immunogenic form without adjuvant after starting Linomide treatment. The primary anti-SRBC antibody response was also suppressed by high dose Linomide given prior to immunization although normal secondary responses were retained. It is worth noting that no immunosuppressive effects on antibody responses were found at low dose ranges which effectively reversed T cell dependent autoimmune manifestation.

Animals↗
PubMed1997

Role of C3a and C5a in the activation of mast cells.

Mast cells and basophils are known to be triggered by allergens via cross-linking with their high-affinity IgE-binding receptors, Fc epsilon RI. The anaphylatoxic activity of the complement-derived peptides C3a and C5a has been known for a long time; however, it has also been reported that serosal- and mucosal-type mast cells respond differently to peptidergic stimuli. The mechanism of mast cell activation by cross-linking of Fc epsilon RI has been the subject of intensive studies in the past few years, while the action mode of the anaphylatoxic complement peptides has been revealed only recently. We report about a novel function of C3a: its inhibitory activity on IgE-mediated triggering of the mucosal RBL-2H3 cells. Surprisingly, the other anaphylatoxic peptide C5a, which has been shown to be significantly more effective in several biological assays, did not influence antigen-induced triggering of the RBL-2H3 cell line at all.

Animals↗
PubMed1996

Peptide interaction with a class I major histocompatibility complex-encoded molecule: allosteric control of the ternary complex stability.

Thermodynamics and kinetics of interaction between a soluble class I MHC heterodimer composed of the H-2Kd heavy chain (H) and human beta 2microglobulin (beta 2m) with a dansylated peptide series based on residues 147-155 of influenza virus nucleoprotein sequence were studied by means of real-time fluorescence measurements. Peptide-heterodimer binding is a second-order process with specific rates practically independent of peptide structure (3-5 x 10(6) M-1 s-1). The ternary complex assembly involves a rate-limiting step of beta 2m association with H to yield an unstable heterodimer (tau < or = 5 s, 37 degrees C). Peptide binding provides a positive feedback enhancing H's affinity for beta 2m, thus stabilizing the ternary complex. The latter decays by either peptide or beta 2m dissociation. The first-order rate constants of peptide dissociation (0.5 x 10(-2))-(0.4 x 10(-3)) s-1, 37 degrees C) depend on their structures and are faster than that of beta 2m dissociation. The former process decreases the H affinity for beta 2m and induces their dissociation. This dissociation, in turn, drastically lowers H affinity for peptide. Thus, these three components produce a system which is stable as a trimer. This behavior is rationalized by the functional requirements of class I molecules: Peptide structure determines the ternary complex's lifetime, and peptide rebinding on the cell surface is rendered unlikely by the limited stability of the empty heterodimers and the very low peptide affinity of the heavy chains.

Allosteric Regulation↗
PubMed1996

Immobilization of the type I receptor for IgE initiates signal transduction in mast cells.

Clustering of the type I receptor for IgE (Fc(epsilon) RI) on mast cells initiates a cascade of biochemical processes that results in the secretion of inflammatory mediators. We have studied this clustering process in order to obtain information about receptor density and mobility required for initiating that cascade. Specifically, we examined the role of new cluster formation in sustaining the secretory response and the minimal cluster density required for initiating secretion. The experimental protocol adopted for these studies employed photoactivatable antigens and antigen-carrying solid surfaces which enabled us to control the density and mobility of the Fc epsilon RI within the cluster. Our results show that recruitment of new Fc(epsilon) RI into clusters, either by antigen exchange among Fc(epsilon) RI-bound IgE molecules or by IgE-bound Fc(epsilon) RI exchange with vacant receptors, is not required for sustaining the cellular secretory response. Furthermore, we find that the cell's secretory response is very sensitive to the density of immobilized Fc(epsilon) RIs, increasing steeply above a density of ca. 1000 immobilized molecules/microns 2. Taken together, these finding suggest that immobilization of a fraction of the randomly distributed Fc(epsilon) RIs that are in sufficient proximity on the surface of mucosal-type mast cells of the RBL-2H3 line initiates a degranulation signal, and that this is maintained as long as these receptors are kept within this distance. The above conclusions and the experimental protocol presented in this study are expected to have wider applications for the study and understanding of signaling by immuno (as well as other) receptors.

Animals↗
PubMed1996

Proximity relationships between the type I receptor for Fc epsilon (Fc epsilon RI) and the mast cell function-associated antigen (MAFA) studied by donor photobleaching fluorescence resonance energy transfer microscopy.

