Non-functional alternative splicing caused by a Latino pathogenic variant in a case of PMM2-CDG.

We report on a Mexican mestizo with a multisystemic syndrome including neurological involvement and a type I serum transferrin isoelectric focusing (Tf IEF) pattern. Diagnosis of PMM2-CDG was obtained by clinical exome sequencing (CES) that revealed compound heterozygous variants in PMM2, the encoding gene for the phosphomannomutase 2 (PMM2). This enzyme catalyzes the conversion of mannose-6-P to mannose-1-P required for the synthesis of GDP-Man and Dol-P-Man, donor substrates for glycosylation reactions. The identified variants were c.422G>A (R141H) and c.178G>T, the former being the most frequent PMM2 pathogenic mutation and the latter a previously uncharacterized variant restricted to the Latino population with conflicting interpretations of pathogenicity and that we here report causes leaky non-functional alternative splicing (p.V60Cfs*3).

Analysis of expression and function of the co-stimulatory receptor SLAMF1 in immune cells from patients with systemic lupus erythematosus (SLE).

The signaling lymphocytic activation molecule SLAMF1 (CD150) is a co-stimulatory molecule that is expressed by most immune cells, including T regulatory (Treg) lymphocytes. Since different abnormalities have been reported regarding the number and function of Foxp3+ Treg cells in patients with systemic lupus erythematosus (SLE), we decided to analyze the expression and function of CD150 in these regulatory lymphocytes in this condition. We isolated peripheral blood mononuclear cells from 20 patients with SLE, and 20 healthy controls. The expression of SLAMF1 was determined by multi-parametric flow cytometry and the suppressive function of CD4+CD25+ lymphocytes, upon engagement or not of CD150 with an agonistic monoclonal antibody, was analyzed by an assay of inhibition of cell proliferation. We observed a significantly increased expression of SLAMF1 by CD3+CD4+ helper T cells and CD19+ B cells in patients with SLE and active disease. However, similar levels of SLAMF1 expression were detected in Foxp3+ Treg cells from patients and controls. In contrast, a higher proportion of SLE patients increased their suppressive function of Treg cells upon CD150 engagement compared to healthy controls. Our data suggest that SLAMF1 is another significant piece in the intricate defective immune-regulatory function of patients with SLE.

Regulation of Cbl molecular interactions by the co-receptor molecule CD43 in human T cells.

CD43, one of the most abundant glycoproteins on the T cell surface, has been implicated in selection and maturation of thymocytes and migration, adhesion, and activation of mature T cells. The adapter molecule Cbl has been shown to be a negative regulator of Ras. Furthermore, it may also regulate intracellular signaling through the formation of several multi-molecular complexes. Here we investigated the role of Cbl in the CD43-mediated signaling pathway in human T cells. Unlike T cell receptor signaling, the interaction of the adapter protein Cbl with Vav and phosphatidylinositol 3-kinase, resulting from CD43-specific signals, is independent of Cbl tyrosine phosphorylation, suggesting an alternative mechanism of interaction. CD43 signals induced a Cbl serine phosphorylation-dependent interaction with the tau-isoform of 14-3-3. protein. Protein kinase C-mediated Cbl serine phosphorylation was required for this interaction, because the PKC inhibitor RO-31-8220 prevented it, as well as 14-3-3 dimerization. Moreover, mutation of Cbl serine residues 619, 623, 639, and 642 abolished the interaction between Cbl and 14-3-3. Overexpression of Cbl in Jurkat cells inhibited the CD43-dependent activation of the mitogen-activated protein kinase (MAPK) pathway and AP-1 transcriptional activity, confirming nevertheless a negative role for Cbl in T cell signaling. However, under normal conditions, PKC activation resulting from CD43 engagement was required to activate the MAPK pathway, suggesting that phosphorylation of Cbl on serine residues by PKC and its association with 14-3-3 molecules may play a role in preventing the Cbl inhibitory effect on the Ras-MAPK pathway. These data suggest that by inducing its phosphorylation on serine residues, CD43-mediated signals may regulate the molecular associations and functions of the Cbl adapter protein.

Crosstalk: phosphorylation of alpha1b-adrenoceptors induced through activation of bradykinin B2 receptors.

The action of bradykinin was studied in rat-1 fibroblasts stably expressing alpha1b-adrenoceptors. It was observed that bradykinin and kallidin markedly increase cytosol calcium concentration, but that the B1 agonist, des-Arg9-bradykinin, only mimicked this effect to a minimal extent. Antagonists, selective for the B2 subtype, such as Hoe 140, blocked this effect of bradykinin and kallidin. Similarly, bradykinin and kallidin stimulated the production of inositol phosphates and B2 antagonists blocked their actions. The possibility that bradykinin could modulate alpha1b-adrenoceptors was studied. It was observed that bradykinin and kallidin increased alpha1b-adrenoceptor phosphorylation and that such effect was also blocked by Hoe 140. Interestingly, the ability of norepinephrine to increase intracellular calcium concentration was not altered by pretreatment of the cells with bradykinin, i.e. bradykinin induced alpha1b-adrenoceptor phosphorylation but this did not lead to receptor desensitization.

Characterization of the beta 2 adrenoceptors of dog liver.

The beta-adrenoceptors present in dog liver were characterized by radioligand binding techniques. [3H]Dihydroalprenolol bound with high affinity (KD 0.9 nM) to a relatively low number of sites (approximately 68 fmol/mg protein) in partially purified dog liver plasma membranes. These sites were characterized by binding competition; the orders of affinity were: (a) for agonists: (-)isoproterenol > or = salbutamol > epinephrine > or = terbutaline > norepinephrine > (+)isoproterenol and (b) for antagonists: propranolol > ICI 118551 >> metoprolol >> atenolol. These data indicate that dog liver expresses beta-adrenoceptors of the beta2 subtype.