Cholesterol present in the plasma membrane of target cells has been shown to be important for the infection by SARS-CoV. We show that cholesterol depletion by treatment with methyl-beta-cyclodextrin (m beta CD) affects infection by SARS-CoV to the same extent as infection by vesicular stomatitis virus-based pseudotypes containing the surface glycoprotein S of SARS-CoV (VSV-Delta G-S). Therefore, the role of cholesterol for SARS-CoV infection can be assigned to the S protein and is unaffected by other coronavirus proteins. There have been contradictory reports whether or not angiotensin-converting enzyme 2 (ACE2), the cellular receptor for SARS-CoV, is present in detergent-resistant membrane domains. We found that ACE2 of both Vero E6 and Caco-2 cells co-purifies with marker proteins of detergent-resistant membranes supporting the notion that cholesterol-rich microdomains provide a platform facilitating the efficient interaction of the S protein with the cellular receptor ACE2. To understand the involvement of cholesterol in the initial steps of the viral life cycle, we applied a cell-based binding assay with cells expressing the S protein and cells containing angiotensin-converting enzyme 2 (ACE2). Alternatively, we used a soluble S protein as interaction partner. Depletion of cholesterol from the ACE2-expressing cells reduced the binding of S-expressing cells by 50% whereas the binding of soluble S protein was not affected. This result suggests that optimal infection requires a multivalent interaction between viral attachment protein and cellular receptors.

The plasma membrane of polarised epithelial cells is characterised by two structurally and functionally different domains, the apical and basolateral domains. These domains contain distinct protein and lipid constituents that are sorted by specific signals to the correct surface domain [1]. The best characterised apical sorting signal is that of glycophosphatidylinositol (GPI) membrane anchors [2], although N-linked glycans on some secreted proteins [3] and O-linked glycans [4] also function as apical sorting signals. In the latter cases, however, the underlying sorting mechanisms remain obscure. Here, we have analysed the role of O-glycosylation in the apical sorting of sucrase-isomaltase (SI), a highly polarised N- and O-glycosylated intestinal enzyme, and the mechanisms underlying this process. Inhibition of O-glycosylation by benzyl-N-acetyl-alpha-D-galactosaminide (benzyl-GalNAc) was accompanied by a dramatic shift in the sorting of SI from the apical membrane to both membranes. The sorting mechanism of SI involves its association with sphingolipid- and cholesterol-rich membrane rafts because this association was eliminated when O-glycosylation was inhibited by benzyl-GaINAc. The results demonstrate for the first time that O-linked glycans mediate apical sorting through association with lipid rafts.

The primary target of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is epithelial cells in the respiratory and intestinal tract. The cellular receptor for SARS-CoV, angiotensin-converting enzyme 2 (ACE2), has been shown to be localized on the apical plasma membrane of polarized respiratory epithelial cells and to mediate infection from the apical side of these cells. Here, these results were confirmed and extended by including a colon carcinoma cell line (Caco-2), a lung carcinoma cell line (Calu-3) and Vero E6 cells in our analysis. All three cell types expressed human ACE2 on the apical membrane domain and were infected via this route, as determined with vesicular stomatitis virus pseudotypes containing the S protein of SARS-CoV. In a histological analysis of the respiratory tract, ACE2 was detected in the trachea, main bronchus and alveoli, and occasionally also in the small bronchi. These data will help us to understand the pathogenesis of SARS-CoV infection.Epithelia are a primary barrier to infection by microorganisms entering their host via body cavities such as the respiratory or intestinal tract (reviewed by Compans & Herrler, 2005). Epithelial cells are organized in a polarized fashion that involves the separation of the plasma membrane into an apical and a basolateral domain. The polarity of these cells affects both the early and late stages of infection, i.e. viruses may enter into and exit from a cell either via the apical membrane facing the external environment or via the basolateral membrane directed to the internal milieu of the organism. An important determinant of the virus infection is the presence of suitable receptors on the cell surface that allow attachment to and penetration through the plasma membrane. For viruses entering their host via the respiratory or gastrointestinal route, infection is understood most easily when the virus receptor is expressed on the apical surface.The primary target of the coronavirus associated with severe acute respiratory syndrome (SARS-CoV) is the respiratory tract. In addition to respiratory complications, some patients show intestinal symptoms, indicating that not only the respiratory but also the intestinal epithelium is susceptible to infection. It has been shown recently that the receptor for SARS-CoV, angiotensin-converting enzyme 2 (ACE2; Li et al., 2003;Wang et al., 2004), is localized on and mediates infection through the apical plasma membrane of respiratory epithelial cells (Jia et al., 2005;Sims et al., 2005; Tseng et al., 2005). On the other hand, ACE2 has been reported to be absent from enterocytes of the colon (Hamming et al., 2004), despite active replication of SARS-CoV in this portion of the intestine (Leung et al., 2003).To determine whether epithelial cells of different origin differ in the expression of ACE2, we included in our analysis three cell lines that form a highly polarized epithelial monolayer when grown on microporous filters: (i) Calu-3 (human lung carcinoma cells), (ii) Caco-2 (human colon carcinoma ce...

