Siglec-9 is an inhibitory receptor on human mast cells in vitro.

BACKGROUND: Mast cell activation is critical for the development of allergic diseases. Ligation of sialic acid-binding immunoglobin-like lectins (Siglecs), such as Siglec-6, -7, and -8 as well as CD33, have been shown to inhibit mast cell activation. Recent studies showed that human mast cells express Siglec-9, an inhibitory receptor also expressed by neutrophils, monocytes, macrophages, and dendritic cells. OBJECTIVE: We aimed to characterize Siglec-9 expression and function in human mast cells in vitro. METHODS: We assessed the expression of Siglec-9 and Siglec-9 ligands on human mast cell lines and human primary mast cells by real-time quantitative PCR, flow cytometry, and confocal microscopy. We used a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing approach to disrupt the SIGLEC9 gene. We evaluated Siglec-9 inhibitory activity on mast cell function by using native Siglec-9 ligands, glycophorin A (GlycA), and high-molecular-weight hyaluronic acid, a monoclonal antibody against Siglec-9, and coengagement of Siglec-9 with the high-affinity receptor for IgE (FcεRI). RESULTS: Human mast cells express Siglec-9 and Siglec-9 ligands. SIGLEC9 gene disruption resulted in increased expression of activation markers at baseline and increased responsiveness to IgE-dependent and IgE-independent stimulation. Pretreatment with GlycA or high-molecular-weight hyaluronic acid followed by IgE-dependent or -independent stimulation had an inhibitory effect on mast cell degranulation. Coengagement of Siglec-9 with FcεRI in human mast cells resulted in reduced degranulation, arachidonic acid production, and chemokine release. CONCLUSIONS: Siglec-9 and its ligands play an important role in limiting human mast cell activation in vitro.

Mast cell surfaceome characterization reveals CD98 heavy chain is critical for optimal cell function.

BACKGROUND: Mast cells are involved in many distinct pathologic conditions, suggesting that they recognize and respond to various stimuli and thus require a rich repertoire of cell surface proteins. However, mast cell surface proteomes have not been comprehensively characterized. OBJECTIVE: We aimed to further characterize the mast cell surface proteome to obtain a better understanding of how mast cells function in health and disease. METHODS: We enriched for glycosylated surface proteins expressed in mouse bone marrow-derived cultured mast cells (BMCMCs) and identified them using mass spectrometry analysis. The presence of novel surface proteins in mast cells was validated by real-time quantitative PCR and flow cytometry analysis in BMCMCs and peritoneal mast cells (PMCs). We developed a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing approach to disrupt genes of interest in BMCMCs. RESULTS: The glycoprotein enrichment approach resulted in the identification of 1270 proteins in BMCMCs, 378 of which were localized to the plasma membrane. The most common protein classes among plasma membrane proteins were small GTPases, receptors, and transporters. One such cell surface protein was CD98 heavy chain (CD98hc), encoded by the Slc3a2 gene. Slc3a2 gene disruption resulted in a significant reduction in CD98hc expression, adhesion, and proliferation. CONCLUSIONS: Glycoprotein enrichment coupled with mass spectrometry can be used to identify novel surface molecules in mast cells. Moreover, CD98hc plays an important role in mast cell function.

Defective nucleotide-dependent assembly and membrane fusion in Mfn2 CMT2A variants improved by Bax.

Mitofusins are members of the dynamin-related protein family of large GTPases that harness the energy from nucleotide hydrolysis to remodel membranes. Mitofusins possess four structural domains, including a GTPase domain, two extended helical bundles (HB1 and HB2), and a transmembrane region. We have characterized four Charcot-Marie-Tooth type 2A-associated variants with amino acid substitutions in Mfn2 that are proximal to the hinge that connects HB1 and HB2. A functional defect was not apparent in cells as the mitochondrial morphology of Mfn2-null cells was restored by expression of any of these variants. However, a significant fusion deficiency was observed in vitro, which was improved by the addition of crude cytosol extract or soluble Bax. All four variants had reduced nucleotide-dependent assembly in cis, but not trans, and this was also improved by the addition of Bax. Together, our data demonstrate an important role for this region in Mfn2 GTP-dependent oligomerization and membrane fusion and is consistent with a model where cytosolic factors such as Bax are masking molecular defects associated with Mfn2 disease variants in cells.