Organization and chromosomal localization of the human and mouse genes coding for LanC-like protein 1 (LANCL1).

We have determined the organization and chromosome location of the human LANCL1 and mouse Lancl1 genes encoding LANCL1, the lanthionine synthetase component C (LanC)-like protein 1. LANCL1 is related to the bacterial LanC family which is involved in the biosynthesis of antimicrobial peptides. The human and mouse genes span 45 kb and 38 kb, respectively, each comprising ten exons. Within the potential promoter regions, several consensus sequences for ubiquitous and tissue-specific transcription factors are present, reflecting the expression data. The nucleotide sequence of the previously unknown mouse full-length transcript is also reported here. Fluorescence in situ hybridization analyses assigned the LANCL1 gene to human chromosome 2q34 and the Lancl1 gene to mouse chromosome 1, region C2-C5, in accordance with the known homology.

Characterization of rat LANCL1, a novel member of the lanthionine synthetase C-like protein family, highly expressed in testis and brain.

We isolated and characterized the cDNA coding for rat LANCL1, a new member of the eukaryotic LanC-like protein family which is related to the bacterial lanthionine synthetase components C (LanC). LanC is involved in the synthesis of antimicrobial peptides. Rat LANCL1 showed 91.5% and 96% identity when compared with the previously characterized human and mouse orthologs, respectively. Northern blot analysis revealed the presence of two major transcripts, at 1.5 kb and 5 kb, probably arising from the usage of two different polyadenylation signals. The 1.5 kb mRNA is massively expressed in testis, whereas the 5 kb transcript is most abundant in brain. The high level of expression of rat LANCL1 in these tissues was confirmed by Western blotting. In situ hybridization analyses of various rat tissues revealed a strong signal in the germinal cells of the seminiferous tubules in testis, in the neurons of the cerebellum, in liver hepatocytes, and in cardiac myocytes. The clear relationship between LANCL1 and bacterial LanC proteins suggests similar functions as peptide-modifying enzymes synthesizing antimicrobial peptides. In particular, the high expression of LANCL1 in testis and brain, organs separated by blood-tissue barriers, may hint at a role in the immune surveillance of these organs.

Overexpression of stomatin depresses GLUT-1 glucose transporter activity.

We showed previously that GLUT-1 glucose transporter is associated with stomatin (band 7.2b) in human red blood cell membranes and in Clone 9 cells. We show here that in a mixed population of stably transfected cells, overexpression of either murine or human stomatin resulted in 35-50% reduction in the basal rate of glucose transport. Moreover, there was a correlation between increased expression of stomatin and depression in the rate of glucose transport. In two clones chosen for further study, the ~10% and ~70% reduction in basal rate of glucose transport was associated with increases in stomatin mRNA and protein expression without a detectable change in GLUT-1 content in plasma membranes of either clone. In the clone overexpressing high levels of stomatin, immunoprecipitated GLUT-1 was associated with a large amount of stomatin as a coimmunoprecipitant. Employing extracts of cells overexpressing human stomatin, we found that stomatin bound to the glutathione-S-transferase (GST) fusion protein containing the COOH-terminal 42-amino acid segment of GLUT-1 but not to GST alone or a GST fusion protein containing the 66-amino acid central loop of GLUT-1. Rat stomatin cDNA was cloned by RT-PCR and found to be highly homologous to mouse (97%) and human (86%) stomatins. These results suggest that overexpression of stomatin results in a depression in the basal rate of glucose transport by decreasing the "intrinsic" activity of GLUT-1, probably through protein-protein interaction.

Stomatin, flotillin-1, and flotillin-2 are major integral proteins of erythrocyte lipid rafts.

