The role of ß2-glycoprotein I (ß2GPI) carbohydrate chains in the reactivity of anti-ß2GPI antibodies from patients with primary antiphospholipid syndrome and in the activation and differentiation of U937 cells.

Several studies have shown that conformational changes of ß(2)-glycoprotein I (ß(2)GPI) when bound to negatively charged components expose cryptic epitopes and subsequent binding of anti-ß(2)GPI from patients with antiphospholipid syndrome (APS). However, the role of the carbohydrate chains of ß(2)GPI in this anti-ß(2)GPI reactivity is poorly understood. We therefore studied the reactivity and inhibition of anti-ß(2)GPI antibodies from APS patients with native, partially glycosylated ß(2)GPI (pdß(2)GPI; without sialic acid) and completely deglycosylated ß(2)GPI (cdß(2)GPI). To determine the potential biologic importance of these glycoforms and their interaction with anti-ß(2)GPI in vitro, stimulation assays were performed with the U937 cell line. Circular dichroism (CD) and fluorescence analysis of the three ß(2)GPI forms were also studied. We found an increased reactivity of anti-ß(2)GPI against pdß(2)GPI and cdß(2)GPI compared to native ß(2)GPI. Both deglycosylated ß(2)GPI isoforms showed higher inhibition of the anti-ß(2)GPI reactivity than the native protein in soluble-phase. Likewise, the antibody/ß(2)GPI/glycoform complexes increased the synthesis of IL-6, IFNγ and TNFα and the expression of HLA-DR, CD14 and CD11c in U937 cells. CD and fluorescence studies of the glycoforms yielded considerable changes in the fluorescence signals. Our work suggests that the partial or complete removal of the carbohydrate chains uncover cryptic epitopes present in ß(2)GPI. The differentiation and increased synthesis of pro-inflammatory cytokines by U937 cells in vitro may have pathogenetic implications.

Heat capacity changes in carbohydrates and protein-carbohydrate complexes.

Carbohydrates are crucial for living cells, playing myriads of functional roles that range from being structural or energy-storage devices to molecular labels that, through non-covalent interaction with proteins, impart exquisite selectivity in processes such as molecular trafficking and cellular recognition. The molecular bases that govern the recognition between carbohydrates and proteins have not been fully understood yet. In the present study, we have obtained a surface-area-based model for the formation heat capacity of protein-carbohydrate complexes, which includes separate terms for the contributions of the two molecular types. The carbohydrate model, which was calibrated using carbohydrate dissolution data, indicates that the heat capacity contribution of a given group surface depends on its position in the saccharide molecule, a picture that is consistent with previous experimental and theoretical studies showing that the high abundance of hydroxy groups in carbohydrates yields particular solvation properties. This model was used to estimate the carbohydrate's contribution in the formation of a protein-carbohydrate complex, which in turn was used to obtain the heat capacity change associated with the protein's binding site. The model is able to account for protein-carbohydrate complexes that cannot be explained using a previous model that only considered the overall contribution of polar and apolar groups, while allowing a more detailed dissection of the elementary contributions that give rise to the formation heat capacity effects of these adducts.

Multithermal titration calorimetry: a rapid method to determine binding heat capacities.

Herein a new method that allows binding DeltaCp to be determined with a single experiment is presented. Multithermal titration calorimetry (MTC) is a simple extension of isothermal titration calorimetry (ITC) that explicitly takes into account the thermal dependences of DeltaH and the binding constant. Experimentally, this is accomplished by performing a single stepwise titration with ITC equipment, allowing temperature re-adjustments of the system at intermediate states of the titration process. Thus, from the resulting multitherm, DeltaCp can also be determined. The experimental feasibility of MTC was tested by using the well-characterized lysozyme-chitotriose complex as a model system.