Jeff L. Ellsworth

Phosphatidylcholine Hydrolysis Is Required for Pancreatic Cholesterol Esterase- and Phospholipase A2-facilitated Cholesterol Uptake into Intestinal Caco-2 Cells

Pancreatic secretion is required for efficient cholesterol absorption by the intestine, but the factors responsible for this effect have not been clearly defined. To identify factors involved and to investigate their role in cholesterol uptake, we studied the effect of Viokase®, a porcine pancreatic extract, on cholesterol uptake into human intestinal Caco-2 cells. Viokase is capable of facilitating cholesterol uptake into these cells such that the level of uptake is 5-fold higher in the presence of solubilized Viokase. This stimulation is time-dependent and is dependent on the presence of bile salt. However, bile salt-stimulated pancreatic cholesterol esterase, which has been proposed to mediate cholesterol uptake, is not fully responsible. The major cholesterol transport activity was purified and identified as pancreatic phospholipase A2. Anti-phospholipase A2 antibodies abolished virtually all of the phospholipase A2 and cholesterol transport activity of solubilized Viokase. We demonstrate that both phospholipase A2 and cholesterol esterase increase cholesterol uptake by hydrolyzing the phosphatidylcholine that is used to prepare the cholesterol-containing micelles. In the absence of cholesterol esterase or phospholipase A2, uptake of cholesterol from micelles containing phosphatidylcholine is not as efficient as uptake from micelles containing phospholipase A2-hydrolytic products. These results indicate that phospholipase A2 may mediate cholesterol absorption by altering the physical-chemical state of cholesterol within the intestine.

Crystal Structure of Fcγ Receptor I and Its Implication in High Affinity γ-Immunoglobulin Binding

Background: FcγRI plays important roles in antibody functions. Results: We report the first crystal structure of the extracellular human FcγRI. Conclusion: The receptor D3 domain is positioned away from the IgG binding site, and its shorter D2 domain FG-loop is important for its high affinity. Significance: This work provides insights to the mechanism of FcγRI function and helps to design therapeutic reagents. Fcγ receptors (FcγRs) play critical roles in humoral and cellular immune responses through interactions with the Fc region of immunoglobulin G (IgG). Among them, FcγRI is the only high affinity receptor for IgG and thus is a potential target for immunotherapy. Here we report the first crystal structure of an FcγRI with all three extracellular Ig-like domains (designated as D1, D2, and D3). The structure shows that, first, FcγRI has an acute D1-D2 hinge angle similar to that of FcϵRI but much smaller than those observed in the low affinity Fcγ receptors. Second, the D3 domain of FcγRI is positioned away from the putative IgG binding site on the receptor and is thus unlikely to make direct contacts with Fc. Third, the replacement of FcγRIII FG-loop (171LVGSKNV177) with that of FcγRI (171MGKHRY176) resulted in a 15-fold increase in IgG1 binding affinity, whereas a valine insertion in the FcγRI FG-loop (171MVGKHRY177) abolished the affinity enhancement. Thus, the FcγRI FG-loop with its conserved one-residue deletion is critical to the high affinity IgG binding. The structural results support FcγRI binding to IgG in a similar mode as its low affinity counterparts. Taken together, our study suggests a molecular mechanism for the high affinity IgG recognition by FcγRI and provides a structural basis for understanding its physiological function and its therapeutic implication in treating autoimmune diseases.

Inhibition of acyl coenzyme A: Cholesterol acyltransferase blocks esterification but not uptake of cholesterol in Caco-2 cells

The effects of cholesterol esterase (CEase) and acyl coenzyme A:cholesterol acyltransferase (ACAT) inhibitors on the uptake and esterification of cholesterol in Caco-2 cells were examined. CEase increased the uptake of [3H]cholesterol from bile salt mixed-micelles by 2.5- to 3.0-fold and its esterification by greater than 25-fold. Inhibition of cellular ACAT activity with CL277082 or CP113818 had little or no effect on cholesterol uptake measured in the presence or absence of CEase. The subsequent esterification of [3H]cholesterol was reduced greater than 90% by each ACAT inhibitor. Similar results were obtained in cells in which ACAT activity was induced by preincubation either with 25-hydroxycholesterol and mevalonic acid or with CEase and bile salt mixed-micelles containing 100 micromol/L cholesterol. Neither ACAT inhibitor had an effect on CEase-mediated synthesis or hydrolysis of cholesteryl oleate in vitro. Thus, the uptake of cholesterol from bile salt mixed-micelles in the presence or absence of CEase was not regulated by the level of cellular ACAT expression. The subsequent esterification of exogenous sterol was not due to CEase, but was completely dependent on ACAT activity. The dissociation of cholesterol uptake from ACAT activity suggests that the factors controlling the transfer of sterol from extracellular media to the cell are different from the factors regulating the cellular level of cholesterol esterification.