Structural features of GPI-specific phospholipase D revealed by proteolytic fragmentation and Ca2+ binding studies.

J Y Li,K Hollfelder,K S Huang,M G Low

doi:10.1016/s0021-9258(19)62000-4

Abstract

Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) is abundant in plasma and is potentially capable of degrading the anchor utilized by many cell surface proteins. The goal of this work was to study structural features of the GPI-PLD that might be involved in regulation of its activity. Trypsin cleaved the 100-110 kDa GPI-PLD polypeptide into three major fragments (two of approximately 40 kDa and a carboxyl-terminal fragment of 30 kDa) which were relatively resistant to further proteolysis. Pretreatment of the GPI-PLD with chelators resulted in complete degradation. During the cleavage process the GPI-PLD enzymatic activity increased approximately 3-4-fold but no other major change in its properties (e.g. inhibition by chelators and lipids, thermal stability, oligomerization, etc.) was observed. Intact or trypsinized GPI-PLD bound 45Ca2+ (approximately 5.5 ions/molecule GPI-PLD; Kd approximately 16.1 microM as determined by equilibrium dialysis) which could not be blocked by the addition of other divalent metal ions. However, inhibition of enzymatic activity by divalent cation chelators appeared to involve removal of bound Zn2+ rather than Ca2+. A metal analysis of GPI-PLD revealed approximately 5 and 10 atom/molecule of calcium and zinc, respectively. The data suggest that the predicted integrin E-F hand-like sites in GPI-PLD are functional but not directly involved in enzymatic activity.

Highlights

Glycosylphosphatidylinositol-specificphospholipase they are unablet o hydrolyze these substrates whenlocated in D (GPI-PLD)is abundant in plasma and is potentially membranes [11, 12]
The structural featuresof the membrane capable of degrading the anchor utilized by many cell which restrict theability of GPI-PLD to hydrolyze its substrate surface proteins
GPI-PLD; Kd 16.1 p~ as determined by equilibrium GPI-anchored proteins and GPI-PLD have been cotransfected dialysis) which could not be blockbeyd the addition of into COS cells or CHO fibroblasts provides some support for other divalent metal ions

Summary

Introduction

Glycosylphosphatidylinositol-specificphospholipase they are unablet o hydrolyze these substrates whenlocated in D (GPI-PLD)is abundant in plasma and is potentially membranes [11, 12]. The structural featuresof the membrane capable of degrading the anchor utilized by many cell which restrict theability of GPI-PLD to hydrolyze its substrate surface proteins. The data suggest thatavailable peptide andcDNA sequences do reveal four repeats the predicted integrin E-F hand-like sites in GPI-PLD with a high degree of similarity t o metal ion-binding domains are functional but not directly involved in enzymatic in thea subunits of proteins belonging to the integrifnamily of activity. Cell surface adhesion molecules [4, 17, 19] These repeats (in both GPI-PLD and integrins) contain within them sequences which exhibit some similarity with the E-F hand found in a Glycosylphosphatidylinositol-specific phospholipase D (GPIPLD),’ hydrolyzes the covalently attached GPImoiety used as a membrane anchorby a large numberof cell surface proteins.

Objectives

Results

Conclusion