Exofacial Domain Research Articles

In Situ Self‐Assembly of Quantum Dots at the Plasma Membrane Mediates Energy Transfer‐Based Activation of Channelrhodopsin (Part. Part. Syst. Charact. 7/2021)

In article 2100053, Delehanty and co-workers describe an energy transfer system for the indirect activation of channelrhodopsin ion channels. A light-harvesting quantum dot (QD) nanoparticle is self-assembled to the exofacial domain of the ion channel. Upon photoexcitation, the QD transfers energy to the retinal moiety in the ion channel, resulting in its photoisomerization and channel opening.

Reductive Reprogramming: A Not-So-Radical Hypothesis of Neurodegeneration Linking Redox Perturbations to Neuroinflammation and Excitotoxicity.

Free radical-mediated oxidative stress, neuroinflammation, and excitotoxicity have long been considered insults relevant to the progression of Alzheimer's disease and other aging-related neurodegenerative disorders (NDD). Among these phenomena, the significance of oxidative stress and, more generally, redox perturbations, for NDD remain ill-defined and unsubstantiated. Here, I argue that (i) free radical-mediated oxidations of biomolecules can be dissociated from the progression of NDD, (ii) oxidative stress fails as a descriptor of cellular redox states under conditions relevant to disease, and (iii) aberrant upregulation of compensatory reducing activities in neural cells, resulting in reductive shifts in thiol-based redox potentials, may be an overlooked and paradoxical contributor to disease progression. In particular, I summarize evidence which supports the view that reductive shifts in the extracellular space can occur in response to oxidant and inflammatory signals and that these have the potential to reduce putative regulatory disulfide bonds in exofacial domains of the N-methyl-D-aspartate receptor, leading potentially to aberrant increases in neuronal excitability and, if sustained, excitotoxicity. The novel reductive reprogramming hypothesis of neurodegeneration presented here provides an alternative view of redox perturbations in NDD and links these to both neuroinflammation and excitotoxicity.

GLUT4 Translocation in Single Muscle Cells in Culture: Epitope Detection by Immunofluorescence.

GLUT4 is the major glucose transporter in skeletal muscle. GLUT4 cycles to and from the plasma membrane and its exocytic rate is accelerated by insulin and muscle contraction to achieve a new steady state with more GLUT4 proteins at the muscle cell surface. To gain a better understanding of the molecular and cellular mechanisms that govern GLUT4 protein recycling, we developed an in vitro model in which myc-epitope-tagged GLUT4 or GLUT4-GFP is expressed in L6 skeletal muscle cells. The myc-epitope is inserted into an exofacial domain that is accessible to anti-myc antibodies from the outside of non-permeabilized cells, allowing one to count the number of transporters at the cell surface. This enables one to perform single-cell analysis using confocal fluorescence microscopy to quantify cell surface GLUT4myc or GLUT4myc-GFP in cells co-transfected with diverse cDNA constructs, treated with siRNAs, or co-stained with antibodies for other proteins of interest. Herein, we describe the methodology to perform these experimental approaches in insulin-stimulated L6 muscle cells.

The Small Tellurium Compound AS101 Ameliorates Rat Crescentic Glomerulonephritis: Association with Inhibition of Macrophage Caspase-1 Activity via Very Late Antigen-4 Inactivation.

Crescentic glomerulonephritis (CGN) is the most aggressive form of GN and, if untreated, patients can progress to end-stage renal failure within weeks of presentation. The α4β1 integrin very late antigen-4 (VLA-4) is an adhesion molecule of fundamental importance to the recruitment of leukocytes in inflammation. We addressed the role of VLA-4 in mediating progressive renal injury in a rat model of CGN using a small tellurium compound. AS101 [ammonium trichloro(dioxoethylene-o,o′)tellurate]. This compound has been previously shown to uniquely inhibit VLA-4 activity by redox inactivation of adjacent thiols in the exofacial domain of VLA-4. The study shows that administration of AS101 either before or after glomerular basement membrane anti-serum injection ameliorates crescent formation or preserves renal function. This was associated with profound inhibition of critical inflammatory mediators, accompanied by decreased glomerular infiltration of macrophages. Mechanistic studies demonstrated vla-4 inactivation on glomerular macrophages both in vitro and in vivo as well as inhibition of caspase-1 activity. Importantly, this cysteine protease activity modification was dependent on VLA-4 inactivation and was associated with the anti-inflammatory activity of AS101. We propose that inactivation of macrophage VLA-4 by AS101 in vivo results in a decrease of inflammatory cytokines and chemokines produced in the glomeruli of diseased rats, resulting in decreased further macrophage recruitment and decreased extracellular matrix expansion. Thus, AS101, which is currently in clinical trials for other indications, might be beneficial for treatment of CGN.

