scholarly journals Biochemical analysis of ligand-induced receptor patching and capping using a novel immunolactoperoxidase iodination technique.

1979 ◽  
Vol 83 (3) ◽  
pp. 649-656 ◽  
Author(s):  
L Y Bourguignon
Keyword(s):  
Membrane Proteins ◽  
Biochemical Analysis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Histocompatibility Antigen ◽  
Surface Proteins ◽  
Cell Surface Proteins ◽  
Cytochemical Analysis ◽  
Novel Approach ◽  
Surface Receptor ◽  
Specific Ligand

A novel approach for the analysis of membrane proteins involved in ligand-induced surface receptor patching and capping is described. The technique is based on the use of immunolactoperoxidase (immuno-LPO) conjugates which catalyze the iodination of those surface proteins with available tyrosine groups that are located in the immediate vicinity of the patch or cap of a particular antigen. We have used the patching and capping of the H-2 (histocompatibility) antigen on mouse thymocytes to illustrate this method. However, this technique should be generally applicable to any cell surface proteins which can be induced to form patches or caps by a specific ligand. Cytochemical analysis indicates that the immuno-LPO conjugates induce the same patching and capping of the H-2 antigen as does the unconjugated antibody. Biochemical analysis of the 125I-labeled proteins by SDS polyacrylamide gel electrophoresis indicates that a large membrane protein (mol wt of approximately 200,000 daltons) is closely associated with H-2 patches and caps. Since a number of other prominent membrane proteins are not labeled by this procedure, selective redistribution of certain surface proteins must be occurring during H-2 antibody-induced patching and capping.

scholarly journals Alterations in the chondrocyte surfaceome in response to pro-inflammatory cytokines

2019 ◽  
Author(s):  
Bernadette Jeremiasse ◽  
Csaba Matta ◽  
Christopher R. Fellows ◽  
David J. Boocock ◽  
Julia R. Smith ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Cell Surface ◽  
Inflammatory Cytokines ◽  
Ionic Composition ◽  
Interleukin 1 ◽  
Surface Proteins ◽  
Low Grade ◽  
Necrosis Factor Alpha ◽  
Cell Surface Proteins ◽  
Pro Inflammatory Cytokines

Abstract Background: Chondrocytes are exposed to an inflammatory micro-environment in the extracellular matrix (ECM) of articular cartilage in joint diseases such as osteoarthritis (OA) and rheumatoid arthritis (RA). In OA, degenerative changes and low-grade inflammation within the joint transform the behaviour and metabolism of chondrocytes, disturb the balance between ECM synthesis and degradation, and alter the osmolality and ionic composition of the micro-environment. We hypothesize that chondrocytes adjust their physiology to the inflammatory microenvironment by modulating the expression of cell surface proteins, collectively referred to as the ‘surfaceome’. Therefore, the aim of this study was to characterize the surfaceome of primary equine chondrocytes isolated from healthy joints following exposure to the pro-inflammatory cytokines interleukin-1-beta (IL-1β) and tumour necrosis factor-alpha (TNF-α). Methods: We employed combined methodology that we recently developed for investigating the surfaceome in stem cells. Membrane proteins were isolated using an aminooxy-biotinylation technique and analysed by mass spectrometry using high throughput shotgun proteomics. Results: Amongst the 431 unique cell surface proteins identified, a high percentage of low-abundance proteins, such as ion channels, receptors and transporter molecules were detected. Data are available via ProteomeXchange with identifier PXD014773. A high number of proteins exhibited different levels of expression following chondrocyte stimulation with pro-inflammatory cytokines.LPR-1, thrombospondin, VDAC1-2 and annexin A1 were considered to be of special interest and were analysed further and validated by western blotting. Conclusions: Our results provide, for the first time, a repository for proteomic data on differentially expressed low-abundance membrane proteins on the surface of chondrocytes in response to pro-inflammatory stimuli.

scholarly journals ANTH domains within CALM, HIP1R, and Sla2 recognize ubiquitin internalization signals

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Natalya Pashkova ◽  
Lokesh Gakhar ◽  
Liping Yu ◽  
Nicholas J Schnicker ◽  
Annabel Y Minard ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Cell Surface ◽  
Terminal Region ◽  
Surface Proteins ◽  
Structural Studies ◽  
Cell Surface Proteins ◽  
Functional Studies ◽  
Binding Domains ◽  
Endocytic Machinery

