alpha(2)Macroglobulin: Kinetic analysis of nonproteolytic antigen incorporation and characterization of an analog in rabbits.

摘要

Human α₂-macroglobulin (α₂M) is a 720 kDa tetrameric glycoprotein that inhibits proteinases of all classes and specificities through a unique mechanism involving both steric entrapment and covalent binding. Though classically known as a proteinase inhibitor, α ₂M has also been implicated in the regulation of the immune system and has been identified as a major serum carrier of cytokines and growth factors. α ₂M facilitates in vitro receptor-mediated delivery of antigen to macrophages, enhance antigen presentation to T cells 100- to 1000-fold, and amplify the adaptive immune response. These properties likely explain α₂M's potency as a vaccine adjuvant. Although α ₂M conventionally reacts with and binds to proteinases, nonproteolytic proteins can be covalently incorporated into α₂M with the addition of exogenous energy to the activated form (α₂M*). This dissertation examines the reaction kinetics of the nonproteolytic incorporation of ligands into α₂M and provides greater insight into the mechanism of this reaction. Additionally, this dissertation characterizes rabbit almacroglobulin, compares its similarity to human α₂M, and evaluates the validity of predicting the effect of α₂M on human immune responses based on experimental models in rabbits.; To characterize the mechanism of nonproteolytic ligand incorporation into receptor-recognized α₂-macroglobulin (α₂ M*), we analyzed the chemical kinetics, thermodynamics, and pH-dependence of the incorporation of hen egg lysozyme (HEL) into α₂M*. The reaction was a mixed bimolecular reaction following second-order kinetics with a specific rate constant of 91.0 ± 6.9 M−1 s−1 at 50.0°C, pH 7.4. From the Arrhenius plot of the specific rate constants at temperatures from 47.5°C to 55.0°C, an activation energy of 156 kJ mol−1, an activation entropy of 266 J mol−1 K−1, and a Gibbs' free energy of 70 kJ mol−1 at 50.0°C were determined. The rate of incorporation increased as a function of pH from pH 5.0 to 7.0 and was unchanged from pH 7.0 to 9.0. In contrast to the reaction between α ₂M and NH₃, the reaction between α₂M and HEL could not be reversed by heating at 50.0°C for 180 min. The data are consistent with a two-step mechanism of incorporation. In the first step, α ₂M* reforms its thiolester bond, entering a reactive state which mimics the proteolytically-induced nascent state. The reformed bond is quickly subjected to nucleophilic attack by HEL in the rate-limiting second step. The irreversibility of HEL incorporation suggests that α₂M*-ligand complexes are stabilized through protein-protein interactions between the ligand and the α₂M* cage. (Abstract shortened by UMI.)