Lys 43 and Asp 46 in alpha-helix 3 of uteroglobin are essential for its phospholipase A2 inhibitory activity.
Uteroglobin (UG) is an anti-inflammatory, secreted protein with soluble phospholipase A2 (sPLA2)-inhibitory activity. However, the mechanism by which UG inhibits sPLA2 activity is unknown. UG is a homodimer in which each of the 70-amino acid subunits forms four alpha-helices. We previously reported that sPLA2-inhibitory activity of UG may reside in a segment of alpha-helix 3 that is exposed to the solvent. In addition, it has been suggested that UG may inhibit sPLA2 activity by binding and sequestering Ca++, essential for sPLA2 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 sPLA2-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 sPLA2-inhibitory activity of UG and (ii) Ca++-sequestration by rhUG is not likely to be one of the mechanisms responsible for its sPLA2-inhibitory activity.
Section on Developmental Genetics, Heritable Disorders Branch, NICHD, Bethesda, MD 20892-1830, USA.
Bhabadeb
B
Chowdhury
Giuditta
G
Mantile-Selvaggi
Lucio
L
Miele
Eleonora
E
Cordella-Miele
Zhongjian
Z
Zhang
Anil B
AB
Mukherjee
D001224:Aspartic Acid / Q000737:chemistry; D002118:Calcium / Q000378:metabolism; D002855:Chromatography, Thin Layer; D018076:DNA, Complementary / Q000378:metabolism; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D018698:Glutamic Acid / Q000378:metabolism; D008239:Lysine / Q000737:chemistry* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D014598:Uteroglobin / Q000737:chemistry* / Q000302:isolation & purification / Q000502:physiology*
D018076:DNA, Complementary; D011994:Recombinant Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; D014598:Uteroglobin; D010741:Phospholipases A; D054467:Phospholipases A2; D008239:Lysine; D002118:Calcium