Identification of quercetin glucuronides in human plasma by high-performance liquid chromatography–tandem mass spectrometry

After intake of food or herbal medicinal products containing quercetin glycosides, the systemic availability of the genuine glycoside, as well as the systemic occurrence of the aglycone or conjugates of this polyphenol has been a matter of dispute. Consequently, we designed this study to develop a reliable method for determination of quercetin and its metabolites. Following consumption of fried onions five different glucuronides of quercetin could be identified in human plasma samples by means of HPLC–UV–MS/MS. Selective determination of the target compounds was achieved by simultaneous UV (254 nm) and MS/MS detection with selected reaction monitoring experiments using positive mode electrospray ionisation. In contrast, neither the free flavonol nor the genuine glycoside could be detected in plasma. Identification of the quercetin glucuronides detected in vivo was confirmed by comparison with authentic reference compounds synthesised enzymatically using glucuronyl transferase from rabbit liver.     

Tao controls epithelial morphogenesis by promoting Fasciclin 2 endocytosis

Regulation of epithelial cell shape, for example, changes in relative sizes of apical, basal, and lateral membranes, is a key mechanism driving morphogenesis. However, it is unclear how epithelial cells control the size of their membranes. In the epithelium of the Drosophila melanogaster ovary, cuboidal precursor cells transform into a squamous epithelium through a process that involves lateral membrane shortening coupled to apical membrane extension. In this paper, we report a mutation in the gene Tao, which resulted in the loss of this cuboidal to squamous transition. We show that the inability of Tao mutant cells to shorten their membranes was caused by the accumulation of the cell adhesion molecule Fasciclin 2, the Drosophila N-CAM (neural cell adhesion molecule) homologue. Fasciclin 2 accumulation at the lateral membrane of Tao mutant cells prevented membrane shrinking and thereby inhibited morphogenesis. In wild-type cells, Tao initiated morphogenesis by promoting Fasciclin 2 endocytosis at the lateral membrane. Thus, we identify here a mechanism controlling the morphogenesis of a squamous epithelium.     

The transient receptor potential channel TRPV6 is dynamically expressed in bone cells but is not crucial for bone mineralization in mice

Bone is the major store for Ca(2+) in the body and plays an important role in Ca(2+) homeostasis. During bone formation and resorption Ca(2+) must be transported to and from bone by osteoblasts and osteoclasts, respectively. However, little is known about the Ca(2+) transport machinery in these bone cells. In this study, we examined the epithelial Ca(2+) channel TRPV6 in bone. TRPV6 mRNA is expressed in human and mouse osteoblast-like cells as well as in peripheral blood mononuclear cell-derived human osteoclasts and murine tibial bone marrow-derived osteoclasts. Also other transcellular Ca(2+) transport genes, calbindin-D(9k) and/or -D(28K), Na(+)/Ca(2+) exchanger 1, and plasma membrane Ca(2+) ATPase (PMCA1b) were expressed in these bone cell types. Immunofluorescence and confocal microscopy on human osteoblasts and osteoclasts and mouse osteoclasts revealed TRPV6 protein at the apical domain and PMCA1b at the osteoidal domain of osteoblasts, whereas in osteoclasts TRPV6 was predominantly found at the bone-facing site. TRPV6 was dynamically expressed in human osteoblasts, showing maximal expression during mineralization of the extracellular matrix. 1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) did not change TRPV6 expression in both mineralizing and non-mineralizing SV-HFO cultures. Lentiviral transduction-mediated overexpression of TRPV6 in these cells did not alter mineralization. Bone microarchitecture and mineralization were unaffected in Trpv6(D541A/D541A) mice in which aspartate 541 in the pore region was replaced with alanine to render TRPV6 channels non-functional. In summary, TRPV6 and other proteins involved in transcellular Ca(2+) transport are dynamically expressed in bone cells, while TRPV6 appears not crucial for bone metabolism and matrix mineralization in mice.     

