Functional effects of coxsackievirus and adenovirus receptor glycosylation on homophilic adhesion and adenoviral infection.

The coxsackievirus and adenovirus receptor (CAR) is both a viral receptor and homophilic adhesion protein. The extracellular portion of CAR consists of two immunoglobulin (Ig)-like domains, each with a consensus sequence for N-glycosylation. We used chemical, genetic, and biochemical studies to show that both sites are glycosylated and contribute to the function of CAR. Although the glycosylation of CAR does not alter cell surface levels or junctional localization, it affects both adhesion and adenovirus infection in unique ways. CAR-mediated adhesion appears to require at least one site of glycosylation since cells expressing CAR without glycosylation do not cluster with each other. In contrast, glycosylation of the Ig-like domain proximal to the membrane is key to the cooperative behavior of adenovirus binding and infection. Contrary to the hypothesis that cooperativity improves viral infection, our data show that although glycosylation of the D2 domain is required for adenovirus cooperative binding, it has a negative consequence upon infection. This is the first report dissecting the adhesion and receptor activities of CAR, revealing that factors other than the binding interface play a significant role in the function of CAR. These data have important implications for both cancers with altered glycosylation states and cancer treatments using oncolytic adenovirus.

Complete rescue of lipoprotein lipase-deficient mice by somatic gene transfer of the naturally occurring LPLS447X beneficial mutation.

The naturally occurring human lipoprotein lipase S447X variant (LPLS447X) exemplifies a gain-of function mutation with significant benefits including decreased plasma triglycerides (TG), increased high-density lipoprotein (HDL) cholesterol, and reduced risk of coronary artery disease. The S447X variant may be associated with higher LPL catalytic activity; however, in vitro data supporting this hypothesis are contradictory. We wanted to investigate the in vivo mechanism by which the LPLS447X variant improves the lipid profile of S447X carriers. We conducted a functional assessment of human LPLS447X compared with LPLWT in mice. LPL variants were compared in the absence of endogenous mouse LPL in newborn LPL(-/-) mice by adenoviral-mediated gene transfer. LPL(-/-) mice normally exhibit severe hypertriglyceridemia and die within 48 hours of birth. LPLWT gene transfer prolonged the survival of mice up to 21 days. In contrast, LPLS447X completely rescued 95% of the mice to adulthood and increased LPL catalytic activity in postheparin plasma 2.1-fold compared with LPLWT at day 3 (P=0.003). LPLS447X also reduced plasma TG 99% from baseline (P<0.001), 2-fold more than LPLWT, (P<0.01) and increased plasma HDL cholesterol 2.9-fold higher than LPLWT (P<0.01). These data provide in vivo evidence that the increased catalytic activity of LPLS447X improves plasma TG clearance and increases the HDL cholesterol pool compared with LPLWT.

A role for the PDZ-binding domain of the coxsackie B virus and adenovirus receptor (CAR) in cell adhesion and growth.

The coxsackie and adenovirus receptor (CAR) plays a role in viral infection, maintenance of the junction adhesion complex in polarized epithelia, and modulation of cellular growth properties. As a viral receptor, the C-terminus appears to play no role indicating that the major function of CAR is to tether the virus to the cell. By contrast, the C-terminus is known to play a role in cellular localization and probably has a significant function in CAR-mediated adhesion and cell growth properties. We hypothesized that the CAR PDZ (PSD-95/Disc-large/ZO-1) binding motif interacts with PDZ-domain-containing proteins to modulate the cellular phenotype. CAR was modified by deleting the last four amino acids (CARDeltaGSIV) and evaluated for cell-cell adhesion in polarized primary human airway epithelia and growth characteristics in stably transfected L-cells. Although ablation of the CAR PDZ-binding motif did not affect adenoviral infection, it did have a significant effect both on cell-cell adhesion and on cell growth. Expression of CARDeltaGSIV failed to increase the transepithelial resistance in polarized epithelia to the same degree as wild-type CAR and failed to act as a growth modulator in L-cells. Furthermore, we provide evidence for three new CAR interacting partners, including MAGI-1b, PICK1 and PSD-95. CAR appears to interact with several distinct PDZ-domain-containing proteins and may exert its biological function through these interactions.

Histamine alters E-cadherin cell adhesion to increase human airway epithelial permeability.

During the immediate response to an inhaled allergen, there is an increase in the paracellular permeability of the airway epithelium.1 Histamine is an important agonist released during the immediate response to inhaled allergen. We hypothesized that histamine would increase human airway epithelial paracellular permeability and that it would do this by interrupting E-cadherin-based cell adhesion. Histamine, applied to the basolateral surface, increased the paracellular permeability of cultured human airway epithelia, and this effect of histamine was blocked by the histamine receptor antagonist promethazine. ECV304 cells express a histamine receptor, N-cadherin, and elements of the tight junction, including claudins, but they do not express E-cadherin. Histamine increased the paracellular permeability of ECV304 cells transfected with a vector and expressing E-cadherin but not ECV304 cells expressing lac-Z in the same vector. L cells do not express the histamine receptor, cadherins, or claudins. Histamine decreased adhesion of L cells expressing the human histamine receptor and E-cadherin to an E-cadherin-Fc fusion protein. Histamine did not alter the adhesion to the E-cadherin fusion protein of L cells expressing either the histamine receptor or E-cadherin alone. When applied to the apical surface, adenovirus poorly infects airway epithelial cells because its receptor, CAR, is restricted to the basolateral surface of the cells. When histamine was applied to the basolateral surface of airway epithelial cells, infection of the cells by adenovirus increased by approximately one log. This effect of histamine was also blocked by promethazine. Histamine increases airway paracellular permeability and increases susceptibility of airway epithelial cells to infection by adenovirus by interrupting E-cadherin adhesion.