Proteomic Analysis of Lipid Rafts from RBL-2H3 Mast Cells

Abstract

Lipid rafts are highly ordered membrane microdomains enriched in cholesterol, glycosphingolipids, and certain proteins. They are involved in the regulation of cellular processes in diverse cell types, including mast cells (MCs). The MC lipid raft protein composition was assessed using qualitative mass spectrometric characterization of the proteome from detergent-resistant membrane fractions from RBL-2H3 MCs. Using two different post-isolation treatment methods, a total of 949 lipid raft associated proteins were identified. The majority of these MC lipid raft proteins had already been described in the RaftProtV2 database and are among highest cited/experimentally validated lipid raft proteins. Additionally, more than half of the identified proteins had lipid modifications and/or transmembrane domains. Classification of identified proteins into functional categories showed that the proteins were associated with cellular membrane compartments, and with some biological and molecular functions, such as regulation, localization, binding, catalytic activity, and response to stimulus. Furthermore, functional enrichment analysis demonstrated an intimate involvement of identified proteins with various aspects of MC biological processes, especially those related to regulated secretion, organization/stabilization of macromolecules complexes, and signal transduction. This study represents the first comprehensive proteomic profile of MC lipid rafts and provides additional information to elucidate immunoregulatory functions coordinated by raft proteins in MCs.

Lipid rafts, Rab GTPases, and a late endosomal checkpoint for plasma membrane recycling

“…propose palmitoylation of Rab3 (and, to a smaller extent, of Rab7) as a mechanism for its reversible association with rafts and tested this idea with a point mutation of a putative palmitoylation site. The association of these Rab proteins with ordered membranes is consistent with previous proteomic analyses of detergent-resistant membrane fractions that identified several Rab GTPases, including Rab3D and Rab7 ( 22 ). On the other hand, Rab GTPases have been shown to preferentially insert in disordered or curved membranes (e.g., highly curved transport vesicles) through their prenyl groups, including Rab1, Rab5, and Rab6 ( 23 ).…”

Human Mast Cell Proteome Reveals Unique Lineage, Putative Functions, and Structural Basis for Cell Ablation

“…To aid future studies into mast cell functions, comprehensive proteome data are a valuable resource. Proteome analyses of mast cells have been previously reported for proteins released from mouse mast cells (Shubin et al, 2017), for human short-term cultured skin mast cells (Gschwandtner et al, 2017), for phosphoproteins (Cao et al, 2007), and for lipid rafts (Freitas Filho et al, 2019) in cultured rodent mast cells. Here, we isolated primary human connective tissue mast cells from skin and fat from healthy individuals and analyzed the mast cell proteome by quantitative mass spectrometry.…”

Lipid Metabolism Reprogramming of Immune Cells in Acne: An Update

“… 54 Mast cells are rich in lipid rafts, highly ordered membrane microdomains enriched in cholesterol, glycosphingolipids, and certain proteins, lipid modification is tightly linked to the degranulation, endocytosis of mast cells. 55 Lipid reprogramming exerted a dependent role in mast cell activation. The phosphorylation of immunoreceptor tyrosine-based activation motif activated the mitogen promoter protein (MAK) kinase signaling pathway to decompose membrane phospholipid choline (PC) to produce arachidonic acid, then the arachidonic acid was the essential substance for the synthesis of arachidonic acids such as prostaglandin D2 (PGD2), leukotrienes, and platelet-activating factor, which induced the inflammatory response.…”