Cellular localization and trafficking of vascular adhesion protein-1 as revealed by an N-terminal GFP fusion protein

• Published in: Journal of Neural Transmission Vol. 120; no. 6; pp. 951 - 961
• Main Authors: Weston, Chris J., Shepherd, Emma L., Adams, David H.
• Format: Journal Article
• Language: English
• Published: Vienna Springer Vienna 01.06.2013; Springer Nature B.V
• Subjects: Amine Oxidase (Copper-Containing) - genetics; Amine Oxidase (Copper-Containing) - metabolism; Cell Adhesion Molecules - genetics; Cell Adhesion Molecules - metabolism; Cells, Cultured; Endoplasmic Reticulum - drug effects; Endoplasmic Reticulum - metabolism; Enzyme Inhibitors - pharmacology; Ethylamines - pharmacology; Golgi Apparatus - drug effects; Golgi Apparatus - metabolism; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; Humans; Liver - cytology; Lysosomal Membrane Proteins - metabolism; Medicine; Medicine & Public Health; Myoblasts - drug effects; Myoblasts - metabolism; Neurology; Neurosciences; Protein Transport - drug effects; Protein Transport - physiology; Psychiatry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Transfection; Translational Neurosciences - Original; Translational Neurosciences - Original Article; Vesicular Transport Proteins - metabolism; Vascular adhesion protein-1; Amine oxidase; Trafficking; Imaging; Liver
• ISSN: 0300-9564; 1435-1463
• DOI: 10.1007/s00702-013-1003-3

Recent studies of vascular adhesion protein-1 (VAP-1) have greatly advanced our understanding of the important role this protein plays in the establishment and progression of inflammatory disease. To facilitate more detailed studies on the function of VAP-1, we developed a GFP-fusion protein that enabled us to monitor the trafficking of the protein in three selected cell types: hepatic sinusoidal endothelial cells, liver myofibroblasts and an hepatic stellate cell line (LX-2). The fusion protein was detected as punctate cytoplasmic GFP staining, but was present only at low levels at the cell surface in all cell types studied. The subcellular distribution of the protein was not altered in a catalytically inactive mutant form of the protein (Tyr471Phe) or in the presence of exogenous VAP-1 substrate (methylamine) or inhibitor (semicarbazide). The GFP-VAP-1 protein was localized to the Golgi apparatus (GM-130), endoplasmic reticulum (GRP94) and early endosomes (EEA-1). Additional staining for VAP-1 revealed that the overexpressed protein was also present in vesicles that were negative for GFP fluorescent signal and did not express EEA-1. We propose that these vesicles are responsible for recycling the fusion protein and that the fluorescence of the GFP moiety is quenched at the low pH within these vesicles. This feature of the protein makes it well suited for live cell imaging studies where we wish to track protein that is being actively trafficked within the cell in preference to that which is being recycled.