The Neuronal Cell‐Adhesion Molecule Axonin‐1 is Specifically Released by an Endogenous Glycosylphosphatidylinositol‐Specific Phospholipase

Abstract

Axonin-I , a member of the immunoglobulidfibronectin type-I11 family of cell-adhesion molecules, occurs both as a glycosylphosphatidylinositol-(glycosylPtdIns)-anchored membrane-bound and a soluble form. In vivo observations show that the major part of axonin-1 is found in the soluble fraction and that soluble axonin-I perturbs neurite fasciculation and pathfinding in the developing chicken embryo. This has prompted further investigations into the mechanism of the axonin-I release. We demonstrate here that axonin-1 released from dorsal root ganglion neurons contains ethanolamine and inositol, components of the glycosylPtdIns anchor. Secreted axonin-1 does not exhibit the cross-reacting determinant epitope, an indication that the cleavage of the anchor is not mediated by a phosphatidylinositol-specific phospholipase C. Treatment of dorsal root ganglion neurons with 1,lO-phenanthroline, an inhibitor of glycosylPtdIns-specific phospholipase D, reduces the release of axonin-I by 56 %. Moreover, glycosylPtdIns-specific phospholipase D activity was detected in dorsal root ganglion neurons and brain. These results suggest that axonin-1 is released from the membrane by an endogenously expressed glycosylPtdIns-specific phospholipase D in viva With domain-swaping experiments between axonin-1 and its non-released relative F1 1, deletion mutants and monoclonal antibodies, we demonstrate that the fourth fibronectin type-111-like domain of axonin-I is required for the generation of the soluble form of axonin-1.

Release of the glycosylphosphatidylinositol-anchored enzyme ecto-5′-nucleotidase by phospholipase C: catalytic activation and modulation by the lipid bilayer

“…Release of GPI-anchored proteins by endogenous phospholipases has been proposed to play an important role in regulation of their surface activity, and may also generate second messengers that initiate transmembrane signalling processes [7,10,11,25]. It is, therefore, important to have a detailed understanding of the potential consequences of such release.…”

Membrane proteomics: Use of additive main effects with multiplicative interaction model to classify plasma membrane proteins according to their solubility and electrophoretic properties

“…Those proteins were shown to be anchored to the membrane via a glycosylphosphatidylinositol (GPI) anchor, commonly employed as a mode of membrane attachment in a wide range of eukaryotes [39]. The release of GPI anchor, mediated by phospholipase C or D, has been proposed to play an important role in the regulation of their surface activity, and may also generate second messengers that initiate transmembrane signaling processes [40]. Interestingly, a phospholipase C could be identified in this group.…”

Mass spectrometric characterisation of primary structure, sequence heterogeneity, and intramolecular disulfide loops of the cell adhesion protein axonin-1 from chicken

“…4 Initial characterisation by amino acid analysis and determination of non-protein components yielded significant amounts of myo-inositol and ethanolamine. 9 Since myo-inositol is not a constituent in regular protein glycosylation, this suggested a GPI-modification. The soluble form of axonin-1 is eventually released by a specific mechanism which might play a role as a regulatory element in growth cone-neurite interactions.…”