Author Correction: A spatial similarity of stereochemical environments formed by amino acid residues defines a common epitope of two non-homologous proteins.

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A spatial similarity of stereochemical environments formed by amino acid residues defines a common epitope of two non-homologous proteins.

It is critical for development of high-quality antibodies in research and diagnostics to predict accurately their cross-reactivities with "off-target" molecules, which potentially induce false results. Herein, we report a good example of such a cross-reactivity for an off-target due to a stereochemical environment of epitopes, which does not simply depend on amino acid sequences. We found that significant subpopulation of a polyclonal peptide antibody against Bcnt (Bucentaur) (anti-BCNT-C antibody) cross-reacted with a completely different protein, glutamine synthetase (GS), and identified four amino acids, GYFE, in its C-terminal region as the core amino acids for the cross-reaction. Consistent with this finding, the anti-BCNT-C antibody strongly recognized endogenously and exogenously expressed GS in tissues and cultured cells by Western blotting and immunohistochemistry. Furthermore, we elucidated that the cross-reaction is caused by a spatial similarity between the stereochemical environments formed by amino acid residues, including the GYFE of GS and the GYIE of Bcnt, rather than by their primary sequences. These results suggest it is critical to comprehensively analyze antibody interactions with target molecules including off-targets with special attention to the physicochemical environments of epitope-paratope interfaces to decrease the risk of false interpretations of results using antibodies in science and clinical applications.

Mammalian Bcnt/Cfdp1, a potential epigenetic factor characterized by an acidic stretch in the disordered N-terminal and Ser250 phosphorylation in the conserved C-terminal regions.

The BCNT (Bucentaur) superfamily is classified by an uncharacteristic conserved sequence of ∼80 amino acids (aa) at the C-terminus, BCNT-C (the conserved C-terminal region of Bcnt/Cfdp1). Whereas the yeast Swc5 and Drosophila Yeti homologues play crucial roles in chromatin remodelling organization, mammalian Bcnt/Cfdp1 (craniofacial developmental protein 1) remains poorly understood. The protein, which lacks cysteine, is largely disordered and comprises an acidic N-terminal region, a lysine/glutamic acid/proline-rich 40 aa sequence and BCNT-C. It shows complex mobility on SDS/PAGE at ∼50 kDa, whereas its calculated molecular mass is ∼33 kDa. To characterize this mobility discrepancy and the effects of post-translational modifications (PTMs), we expressed various deleted His-Bcnt in E. coli and HEK cells and found that an acidic stretch in the N-terminal region is a main cause of the gel shift. Exogenous BCNT/CFDP1 constitutively expressed in HEK clones appears as a doublet at 49 and 47 kDa, slower than the protein expressed in Escherichia coli but faster than the endogenous protein on SDS/PAGE. Among seven in vivo phosphorylation sites, Ser(250), which resides in a region between disordered and ordered regions in BCNT-C, is heavily phosphorylated and detected predominantly in the 49 kDa band. Together with experiments involving treatment with phosphatases and Ser(250) substitutions, the results indicate that the complex behaviour of Bcnt/Cfdp1 on SDS/PAGE is caused mainly by an acidic stretch in the N-terminal region and Ser(250) phosphorylation in BCNT-C. Furthermore, Bcnt/Cfdp1 is acetylated in vitro by CREB-binding protein (CBP) and four lysine residues including Lys(268) in BCNT-C are also acetylated in vivo, revealing a protein regulated at multiple levels.

Clot retraction is mediated by factor XIII-dependent fibrin-αIIbß3-myosin axis in platelet sphingomyelin-rich membrane rafts.