Redox Modulation of Adjacent Thiols in VLA-4 by AS101 Converts Myeloid Leukemia Cells from a Drug-Resistant to Drug-Sensitive State

Interaction between the integrin VLA-4 on acute myelogenous leukemia (AML) cells with stromal fibronectin is a decisive factor in chemotherapeutic resistance. In this study, we provide a rationale for a drug repositioning strategy to blunt integrin activation in AML cells and restore their sensitivity to chemotherapy. Specifically, we demonstrate that the nontoxic tellurium compound AS101, currently being evaluated in clinical trials, can abrogate the acquired resistance of AML. Mechanistic investigations revealed that AS101 caused redox inactivation of adjacent thiols in the exofacial domain of VLA-4 after its ligation to stromal fibronectin. This effect triggered cytoskeletal conformational changes that decreased PI3K/Akt/Bcl2 signaling, an obligatory step in chemosensitization by AS101. In a mouse xenograft of AML derived from patient leukemic cells with high VLA-4 expression and activity, we demonstrated that AS101 abrogated drug resistance and prolonged survival in mice receiving chemotherapy. Decreased integrin activity was confirmed on AML cells in vivo. The chemosensitizing activity of AS101 persisted in hosts with defective adaptive and innate immunity, consistent with evidence that integrin deactivation was not mediated by heightening immune attack. Our findings provide a mechanistic rationale to reposition the experimental clinical agent, AS101, to degrade VLA-4-mediated chemoresistance and improve clinical responses in patients with AML.

Ero1α Is Expressed on Blood Platelets in Association with Protein-disulfide Isomerase and Contributes to Redox-controlled Remodeling of αIIbβ3

Recent evidence supports a role of protein-disulfide isomerase (PDI) in redox-controlled remodeling of the exofacial domains of α(IIb)β(3) in blood platelets. The aim of this study was to explain whether Ero1α can be responsible for extracellular reoxidation of the PDI active site. We showed that Ero1α can be found on platelets and is rapidly recruited to the cell surface in response to platelet agonists. It is physically associated with PDI and α(IIb)β(3), as suggested by colocalization analysis in confocal microscopy and confirmed by immunoprecipitation experiments. Apart from monomeric oxidized Ero1α, anti-α(IIb)β(3) immunoprecipitates showed the presence of several Ero1α-positive bands that corresponded to the complexes α(IIb)β(3)-PDI-Ero1α, PDI-Ero1α, and Ero1α-Ero1α dimers. It binds more efficiently to the activated α(IIb)β(3) conformer, and its interaction is inhibited by RGD peptides. Ero1α appears to be involved in the regulation of α(IIb)β(3) receptor activity because of the following: (a) blocking the cell surface Ero1α by antibodies leads to a decrease in platelet aggregation in response to agonists and a decrease in fibrinogen and PAC-1 binding, and (b) transfection of MEG01 with Ero1α increases α(IIb)β(3) receptor activity, as indicated by increased binding of fibrinogen.

High-Pressure-Induced Hemolysis in Papain-Digested Human Erythrocytes Is Suppressed by Cross-Linking of Band 3 via Anti-Band 3 Antibodies

