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F Sinigaglia

Publications and source records attributed to F Sinigaglia.

At least 19 recordsLinked to original sources
PubMed1996

Expression of cyclic ADP-ribose-synthetizing CD38 molecule on human platelet membrane.

CD38 is a cell surface molecule widely used as a marker for immature and activated lymphocytes. It has been recently shown that CD38 displays three enzymatic activities: hydrolysis of NAD+ to ADP-ribose, synthesis of cyclic ADP-ribose from NAD+, and hydrolysis of cyclic ADP-ribose to ADP-ribose. Thus, CD38 plays a key role in the synthesis of cyclic ADP-ribose, a calcium-mobilizing compound. We investigate here the expression and cellular localization of CD38 in human platelets using a specific monoclonal antibody. Results showed that CD38 is expressed by human platelet membranes. Moreover, we show that platelet CD38 possesses NAD glycohydrolase, ADP-ribose cyclase, and cyclic ADP-ribose hydrolase activities. This finding indicates that the calcium-mobilizing agent cyclic ADP-ribose can be synthetized by human platelets and raises the question about the possible role of CD38 expression and enzymatic activities in the signal transduction pathways leading to platelet activation.

ADP-ribosyl Cyclase↗
PubMed1996

The insulin-dependent diabetes mellitus-associated ICA 105 autoantigen in stiff-man syndrome patients.

The pathogenetic role of anti-glutamic acid decarboxylase (GAD) antibodies found in up to 60% of patients with stiff-man syndrome (SMS) is still controversial. GAD, in fact, is also one of the major target antigen of insulin-dependent diabetes mellitus (IDDM), a disease affecting one third of anti-GAD antibody-positive patients with SMS. To better define the role of autoimmunity in SMS we looked for molecular and immunological evidence of an autoimmune recognition of a second IDDM-associated autoantigen, the pancreatic 37/40 kDa IDDM-autoantigen, whose gene called ICA 105 has been recently cloned. By Northern blot analysis we found that tissue distribution of human ICA 105 is restricted to pancreas and brain and within the central nervous system (CNS) its distribution is similar to GAD. We also measured anti-ICA 105 antibodies in 11 SMS patients and 56 control patients with other neurological diseases (OND). Anti-ICA 105 antibodies were found in 4/11 (36%) patients with SMS (a frequency similar to that of anti-GAD-antibodies in our SMS population) but in only 2/56 (3%) patients with OND (P < 0.001). Anti-ICA 105 and anti-GAD antibodies were associated in 3/4 (75%) patients with SMS but in none of the 2 anti-ICA 105 antibody-positive OND patients. Among anti-ICA 105 antibody-positive patients with SMS, only 1 suffered also from IDDM. In contrast, the only 2 anti-ICA 105 antibody-positive with OND had IDDM. Our results indicate that ICA 105 represents another putative neuroendocrine autoantigen in SMS. The presence of circulating anti-GAD and/or anti-ICA 105 antibodies might help the diagnosis of SMS. The absence, however, of antibodies recognising specific CNS autoantigens (e.g. GAD, ICA 105) does not rule out SMS.

Adult↗
PubMed1996

Analysis of peptide-binding motifs for two disease associated HLA-DR13 alleles using an M13 phage display library.

Major histocompatibility complex (MHC) molecules bind peptides bearing an appropriate 'sequence motif' for MHC binding. The use of phage display libraries exploits the ability of MHC class II molecules to exchange peptides in solution and thus select out peptide sequences with high-affinity binding from a large array of random peptides. We have analysed the peptide binding motifs of HLA-DRB1*1301 and *1302 using affinity purified HLA-DR13 molecules to purify sequentially HLA-DR13-binding peptides from a large random library of M13 phage containing nonamer inserts in the pIII coat protein. These DR13 alleles differ only at position 86 of the HLA-DR beta chain, where they contain valine and glycine residues respectively. These alleles were chosen because of their association with protection from severe malaria and chronic hepatitis B virus infection in West Africa. Analysis of the phage bound to these DR molecules suggests binding motifs. We compare the results derived from the use of the phage display library with results obtained from analysis of eluted peptides and peptide-binding studies. This analysis shows that although there is a common theme to motifs derived using different methods, there are also subtle variations between them.

Alleles↗
PubMed1996

Activation of human platelets by lysophosphatidic acid.