Membrane rafts are spatially and functionally heterogenous in the cell membrane. We observed that lysenin-positive sphingomyelin (SM)-rich rafts are identified histochemically in the central region of adhered platelets where fibrin and myosin are colocalized on activation by thrombin. The clot retraction of SM-depleted platelets from SM synthase knockout mouse was delayed significantly, suggesting that platelet SM-rich rafts are involved in clot retraction. We found that fibrin converted by thrombin translocated immediately in platelet detergent-resistant membrane (DRM) rafts but that from Glanzmann's thrombasthenic platelets failed. The fibrinogen γ-chain C-terminal (residues 144-411) fusion protein translocated to platelet DRM rafts on thrombin activation, but its mutant that was replaced by A398A399 at factor XIII crosslinking sites (Q398Q399) was inhibited. Furthermore, fibrin translocation to DRM rafts was impaired in factor XIII A subunit-deficient mouse platelets, which show impaired clot retraction. In the cytoplasm, myosin translocated concomitantly with fibrin translocation into the DRM raft of thrombin-stimulated platelets. Furthermore, the disruption of SM-rich rafts by methyl-ß-cyclodextrin impaired myosin activation and clot retraction. Thus, we propose that clot retraction takes place in SM-rich rafts where a fibrin-αIIbß3-myosin complex is formed as a primary axis to promote platelet contraction.

A critical role for the cholesterol-associated proteolipids PLP and M6B in myelination of the central nervous system.

The formation of central nervous system myelin by oligodendrocytes requires sterol synthesis and is associated with a significant enrichment of cholesterol in the myelin membrane. However, it is unknown how oligodendrocytes concentrate cholesterol above the level found in nonmyelin membranes. Here, we demonstrate a critical role for proteolipids in cholesterol accumulation. Mice lacking the most abundant myelin protein, proteolipid protein (PLP), are fully myelinated, but PLP-deficient myelin exhibits a reduced cholesterol content. We therefore hypothesized that "high cholesterol" is not essential in the myelin sheath itself but is required for an earlier step of myelin biogenesis that is fully compensated for in the absence of PLP. We also found that a PLP-homolog, glycoprotein M6B, is a myelin component of low abundance. By targeting the Gpm6b-gene and crossbreeding, we found that single-mutant mice lacking either PLP or M6B are fully myelinated, while double mutants remain severely hypomyelinated, with enhanced neurodegeneration and premature death. As both PLP and M6B bind membrane cholesterol and associate with the same cholesterol-rich oligodendroglial membrane microdomains, we suggest a model in which proteolipids facilitate myelination by sequestering cholesterol. While either proteolipid can maintain a threshold level of cholesterol in the secretory pathway that allows myelin biogenesis, lack of both proteolipids results in a severe molecular imbalance of prospective myelin membrane. However, M6B is not efficiently sorted into mature myelin, in which it is 200-fold less abundant than PLP. Thus, only PLP contributes to the high cholesterol content of myelin by association and co-transport.

Visualization of sterol-rich membrane domains with fluorescently-labeled theonellamides.

Cholesterol plays important roles in biological membranes. The cellular location where cholesterol molecules work is prerequisite information for understanding their dynamic action. Bioimaging probes for cholesterol molecules would be the most powerful means for unraveling the complex nature of lipid membranes. However, only a limited number of chemical or protein probes have been developed so far for cytological analysis. Here we show that fluorescently-labeled derivatives of theonellamides act as new sterol probes in mammalian cultured cells. The fluorescent probes recognized cholesterol molecules and bound to liposomes in a cholesterol-concentration dependent manner. The probes showed patchy distribution in the plasma membrane, while they stained specific organelle in the cytoplasm. These data suggest that fTNMs will be valuable sterol probes for studies on the role of sterols in the biological membrane under a variety of experimental conditions.

A role for sphingomyelin-rich lipid domains in the accumulation of phosphatidylinositol-4,5-bisphosphate to the cleavage furrow during cytokinesis.