Upon exposure of human erythrocytes to a high pressure of 200 mPa, both hemolysis and vesiculation occur. The hemolysis of erythrocytes at 200 mPa was enhanced by removal of sialic acids from the membrane surface with papain. However, such enhancement was suppressed by cross-linking of band 3 via an anti-band 3 antibody (AB3A), which recognizes the exofacial domain of band 3, or by clustering of band 3 via Zn2+. On the other hand, the size of high-pressure-induced vesicles increased from 423 to 525 nm in diameter upon exposure to papain of erythrocytes, but decreased to 444 nm with following treatment with AB3A. In these vesicles, the content of spectrin relative to band 3 was almost the same. Furthermore, the band 3-cytoskeleton interactions in erythrocyte membranes remained unaltered upon treatment with papain and AB3A. Flow cytometric analysis demonstrated that papain-pretreated erythrocytes mainly produce open ghosts at 200 mPa and that the production of such open ghosts is suppressed by AB3A. Thus, upon removal of negative charges from the membrane surface, open ghosts are readily produced due to the release of larger vesicles under pressure. Upon cross-linking of band 3 via AB3A, however, the release of smaller vesicles at 200 mPa is facilitated so that high-pressure-induced hemolysis is suppressed.

Sustained integrin ligation involves extracellular free sulfhydryls and enzymatically catalyzed disulfide exchange

Studies have suggested a pivotal role for free sulfhydryls in platelet integrin function, and enzyme-mediated reduction of disulfide bonds on platelets has been implicated. The platelet fibrinogen receptor alpha(IIb)beta(3) is the best-studied platelet integrin and serves as a model system for studying the structure-function relation in this family of adhesion receptors. The demonstration of free sulfhydryls on the exofacial domain of purified alpha(IIb)beta(3), specifically in its activated conformation, prompted us to explore the potential for activation-dependent, enzymatically catalyzed thiol expression on intact platelets and the possible role of surface-associated protein disulfide isomerase (PDI) in alpha(IIb)beta(3) ligation. Using the membrane-impermeant sulfhydryl blocker para-chloromercuriphenyl sulfonate, the inhibitor of disulfide exchange bacitracin, and the monoclonal anti-PDI antibody RL90, we examined fibrinogen binding to alpha(IIb)beta(3) as well as ligation-induced allosteric changes in the conformation of alpha(IIb)beta(3). We sought to distinguish the possible involvement of disulfide exchange in agonist-induced platelet stimulation from its role in integrin ligation. Analysis of the role of free thiols in platelet aggregation suggested a thiol-independent initial ligation followed by a thiol-dependent stabilization of binding. Flow cytometric analysis showed that sustained binding of fibrinogen, as well as expression of ligand-induced binding site epitopes and ligand-bound conformation, depended on free thiols and disulfide exchange. Expression of P-selectin was minimally affected, even with complete inhibition of alpha(IIb)beta(3) function. These data indicate that although agonist-induced platelet stimulation is independent of ecto-sulfhydryls, engagement of integrin alpha(IIb)beta(3) on the intact platelet depends totally on their enzymatically catalyzed surface expression.

Site-directed mutagenesis of the human D antigen: definition of D epitopes on the sixth external domain of the D protein expressed on K562 cells.

The antigens of the human Rh system are of great clinical significance in transfusion medicine and pregnancy. Of the Rh system antigens, D is clinically the most important, being one of the most immunogenic structures arising from human cells. The human D antigen represents a collection of epitopes expressed on a red cell membrane protein that is predicted to have 12 membrane-spanning segments giving rise to six exofacial domains. By site-directed mutagenesis using the method of inverse polymerase chain reaction, cE and D cDNA mutant constructs were generated with changes to the RHD-specific residues 350, 353, and 354 in the predicted sixth exofacial loop. Each mutant cDNA was subcloned into the pBabe puromycin retroviral vector, and supernatants were used to transduce K562 cells. Puromycin-resistant K562 clones were screened by flow cytometric analysis using a panel of monoclonal antibodies with specificities to ep (epitope) D1 through epD9. De novo expression of epD3 and epD9 was generated in the K562 cell lines expressing the mutated cE polypeptide (cE-Asp350His, Gly353Trp, Ala354Asn). Expression of c and E was unaffected. Conversely, the cells expressing the mutated D polypeptide demonstrated loss of expression of epD1, epD2, epD3, epD4, and epD9. The data provide strong evidence for the critical involvement of three amino acids, Asp350, Gly353, and Ala354, in the expression of epD3 and epD9 on the predicted sixth external domain of the D protein. This domain also appears to be essential for the expression of epD1, epD2, and epD4, as a loss of expression of these epitopes was observed in K562 cells transduced with the Dmut construct (encoding His350, Trp353, and Asn354). The K562/Dmut cell line has an identical molecular and serologic profile as the red cell D(IVb) phenotype, which confirms that retroviral gene transfer of Rh cDNA into K562 cells provides us with a powerful means by which to further map epitopes of D.

