Lys 43 and Asp 46 in α-helix 3 of uteroglobin are essential for its phospholipase A 2 inhibitory activity

Abstract

Uteroglobin (UG) is an anti-inflammatory, secreted protein with soluble phospholipase A 2 (sPLA 2)-inhibitory activity. However, the mechanism by which UG inhibits sPLA 2 activity is unknown. UG is a homodimer in which each of the 70-amino acid subunits forms four α-helices. We previously reported that sPLA 2-inhibitory activity of UG may reside in a segment of α-helix 3 that is exposed to the solvent. In addition, it has been suggested that UG may inhibit sPLA 2 activity by binding and sequestering Ca ++, essential for sPLA 2 activation. By site-specific mutation, we demonstrate here that Lys 43 Glu, Asp 46 Lys or a combination of the two mutations in the full-length, recombinant human UG (rhUG) abrogates its sPLA 2-inhibitory activity. We demonstrate further that recombinant UG does not bind Ca ++ although when it is expressed with histidine-tag (H-tag) it is capable of binding Ca ++. Taken together our results show that: (i) Lys 43 and Asp 46 in rhUG are critical residues for the sPLA 2-inhibitory activity of UG and (ii) Ca ++-sequestration by rhUG is not likely to be one of the mechanisms responsible for its sPLA 2-inhibitory activity.