Cytokinesis is a crucial step in the creation of two daughter cells by the formation and ingression of the cleavage furrow. Here, we show that sphingomyelin (SM), one of the major sphingolipids in mammalian cells, is required for the localization of phosphatidylinositol-4,5-bisphosphate (PIP(2)) to the cleavage furrow during cytokinesis. Real-time observation with a labeled SM-specific protein, lysenin, revealed that SM is concentrated in the outer leaflet of the furrow at the time of cytokinesis. Superresolution fluorescence microscopy analysis indicates a transbilayer colocalization between the SM-rich domains in the outer leaflet and PIP(2)-rich domains in the inner leaflet of the plasma membrane. The depletion of SM disperses PIP(2) and inhibits the recruitment of the small GTPase RhoA to the cleavage furrow, leading to abnormal cytokinesis. These results suggest that the formation of SM-rich domains is required for the accumulation of PIP(2) to the cleavage furrow, which is a prerequisite for the proper translocation of RhoA and the progression of cytokinesis.

Fluorescence image screening for chemical compounds modifying cholesterol metabolism and distribution.

An automated fluorescence microscopy assay using a nontoxic cholesterol binding protein, toxin domain 4, (D4), was developed in order to identify chemical compounds modifying intracellular cholesterol metabolism and distribution. Using this method, we screened a library of 1,056 compounds and identified 35 compounds that decreased D4 binding to the cell surface. Among them, 8 compounds were already reported to alter the biosynthesis or the intracellular distribution of cholesterol. The remaining 27 hit compounds were further analyzed biochemically and histochemically. Cell staining with another fluorescent cholesterol probe, filipin, revealed that 17 compounds accumulated cholesterol in the late endosomes. Five compounds decreased cholesterol biosynthesis, and two compounds inhibited the binding of D4 to the membrane. This visual screening method, based on the cholesterol-specific probe D4 in combination with biochemical analyses, is a cell-based, sensitive technique for identifying new chemical compounds and modifying cholesterol distribution and metabolism. Furthermore, it is suitable for high-throughput analysis for drug discovery.

Plasma membrane microdomains in aging and disease.

The plasma membrane of eukaryotic cells participates in signal transduction and many other cellular events to maintain the physiological state of cells. In recent decades, much attention has been paid to membrane microdomains, called lipid rafts or membrane rafts, as signaling platforms in the plasma membrane. Lipid rafts are lateral lipid clusters enriched in cholesterol and sphingolipids in which particular molecules are concentrated and participate in membrane-mediated signaling events. Recent studies have shown a close relationship between lipid rafts and the age-associated decline and dysregulation of cellular signaling pathways, such as T-cell receptor signaling and cellular senescence-related signaling. Lipid rafts have also been implicated in senile diseases and in lifestyle-related diseases whose incidences increase with age.

Cholesterol-binding toxins and anti-cholesterol antibodies as structural probes for cholesterol localization.

Cholesterol is one of the major constituents of mammalian cell membranes. It plays an indispensable role in regulating the structure and function of cell membranes and affects the pathology of various diseases. In recent decades much attention has been paid to the existence of membrane microdomains, generally termed lipid "rafts", and cholesterol, along with sphingolipids, is thought to play a critical role in raft structural organization and function. Cholesterol-binding probes are likely to provide useful tools for analyzing the distribution and dynamics of membrane cholesterol, as a structural element of raft microdomains, and elsewhere within the cell. Among the probes, non-toxic derivatives of perfringolysin O, a cholesterol-binding cytolysin, bind cholesterol in a concentration-dependent fashion with a strict threshold. They selectively recognize cholesterol in cholesterol-enriched membranes, and have been used in many studies to detect microdomains in plasma and intracellular membranes. Anti-cholesterol antibodies that recognize cholesterol in domain structures have been developed in recent years. In this chapter, we describe the characteristics of these cholesterol-binding proteins and their applications to studies on membrane cholesterol localization.

Use of dansyl-cholestanol as a probe of cholesterol behavior in membranes of living cells.

