Molecular interactions of the neuronal GPI‐anchored lipocalin Lazarillo

Abstract

Lazarillo, a glycoprotein involved in axon growth and guidance in the grasshopper embryo, is the only member of the lipocalin family that is attached to the cell surface by a GPI anchor. Recently, the study of Lazarillo homologous genes in Drosophila and mouse has revealed new functions in the regulation of lifespan, stress resistance and neurodegeneration. Here we report an analysis of biochemical properties of Lazarillo to gain insight into the molecular basis of its physiological function. Recombinant forms of the grasshopper protein were expressed in two different systems to test: (1) potential binding of several hydrophobic ligands; (2) protein-protein homophilic interactions; and (3) whether interaction with the function-blocking mAb 10E6 interferes with ligand binding. We tested 10 candidate ligands (retinoic acid, heme, bilirubin, biliverdin, ecdysterone, juvenile hormone, farnesol, arachidonic acid, linoleic acid and palmitic acid), and monitored binding using electrophoretic mobility shift, absorbance spectrum, and fluorimetry assays. Our work indicates binding to heme and retinoic acid, resulting in increased electrophoretic mobility, as well as to fatty acids, resulting in multimerization. Retinoic acid and fatty acids binding were confirmed by fluorescence titration, and heme binding was confirmed with absorbance spectrum assays. We demonstrate that Lazarillo oligomerizes in solution and can form clusters in the plasma membrane when expressed and GPI-anchored to the cell surface, however it is unable to mediate cell-cell adhesion. Finally, by ligand-mAb competition experiments we show that ligand-binding alone cannot be the key factor for Lazarillo to perform its function during axonal growth in the grasshopper embryo.

Schwann cell-derived Apolipoprotein D controls the dynamics of post-injury myelin recognition and degradation

“…Ligand binding to ApoD was performed as previously described (Sanchez et al, 2008; Ruiz et al, 2013). Fluorescence measurements were conducted with a Shimadzu RF-5301PC spectrofluorometer with a quartz cuvette (Hellma 105.251-QS, 3 mm path length).…”

“…Ethanol final concentration was ≤0.004%. The corrected fluorescence at 340 nm vs. the total concentration was fitted as previously described (Sanchez et al, 2008). K D was calculated under the assumption of a single binding site, consistent with known Lipocalin structural properties.…”

Tear Lipocalin: structure and Function

“…Lazarillo is a glycoprotein involved in axon growth and guidance in the grasshopper embryo 16 . Lazarillo is a unique lipocalin in that it is anchored to the cell surface by GPI.…”

Grasshopper Lazarillo, a GPI-anchored Lipocalin, increases Drosophila longevity and stress resistance, and functionally replaces its secreted homolog NLaz

“…A fragment of the Lazarillo cDNA, translating into residues 1e192 of the precursor (Uniprot reference P49291), and thus missing the GPI signal peptide, was subcloned using the EcoRI and NotI sites into the pRmHa3 vector (Bunch et al, 1988;Sanchez et al, 2008). This system expresses the cloned sequence under the control of the inducible Drosophila metallothionein promoter, and the presence of a C-terminal His-tag sequence allows for protein purification from the culture medium.…”