The biochemical mechanisms of platelet activation by lysophosphatidic acid were investigated. Lysophosphatidic acid interacts with a membrane receptor coupled to the inhibitory GTP-binding protein Gi and produces a rapid decrease of the intracellular concentration of cAMP. Aggregation of gel-filtered platelets by lysophosphatidic acid requires the presence of extracellular CaCl2, as this phospholipid does not induce secretion of platelet dense granules. Platelet activation by lysophosphatidic acid in the absence of extracellular CaCl2 does not involve phospholipase C activation, as evaluated by measuring mobilization of Ca2+ from internal stores and pleckstrin phosphorylation, but causes the rapid tyrosine phosphorylation of several intracellular proteins. Our results indicate that activation of intracellular tyrosine kinases is not secondary to Ca2+ mobilization and protein kinase C activation in lysophosphatidic acid-stimulated platelets.

Affinity Labels↗
PubMed1996

Agonist-induced actin polymerization is required for the irreversibility of platelet aggregation.

Cytochalasin D was used to investigate the role of intracellular cytoskeleton in the stabilization of platelet aggregation induced by strong platelet agonists. Incubation of gel-filtered platelets with increasing concentrations of cytochalasin D resulted in a dose-dependent inhibition of actin polymerization and association of actin-binding proteins with the Triton X-100-insoluble material induced by the thromboxane analogue, U46619, and the thrombin receptor activating peptide, TRAP. The same concentrations of cytochalasin D did not significantly inhibit platelet aggregation promoted by the two agonists. The addition of the chelating agent EDTA to fully aggregated platelets, that had been treated with cytochalasin D, resulted in the rapid and almost complete disaggregation. EDTA did not cause disaggregation of control, solvent-treated, aggregated platelets. The degree of platelet disaggregation induced by EDTA was dependent on the dose of cytochalasin D used, and was correlated with the inhibition of the cytoskeletal reorganization. Aggregation of cytochalasin D-treated platelets stimulated with U46619 or TRAP was also reverted by the addition of the tetrapeptide RGDS or the fibrinogen gamma-chain dodecapeptide, which competitively interfere with fibrinogen binding to the glycoprotein IIb-IIIa complex. These results indicate that the intracellular cytoskeleton plays an essential role in the stabilization of the fibrinogen-platelet interaction, and is necessary for the irreversibility of platelet aggregation induced by strong agonists.

Actins↗
PubMed1995

The 37/40-kilodalton autoantigen in insulin-dependent diabetes mellitus is the putative tyrosine phosphatase IA-2.

Major targets for autoantibodies associated with the development of insulin-dependent diabetes mellitus (IDDM) include tryptic fragments with a molecular mass of 37 kDa and/or 40 kDa of a pancreatic islet cell antigen of unknown identity. The assay identifying autoantibodies against the 37/40-kDa antigen in human sera is based on the immunoprecipitation of 35S-labeled rat insulinoma cell proteins with sera from IDDM patients, followed by limited trypsin digestion of the immunoprecipitated material. To identify cDNA clones coding for the 37/40-kDa antigen, we have screened a cDNA expression library from rat insulinoma cells with a serum from an IDDM patient that precipitated the 37/40-kDa antigen in our assay. Among the cDNA products that reacted with the IDDM serum, we identified one cDNA clone whose open reading frame encodes a protein with a predicted mass of 105 kDa that we termed "ICA105" for 105-kDa islet cell antibody. The deduced amino acid sequence has high homology to a recently cloned putative tyrosine phosphatase IA-2 from human and mouse cDNA libraries. Translation of the cDNA in vitro results in a polypeptide with the expected molecular mass of 105 kDa. The evidence that ICA105 is indeed the precursor of the 37/40-kDa tryptic fragments is based on the following three results: (i) Sera from IDDM patients containing autoantibodies to the 37/40-kDa antigen precipitate the in vitro translated polypeptide, whereas sera from healthy subjects as well as sera from IDDM patients not reactive with the 37/40-kDa antigen do not precipitate the cDNA product. (ii) Immunoprecipitation of the in vitro translated protein with sera containing autoantibodies to the 37/40-kDa antigen followed by limited trypsin digestion of the precipitated proteins results in a 40-kDa polypeptide. (iii) The protein derived from our cDNA but not from an unrelated control cDNA clone can block immunoprecipitation of the 37/40-kDa antigen from a labeled rat insulinoma cell extract. The availability of the cloned 37/40-kDa antigen should facilitate the identification of individuals at risk of IDDM with increased accuracy. Furthermore, the identification of the 37/40-kDa antigen as the putative tyrosine phosphatase IA-2 is of relevance in elucidating the role of this antigen in the development of IDDM.