While plasma membrane cholesterol-rich microdomains play a role in cholesterol trafficking, little is known about the appearance and dynamics of cholesterol through these domains in living cells. The fluorescent cholesterol analog 6-dansyl-cholestanol (DChol), its biochemical fractionation, and confocal imaging of L-cell fibroblasts contributed the following new insights: i) fluorescence properties of DChol were sensitive to microenvironment polarity and mobility; (ii) DChol taken up by L-cell fibroblasts was distributed similarly as cholesterol and preferentially into cholesterol-rich vs. -poor microdomains resolved by affinity chromatography of purified plasma membranes; iii) DChol reported similar polarity (dielectric constant near 18) but higher mobility near phospholipid polar head group region for cholesterol in purified cholesterol-rich versus -poor microdomains; and iv) real-time confocal imaging, quantitative colocalization analysis, and fluorescence resonance energy transfer with cholesterol-rich and -poor microdomain markers confirmed that DChol preferentially localized in plasma membrane cholesterol-rich microdomains of living cells. Thus, DChol sensed a unique, relatively more mobile microenvironment for cholesterol in plasma membrane cholesterol-rich microdomains, consistent with the known, more rapid exchange dynamics of cholesterol from cholesterol-rich than -poor microdomains.

Age-associated up-regulation of a negative co-stimulatory receptor PD-1 in mouse CD4+ T cells.

To explore whether any co-stimulatory receptor(s) for TCR signaling is involved in the age-associated decline in T-cell function, we analyzed changes in these receptors in freshly isolated mouse CD4(+) T cells during aging. Both the mRNA and protein expression levels of CTLA-4 and PD-1, negative co-stimulatory receptors, increase with aging. No such changes are observed for CD28, a positive regulatory receptor. PD-1 is highly expressed on the surface of old, but not young, mouse T cells, while the level of surface-expressed CTLA-4 is very low regardless of age. PD-1 is preferentially expressed on the surface of effector-memory (CD44(hi)CD62L(lo)) T cells, a subset that increases with aging. CD4(+)PD-1(+) T cells from old mice exhibit proliferative hyporesponsiveness. These results suggest that the up-regulation of surface-expressed PD-1 may cause the age-dependent functional decline in effector-memory T cells.

Partial blockage of sterol biosynthesis with a squalene synthase inhibitor in early postnatal Niemann-Pick type C npcnih null mice brains reduces neuronal cholesterol accumulation, abrogates astrogliosis, but may inhibit myelin maturation.

Niemann-Pick C disease (NPC) is a fatal, neurovisceral genetic disorder. Cell culture studies showed that NPC1 or NPC2 mutations cause malfunctions in cellular cholesterol trafficking and lead to accumulation of cholesterol and other lipids in the late endo/lysosomes. Previous work showed that neuronal cholesterol accumulation occurs in the brains of young postnatal NPC1-/- mice. Here, to evaluate the potential of partial blockage of cholesterol biosynthesis as a therapy for the NPC disease, we first developed a simple method to monitor the relative rates of lipid biosynthesis in mice brains. We next administered squalene synthase inhibitor (SSI) CP-340868 to young mice. The results show that treating 8-day-old NPC1-/- mice with CP-340868 for 6 days significantly inhibits cholesterol biosynthesis in the mice brains. It reduces neuronal cholesterol accumulation, reduces GM3 ganglioside accumulation, and diminishes astrogliosis in the brain. These results suggest that neuronal cholesterol accumulation contributes to early pathogenesis in the NPC1-/- mice brains. The SSI treatment also reduced brain galactolipid content, suggesting that blocking endogenous cholesterol synthesis in the young mice brains may disrupt the normal myelin maturation processes. The methods described in the current work have general applicability for lipid metabolism studies in mice brains in various pathophysiological conditions.

Impairment in a negative regulatory system for TCR signaling in CD4+ T cells from old mice.

To examine the involvement of lipid rafts in an age-associated decline in T cell function, we analyzed the effect of aging on the constituents of lipid rafts in resting mouse CD4(+) T cells. We found a pronounced, age-dependent reduction in PAG/Cbp, which is involved in the regulation of Src family kinases (SFKs) by recruiting Csk (a negative regulator of SFKs) to lipid rafts. This reduction is specific for T cells and is attributed, at least in part, to the reduction in its mRNA level. The reduction of PAG accompanies marked impairment in recruiting Csk to lipid rafts and a concomitant decrease in the inactive forms of SFKs. These findings indicate that old mouse CD4(+) T cells have a defect in a negative SFK regulatory system.

