Distinct enzymatic and cellular characteristics of two secretory phospholipases A2 in the filamentous fungus Aspergillus oryzae

Abstract

Summary Microbial secretory phospholipases A2 (sPLA2s) are among the last discovered and least known members of this functionally diverse family of enzymes. We analyzed here two sPLA2s, named sPlaA and sPlaB, of the filamentous ascomycete Aspergillus oryzae. sPlaA and sPlaB consist of 222 and 160 amino acids, respectively, and share the conserved Cys and catalytic His-Asp residues typical of microbial sPLA2s. Two sPLA2s differ in pH optimum, Ca2+ requirement and expression profile. The splaA mRNA was strongly upregulated in response to carbon starvation, oxidative stress and during conidiation, while splaB was constitutively expressed at low levels and was weakly upregulated by heat shock. Experiments with sPLA2-overexpressing strains demonstrated that two enzymes produce subtly different phospholipid composition variations and also differ in their subcellular localization: sPlaA is most abundant in hyphal tips and secreted to the medium, whereas sPlaB predominantly localizes to the ER-like intracellular compartment. Both sPLA2-overexpressing strains were defective in conidiation, which was more pronounced for sPlaB overexpressors. Although no major morphological abnormality was detected in either ΔsplaA or ΔsplaB mutants, hyphal growth of ΔsplaB, but not that of ΔsplaA, displayed increased sensitivity to H2O2 treatment. These data indicate that two A. oryzae sPLA2 enzymes display distinct, presumably non-redundant, physiological functions.

Filaggrin inhibits generation of CD1a neolipid antigens by house dust mite–derived phospholipase

“…Indeed phospholipases are known to be present in many other allergens including pollens and fungal allergens (75, 76) and therefore activation of this pathway may facilitate the allergenic process. By producing type 2 cytokines, CD1a-reactive lipid-specific T cells may lead to down-regulation of filaggrin and anti-microbial peptide expression and thus compound physical and antimicrobial barrier dysfunction.…”

Autoproteolytic Activation of a Symbiosis-regulated Truffle Phospholipase A2

“…The lysate was clarified by centrifugation, and inclusion bodies, recovered in the pellet, were subjected to a second round of sonication in washing buffer (20 mM Tris-HCl, 500 mM NaCl, 2 M urea, 2% Triton X-100, pH 8.0), followed by an additional centrifugation step. The pellet was resuspended in solubilization buffer (20 mM Tris-HCl, 500 mM NaCl, 1 mM ␤-mercaptoethanol, 6 M guanidine hydrochloride, pH 8.0) and stirred at room temperature for 30 min, and the resulting solution was then dialyzed against urea-containing buffer (20 mM Tris-HCl, 500 mM NaCl, 1 mM (19); p15 (50); and S. violaceoruber PLA 2 (51)) is represented on a white background; the active site consensus peptide and positionally conserved, disulfide-bonded Cys residues flanking this site are indicated as black boxes and black vertical lines, respectively. N-terminal polypeptide extensions (interposed between the conserved catalytic domain region and the secretion signal peptide in the T. borchii and T. melanosporum sPLA 2 s) and C-terminal polypeptide extensions (Aspergillus oryzae and Helicosporium sPLA 2 s) are shown as striped and dark gray bars, respectively.…”

“…Based on their primary structures and enzymatic and subcellular localization properties, members of the PLA 2 superfamily are classified into five main subfamilies: Ca 2ϩ -dependent secreted and cytosolic PLA 2 s, Ca 2ϩ -independent PLA 2 s, platelet-activating factor acetylhydrolases, and lysosomal PLA 2 s (1, 2). Secreted PLA 2 s (sPLA 2 s), which comprise 14 different groups of enzymes from a variety of organisms and tissues, are the most diverse and populated PLA 2 subfamily (1, 7) sPLA 2 s are glycerophospholipid hydrolases with a His-Asp catalytic dyad, characterized by a relatively low molecular mass (13)(14)(15)(16)(17)(18)(19) and by a variable number (from 2 to 8) of disulfide bonds (2,7,8). In accordance with their extracellular localization, most sPLA 2 s have an N-terminal secretion signal peptide that is cleaved upon internalization into the endoplasmic reticulum (or into the periplasmic space in the case of bacterial enzymes) (2).…”

“…1). Three additional fungal sPLA 2 s homologous to TbSP1 but lacking the above mentioned polypeptide insertion have been characterized in recent years (19,20) (see Fig. 1).…”

Secretory expression of a phospholipase A2 from Lactobacillus casei DSM20011 in Kluyveromyces lactis

“…The first microbial secreted Ca 2þ -dependent PLA 2 was identified in the mycorrhizal ascomycete Tuber borchii, and then some homologous PLA 2 s were found in fungi (15). sPLA 2 s gene from the filamentous ascomycete Aspergillus orzae were cloned and expressed in E. coli and extracted from inclusion bodies (16). The first phospholipase A 2 identified in prokaryotes was from Streptomyces violaceoruber.…”