Effects of intracellular pH on high pressure-induced hemolysis of anion transport inhibitor-treated erythrocytes

Effects of anion transport inhibitors such as 4,4′-diisothiocyanostilbene-2,2′-disulfonate (DIDS) and 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonate on hemolysis of human erythrocytes at 200 MPa were examined by changing intracellular pH (7.2–7.9). These inhibitors suppressed the hemolysis at neutral pH but enhanced it at alkaline pH. However, such an enhancement was suppressed by cross-linking of membrane proteins using diamide. From the near-UV CD spectra of band 3 and the relation between hemolysis and anion transport in intact or trypsin-treated erythrocytes, it was found that such hemolytic properties were characterized by the binding of inhibitors to band 3. In addition, spectrin detachment from the erythrocyte membrane by high pressure was considerably suppressed by DIDS treatment at neutral pH, but not by DIDS labeling at alkaline pH. These results suggest that the interaction of the cytoplasmic domain of band 3 with the cytoskeleton, which is induced by the binding of ligands to the exofacial domain of band 3, is dependent on the intracellular pH, i.e., the linking is tightened at neutral pH but relaxed at alkaline pH.

Effects of anion transport inhibitors on hemolysis of human erythrocytes under hydrostatic pressure.

Effects of anion transport inhibitors on hemolysis of human erythrocytes at 200 mPa were examined. The degree of hemolysis was decreased by treating intact cells with 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS), 4,4'-dinitrostilbene-2,2'-disulfonate and pyridoxal 5'-phosphate (PLP) had little effect on the hemolysis. In contrast, the degree of hypotonic hemolysis increased upon treatment with anion transport, inhibitors. From the relationship between the hemolysis at 200 mPa and anion transport, it was found that high-pressure-induced hemolysis was suppressed by the covalent binding of anion transport inhibitors to band 3. This idea was supported by the finding that the hemolysis at 200 mPa of trypsin-treated erythrocytes was suppressed by DIDS. Furthermore, the spectrin content in vesicles which are released from erythrocyte ghost by dimyristoyl-phosphatidylcholine decreased upon DIDS labeling of band 3, but did not change upon PLP labeling. These results suggest that the interaction of the cytoplasmic domain of band 3 with spectrin, perhaps via ankyrin, is tightened by the covalent binding of bulky ligands to the exofacial domain of band 3.

Loss of rotational mobility of band 3 proteins in human erythrocyte membranes induced by antibodies to glycophorin A

The effect of antibodies to glycophorin A on the rotational diffusion of band 3 in human erythrocyte membranes was investigated by transient dichrosim. Three antibodies that recognize different epitopes on the exofacial domain of glycophorin A all strongly reduce the rotational mobility of band 3. The effect is at most only weakly dependent on the distance of the epitope from the membrane surface. The degree of immobilization obtained with two of the antibodies, BRIC14 and R18, is very similar to that produced by antibodies to band 3 itself. Similar results were obtained with membranes stripped of skeletal proteins. Fab fragments and an antibody to glycophorin C had no effect on band 3 rotational mobility. These results rule out a mechanism whereby band 3 rotational immobilization results from enhanced interactions with the membrane skeleton that are mediated by a conformational change in glycophorin A. Rather, they strongly indicate that the antibodies to glycophorin A cross-link existing band 3-glycophorin A complexes that have lifetimes that are long compared with the millisecond time scale of the transient dichroism measurements.

Topology of the Glut 1 glucose transporter deduced from glycosylation scanning mutagenesis.