Lipid rafts are sphingolipid- and cholesterol-rich membrane microdomains that are insoluble in nonionic detergents, have a low buoyant density, and preferentially contain lipid-modified proteins, like glycosyl phosphatidylinositol (GPI)-anchored proteins. The lipid rafts were isolated from human erythrocytes and major protein components were identified. Apart from the GPI-anchored proteins, the most abundant integral proteins were found to be the distantly related membrane proteins stomatin (band 7.2b), flotillin-1, and flotillin-2. Flotillins, already described as lipid raft components in neurons and caveolae-associated proteins in A498 kidney cells, have not been recognized as red cell components yet. In addition, it was shown that the major cytoskeletal proteins, spectrin, actin, band 4.1, and band 4.2, are partly associated with the lipid rafts. Stomatin and the flotillins are present as independently organized high-order oligomers, suggesting that these complexes act as separate scaffolding components at the cytoplasmic face of erythrocyte lipid rafts.

Molecular cloning, characterization, and tissue-specific expression of human LANCL2, a novel member of the LanC-like protein family.

We identified and characterized the cDNA coding for human LANCL2, a new member of the eukaryotic LanC-like protein family which is related to the bacterial lanthionine synthetase components C (LanC). The composite nucleotide sequence revealed a coding region of 1353 bp, a 5'-UTR of 186 bp and a 3'-UTR of 2421 bp. The deduced sequence of 450 amino acids showed 57.9% identity (74.7% similarity) when compared with the human LANCL1 homologue. In contrast to LANCL1, a unique ATP/GTP-binding site motif A was found in LANCL2. Northern blot analysis revealed the presence of two major transcripts in the brain, 4.7 kb and 4.1 kb in size, and a major 1.8 kb transcript in testis. Accordingly, expression array analysis showed prominent signals in these tissues. Because of the structural similarity to LanC, we postulate that LANCL2 may play a role as a component of a peptide-modifying complex.

Characterization of p40/GPR69A as a peripheral membrane protein related to the lantibiotic synthetase component C.

The 40 kDa erythrocyte membrane protein p40/GPR69A, previously assigned to the G-protein-coupled receptor superfamily, was now identified by peptide-antibodies and characterized as a loosely associated peripheral membrane protein. This result is in striking contrast to the proposed seven-transmembrane protein structure and function and therefore we wish to correct our previous proposal. p40 is located at the cytoplasmic side of the membrane and is neither associated with the cytoskeleton nor lipid rafts. Refined sequence analysis revealed that p40 is related to the LanC family of bacterial membrane-associated proteins which are involved in the biosynthesis of antimicrobial peptides. Therefore, we rename p40 to LanC-like protein 1 (LANCL1) and suggest that it may play a similar role as a peptide-modifying enzyme component in eukaryotic cells.

Endothelial cells downregulate expression of the 70 kDa heat shock protein during hypoxia.

Hsp70 is induced by hypoxia in most mammalian cell types and contributes to their ability to survive hypoxic episodes. However, little is known about Hsp70 expression in the hypoxia-tolerant endothelial cells (ECs). We investigated the effect of hypoxia on Hsp70 in human microvascular endothelial HMEC-1 cells. Reduction of pO(2) to 2.5% of normal for 20 h stimulated lactate production and the activity of glycolytic enzymes. This metabolic adaptation to hypoxia was accompanied by a remarkable reduction of Hsp70 on the protein level and on the mRNA level. Approximately 12 h after the hypoxic period Hsp70 expression reached pre-hypoxia levels again. Since ECs are adapted to the low oxygen tension of the vasculature they are confronted with a supraphysiological oxygen level during in vitro culture. We suppose that the high Hsp70 under these conditions reflects a stress response which disappears at the more physiological reduced oxygen tension during hypoxia.

Association of stomatin with lipid-protein complexes in the plasma membrane and the endocytic compartment.