Clustering of the mast cell function-associated antigen (MAFA) on the surface of rat mucosal type mast cells line 2H3 (RBL-2H3) leads to suppression of the secretory response induced by the type I Fc epsilon receptor (Fc epsilon RI). In order to establish a possible association between MAFA and Fc epsilon RI we measured fluorescence resonance energy transfer (FRET) between the MAFA-specific monoclonal antibody (mAb) G63 and Fc epsilon RI-bound ligands as well as between Fc epsilon RI-bound ligands themselves using the donor photobleaching FRET (pbFRET) technique. Average FRET efficiencies between 6 and 9% were determined after low-temperature incubation with fluorescent dye conjugated mAb G63 bound to MAFA (donor) and IgE bound to Fc epsilon RI (acceptor) on RBL-2H3 cells. Subsequent cross-linking of IgE by a polyvalent antigen caused no change in FRET efficiencies. These results suggest that the MAFA is located in the vicinity of the Fc epsilon RI on resting cells, and that clustering of the Fc epsilon RI leads to no significant change in the proximity of the two molecular species. In view of the sequence motif identified in the cytosolic tail of the MAFA and the observed changes in its phosphorylation upon antigen stimulation (Guthmann et al., Proc. Natl. Acad. Sci. USA 1995, 92: 9397-9401), the present study suggests that the secretory response inhibition by MAFA interferes with the signal transduction cascade initiated via the Fc epsilon RI. An additional finding was that clustering of the Fc epsilon RI by antigen showed a clear increase in the efficiency of FRET between Fc epsilon RI-bound IgE molecules conjugated with fluorescent donor and acceptor.

Animals↗
PubMed1996

Clustering the mast cell function-associated antigen (MAFA) induces tyrosyl phosphorylation of the Fc epsilonRI-beta subunit.

The mast cell function associated antigen (MAFA) is a membranal glycoprotein identified on the surface membranes of rat mucosal-type mast cells of the RBL-2H3 line by a monoclonal antibody (G63) binding to it. MAFA clustering by mAb G63 causes a dose-dependent inhibition of these mast cells' response to immunological stimulus provided by the type 1 Fc epsilon receptor (Fc epsilonRI) suppressing the biochemical processes coupling it to mediator secretion. The inhibition was found to take place upstream to the production of inositol phosphates and the transient increase in free cytosolic Ca2+ ion concentration, hence it probably interferes with the cascade at the level of the protein tyrosyl kinases (PTK) activity. We have therefore examined whether MAFA clustering affects protein tyrosyl phosphorylation of cell components and found that a time-dependent increase is caused in this modification of the Fc epsilonRI-beta chain. This constitutes the first evidence for the capacity of the clustered MAFA to enhance, on its own, biochemical changes in the mast cells, changes that are most probably related to its inhibitory signaling capacity. Moreover, that the observed phosphorylation changes are in the Fc epsilonRI-beta chain clearly indicates possible cross-talk between these two membrane components.

Animals↗
PubMed1996

Complement peptides and mast cell triggering.

Mucosal type mast cells have been earlier shown to be unresponsive to the so called 'peptidergic' stimulus provided by cationic agents, such as anaphylatoxins, neuropeptides or polyamines. We studied the relationship between mast cells' secretory response to stimulation via their type I Fc epsilon receptors (Fc epsilonRI) and that provided by C5a and C3a fragments of the complement system, in the rat mucosal-type mast cell line RBL-2H3. Our results shown here reveal a novel function of C3a, its inhibitory capacity on IgE-mediated triggering of mucosal mast cells. This activity of C3a is most probably mediated by its interaction with the beta-chain of Fc epsilonRI. While connective tissue type mast cells are known to be activated by micromolar concentrations of the complement peptides C3a and C5a, the amount of C3a necessary for the inhibition of antigen-induced degranulation of mucosal cells in our assays is in the nanomolar range. Interestingly, the other anaphylatoxic peptide C5a, which is known to be much more effective in several biological assays, did not show any activity in the same test-system.

Animals↗
PubMed1995

A secretion inhibitory signal transduction molecule on mast cells is another C-type lectin.

Secretion of inflammatory mediators by rat mast cells (line RBL-2H3) was earlier shown to be inhibited upon clustering a membrane glycoprotein by monoclonal antibody G63. This glycoprotein, named mast cell function-associated antigen (MAFA), was also shown to interfere with the coupling cascade of the type 1 Fc epsilon receptor upstream to phospholipase C gamma 1 activation by protein-tyrosine kinases. Here we report that the MAFA is expressed as both a monomer and a homodimer. Expression cloning of its cDNA shows that it contains a single open reading frame, encoding a 188-amino acid-long type II integral membrane protein. The 114 C-terminal amino acids display sequence homology with the carbohydrate-binding domain of calcium-dependent animal lectins, many of which have immunological functions. The cytoplasmic tail of MAFA contains a YXXL (YSTL) motif, which is conserved among related C-type lectins and is an essential element in the immunoreceptor tyrosine-based activation motifs. Finally, changes in the MAFA tyrosyl- and seryl-phosphorylation levels are observed in response to monoclonal antibody G63 binding, antigenic stimulation, and a combination of both treatments.

Amino Acid Sequence↗