Attachment of ubiquitin (Ub) to cell surface proteins serves as a signal for internalization via clathrin-mediated endocytosis (CME). How ubiquitinated membrane proteins engage the internalization apparatus remains unclear. The internalization apparatus contains proteins such as Epsin and Eps15, which bind Ub, potentially acting as adaptors for Ub-based internalization signals. Here we show that additional components of the endocytic machinery including CALM, HIP1R, and Sla2 bind Ub via their N-terminal ANTH domain, a domain belonging to the superfamily of ENTH and VHS domains. Structural studies revealed that Ub binds with µM affinity to a unique C-terminal region within the ANTH domain not found in ENTH domains. Functional studies showed that combined loss of Ub-binding by ANTH-domain proteins and other Ub-binding domains within the yeast internalization apparatus caused defects in the Ub-dependent internalization of the GPCR Ste2 that was engineered to rely exclusively on Ub as an internalization signal. In contrast, these mutations had no effect on the internalization of Ste2 engineered to use an alternate Ub-independent internalization signal. These studies define new components of the internalization machinery that work collectively with Epsin and Eps15 to specify recognition of Ub as an internalization signal.

scholarly journals Characterization of plasma menbrane polypeptides of trypanosoma from bats

1989 ◽  
Vol 84 (1) ◽  
pp. 13-18 ◽  
Author(s):  
R. T. Pinho ◽  
Giovanni de Simone
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Surface Proteins ◽  
Trypanosome Species ◽  
Cell Surface Proteins ◽  
Sds Page ◽  
Trypanosoma Dionisii ◽  
Triton X

Cell surface proteins of Trypanosoma dionisii, Trypanosoma vespertilionis and Trypanosoma sp. (M238) were radiodinated and their distribution both in the detergent-poor (DPP) and dertergent-enriched phase (DRP) was studied using a phase separation technique in Triton X-114 as well as polyacrylamide gel electrophoresis in sodium dodecyl sulphate (SDS-PAGE). Significant differences were observed in the proteins present in the DRP when the three species of trypanosoma were compared. Two major bands with 88 and 70 KDa were observed in T. sp. (M238) but were not detectable in T. dionisii and T. vespertilionis. Three polypeptides whith 96, 77 and 60 KDa were identified in the DRP of T. vespertilionis. Three major bands with 84, 72 and 60 KDa were observed in the DRP of T. dionisii. Two polypeptides with 34-36 KDa present in the DPP, were observed in the three Trypanosome species analyzed. Our observations show that T. sp. (M238) has characteristic surface polypeptides not found in T. vespertilionis.

scholarly journals The three cortical membranes of the gregarines (parasitic protozoa). Characterization of the membrane proteins of Gregarina blaberae

1982 ◽  
Vol 201 (3) ◽  
pp. 455-464 ◽  
Author(s):  
M. Philippe ◽  
J. Schrével
Keyword(s):  
Electron Microscopy ◽  
Membrane Proteins ◽  
Cell Surface ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Surface Proteins ◽  
Parasitic Protozoa ◽  
Whole Cells ◽  
Cortical Membranes

Gregarines, which are parasitic protozoa living in invertebrates, possess a cortical structure specific to their vegetative stage: namely two additional cytomembranes are lying just under the plasma membrane. This cortical complex has been isolated by centrifugation on discontinuous sucrose gradients and characterized chemically. Its integrity was tested by electron microscopy. Ghost proteins were resolved by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. About 30 polypeptides of mol.wt. 15000–300000 were present in this fraction and four glycoproteins were detected after periodate/Schiff staining. Ten major proteins were labelled after lactoperoxidase-catalysed iodination. The GP2 glycoprotein (41000–49000 apparent mol.wt.) appears to be a major component of the cell surface. Effects of trypsin and Pronase digestion on ghosts and cells were monitored by gel electrophoresis and by electron microscopy. Ghosts treated with low trypsin or Pronase concentrations (10–25μg/ml) became drastically disorganized; many proteins were vigorously attacked in comparison with those of control ghosts. Variations in proteinase-sensitivity of proteins are pointed out. The GP3 glycoprotein (130000–160000 apparent mol.wt.) seemed to be the only glycoprotein released from the cell surface by trypsin. Whole cells treated under the same conditions or with higher proteinase concentrations (up to 1mg/ml) do not exhibit morphological modifications of the cell surface; furthermore, no discernible cleavage of membrane proteins was indicated by electrophoretograms. It is postulated that cell-surface proteins are protected by the dense carbohydrate cell coat. By using various different methods (change of ionic strength, detergent, denaturing agent, labelling experiment) it was possible to localize several major proteins within the protozoon cortical membranes.

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