The erythrocyte glucose transporter (Glut 1) is predicted to contain 12 membrane-spanning domains based on the hydropathy plot of its deduced amino acid sequence. The membrane topology of Glut 1 was analyzed by a scanning mutagenesis procedure in which the glycosylated exofacial domain of Glut 4 was inserted independently into each of the putative hydrophilic soluble domains of an aglyco-Glut 1 construct. The transporter mutants were expressed both in vitro using a rabbit reticulocyte lysate translation system and in vivo in Xenopus oocytes. The cytoplasmic or exofacial orientation of each soluble domain was inferred from the glycosylation state of the corresponding insertion mutant. The results from the cell-free system were aberrant in that two topological orientations were observed when the epitope was inserted into any of the short cytoplasmic loops or the NH2 terminus. The in vivo data, however, were in complete agreement with the proposed 12-helix model. Therefore, the multiple topologies observed in vitro probably resulted from the inability of the cell-free system to facilitate the proper folding of the insertion mutants into the membrane. 2-Deoxyglucose uptake data on the glycosylation mutants indicated that epitope insertion into the NH2 terminus, the large central loop, or the second, third, or fifth exofacial loop had no dramatic effect on the activity of the transporter. However, insertion into the other soluble domains either completely abolished or significantly reduced transport activity.

Hemolytic properties under hydrostatic pressure of neuraminidase- or protease-treated human erythrocytes.

We investigated the hemolytic properties under high pressure (200 MPa) of human erythrocytes in which sialic acids and glycopeptides had been removed from membrane surface by using neuraminidase and proteolytic enzymes such as trypsin and chymotrypsin, respectively. The degree of hemolysis increased in proportion to the amounts of sialic acids or glycopeptides released from intact erythrocytes. Studies of the time course of hemolysis showed that upon enzymatic digestion erythrocyte membranes became more fragile against high pressure. Such fragility decreased in the presence of chlorpromazine and trifluoperazine but was unaffected by chlorpromazine methiodide or indomethacin. Furthermore, the effect of cross-linking of membrane proteins by diamide on the fragility was examined. The degree of hemolysis at 200 MPa increased upon removal of sialic acids from red cells in which spectrin is mainly cross-linked, but did not upon enzymatic digestion of red cells in which glycophorins, in addition to cross-linking of themselves, are included in the large-molecular-weight aggregates formed by cross-linking of the membrane skeleton with transmembrane proteins. In the latter case, however, upon reduction of the cross-linking by dithiothreitol the effect of enzymatic digestion appeared again. On the other hand, such an enzymatic digestion effect on osmotic hemolysis was not observed either in intact erythrocytes or in diamide-treated red cells. These results suggest that the interaction of the cytoplasmic domains of glycophorins with cytoskeletal proteins may be weakened by enzymatic digestion of the exofacial domains of glycophorins.

Origin of a Soluble Truncated Transferrin Receptor

It has recently become evident that elevation of reticulocytes in the circulation of several species, including humans, leads to the formation of a noncellular transferrin receptor (TFR). In humans, the majority of the released receptor is in truncated form (Shih et al: J Biol Chem 265:19077, 1990). In other species (sheep, rat, chicken) the receptor is associated with a vesicle (exosome) and is full length (Johnstone et al: J Cell Physiol 147:27, 1991). In this report we show that in sheep reticulocytes incubated in vitro, the majority (approximately 75%) of the released receptor is of native size and is exosome associated. A fraction (approximately 25%) is a truncated form of approximately 80 Kd corresponding to the exofacial domain of the TFR. Herein we also address the question of whether the truncated receptor originates by proteolytic cleavage directly from the cell surface or by cleavage from exosomes. Using surface 125I-labeled sheep reticulocytes as the experimental model, we show that during in vitro maturation, 125I-TFR of native size appears in exosomes before the soluble, truncated, exofacial domain of the receptor is detected in the medium. Because cleavage and release of the exofacial domain would likely leave the truncated cytoplasmic and transmembrane domains in the originating membrane (plasma membranes or exosomes), both fractions were probed with antibodies specifically generated against the cytoplasmic domain of the receptor. Only exosomes, not plasma membranes, show the presence of a approximately 17-Kd peptide recognized by the antibody to the cytoplasmic domain of the transferrin receptor. Thus, it is concluded that the truncated, soluble receptor originates from exosomes in sheep. A 17-Kd cytoplasmic domain of the TFR was also detected in exosomes from the reticulocytes of an anemic man, suggesting that the truncated receptor in man may also originate from exosomes. Using in vitro cultures of surface 125I-labeled sheep reticulocytes, it is concluded that exosome formation is the principal route for maturation-associated loss of the TFR. A similar conclusion was made earlier (Johnstone et al: J Cell Physiol 147:27, 1991) for the nucleoside transporter of maturing sheep reticulocytes.

