Inflammation and Noninhibitor Serpins

Abstract

Pigment epithelium-derived factor (PEDF) is a member of the serpin family with neurotrophic activity. However, PEDF belongs to the non-inhibitory subgroup of serpins and its neurotrophic activity does not require inhibition of proteases. PEDF induces neuronal survival on cerebellar granule cell neurons and neurite-outgrowth on retinoblastoma cells. These PEDF-induced neurotrophic activities are blocked by antiserum specific for PEDF, Ab-rPEDF. In order to identify a binding activity for PEDF we have studied the interactions of PEDF to retinoblastoma cell surfaces. Binding activity was determined in retinoblastoma cells treated as for the biological neurite outgrowth assay. Retinoblastoma Y-79 cell cultures were grown in suspension at 1.25 × 10⁵ cell per ml of medium without serum. Radiolabelled I¹²⁵-PEDF was added to the suspension cultures and incubated at 4°C for 60 min. Free ligand was separated from bound PEDF by filtration through glass fibre filters under vacuum. Increasing amounts of unlabelled PEDF were used to compete with labelled proteins. Saturation binding curves indicated that PEDF-binding to retinoblastoma cells reaches a plateau at 15nM PEDF with a linear relationship up to 10nM PEDF. We have used antiserum Ab-rPEDF and ovalbumin to compete for the I¹²⁵-PEDF binding to retinoblastoma cells. PEDF binding in the presence of Ab-rPEDF, excess of cold PEDF (15 nM) or ovalbumin (15 nM) was 30%, 28% and 90%, respectively, of the assays without these competitors.