Abstract
Human lipocalin-1 (Lcn-1, also called tear lipocalin), a member of the lipocalin structural superfamily, is produced by a number of glands and tissues and is known to bind an unusually large array of hydrophobic ligands. Apart from its specific function in stabilizing the lipid film of human tear fluid, it is suggested to act as a physiological scavenger of potentially harmful lipophilic compounds, in general. To characterize proteins involved in the reception, detoxification, or degradation of these ligands, a cDNA phage-display library from human pituitary gland was constructed and screened for proteins interacting with Lcn-1. Using this method an Lcn-1 interacting phage was isolated that expressed a novel human protein. Molecular cloning and analysis of the entire cDNA indicated that it encodes a 55-kDa protein, lipocalin-1 interacting membrane receptor (LIMR), with nine putative transmembrane domains. The cell membrane location of this protein was confirmed by immunocytochemistry and Western blot analysis of membrane fractions of human NT2 cells. Independent biochemical investigations using a recombinant N-terminal fragment of LIMR also demonstrated a specific interaction with Lcn-1 in vitro. Based on these data, we suggest LIMR to be a receptor of Lcn-1 ligands. These findings constitute the first report of cloning of a lipocalin interacting, plasma membrane-located receptor, in general. In addition, a sequence comparison supports the biological relevance of this novel membrane protein, because genes with significant nucleotide sequence similarity are present in Takifugu rubripes, Drosophila melanogaster, Caenorhabditis elegans, Mus musculus, Bos taurus, and Sus scrofa. According to data derived from the human genome sequencing project, the LIMR-encoding gene has to be mapped on human chromosome 12, and its intron/exon organization could be established. The entire LIMR-encoding gene consists of about 13.7 kilobases in length and contains 16 introns with a length between 91 and 3438 base pairs.
Highlights
The protein superfamily of lipocalins consists of small, mainly secretory proteins defined on the basis of conserved amino acid sequence motifs and their common structure
A sequence comparison supports the biological relevance of this novel membrane protein, because genes with significant nucleotide sequence similarity are present in Takifugu rubripes, Drosophila melanogaster, Caenorhabditis elegans, Mus musculus, Bos taurus, and Sus scrofa
Whereas a large number of various lipophilic ligands able to bind to lipocalins are known, only limited data are available concerning the identity of lipocalin receptors
Summary
General Methods—Total RNA was extracted by RNazolTMB (Cinna/ Biotecx), which is based on the method developed by Chomczynski and Sacchi [26]. Enrichment of phages was performed in MaxiSorb microtiter plates (Nunc, Roskilde, Denmark) under the following conditions: wells were precoated with 2 g of purified Lcn-1 for 2 h at room temperature, washed, and blocked with 1% BSA. For purification of the recombinant protein, E. coli cultures were centrifuged, and the pellet was resuspended in 50 mM Na2HPO4 (pH 8.0)/300 mM NaCl at 2–5 volumes/g wet weight and passed twice through a French pressure cell (at 97 megapascal). The cell extract was centrifuged at 9000 ϫ g for 20 min, and the recombinant protein present in the supernatant was purified by affinity chromatography on Ni-NTA resin (Qiagen) as described by the supplier. Biochemical Assays for Protein-Protein Interaction—For a solid phase-based assay of LIMR-Lcn-1 interaction wells of a microtiter plate were precoated with purified recombinant LIMR fragment (2 g per well). The coverslips were washed in PBS overnight at 4 °C and mounted with a drop of glycerine and sealed with clear nail polish
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