Origin of a soluble truncated transferrin receptor

It has recently become evident that elevation of reticulocytes in the circulation of several species, including humans, leads to the formation of a noncellular transferrin receptor (TFR). In humans, the majority of the released receptor is in truncated form (Shih et al: J Biol Chem 265:19077, 1990). In other species (sheep, rat, chicken) the receptor is associated with a vesicle (exosome) and is full length (Johnstone et al: J Cell Physiol 147:27, 1991). In this report we show that in sheep reticulocytes incubated in vitro, the majority (approximately 75%) of the released receptor is of native size and is exosome associated. A fraction (approximately 25%) is a truncated form of approximately 80 Kd corresponding to the exofacial domain of the TFR. Herein we also address the question of whether the truncated receptor originates by proteolytic cleavage directly from the cell surface or by cleavage from exosomes. Using surface 125I-labeled sheep reticulocytes as the experimental model, we show that during in vitro maturation, 125I-TFR of native size appears in exosomes before the soluble, truncated, exofacial domain of the receptor is detected in the medium. Because cleavage and release of the exofacial domain would likely leave the truncated cytoplasmic and transmembrane domains in the originating membrane (plasma membranes or exosomes), both fractions were probed with antibodies specifically generated against the cytoplasmic domain of the receptor. Only exosomes, not plasma membranes, show the presence of a approximately 17-Kd peptide recognized by the antibody to the cytoplasmic domain of the transferrin receptor. Thus, it is concluded that the truncated, soluble receptor originates from exosomes in sheep. A 17-Kd cytoplasmic domain of the TFR was also detected in exosomes from the reticulocytes of an anemic man, suggesting that the truncated receptor in man may also originate from exosomes. Using in vitro cultures of surface 125I-labeled sheep reticulocytes, it is concluded that exosome formation is the principal route for maturation-associated loss of the TFR. A similar conclusion was made earlier (Johnstone et al: J Cell Physiol 147:27, 1991) for the nucleoside transporter of maturing sheep reticulocytes.

Suppressed intrinsic catalytic activity of GLUT1 glucose transporters in insulin-sensitive 3T3-L1 adipocytes.

Previous studies indicated that the erythroidtype (GLUT1) glucose transporter isoform contributes to basal but not insulin-stimulated hexose transport in mouse 3T3-L1 adipocytes. In the present studies it was found that basal hexose uptake in 3T3-L1 adipocytes was about 50% lower than that in 3T3-L1 or CHO-K1 fibroblasts. Intrinsic catalytic activities of GLUT1 transporters in CHO-K1 and 3T3-L1 cells were compared by normalizing these hexose transport rates to GLUT1 content on the cell surface, as measured by two independent methods. Cell surface GLUT1 levels in 3T3-L1 fibroblasts and adipocytes were about 10- and 25-fold higher, respectively, than in CHO-K1 fibroblasts, as assessed with an anti-GLUT1 exofacial domain antiserum, delta. The large excess of cell surface GLUT1 transporters in 3T3-L1 adipocytes relative to CHO-K1 fibroblasts was confirmed by GLUT1 protein immunoblot analysis and by photoaffinity labelling (with 3-[125I]iodo-4-azidophenethylamido-7-O-succinyldeacetylforskoli n) of glucose transporters in isolated plasma membranes. Thus, GLUT1 intrinsic activity is markedly reduced in 3T3-L1 fibroblasts compared with the CHO-K1 fibroblasts, and further reduction occurs upon differentiation to adipocytes. Intrinsic catalytic activities specifically associated with heterologously expressed human GLUT1 protein in transfected CHO-K1 versus 3T3-L1 cells were determined by subtracting appropriate control cell values for hexose transport and delta-antibody binding from those determined in the transfected cells expressing high levels of human GLUT1. The results confirmed a greater than 90% inhibition of the intrinsic catalytic activity of human GLUT1 transporters on the surface of mouse 3T3-L1 adipocytes relative to CHO-K1 fibroblasts. We conclude that a mechanism that markedly suppresses basal hexose transport catalyzed by GLUT1 is a major contributor to the dramatic insulin sensitivity of glucose uptake in 3T3-L1 adipocytes.