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Members of the chloride channels, calcium-activated (CLCA) family of proteins and in particular the murine mCLCA3 (alias gob-5) and its human ortholog hCLCA1 have been identified as clinically relevant molecules in diseases with secretory dysfunctions including asthma and cystic fibrosis. Initial studies have indicated that these proteins evoke a calcium-activated chloride conductance when transfected into human embryonic kidney cells 293 cells. However, it is not yet clear whether the CLCA proteins form chloride channels per se or function as mediators of other, yet unknown chloride channels. Here, we present a systematic biochemical analysis of the posttranslational processing and intracellular trafficking of the mCLCA3 protein.Pulse-chase experiments after metabolic protein labeling of mCLCA3-transfected COS-1 or human embryonic kidney 293 cells revealed cleavage of a primary 110-kDa mCLCA3 translation product in the endoplasmic reticulum into a 75-kDa amino-terminal and a 35-kDa carboxyl-terminal protein that were glycosylated and remained physically associated with each other. Confocal fluorescent analyses identified both cleavage products in vesicles of the secretory pathway. Neither cleavage product was associated with the cell membrane at any time. Instead, both subunits were fully secreted into the extracellular environment as a soluble complex of two glycoproteins. These results suggest that the two mCLCA3 cleavage products cannot form an anion channel on their own but may instead act as extracellular signaling molecules. Furthermore, our results point toward significant structural differences between mCLCA3 and its human ortholog, hCLCA1, which is thought to be a single, non-integral membrane protein.Several members of the CLCA 2 gene family have been shown to mediate an anion current that is activated by intracellular calcium. Electrophysiological data derived from whole-cell or single-channel patch clamp analyses of HEK 293 or COS-1 cells heterologously transfected with different CLCA homologues consistently identified a transmembrane current that was activated by calcium ionophores including ionomycin. These currents were blocked by several chloride channel blockers including 4,4Ј-diisothiocyanatostilbene-2,2Ј-disulfonic acid (1, 2-4, 6, 8, 12-16, 18 -21). Hence, the gene products have been tentatively designated as chloride channels, calcium-activated (1).At present, 15 CLCA members are known in 6 mammalian species. These include four human homologues (hCLCA1 (2, 3), hCLCA2 (4, 5), hCLCA3 (6), and hCLCA4 alias hCaCC2 (3)), six murine homologues (mCLCA1 (6, 7), mCLCA2 (8), mCLCA3 alias gob-5 (9 -11), mCLCA4 (13), mCLCA5, and mCLCA6 (14)), two bovine members (bCLCA1 alias CaCC (15, 16) and bCLCA2 alias Lu-ECAM-1 (17, 18)), one porcine member (pCLCA1 (19)), one equine member (eCLCA1 (20)), and one rat homologue (21).Initial protein analyses have identified for the majority of CLCA members a consensus protein model of a 100-kDa primary translation product that contains an amino-terminal signal peptid...

The distinct protein and lipid constituents of the apical and basolateral membranes in polarized cells are sorted by specific signals. O-Glycosylation of a highly polarized intestinal brush-border protein sucrase isomaltase is implicated in its apical sorting through interaction with sphingolipid-cholesterol microdomains. We characterized the structural determinants required for this mechanism by focusing on two major domains in pro-SI, the membrane anchor and the Ser/Thr-rich stalk domain. Deletion mutants lacking either domain, pro-SI ⌬ST (stalk-free) and pro-SI ⌬MA (membrane anchorfree), were constructed and expressed in polarized Madin-Darby canine kidney cells. In the absence of the membrane anchoring domain, pro-SI ⌬MA does not associate with lipid rafts and the mutant is randomly delivered to both membranes. Therefore, the O-glycosylated stalk region is not sufficient per se for the high fidelity of apical sorting of pro-SI. Pro-SI ⌬ST does not associate either with lipid rafts and its targeting behavior is similar to that of pro-SI ⌬MA . Only wild type pro-SI containing both determinants, the stalk region and membrane anchor, associates with lipid microdomains and is targeted correctly to the apical membrane. However, not all sequences in the stalk region are required for apical sorting. Only O-glycosylation of a stretch of 12 amino acids (Ala 37 -Pro 48 ) juxtapose the membrane anchor is required in conjunction with the membrane anchoring domain for correct targeting of pro-SI to the apical membrane. Other O-glycosylated domains within the stalk (Ala 49 -Pro 57 ) are not sufficient for apical sorting. We conclude that the recognition signal for apical sorting of pro-SI comprises O-glycosylation of the Ala 37 -Pro 48 stretch and requires the presence of the membrane anchoring domain.

The temporal association between O-glycosylation and processing of N-linked glycans in the Golgi apparatus as well as the implication of these events in the polarized sorting of three brush border proteins has been the subject of the current investigation. O-Glycosylation of pro-sucrase-isomaltase (pro-SI),