Structural and functional analysis of p40, a novel putative G-protein-coupled receptor

The project "Structure and function of p40" comprises biochemical, cell biological, cell biological and molecular biological investigations of a novel human red cell membrane protein that had been isolated in our laboratory. We found that this protein is mainly expressed in the neurons of the brain and that it has structural similarity with the large family of receptors which are responsible for the signal tranduction of light, taste, odor and several hormones. Rhodopsin is a well known member of this family of so-called "G protein-coupled receptors". In this project we will prepare antibodies against this protein p40, in order to identify it by immunochemical methods, like Western blot analysis and immuno fluorescence microscopy. This way we can also identify proteins which may be associated with p40, possibly the membrane protein stomatin. Because of its localization at the red cell surface, it is well possible that p40 gave rise to a blood group-specific antigen by mutation. In a cooperation with a British blood group reference laboratory we will search for such an associated blood group. In our laboratory we will investigate the functional and structural properties of this receptor and will search for the substance which will specifically interact with p40. Moreover, we will investigate the signaling pathway induced by this substance and thereby determine the biological function of the receptor. In another cooperation with an American group we will analyse samples of members of a family with a hereditary disease of the red cells, called xerocytosis. It is possible that p40 plays a role in this disease. Investigation of the DNA samples will show, if this is the case.

In this project, the putative G-protein-coupled receptor (GPCR) p40/GPR69A, which had been isolated from human red cell membranes by our group, was characterized at the protein and gene level. Although a characteristic seven-transmembrane-protein structure had been clearly predicted and thereby suggested the GPCR function, our subsequent biochemical results showed that p40 is not an integral but rather a peripheral membrane protein and therefore could not be a GPCR. Re-evaluation of the p40 structure, in cooperation with Aron Marchler-Bauer (NCBI), revealed a similarity with bacterial proteins collectively termed LanC, which are involved in the biosynthesis of "lantibiotics". These are anti-bacterial peptides which are synthesized from prepeptides by dehydration and formation of a cyclic thioether. Because of the evident structural relationship to LanC proteins, we renamed p40 to LanC-like protein 1 (LANCL1). We isolated and characterized the mouse, rat, and zebrafish orthologues and found more than 90 % identity to the human protein. Whereas the major expression of human LANCL1 is found in brain, the highest expression of mouse and rat LANCL1 is found in testis and brain, with high expression also in heart and liver. Interestingly, there is a tissue-specific size variation in the mRNAs, suggesting a tissue-specific regulation of expression. At the single cell level, our immunofluorescence studies showed that LANCL1 is located in the cytoplasm, however, when the cells are first trypsinized in order to detach them, a strong staining of the plasma membrane was observed. Possibly, trypsin treatment may convey a signal to the cell which results in the translocation of cytosolic LANCL1 to the membrane. More recently, LANCL1 was also found in vesicles near the plasma membrane. When we searched for more LANCL proteins, we identified and subsequently characterized a related human protein which we termed LANCL2. This protein shows 58 % identity to LANCL1 and a similar expression pattern, with the major expression in brain and testis. The model plant Arabidopsis thaliana also expresses two LANCL proteins, one being similar to LANCL1 and the other to LANCL2. This result shows that these proteins are important in both the animal and plant kingdoms. In order to build the basis for a LANCL1 knockout, we studied the organization of the respective human and mouse genes. Both genes span about 40 kb and comprise ten exons. Interestingly, most of the seven hydrophobic domains are encoded by single exons. This finding, together with the repeat character of the respective sequences, suggests that the LANCL1 genes evolved by duplications. Fluorescence in situ hybridization analyses assigned the LANCL1 gene to human chromosome 2q34 and mouse chromosome 1, region C2-C5. In summary, our data suggest that proteins of the LANCL family have a function similar to the bacterial LanC proteins in the modification of antimicrobial peptides. These peptides are a highly interesting research topic with a great potential of practical applicability in medicine and food industry.