Adolescent↗
PubMed1995

Evidence for a glycoprotein IIb-IIIa- and aggregation-independent mechanism of phosphatidylinositol 3',4'-bisphosphate synthesis in human platelets.

The synthesis of phosphatidylinositol 3',4'-bisphosphate (PtdIns(3,4)P2) in 32P-labeled human platelets induced by the tetrameric lectin concanavalin A and the physiological agonist thrombin were compared. Like thrombin, concanavalin A stimulated a time-dependent accumulation of PtdIns(3,4)P2, which reached maximal levels after 5 min of stimulation. However, while synthesis of PtdIns(3,4)P2 induced by thrombin was dependent on platelet aggregation, the production of PtdIns(3,4)P2 induced by concanavalin A was unchanged when aggregation was prevented by the omission of stirring or when fibrinogen binding to platelets was inhibited by the tetrapeptide RGDS. Accumulation of PtdIns(3,4)P2 was not observed in platelets stimulated with succinyl-concanavalin A, a dimeric derivative of the lectin that binds to the same receptors on the platelet surface but does not promote clustering of membrane glycoproteins. The synthesis of PtdIns(3,4)P2 induced by concanavalin A was also independent of the membrane glycoprotein IIb-IIIa, as normal accumulation of this lipid was observed in platelets from two patients affected by Glanzmann thrombasthenia. In contrast, thrombin showed a strongly reduced ability to stimulate PtdIns(3,4)P2 production in thrombasthenic platelets. Although concanavalin A was able to induce association of the regulatory subunit of the phosphatidylinositol 3-kinase with tyrosine-phosphorylated proteins, the tyrosine kinase inhibitor tyrphostin AG-213 did not inhibit the lectin-induced synthesis of PtdIns(3,4)P2. These results demonstrate the existence of a novel mechanism of PtdIns(3,4)P2 synthesis in human platelets, which is independent of glycoprotein IIb-IIIa and aggregation, but requires clustering of membrane glycoproteins. As clustering events occur during platelet aggregation promoted by physiological agonists, this new mechanism may also be involved in the aggregation-dependent production of PtdIns(3,4)P2 in thrombin-stimulated platelets.

Blood Platelets↗
PubMed1995

Peptide binding specificity of HLA-DR4 molecules: correlation with rheumatoid arthritis association.

We have investigated whether sequence 67 to 74 shared by beta chains of rheumatoid arthritis (RA)-associated HLA-DR molecules imparts a specific pattern of peptide binding. The peptide binding specificity of the RA-associated molecules, DRB1*0401, DRB1*0404, and the closely related, RA nonassociated DRB1*0402 was, therefore, determined using designer peptide libraries. The effect of single key residues was tested with site-directed mutants of DRB1*0401. The results have demonstrated striking differences between RA-linked and unlinked DR allotypes in selecting the portion of peptides that interacts with the 67-74 area. Most differences were associated with a single amino acid exchange at position 71 of the DR beta chain, and affected the charge of residues potentially contacting position 71. The observed binding patterns permitted an accurate prediction of natural protein derived peptide sequences that bind selectively to RA-associated DR molecules. Thus, the 67-74 region, in particular position 71, induces changes of binding specificity that correlate with the genetic linkage of RA susceptibility. These findings should facilitate the identification of autoantigenic peptides involved in the pathogenesis of RA.

Amino Acid Sequence↗
PubMed1995

Cytotoxic T cells specific for glutamic acid decarboxylase in autoimmune diabetes.

Insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease that results in the destruction of the pancreatic islet beta cells. Glutamic acid decarboxylase (GAD) has been recently indicated as a key autoantigen in the induction of IDDM in nonobese diabetic mice. In human diabetes, the mechanism by which the beta cells are destroyed is still unknown. Here we report the first evidence for the presence of GAD-specific cytotoxic T cells in asymptomatic and recent diabetic patients. GAD65 peptides displaying the human histocompatibility leukocyte antigen (HLA)-A*0201 binding motif have been synthesized. One of these peptides, GAD114-123, binds to HLA-A*0201 molecules in an HLA assembly assay. Peripheral blood mononuclear cells from individuals with preclinical IDDM, recent-onset IDDM, and from healthy controls were stimulated in vitro with the selected peptide in the presence of autologous antigen-presenting cells. In three cases (one preclinical IDDM and two recent-onset IDDM), we detected specific killing of autologous antigen-presenting cells when incubated with GAD114-123 peptide or when infected with a recombinant vaccinia virus expressing GAD65. These patients were the only three carrying the HLA-A*0201 allele among the subjects studied. Our finding suggests that GAD-specific cytotoxic T lymphocytes may play a critical role in the initial events of IDDM.