Excision of Trpv6 gene leads to severe defects in epididymal Ca2+ absorption and male fertility much like single D541A pore mutation

Replacement of aspartate residue 541 by alanine (D541A) in the pore of TRPV6 channels in mice disrupts Ca(2+) absorption by the epididymal epithelium, resulting in abnormally high Ca(2+) concentrations in epididymal luminal fluid and in a dramatic but incomplete loss of sperm motility and fertilization capacity, raising the possibility of residual activity of channels formed by TRPV6(D541A) proteins (Weissgerber, P., Kriebs, U., Tsvilovskyy, V., Olausson, J., Kretz, O., Stoerger, C., Vennekens, R., Wissenbach, U., Middendorff, R., Flockerzi, V., and Freichel, M. (2011) Sci. Signal. 4, ra27). It is known from other cation channels that introducing pore mutations even if they largely affect their conductivity and permeability can evoke considerably different phenotypes compared with the deletion of the corresponding protein. Therefore, we generated TRPV6-deficient mice (Trpv6(-/-)) by deleting exons encoding transmembrane domains with the pore-forming region and the complete cytosolic C terminus harboring binding sites for TRPV6-associated proteins that regulate its activity and plasma membrane anchoring. Using this strategy, we aimed to determine whether the TRPV6(D541A) pore mutant still contributes to residual channel activity and/or channel-independent functions in vivo. Trpv6(-/-) males reveal severe defects in fertility and motility and viability of sperm and a significant increase in epididymal luminal Ca(2+) concentration that is mirrored by a lack of Ca(2+) uptake by the epididymal epithelium. Therewith, Trpv6 excision affects epididymal Ca(2+) handling and male fertility to the same extent as the introduction of the D541A pore mutation, arguing against residual functions of the TRPV6(D541A) pore mutant in epididymal epithelial cells.     

Expression of type XXIII collagen mRNA and protein

Collagen XXIII is a member of the transmembranous subfamily of collagens containing a cytoplasmic domain, a membrane-spanning hydrophobic domain, and three extracellular triple helical collagenous domains interspersed with non-collagenous domains. We cloned mouse, chicken, and humanalpha1(XXIII) collagen cDNAs and showed that this non-abundant collagen has a limited tissue distribution in non-tumor tissues. Lung, cornea, brain, skin, tendon, and kidney are the major sites of expression. In contrast, five transformed cell lines were tested for collagen XXIII expression, and all expressed the mRNA. In vivo the alpha1(XXIII) mRNA is found in mature and developing organs, the latter demonstrated using stages of embryonic chick cornea and mouse embryos. Polyclonal antibodies were generated in guinea pig and rabbit and showed that collagen XXIII has a transmembranous form and a shed form. Comparison of collagen XXIII with its closest relatives in the transmembranous subfamily of collagens, types XIII and XXV, which have the same number of triple helical and non-collagenous regions, showed that there is a discontinuity in the alignment of domains but that striking similarities remain despite this.     

Trafficking and assembly of the cold-sensitive TRPM8 channel

TRPM (transient receptor potential melastatin-like) channels are distinct from many other members of the transient receptor potential family in regard to their overall size (>1000 amino acids), the lack of N-terminal ankyrin-like repeats, and hydrophobicity predictions that may allow for more than six transmembrane regions. Common to each TRPM member is a prominent C-terminal coiled coil region. Here we have shown that TRPM8 channels assemble as multimers using the putative coiled coil region within the intracellular C terminus and that this assembly can be disturbed by a single point mutation within the coiled coil region. This mutant neither gives rise to functional channels nor do its subunits interact or form protein complexes that correspond to a multimer. However, they are still transported to the plasma membrane. Furthermore, wild-type currents can be suppressed by expressing the membrane-attached C-terminal region of TRPM8. To separate assembly from trafficking, we investigated the maturation of TRPM8 protein by identifying and mutating the relevant N-linked glycosylation site and showing that glycosylation is neither essential for multimerization nor for transport to the plasma membrane per se but appears to facilitate efficient multimerization and transport.     

Lateral Distribution of the Transmembrane Domain of Influenza Virus Hemagglutinin Revealed by Time-resolved Fluorescence Imaging

