Interactions of calmodulin with metal ions and with its target proteins revealed by conformation-sensitive monoclonal antibodies

Abstract

Two monoclonal antibodies (mAbs) raised against bovine calmodulin (CaM), CAM1 and CAM4, enable one to monitor conformational changes that occur in the molecule. The interaction of CAM1 with CaM depends on the Ca2+ occupancy of its Ca(2+)-binding sites. CAM4, in contrast, interacts with CaM in a Ca(2+)-independent manner, interacting with both holoCaM and EGTA-treated CaM to a similar extent. Their interaction with various CaMs, CaM tryptic fragments and chemically modified CaM, as well as molecular graphics, led to identification of the CAM1 and CAM4 epitopes on the C- and N-terminal lobes of CAM respectively. The two mAbs were used as macromolecular probes to detect conformational changes occurring in the CaM molecule upon binding of metal ions and target proteins and peptides. MAb CAM1 successfully detected changes associated with Al3+ binding even in the presence of Ca2+, indicating that Al3+ and Ca2+ ions may bind to the protein simultaneously, leading to a new conformation of the molecule. MAbs CAM1 and CAM4 were used to follow the interactions of CaM with its target peptides and proteins. Complexes with melittin, mastoparan, calcineurin and phosphodiesterase showed different immunological properties on an immuno-enzyme electrode, indicating unique structural properties for each complex.

Aluminum Disruption of Calcium Homeostasis and Signal Transduction Resembles Change that Occurs in Aging and Alzheimer's Disease

“…One article [160] has disputed the original report [161] that Al binds to spin-labeled CaM. However, Al-CaM interactions have since been confirmed by many techniques including the use of steady-state and timedependent fluorescence spectroscopy, circular dicroic spectroscopy, equilibrium dialysis, electron paramagnetic resonance, heteronuclear 2-dimensional NMR, surface plasma resonance, calorimetry, monoclonal antibody (non)recognition and by chelation studies using citric acid or desferrioxamine B which can both chelate Al and reverse the effects of Al on CaM conformational change that occurs with Al exposure [157,159,[162][163][164][165][166][167][168][169]. These studies and others have helped to define conditions under which Al binds to CaM, and how Al interferes with signal transduction.…”

“…The monoclonal antibody CAM-4, which normally recognizes CaM with or without bound Ca 2+ , recognizes epitopes on the terminal lobes. In the presence of Al, CAM-4 recognises CaM complexes bound to some target proteins (e.g., calcineurin), partially recognizes CaM-binding to other target proteins (e.g., phosphodiesterase), and shows little recognition, if any, of CaM-binding to yet other proteins (e.g., mastoparan) [162].…”

A citrate-binding site in calmodulin

“…CAM1 and CAM4 are two conformation-dependent mAbs produced in our laboratory (Wolf et al, 1995). Interaction of CaM with mAb CAM1, which recognizes an epitope on the C-terminal lobe of the protein, was shown to be highly dependent on Ca 2 presence in the molecule (Wolf et al, 1998). Thus, this antibody interacts with holoCaM even after extensive dialysis against doubly distilled water, but not with the EGTA-formed apoCaM.…”

Chronic Aluminum Intake Causes Alzheimer's Disease: Applying Sir Austin Bradford Hill's Causality Criteria

“…(2) Al 3+ disrupts the phosphoinositide and c-AMP signaling pathways and their downstream effects on PKC activity [270,[409][410][411][412][413]. (3) Al 3+ impairs Ca 2+ signaling [313,408,[414][415][416][417][418][419][420][421] and calmodulin-mediated signal transduction [23,178,[422][423][424][425][426][427][428][429][430]. Al 3+ interferes with Ca 2+ signaling by restricting both inositol triphosphate-and caffeine-evoked Ca 2+ release from endoplasmic reticulum stores [313].…”