Ion Binding Properties of a Naturally Occurring Metalloantibody

LT1009 is a humanized version of murine LT1002 IgG1 that employs two bridging Ca(2+) ions to bind its antigen, the biologically active lipid sphingosine-1-phosphate (S1P). We crystallized and determined the X-ray crystal structure of the LT1009 Fab fragment in 10 mM CaCl(2) and found that it binds two Ca(2+) in a manner similar to its antigen-bound state. Flame atomic absorption spectroscopy (FAAS) confirmed that murine LT1002 also binds Ca(2+) in solution and inductively-coupled plasma-mass spectrometry (ICP-MS) revealed that, although Ca(2+) is preferred, LT1002 can bind Mg(2+) and, to much lesser extent, Ba(2+). Isothermal titration calorimetry (ITC) indicated that LT1002 binds two Ca(2+) ions endothermically with a measured dissociation constant (K(D)) of 171 μM. Protein and genome sequence analyses suggested that LT1002 is representative of a small class of confirmed and potential metalloantibodies and that Ca(2+) binding is likely encoded for in germline variable chain genes. To test this hypothesis, we engineered, expressed, and purified a Fab fragment consisting of naïve murine germline-encoded light and heavy chain genes from which LT1002 is derived and observed that it binds Ca(2+) in solution. We propose that LT1002 is representative of a class of naturally occurring metalloantibodies that are evolutionarily conserved across diverse mammalian genomes.