Membrane protein - microvilli - lipid raft - GPI-anchored protein - epithelial cell The 31 kDa integral membrane protein stomatin (protein 7.2b) has a monotopic structure and a cytofacial orientation. We have shown previously that stomatin is located in plasma membrane protruding structures and forms high-order homo-oligomers in the human epithelial cell line UAC, suggesting that this protein has a structural function in the cortical morphogenesis of the cells. It is also present in a pool of juxtanuclear vesicles. In this study, we show that stomatin colocalizes with the GPI-anchored proteins placental alkaline phosphatase (PLAP) and membrane folate receptor alpha (MFRalpha) endogenously expressed in UAC cells. This observation enabled us to demonstrate two different aspects of stomatin. First, using anti-PLAP antibody internalization, we show that the peri-centrosomal vesicles containing stomatin correspond to a subset of endosomes, which can also be labeled with the late endosomal/lysosomal marker LAMP-2. Secondly, we found that stomatin is partially present in detergent-insoluble membrane domains and co-patches with PLAP on the plasma membrane, after cross-linking of PLAP by antibodies. These data indicate that stomatin and GPI-anchored proteins are linked through lipid rafts and undergo the same sorting events. We propose that stomatin, through its affinity for lipid rafts, functions in concentrating GPI-anchored proteins in membrane microvillar structures. Consistent with this hypothesis, we found that stomatin is expressed exclusively in microvilli of the apical membrane in polarized Madin-Darby canine kidney (MDCK) cells.

Association of stomatin (band 7.2b) with Glut1 glucose transporter.

Employing a monoclonal antibody directed against the C-terminal peptide of glucose transporter molecule 1 (Glut1), we identified a approximately 30-kDa polypeptide which coimmunoprecipitated with Glut1 from sample of human red blood cells (RBC) membranes. The approximately 30-kDa polypeptide reacted with an antibody directed against stomatin, an integral plasma membrane protein which is also present at a high abundance in the human RBC plasma membrane. Likewise, employing anti-stomatin antibody, we found that Glut1 coimmunoprecipitated with stomatin from samples of RBC membranes. However, neither band 3, which is the most abundant integral membrane protein in the RBC, nor actin coimmunoprecipitated with Glut1, indicating a specific interaction between Glut1 and stomatin. Similar to the results obtained in the RBC, Glut1 and stomatin immunoprecipitated with each other in lysates of Clone 9 cells, a rat liver cell line in which Glut1 is expressed at approximately 1/200 the level present in RBC. Employing conditions that resulted in immunoprecipitation of approximately 10% of Glut1 in RBC membranes led to a approximately 3% coimmunoprecipitation of stomatin. A mixed population of Clone 9 cells stably transfected with a plasmid overexpressing the mouse stomatin exhibited 30 +/- 3% reduction in the basal rate of glucose transport compared to control cells or cells stably transfected with the empty vector. The above results suggest that stomatin is closely associated with Glut1 in the plasma membrane and that overexpression of stomatin results in a depression in the basal rate of glucose transport.

Isolation, molecular characterization, and tissue-specific expression of ECP-51 and ECP-54 (TIP49), two homologous, interacting erythroid cytosolic proteins.

We isolated two proteins, ECP-51 and ECP-54, from human erythrocyte cytosol by affinity chromatography using a peptide of the integral membrane protein stomatin as bait. Partial amino acid sequence information obtained by microsequencing allowed us to clone the respective cDNAs. Analysis of the nucleotide sequences revealed that ECP-51 and ECP-54 are homologous (44.2% amino acid identity) and contain ATP-binding sites. ECP-54 was identified as TIP49/RUVBL1/NMP238, which is a component of a large nuclear protein complex, possibly the RNA polymerase II holoenzyme; ECP-51 is a novel protein. Using the two-hybrid system, we showed that these proteins interact with each other. The interaction of ECP-51 and ECP-54 with the stomatin peptide and the localization to the nucleus and cytoplasm suggest an additional function for these proteins as chaperone components.

Cysteine 29 is the major palmitoylation site on stomatin.