Increased lipid rafts and accelerated lipopolysaccharide-induced tumor necrosis factor-alpha secretion in Abca1-deficient macrophages.

Lipid rafts on the cell surface are believed to be very important as platforms for various cellular functions. The aim of this study was to know whether defective lipid efflux may influence lipid rafts on the cell surface and their related cellular functions. We investigated macrophages with defective lipid efflux from ATP binding cassette transporter A1-deficient (Abca1-KO) mice. Lipid rafts were evaluated by the following two novel probes: a biotinylated and protease (subtilisin Carlsberg)-nicked derivative of theta-toxin and a fluorescein ester of polyethylene glycol-derived cholesterol. Lipid rafts in Abca1-KO macrophages were increased, as demonstrated by both probes. Moreover, activities of nuclear factor kappaB, mRNA and intracellular distribution, and secretion of tumor necrosis factor-alpha (TNF-alpha) were examined after stimulation by lipopolysaccharides (LPSs). LPS-induced responses of the activation of nuclear factor kappaB and TNF-alpha were more prompt and accelerated in the Abca1-KO macrophages compared with wild-type macrophages. Modification of lipid rafts by cyclodextrin and nystatin corrected the abnormal response, suggesting an association between the increased lipid rafts and abnormal TNF-alpha secretion. We report here that Abca1-KO macrophages with defective lipid efflux exhibited increased lipid rafts on the cell surface and accelerated TNF-alpha secretion.

Detection of cholesterol-rich microdomains in the inner leaflet of the plasma membrane.

The C-terminal domain (D4) of perfringolysin O binds selectively to cholesterol in cholesterol-rich microdomains. To address the issue of whether cholesterol-rich microdomains exist in the inner leaflet of the plasma membrane, we expressed D4 as a fusion protein with EGFP in MEF cells. More than half of the EGFP-D4 expressed in stable cell clones was bound to membranes in raft fractions. Depletion of membrane cholesterol with beta-cyclodextrin reduced the amount of EGFP-D4 localized in raft fractions, confirming EGFP-D4 binding to cholesterol-rich microdomains. Subfractionation of the raft fractions showed most of the EGFP-D4 bound to the plasma membrane rather than to intracellular membranes. Taken together, these results strongly suggest the existence of cholesterol-rich microdomains in the inner leaflet of the plasma membrane.

Detachment-associated changes in lipid rafts of senescent human fibroblasts.

We characterized the effects of in vitro cellular aging on constituents of lipid rafts in human diploid fibroblasts, TIG-1. Cholesterol recovery from lipid rafts of senescent cells was decreased by the detaching treatment, while the decrease was far less obvious in young cells. A probe that binds selectively to cholesterol in lipid rafts revealed that the amount of lipid rafts on the cell surface decreased in senescent cells upon cell detachment. Accompanying this change was the release of the raft-associated molecules caveolin and Fyn from lipid rafts upon cell detachment, suggesting a detachment-associated disorganization of lipid rafts in senescent cells. In addition, our observations showing differential sensitivities of lipid rafts from young and senescent cells to detaching treatment indicate a caution in how to detach cells. Particular attention needs to be paid to interpreting the results when lipid rafts are prepared from mechanically detached cells under detergent-free conditions.

Separation of a cholesterol-enriched microdomain involved in T-cell signal transduction.

