Group IIA and group V secretory phospholipase A(2): quantitative analysis of expression and secretion and determination of the localization and routing in rat mesangial cells.
Mesangial cells can be induced to express group IIA and group V secretory phospholipase A(2) (sPLA(2)) at the mRNA level and at the protein level. In this report we quantitatively analyze the expression of both proteins in stimulated cells by Western blot techniques. We found that 75-80% of the total amount of synthesized group IIA sPLA(2) was secreted. The synthesized group V sPLA(2), however, was present almost exclusively intracellularly. The amount of group V present in the cell was comparable to the intracellular amount of group IIA sPLA(2). We furthermore studied the localization and routing of both proteins. Using fusion proteins of the group IIA or group V pre-sPLA(2) with green fluorescent protein it was established that both presequences are able to direct the proteins to the Golgi system. In immunofluorescence studies group V sPLA(2) expressed by rat mesangial cells was located in a punctate pattern in the cytosol with an enrichment near the nucleus. Immunofluorescent confocal laser scanning microscopy revealed that the group V and IIA sPLA(2) show partial colocalization in a Golgi-like structure in the inner part in the cell, but no colocalization was seen in the vesicles in the cytoplasm. The images also showed that group IIA sPLA(2) was located throughout the cell while group V was mainly present in the inner part of the cell. After treatment of the cells with brefeldin A or monensin the group IIA enzyme could no longer be detected, while group V sPLA(2) was still present although its localization was somewhat dependent on the treatment. Collectively, these results indicate that the two enzymes differ in both localization and routing in the cell, which underscores the hypothesis that the enzymes might have different functions.
van der Helm
H A
HA
Centre for Biomembranes and Lipid Enzymology, Department Biochemistry of Lipids, Institute of Biomembranes, Utrecht University, The Netherlands.
Buijtenhuijs
P
P
van den Bosch
H
H
D000818:Animals; D000911:Antibodies, Monoclonal; D020126:Brefeldin A / Q000494:pharmacology; D005576:Colforsin; D003907:Dexamethasone / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D005920:Glomerular Mesangium / Q000201:enzymology* / Q000378:metabolism / Q000648:ultrastructure; D006056:Golgi Apparatus / Q000201:enzymology; D049452:Green Fluorescent Proteins; D054501:Group II Phospholipases A2; D054505:Group V Phospholipases A2; D066298:In Vitro Techniques; D007375:Interleukin-1; D008164:Luminescent Proteins / Q000235:genetics; D008856:Microscopy, Fluorescence; D008985:Monensin / Q000494:pharmacology; D010741:Phospholipases A / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000032:analysis
D000911:Antibodies, Monoclonal; D007375:Interleukin-1; D008164:Luminescent Proteins; D011500:Protein Synthesis Inhibitors; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D005576:Colforsin; D020126:Brefeldin A; D003907:Dexamethasone; D008985:Monensin; D010741:Phospholipases A; D054501:Group II Phospholipases A2; D054505:Group V Phospholipases A2; C516301:Pla2g5 protein, rat