Colon adenocarcinomas are known to express elevated levels of A2-6 sialylation and increased activity of ST6Gal-I, the Golgi glycosyltransferase that creates A2-6 linkages. Elevated ST6Gal-I positively correlates with metastasis and poor survival, and therefore ST6Gal-I-mediated hypersialylation likely plays a role in colorectal tumor invasion. Previously we found that oncogenic ras (present in roughly 50% of colon adenocarcinomas) up-regulates ST6Gal-I and, in turn, increases sialylation of B 1 integrin adhesion receptors in colon epithelial cells. However, we wanted to know if this pattern held true in vivo and, if so, how B 1 hypersialylation might contribute to colon tumor progression. In the present study, we find that B 1 integrins from colon adenocarcinomas consistently carry higher levels of A2-6 sialic acid. To explore the effects of increased A2-6 sialylation on B 1 -integrin function, we stably expressed ST6Gal-I in a colon epithelial cell line lacking endogenous ST6Gal-I. ST6Gal-I expressors (with A2-6 sialylated B 1 integrins) exhibited up-regulated attachment to collagen I and laminin and increased haptotactic migration toward collagen I, relative to parental cells (with completely unsialylated B 1 integrins). Blockade of ST6Gal-I expression with short interfering RNA reversed collagen binding back to the level of ST6Gal-I nonexpressors, confirming that a2-6 sialylation regulates B 1 integrin function. Finally, we show that B 1 integrins from ST6Gal-I expressors have increased association with talin, a marker for integrin activation. Collectively, these findings suggest that B 1 hypersialylation may augment colon tumor progression by altering cell preference for certain extracellular matrix milieus, as well as by stimulating cell migration. (Cancer Res 2005; 65(11): 4645-52)

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-regulated chloride channel. Mutations in the CFTR gene result in cystic fibrosis (CF). The most common mutation, ∆F508, results in endoplasmic reticulum-associated degradation (ERAD) of CFTR. ∆F508 CFTR has been described as a temperature-sensitive mutation that can be rescued following growth at 27• C. In order to study the processing and function of wild-type and rescued ∆F508 CFTR at the cell surface under non-polarized and polarized conditions, we developed stable cell lines expressing ∆F508 or wild-type CFTR. CFBE41o -is a human airway epithelial cell line capable of forming high resistance, polarized monolayers when cultured on permeable supports, while HeLa cells are normally grown under non-polarizing conditions. Immunoprecipitation, cell surface biotinylation, immunofluorescence, and functional assays confirmed the presence of ∆F508 CFTR at the cell surface in both cell lines after incubating the cells for 48 h at 27• C. However, stimulators of wild-type CFTR such as forskolin, β 2 -adrenergic or A 2B -adenosine receptor agonists failed to activate rescued ∆F508 CFTR in CFBE41o -monolayers. Rescued ∆F508 CFTR could be stimulated with genistein independent of pretreatment with cAMP signalling agonists. Interestingly, rescued ∆F508 CFTR in HeLa cells could be efficiently stimulated with either forskolin or genistein to promote Cl -transport. These results indicate that ∆F508 CFTR, when rescued in CFBE41o -human airway epithelial cells, is poorly responsive to signalling pathways known to regulate wild-type CFTR. Furthermore, the differences in rescue and activation of ∆F508 CFTR in the two cell lines suggest that cell-type specific differences in ∆F508 CFTR processing are likely to complicate efforts to identify potentiators and/or correctors of the ∆F508 defect.

A vaccine that protects against human immunodeficiency virus type 1 (HIV-1) infection should elicit antibodies that bind to the surface envelope glycoproteins on the membrane of the virus. The envelope glycoproteins have an extensive coat of carbohydrates (glycans), some of which are recognized by virus-neutralizing antibodies and some of which protect the virus from neutralizing antibodies. We found that the HIV-1 membrane envelope glycoproteins have a unique pattern of carbohydrates, with many high-mannose glycans and also, in some places, complex glycans. This pattern was very different from the carbohydrate profile seen for a more easily produced soluble version of the envelope glycoprotein. Our results provide a detailed characterization of the glycans on the natural membrane envelope glycoproteins of HIV-1, a carbohydrate profile that would be desirable to mimic with a vaccine.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent protein kinase A-activated chloride channel that resides on the apical surface of epithelial cells. One unusual feature of this protein is that during biogenesis, ϳ75% of wild type CFTR is degraded by the endoplasmic reticulum (ER)-associated degradative (ERAD) pathway. Examining the biogenesis and structural instability of the molecule has been technically challenging due to the limited amount of CFTR expressed in epithelia. Consequently, investigators have employed heterologous overexpression systems. Based on recent results that epithelial specific factors regulate both CFTR biogenesis and function, we hypothesized that CFTR biogenesis in endogenous CFTR expressing epithelial cells may be more efficient. To test this, we compared CFTR biogenesis in two epithelial cell lines endogenously expressing CFTR (Calu-3 and T84) with two heterologous expression systems (COS-7 and HeLa). Consistent with previous reports, 20 and 35% of the newly synthesized CFTR were converted to maturely glycosylated CFTR in COS-7 and HeLa cells, respectively. In contrast, CFTR maturation was virtually 100% efficient in Calu-3 and T84 cells. Furthermore, inhibition of the proteasome had no effect on CFTR biogenesis in Calu-3 cells, whereas it stabilized the immature form of CFTR in HeLa cells. Quantitative reverse transcriptase-PCR indicated that CFTR message levels are ϳ4-fold lower in Calu-3 than HeLa cells, yet steady-state protein levels are comparable. Our results question the structural instability model of wild type CFTR and indicate that epithelial cells endogenously expressing CFTR efficiently process this protein to post-Golgi compartments.

The viral accessory protein Vpx is required for productive in vitro infection of macrophages by simian immunodeficiency virus from sooty mangabey monkeys (SIV(SM)). To evaluate the roles of Vpx and macrophage infection in vivo, we inoculated pigtailed macaques intravenously or intrarectally with the molecularly cloned, macrophage tropic, acutely pathogenic virus SIV(SM) PBj 6.6, or accessory gene deletion mutants (deltaVpr or deltaVpx) of this virus. Both wild-type and SIV(SM) PBj deltaVpx viruses were readily transmitted across the rectal mucosa. A subsequent 'stepwise' process of local amplification of infection and dissemination was observed for wild-type virus, but not for SIV(SM) PBj deltaVpx, which also showed considerable impairment of the overall kinetics and extent of its replication. In animals co-inoculated with equivalent amounts of wild-type and SIV(SM) Pbj deltaVpx intravenously or intrarectally, the deltaVpx mutant was at a strong competitive disadvantage. Vpx-dependent viral amplification at local sites of initial infection, perhaps through a macrophage-dependent mechanism, may be a prerequisite for efficient dissemination of infection and pathogenic consequences after exposure through either mucosal or intravenous routes.