Influenza virus hemagglutinin (HA) has been suggested to be enriched in liquid-ordered lipid domains named rafts, which represent an important step in virus assembly. We employed Förster resonance energy transfer (FRET) via fluorescence lifetime imaging microscopy to study the interaction of the cytoplasmic and transmembrane domain (TMD) of HA with agly co sylphos pha tidyl ino si tol (GPI)-anchored peptide, an established marker for rafts in the exoplasmic leaflet of living mammalian plasma membranes. Cyan fluorescent protein (CFP) was fused to GPI, whereas the HA sequence was tagged with yellow fluorescent protein (YFP) on its exoplasmic site (TMD-HA-YFP), avoiding any interference of fluorescent proteins with the proposed role of the cytoplasmic domain in lateral organization of HA. Constructs were expressed in Chinese hamster ovary cells (CHO-K1) for which cholesterol-sensitive lipid nanodomains and their dimension in the plasma membrane have been described (Sharma, P., Varma, R., Sarasij, R. C., Ira, Gousset, K., Krishnamoorthy, G., Rao, M., and Mayor, S. (2004) Cell 116, 577–589). Upon transfection in CHO-K1 cells, TMD-HA-YFP is partially expressed as a dimer. Only dimers are targeted to the plasma membrane. Clustering of TMD-HA-YFP with GPI-CFP was observed and shown to be reduced upon cholesterol depletion, a treatment known to disrupt rafts. No indication for association of TMD-HA-YFP with GPI-CFP was found when palmitoylation, an important determinant of raft targeting, was suppressed. Clustering of TMD-HA-YFP and GPI-CFP was also observed in purified plasma membrane suspensions by homoFRET. We concluded that the pal mit oy lated TMD-HA alone is sufficient to recruit HA to cholesterol-sensitive nanodomains. The corresponding construct of the spike protein E2 of Semliki Forest virus did not partition preferentially in such domains.Assembly of enveloped viruses requires the selective recruitment of viral components at distinct sites of the host cell membranes from which viruses bud. One of the most intensely studied enveloped viruses with respect to assembly is the influenza virus, in which budding takes place at the plasma membrane of epithelial cells. Three membrane proteins are embedded in the influenza virus envelope: hemagglutinin (HA),3 which mediates binding of the virus to the host cell and fusion with cell target membrane (1); neuraminidase; and the proton channel M2. The inner viral membrane leaflet is covered by the matrix protein M1, which is supposed to mediate binding of the eight viral RNA-nucleoprotein complexes harboring the genetic information of the virus. Several studies support a role of lipid domains as a platform for enrichment of viral components. HA, the most abundant envelope protein of the influenza virus, has been found to be enriched in detergent-resistant membrane (DRM) fractions (2–4). Typical lipid components of those fractions are saturated phospholipids, glycosphingolipids, and cholesterol, which are known to form liquid-ordered domains (5). This has led to the idea that so-called lipid rafts, which resemble liquid-ordered domains, could function as assembly sites. Support for this hypothesis was given by the observation that the lipid composition of the influenza virus envelope is more similar to that of a raft than to the overall plasma membrane (2, 6).As it has been shown that DRM fractions may not represent the native state of lipid domains, in particular of rafts (7, 8), subsequent efforts have focused on other techniques to assess the lateral organization of HA. Electron microscopy studies using immunogold labeling (4, 9), Förster resonance energy transfer (FRET) measurements between fluorescent HA antibodies in fixed cells (9), and investigations on photoactivatable HA in living cells (10) have revealed cholesterol-sensitive clustering of HA in the plasma membrane of mammalian cells at lengths between 20 and 900 nm.A specific problem encountered in studying the lateral organization of proteins in the plasma membrane is that lipid domains as rafts are typically organized at a submicroscopic level. Indeed, several attempts to image raft domains in biological membranes have suggested that rafts are very small and highly dynamic (11, 12). A guiding study in the characterization of lipid domains in biological membranes has been performed by Mayor and colleagues (13) on the plasma membrane of CHO-K1 cells. Based on homoFRET measurements they have shown that about 20–40% of GFP-tagged glycosylphosphatidylinositol (GPI) (for review see Brown and Rose (14)) and other GPI-anchored proteins are organized with about three to four copies in small cholesterol-sensitive clusters. Mathematical modeling of those experimental data is consistent with a domain diameter of about 5 nm (13).In the present study we investigated the lateral organization of the C terminus of HA, corresponding to the transmembrane domain (TMD), and the cytoplasmic tail (CT) of the protein in the plasma membrane of CHO-K1 cells, taking advantage of the well characterized spatial arrangement of the raft marker GPI in those cells (see above). Lateral organization was studied essentially by fluorescence lifetime imaging microscopy (FLIM)-based FRET between CFP (donor) and YFP (acceptor). For this purpose, we replaced the ectodomain of HA by YFP and studied FRET between this construct and GPI-CFP. As a complementary approach, we performed ensemble measurements in suspensions of plasma membranes purified from cells expressing fluorescent GPI and HA constructs. We measured homoFRET by time-resolved fluorescence anisotropy, providing information on the aggregation/clustering state of the fluorescent constructs, which is important in rationalizing the FLIM-FRET data.