We isolated a cholesterol-enriched membrane subpopulation from the so-called lipid raft fractions of Jurkat T-cells by taking advantage of its selective binding to a cholesterol-binding probe, BCtheta. The BCtheta-bound membrane subpopulation has a much higher cholesterol/phospholipid (C/P) molar ratio (approximately 1.0) than the BCtheta-unbound population in raft fractions (approximately 0.3). It contains not only the raft markers GM1 and flotillin, but also some T-cell receptor (TCR) signalling molecules, including Lck, Fyn and LAT. In addition, Csk and PAG, inhibitory molecules of the TCR signalling cascade, are also contained in the BCtheta-bound membranes. On the other hand, CD3epsilon, CD3zeta and Zap70 are localized in the BCtheta-unbound membranes, segregated from other TCR signalling molecules under nonstimulated conditions. However, upon stimulation of TCR, portions of CD3epsilon, CD3zeta and Zap70 are recruited to the BCtheta-bound membranes. The Triton X-100 concentration used for lipid raft preparation affects neither the C/P ratio nor protein composition of the BCtheta-bound membranes. These results show that our method is useful for isolating a particular cholesterol-rich membrane domain of T-cells, which could be a core domain controlling the TCR signalling cascade.

Structure and function of lipid rafts in human activated T cells.

Lipid rafts, specialized membrane microdomains enriched in sphingolipids and cholesterol, have been shown to function as signaling platforms in T cells. Surface raft expression is known to be increased in human T cells upon activation, and this increased raft expression may account for efficient signaling capability and decreased dependency for co-stimulation in effector and/or activated T cells. However, raft-mediated signaling ability in activated T cells remains to be clarified. In this study, we analyzed the structure and function of lipid rafts in human activated T cells. We demonstrated that raft protein constituents are dramatically changed after activation along with an increase in lipid contents. T cells stimulated with anti-CD3 plus anti-CD28 antibodies showed an increase not only in surface monosialoganglioside GM1 expression but also in total amounts of raft-associated lipids such as sphingomyelin, cholesterol and glycosphingolipids. Raft proteins increased after activation include Csk, Csk-binding protein and Fyn, the molecules known to be involved in negative regulation of T cell activation. Consistent with the increase in expression of these proteins, TCR-mediated Ca(2+) response, a response dependent on raft integrity, was clearly inhibited in activated T cells. Thus, the structure and function of lipid rafts in human activated T cells seem to be quite distinct from those in naive T cells. Further, human activated T cells are relatively resistant to signaling, at least transiently, by TCR re-stimulation even though their raft expression is increased.

Role for up-regulated ganglioside biosynthesis and association of Src family kinases with microdomains in retinoic acid-induced differentiation of F9 embryonal carcinoma cells.

Mouse F9 embryonal carcinoma cells have been widely used as a model for studying the mechanism of embryonic differentiation, because they are similar to the inner cell mass of early mouse embryos and can differentiate into primitive endoderm (PrE) following retinoic acid (RA) treatment. During F9 cell differentiation, the carbohydrate chains of glycoproteins and their corresponding glycosyltransferases are known to undergo rapid changes. However, there have been no corresponding reports on the expression of gangliosides. We have developed a custom cDNA array that is highly sensitive for the genes responsible for sphingolipid (SL) biosynthesis and metabolism. Using this, we found that, of the 28 selected genes, 26 exhibited increased expression during F9 differentiation into PrE. Although neutral glycosphingolipids (GSLs) were expressed at similar levels before and after differentiation, a greater than 20-fold increase in total ganglioside content was evident in PrE. Glucosylceramide synthase inhibitors (d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol [d-PDMP] and its analog) depleted gangliosides and this resulted in delayed expression of Disabled-2 (Dab-2), suggesting the involvement of gangliosides in F9 cell differentiation. Disruption of cholesterol-enriched membrane microdomains by methyl-beta-cyclodextrin (MbetaCD) also delayed differentiation. Both MbetaCD and d-PDMP blocked the accumulation of Src family kinases (SFKs) to microdomains. However, d-PDMP did not block flotillin accumulation, yet MbetaCD did. Additionally, confocal laser microscopy revealed the formation of distinct functional microdomains integrating SFKs with gangliosides and cholesterol during PrE differentiation. Thus, we demonstrate the outstanding up-regulation of ganglioside biosynthesis and its importance in the formation of distinct microdomains incorporating SFKs with gangliosides during RA-induced differentiation of F9 cells.