Adolescent↗
PubMed1995

Pocket 4 of the HLA-DR(alpha,beta 1*0401) molecule is a major determinant of T cells recognition of peptide.

To investigate the functional roles of individual HLA-DR residues in T cell recognition, transfectants expressing wild-type or mutant DR(alpha,beta 1*0401) molecules with single amino acid substitutions at 14 polymorphic positions of the DR beta 1*0401 chain or 19 positions of the DR alpha chain were used as antigen-presenting cells for five T cell clones specific for the influenza hemagglutinin peptide, HA307-19. Of the six polymorphic positions in the DR beta floor that were examined, mutations at only two positions eliminated T cell recognition: positions 13 (four clones) and 28 (one clone). In contrast, individual mutations at DR beta positions 70, 71, 78, and 86 on the alpha helix eliminated recognition by each of the clones, and mutations at positions 74 and 67 eliminated recognition by four and two clones, respectively. Most of the DR alpha mutations had minimal or no effect on most of the clones, although one clone was very sensitive to changes in the DR alpha chain, with loss of recognition in response to 10 mutants. Mutants that abrogated recognition by all of the clones were assessed for peptide binding, and only the beta 86 mutation drastically decreased peptide binding. Single amino acid substitutions at polymorphic positions in the central part of the DR beta alpha helix disrupted T cell recognition much more frequently than substitutions in the floor, suggesting that DR beta residues on the alpha helix make relatively greater contributions than those in the floor to the ability of the DR(alpha,beta 1*0401) molecule to present HA307-19. The data indicate that DR beta residues 13, 70, 71, 74, and 78, which are located in pocket 4 of the peptide binding site in the crystal structure of the DR1 molecule, exert a major and disproportionate influence on the outcome of T cell recognition, compared with other polymorphic residues.

Amino Acid Sequence↗
PubMed1995

Dual mechanism of protein-tyrosine phosphorylation in concanavalin A-stimulated platelets.

Treatment of human platelets with the lectin Concanavalin A (Con A) resulted in the tyrosine phosphorylation of several proteins with molecular masses 65, 80, 85, 95, 120, 135, and 150 kDa. These proteins were divided in two groups: the first group included the 65-, 85-, 95-, and 120-kDa bands, which were tyrosine phosphorylated also in thrombin-stimulated platelets; the second group (80-, 135-, and 150-kDa bands) included proteins whose tyrosine phosphorylation was exclusively promoted by Con A, but not by thrombin. Members of the second group were rapidly dephosphorylated when the lectin was displaced from the cell surface by methyl alpha-D-mannopyranoside. Pretreatment of intact platelets with the prostacyclin analog iloprost, inhibited Con A-induced tyrosine phosphorylation of the first group of proteins, but had no effect on the tyrosine phosphorylation of the proteins of the second group. Succinyl-Con A, a dimeric derivative of the lectin, which binds to the platelet surface but does not promote clustering of the receptor, did not induce tyrosine phosphorylation of the second group of proteins, although phosphorylation of some members of the first group was observed. Our results demonstrate the presence of two different mechanisms leading to protein-tyrosine phosphorylation in Con A-stimulated platelets, and identify a new signal transduction pathway, promoted by the clustering of membrane glycoproteins, that produces tyrosine phosphorylation of specific substrates. This new pathway may be activated by platelet interaction with multivalent ligands, such as adhesive proteins, during adhesion, spreading, and aggregation.

Blood Platelets↗
PubMed1994

Precise prediction of major histocompatibility complex class II-peptide interaction based on peptide side chain scanning.

We describe here a new method for predicting class II major histocompatibility complex-binding peptides, based on the preferences observed in a systematic series of peptide binding experiments where each position in a "minimal" peptide was replaced individually by every amino acid. The DRB1*0401 peptide binding preferences were determined and incorporated into a computer program that looks through sequences for potential epitopes and assigns each a score. These scores correlate well with previously determined T cell epitopes of foreign antigens and endogenous peptides from self proteins. Our findings hold implications for the design of subunit vaccines and in the identification of autoantigenic peptide regions within self proteins.

