Lys 43 and Asp 46 in α-helix 3 of uteroglobin are essential for its phospholipase A2 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 a-helices. We previously reported that sPLA 2 -inhibitory activity of UG may reside in a segment of a-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 þþ , 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 þþ although when it is expressed with histidine-tag (H-tag) it is capable of binding Ca þþ . 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 þþ -sequestration by rhUG is not likely to be one of the mechanisms responsible for its sPLA 2 -inhibitory activity. Ó 2002 Elsevier Science (USA). All rights reserved.

Structural Characterization of the Tetrameric form of the Major Cat Allergen Fel d 1

“…Interestingly Chowdhury et al demonstrated that residues Lys43 and Asp46 in the α-helix 3 of uteroglobin are essential for its PLA 2 -inhibitory activity. 29,31 The independent or simultaneous mutation of Lys43 and Asp46 to a glutamate and a lysine, respectively, resulted in the alteration of the capacity of uteroglobin to inhibit PLA 2 . 31 Molecular modeling of the substitution of Asp46 to a lysine in the crystal structure of Fel d 1 suggested that this residue confers the right conformation to the calcium-binding site (data not included).…”

“…29, 30 Chowdhury et al thereafter assessed the critical role of residues Lys43 and Asp46 of uteroglobin for its PLA 2 -inhibitory activity. 31 Modulation of PLA 2 may affect the biosynthesis of eicosanoids and platelet-activating factors, potent mediators of inflammatory and allergic responses. 32 Uteroglobin only inhibits the function of the enzyme PLA 2 when the concentration of Ca 2+ , an essential cofactor for PLA 2 , becomes limiting.…”

“…34 However, the potential binding of Ca 2+ to uteroglobin has been the subject of some controversy, since some groups demonstrated that Ca 2+ was bound by uteroglobin [33][34][35] while others showed the opposite. 31 Although important information was obtained from the crystal structure of Fel d 1 (2 + 1), no tetramer could be identified. 14 Here we have solved the crystal structure of the tetrameric form of Fel d 1 at 1.6 Å resolution, using a Fel d 1 (1 + 2) construct.…”

Stability and Translation of TCR ζ mRNA Are Regulated by the Adenosine-Uridine-Rich Elements in Splice-Deleted 3′ Untranslated Region of ζ-Chain

“…Equal amounts (20 g) of the total protein derived from cell lysate of each sample were loaded in the gel and resolved by electrophoresis using 4 -12% bis-Tris NuPage gel (Invitrogen Life Technologies) under denaturing and reducing conditions and the proteins transferred onto polyvinylidene difluoride membranes (Amersham Biosciences) (36). The immunoblot analysis was then conducted using specific Abs against TCR (clone 6B10.2).…”

Inhibition of lipopolysaccharide‐mediated rat alveolar macrophage activation in vitro by antiflammin‐1

“…This peptide corresponds to the nine C-terminal amino acids of ahelix 3 of CC10 (Chowdhury et al, 2002). It has been implicated in a broad range of cellular functions, including potent PLA2 inhibition, anti-inflammatory activities and regulating the expression of adhesion molecules on human leukocytes (Miele et al, 1990;Miele, 2000;Zouki et al, 2000).…”