| Autor: |
Koduri, Rao S., Baker, Sharon F., Snitko, Yana, Han, Sang Kyou, Cho, Wonhwa, Wilton, David C., Gelb, Michael H. |
| Zdroj: |
Journal of Biological Chemistry; November 1998, Vol. 273 Issue: 48 p32142-32153, 12p |
| Abstrakt: |
Human group IIa phospholipase A2(hIIa-PLA2) is a highly basic protein that is secreted from a number of cells during inflammation and may play a role in arachidonate liberation and in destruction of invading bacteria. It has been proposed that rodent group IIa PLA2is anchored to cell surfaces via attachment to heparan sulfate proteoglycan and that this interaction facilitates lipolysis. hIIa-PLA2 contains 13 lysines, 2 histidines, and 10 arginines that fall into 10 clusters. A panel of 26 hIIa-PLA2 mutants were prepared in which 1–4 basic residues in each cluster were changed to glutamate or aspartate (charge reversal). A detailed analysis of the affinities of these mutants for anionic vesicles and for heparin and heparan sulfate in vitroand of the specific activities of these proteins for hydrolysis of vesiclesin vitroand of living cell membranes reveal the following trends: 1) the affinity of hIIa-PLA2 for heparin and heparan sulfate is modulated not by a highly localized site of basic residues but by diffuse sites that partially overlap with the interfacial binding site. In contrast, only those residues on the interfacial binding site of hIIa-PLA2 are involved in binding to membranes; 2) the relative ability of these mutants to hydrolyze cellular phospholipids when enzymes were added exogenously to CHO-K1, NIH-3T3, and RAW 264.7 cells correlates with their relative in vitroaffinity for vesicles and not with their affinity for heparin and heparan sulfate. 3) The rates of exogenous hIIa-PLA2-catalyzed fatty acid release from wild type CHO-K1 cells and two mutant lines, one lacking glycosaminoglycan and one lacking heparan sulfate, were similar. Thus basic residues that modulate interfacial binding are important for plasma membrane fatty acid release by exogenously added hIIa-PLA2. Binding of hIIa-PLA2 to cell surface heparan sulfate does not modulate plasma membrane phospholipid hydrolysis by exogenously added hIIa-PLA2. |
| Databáze: |
Supplemental Index |
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