The 31 kDa membrane protein stomatin was metabolically labeled with tritiated palmitic acid in the human amniotic cell line UAC and immunoprecipitated. We show that the incorporated palmitate is sensitive to hydroxylamine, indicating the binding to cysteine residues. Stomatin contains three cysteines. By expressing a myc-tagged stomatin and substituting the three cysteines by serine, individually or in combination, we demonstrate that Cys-29 is the predominant site of palmitoylation and that Cys-86 accounts for the remaining palmitate labeling. Disruption of Cys-52 alone does not show any detectable reduction of palmitic acid incorporation. Given the organization of stomatin into homo-oligomers, the presence of multiple palmitate chains is likely to increase greatly the affinity of these oligomers for the membrane and perhaps particular lipid domains within it.

Are bacterial proteins part of the matrix of kidney stones?

An extracellular protein, produced from Pseudomonas fluorescens strain D with molecular mass of 41.5 kDa was partially purified. Its first 12 amino acid sequence shows strong similarity to a sequence reported to belong to a protein isolated from a urate-calcium oxalate stone (Binnette & Binnette, Scan Microsc1994; 2: 233-239). A possible involvement of bacterial proteins in stone matrix is discussed.

Molecular characterization and tissue-specific expression of a murine putative G-protein-coupled receptor.

We isolated by 5'- and 3'-RACE (rapid amplification of cDNA ends) clones from a murine brain cDNA library which encode a putative G-protein-coupled receptor. The composite nucleotide sequence revealed a coding region of 1197 nt; the deduced amino acid sequence of 399 amino acids showed 91.5% identity (95.7% similarity) when compared with the human homolog. An intron-like sequence, possibly involved in the regulation of expression, was found within the 5'-untranslated region. Northern blot analysis showed that the major 1.7-kb transcript is widely expressed, notably in brain and testis. In situ hybridization studies of tissue sections revealed high expression in neurons of the brain, epithelial cells of the lung, kidney and intestine, and in alveolar macrophages.

Oligomeric nature of the integral membrane protein stomatin.

The 31-kDa integral membrane protein stomatin (protein 7.2b) is not only an important component of the red cell membrane but can also be found in abundance in different tissues and cell lines. The protein is thought to be anchored to the membrane by a hydrophobic domain while both N and C termini are exposed to the cytoplasm. We have previously shown in the human cell line UAC that stomatin concentrates preferentially in plasma membrane folds and protrusions. There is also evidence that stomatin is linked to the cortical actin cytoskeleton, suggesting a role in cortical morphogenesis of the cell. In this study, we demonstrate that the fundamental structure of stomatin is oligomeric. Whereas interaction of stomatin with itself was suggested by cross-linking experiments, we show by density gradient centrifugation analysis that soluble homo-oligomeric complexes of this protein are present in Triton X-100 extracts of UAC cells. We also show the existence of these oligomers by co-immunoprecipitation of the endogenous stomatin and a recombinantly expressed myc-tagged stomatin, using an anti-myc antibody. The data indicate that these complexes comprise between 9 and 12 monomers of stomatin. Two C-terminally truncated forms of stomatin do not incorporate into these oligomers, suggesting an involvement of the C terminus in the homo-oligomeric interaction.

Isolation, molecular characterization, and tissue-specific expression of a novel putative G protein-coupled receptor.

We isolated a 40 kDa integral membrane protein (p40) from human erythrocyte ghosts by affinity chromatography, using a C-terminal peptide of stomatin, and obtained partial sequences which enabled us to isolate two full-length cDNAs from human bone marrow and fetal brain cDNA libraries. The cDNA sequences were identical and encoded a novel putative G protein-coupled receptor (399 amino acids). Northern and RNA dot blot analyses demonstrated that the major 4.8 kb-transcript is predominantly expressed in brain. In situ hybridization studies of tissue sections revealed high expression in neurons of the brain and spinal cord, in thymocytes, megakaryocytes, and macrophages.

Molecular cloning of hSLP-1, a novel human brain-specific member of the band 7/MEC-2 family similar to Caenorhabditis elegans UNC-24.

