title: Enrichment of distinct microfilament-associated and GTP-binding-proteins in membrane/microvilli fractions from lymphoid cells.
abstract: Lymphocyte microvilli mediate initial adhesion to endothelium during lymphocyte transition from blood into tissue but their molecular organization is incompletely understood. We modified a shear-based procedure to prepare biochemical fractions enriched for membrane/microvilli (MMV) from both human peripheral blood T-lymphocytes (PBT) and a mouse pre-B lymphocyte line (300.19). Enrichment of proteins in MMV relative to post nuclear lysate was determined by LC/MS/MS analysis and label-free quantitation. Subsequent analysis emphasized the 291 proteins shared by PBT and 300.19 and estimated by MS peak area to be highest abundance. Validity of the label-free quantitation was confirmed by many internal consistencies and by comparison with Western blot analyses. The MMV fraction was enriched primarily for subsets of cytoskeletal proteins, transmembrane proteins and G-proteins, with similar patterns in both lymphoid cell types. The most enriched cytoskeletal proteins were microfilament-related proteins NHERF1, Ezrin/Radixin/Moesin (ERMs), ADF/cofilin and Myosin1G. Other microfilament proteins such as talin, gelsolin, myosin II and profilin were markedly reduced in MMV, as were intermediate filament- and microtubule-related proteins. Heterotrimeric G-proteins and some small G-proteins (especially Ras and Rap1) were enriched in the MMV preparation. Two notable general observations also emerged. There was less overlap between the two cells in their transmembrane proteins than in other classes of proteins, consistent with a special role of plasma membrane proteins in differentiation. Second, unstimulated primary T-lymphocytes have an unusually high concentration of actin and other microfilament related proteins, consistent with the singular role of actin-mediated motility in the immunological surveillance performed by these primary cells.
authors.affiliation: Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Jian-Jiang
authors.initials: JJ
authors.lastname: Hao
authors.forename: Guanghui
authors.initials: G
authors.lastname: Wang
authors.forename: Trairak
authors.initials: T
authors.lastname: Pisitkun
authors.forename: Genaro
authors.initials: G
authors.lastname: Patino-Lopez
authors.forename: Kunio
authors.initials: K
authors.lastname: Nagashima
authors.forename: Mark A
authors.initials: MA
authors.lastname: Knepper
authors.forename: Rong-Fong
authors.initials: RF
authors.lastname: Shen
authors.forename: Stephen
authors.initials: S
authors.lastname: Shaw
mesh_terms: D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry*; D002853:Chromatography, Liquid; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification*; D006801:Humans; D051379:Mice; D008840:Microfilament Proteins / Q000737:chemistry / Q000302:isolation & purification*; D008871:Microvilli / Q000737:chemistry; D020559:Monomeric GTP-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification; D054448:Precursor Cells, B-Lymphoid / Q000737:chemistry* / Q000648:ultrastructure; D013045:Species Specificity; D013601:T-Lymphocytes / Q000737:chemistry* / Q000648:ultrastructure; D053719:Tandem Mass Spectrometry
chemical_list: D008840:Microfilament Proteins; D019204:GTP-Binding Proteins; D020559:Monomeric GTP-Binding Proteins