Human LIMP, a novel lipocalin-interacting membrane protein

Research project P 14850 Human LIMP, a novel lipocalin-interacting membrane protein Bernhard REDL 27.11.2000 Human lcn-1, identically with tear lipocalin and von Ebner`s gland protein, is a remarkable member of the lipocalin superfamily, since it is known to bind a large variety of different classes of lipophilic ligands, including fatty acids, phospholipids, glycolipids, cholesterol, retinol and retinoic acid. In contrast to other lipocalins many of these lipids were found to be physiological ligands, of lcn-1. Although originally isolated as a protein highly produced by the lacrimal and lingual salivary glands, it was afterwards demonstrated to be expressed by several other secretoric tissues, such as prostate, mucosal glands of the tracheobronchial tree, nasal mucosa and sweat glands. The precise biological function of Icn-1 was not fully characterized so far and is still a matter of controversal discussion. In tear fluid it seems to be most important for the integrity of the tear film by removing lipids from the mucinous surface of the eye to the liquid phase. It was also suggested to play a role in taste transduction or olfactation. Interestingly, besides its binding of various lipophilic molecules it was shown to display additional in vitro activities, very unusual among lipocalins, namely inhibition of cysteine proteinases and non-specific endonuclease activity. Nevertheless, especially from our recent investigations there is emerging evidence that its main function is to act as a scavenger of lipophilic, probably harmful molecules, and might thus be protective to tissues and cells. Within the current research proposal we have searched for Icn-1 interacting proteins, mainly to characterize the putative detoxification or recycling of the lipid ligands of Icn-1. Using phage display technology we have isolated two interaction partners, so far. Besides an interaction with thioredoxin, which seems to be of relevance in terms of modulation of ligand binding, we have found another interaction partner, a putative membrane protein called LIMP (lipocalin interacting membrane protein), with a so far unknown function. In the present proposal we will focus our interest on investigations of the biological function of this novel protein. From its interaction with Icn-1 it might be assumed that LIMP is involved in detoxification/ recycling of lipophilic compounds scavenged by lcn-1, probably by delivering these compounds for further modification. Since it is evident from data-base analysis that LIMP homologous proteins are also present in other species, this protein seems to be of general biological relevance. In addition, our investigations, especially concerning the application of phage-display technology which appears to be a reliable tool for isolation of lipocalin receptors in general, could be of relevance for the entire field of lipocalin research,.

All organisms have to deal with a large number of compounds, which are hardly soluble or even insoluble in biological fluids. They have therefore evolved a sophisticated system of transport/binding proteins to handle these molecules. The most well known example are the HDL and LDL plasma lipoproteins. However, another important group are the lipocalins, which are present, in contrast to HDL and LDL, in all organisms including bacteria, plants and mammalians. In addition, they have a much broader ligand specificity, which includes hormones, pheromones, odorants, fatty acids, hydrophobic peptides and others. The structure and biochemical features of lipocalins are well established. However, there is a major disadvantage in understanding the physiological function of many lipocalin members. This is mainly due to a lack of knowledge of lipocalin receptors and the cellular targeting of lipocalin specific ligands. By using a phage-display based technique for interaction screening of a complex cDNA expression library with Lcn-1 as bait to isolate proteins that may be involved in the reception or degradation of Lcn-1 specific ligands, we were able to identify a novel lipocalin receptor (LIMR). These findings set the stage for exploring the molecular mechanism of the lipocalin-receptor interaction in more detail. Additional research demonstrated that this membrane protein is essential for internalization of the lipocalin-ligand complex. Since sequence and structure analysis indicated that proteins similar to LIMR are present in several organisms and at least two closely related orthologous are found in human and mouse, we suggest LIMR to be the prototype of a new family of endocytic receptors, which are topographically characterized by nine putative transmembrane domains and a characteristic large central cytoplasmic loop. Further investigations of the LIMR-Lcn-1 interaction revealed new insights into the structure and physiological relevance of Lcn-1 itself. On the one hand, we could establish the full 3D structure of Lcn-1. On the other hand, we found that Lcn-1 binds in addition to the well known lipophilic ligands, also microbial siderophores, which are in fact hydrophobic peptides. The physiological consequence of this property is a strong antimicrobial activity of Lcn- 1 under iron limiting conditions.