Base Sequence↗
PubMed1994

Glycoprotein IIb-IIIa and the translocation of Rap2B to the platelet cytoskeleton.

The stimulation of human platelets with physiological agonists results in the incorporation of several proteins into the cytoskeleton, fibrinogen binding, and platelet aggregation. We recently demonstrated that the Ras-related low molecular weight GTP-binding protein Rap2B associates with the cytoskeleton in activated platelets and that this interaction requires platelet aggregation. In the present study we demonstrate that agonist-induced actin polymerization is necessary for the translocation of Rap2B to the cytoskeleton, suggesting that Rap2B interacts with the newly formed actin filaments. Moreover, the association of Rap2B with Triton X-100-insoluble material from platelets was totally blocked by treatment of intact platelets with monoclonal antibodies against the fibrinogen receptor glycoprotein IIb-IIIa. Platelets from patients affected by Glanzmann thrombastenia, a genetic disorder in which platelet plasma membranes lack glycoprotein IIb-IIIa but possess normal levels of Ras-related proteins, failed to incorporate Rap2B into the cytoskeleton upon activation by thrombin. Comparative immunoblotting revealed that the translocation of Rap2B to the cytoskeleton during platelet aggregation was accompanied by the simultaneous translocation of glycoprotein IIb-IIIa. Moreover, the cytoskeleton from aggregated platelets contained Rap2B and glycoprotein IIb-IIIa in comparable amounts. These results demonstrate the association of Rap2B and glycoprotein IIb-IIIa and their translocation to the cytoskeleton in aggregated human platelets.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1994

High-affinity binding of short peptides to major histocompatibility complex class II molecules by anchor combinations.

We have previously identified four anchor positions in HLA-DRB1*0101-binding peptides, and three anchors involved in peptide binding to DRB1*0401 and DRB1*1101 molecules, by screening of an M13 peptide display library (approximately 20 million independent nonapeptides) for DR-binding activity. In this study, high stringency screening of the M13 library for DRB1*0401 binding has resulted in identification of three further anchor positions. Taken together, a peptide-binding motif has been obtained, in which six of seven positions show enrichment of certain residues. We have demonstrated an additive effect of anchors in two different ways: (i) the addition of more anchors is shown to compensate for progressive truncation of designer peptides; (ii) the incorporation of an increasing number of anchors into 6- or 7-residue-long designer peptides is shown to result in a gradual increase of binding affinity to the level of 13-residue-long high-affinity epitopes. The anchor at relative position 1 seems to be obligatory, in that its substitution abrogates binding completely, whereas the elimination of other anchors results only in partial loss of binding affinity. The spacing between anchors is critical, since their effect is lost by shifting them one position toward the N or C terminus. The information born out of this study has been successfully used to identify DR-binding sequences from natural proteins.

Amino Acid Sequence↗
PubMed1994

Effect of cAMP on the association of small GTP-binding proteins with the cytoskeleton of human platelets.

Following activation of human platelets changes in cytoskeletal organization occur: some proteins, which are present in the cytosol or membrane-associated in resting platelets, are recovered in the Triton-insoluble residue in activated cells. Assembly and disassembly of complex effector units on the membrane and inside cells is under the control of low molecular weight GTP-binding proteins, particularly those in the ras family. We investigated the interaction of small GTP-binding proteins with the platelet cytoskeleton and the effect of high cAMP levels on these interactions. At least two GTP-binding proteins of 24 and 28 kDa were detected in the Triton-insoluble residue of resting platelets. Stimulation of platelets with thrombin or concanavalin A (Con A), under non-aggregating conditions, resulted in increased 24 kDa protein-bound GTP, which also contained a significant amount of rap1B. High cAMP levels differently affected this interaction depending on the type of agonist used. cAMP increased association of G-proteins with the cytoskeleton following Con A-activation, while it decreased G-proteins interaction after thrombin stimulation. The activation did not influence the cAMP-dependent phosphorylation of rap1B. No phosphoprotein corresponding to rap1B could be detected in the Triton-insoluble residues, however. These findings could be related to the different mechanisms of cytoskeletal protein recruitment in platelets activated with either thrombin or Con A.

Blood Platelets↗