The identification of a phospholipase B precursor in human neutrophils.
A phospholipase B (PLB) precursor was purified from normal human granulocytes using Sephadex G-75, Mono-S cation-exchange and hydroxyapatite columns. The molecular mass of the protein was estimated to be approximately 130 kDa by gel filtration and 22 and 42 kDa by SDS/PAGE. Tryptic peptide and sequence analyses by MALDI-TOF and tandem mass spectrometry (MS/MS) identified the protein as a FLJ22662 (Homo sapiens) gene product, a homologue of the amoeba Dictyostelium discoideum PLB. The native protein needed modifications to acquire deacylation activity against phospholipids including phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine and lysophospholipids. Enzyme activity was associated with fragments derived from the 42 kDa fragment. The enzyme revealed a PLB nature by removing fatty acids from both the sn-1 and sn-2 positions of phospholipids. The enzyme is active at a broad pH range with an optimum of 7.4. Immunoblotting of neutrophil postnuclear supernatant using antibodies against the 42 kDa fragment detected a band at a molecular mass of 42 kDa, indicating a neutrophil origin of the novel PLB precursor. The existence of the PLB precursor in neutrophils and its enzymatic activity against phospholipids suggest a role in the defence against invading microorganisms and in the generation of lipid mediators of inflammation.
Xu
Shengyuan
S
Department of Medical Sciences, Clinical Chemistry, Uppsala University, Sweden. shengyuan.xu@medsci.uu.se
Zhao
Linshu
L
Larsson
Anders
A
Venge
Per
P
D000595:Amino Acid Sequence; D000818:Animals; D002850:Chromatography, Gel; D017124:Conserved Sequence; D004023:Dictyostelium / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000097:blood*; D006098:Granulocytes / Q000201:enzymology; D006801:Humans; D007113:Immunity, Innate; D007249:Inflammation; D008245:Lysophospholipase / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000201:enzymology* / Q000276:immunology; D010446:Peptide Fragments / Q000097:blood / Q000302:isolation & purification; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014357:Trypsin
D004792:Enzyme Precursors; D010446:Peptide Fragments; D008245:Lysophospholipase; D014357:Trypsin