We have isolated and characterized cDNA clones encoding a stomatin-like protein (hSLP-1) from a human cerebral cortex cDNA library. The deduced amino acid sequence (394 residues) revealed that hSLP-1 is a bipartite protein, containing a major stomatin-like part, starting at the N-terminus, and a non-specific lipid transfer protein (nsLTP)-domain at the C-terminal end, similar to the Caenorhabditis elegans protein UNC-24. Therefore, we conclude that hSLP-1 is the human homologue of UNC-24. In addition, the identification of an alternatively spliced variant demonstrated that two exon/intron boundaries are conserved in the hSLP-1 and unc-24 genes. Northern blot and RNA dot blot analyses showed that the 2. 2-kb transcript is mainly expressed in the brain, with the highest levels in the frontal lobe, cerebral cortex, caudate nucleus, amygdala, temporal lobe, putamen, substantia nigra, and hippocampus. This high-level expression of hSLP-1 in the basal ganglia may also reflect the evolutionary link to UNC-24.

Colocalization of stomatin (band 7.2b) and actin microfilaments in UAC epithelial cells.

Cytolocalization of stomatin, an integral membrane protein also called erythrocyte band 7.2b, was investigated in a human epithelial cell line in which the expression of this protein is up-regulated after treatment with interleukin-6 and dexamethasone. A monoclonal antibody against stomatin was used to perform immunofluorescence and immunoelectron microscopy. The data show that stomatin concentrates preferentially in small plasma membrane protrusions. It is also found in abundance in a juxtanuclear structure possibly derived from the Golgi apparatus. Fluorescent double staining using the anti-stomatin antibody and the actin binding drug phalloidin shows a significant degree of colocalization of stomatin and cortical actin microfilaments. This association remains after actin filament disruption disruption by cytochalasin D treatment indicating a strong connection between stomatin and the membrane-associated cytoskeleton.

Cloning and analysis of a cDNA encoding the BALB/c murine erythrocyte band 7 integral membrane protein.

cDNA clones encoding the BALB/c murine erythrocyte band 7 integral membrane protein (also termed protein 7.2b, or 'stomatin') were isolated by the screening of a corresponding bone-marrow lambda gt11 cDNA library with a human cDNA probe, and by 5'-RACE PCR cloning. Comparison of the murine, human and Caenorhabditis elegans protein 7.2b amino acid (aa) sequences revealed overall identities of 88% (human) and 61% (C. elegans), with the N-terminal domains showing only little similarity. The 7.2b protein sequences of the two mouse strains, BALB/c and C57BL/6J (B6), showed six rather conservative aa substitutions, three of them in the hydrophobic domain. The BALB/c murine mRNA, about 3.5 kb in size, is widely expressed in various tissues, most notably in spleen, lung and testis.

Multiple cytokines inhibit interleukin-6-dependent murine hybridoma/plasmacytoma proliferation.

A panel of cytokines was tested for inhibitors of interleukin-6 (IL-6)-dependent cell proliferation. Murine type I and II interferons (mIFNs) strongly inhibited proliferation of IL-6-dependent B9 and 7TD1 cells in a dose-dependent manner. Human tumor necrosis factor-alpha (hTNF-alpha) and human transforming growth factor-beta (hTGF-beta) potently inhibited B9 and to a lesser extent 7TD1 cells, while hIL-11, human oncostatin M (hOSM), and human leukemia inhibitory factor (hLIF) had no inhibitory effects on IL-6-dependent growth. Conversely, IL-11 and OSM but not LIF stimulated B9 and 7TD1 cell growth. However, compared with IL-6, up to 1000-fold higher IL-11 and OSM concentrations were required to induce maximal cell proliferation. Increasing concentrations of IL-6 (up to 100 ng/ml) could not overcome the antiproliferative effects of mIFNs, hTNF-alpha and hTGF-beta. Supernatants from mIFN-gamma and lipopolysaccharide (LPS)-treated mouse macrophages (ANA-1 cell line) were tested in B9 cell assays to identify cytokines among stimulatory and inhibitory biological activities that can inhibit IL-6-dependent proliferation. Undiluted or relatively concentrated supernatants from ANA-1 macrophages treated with mIFN-gamma and/or LPS did not contain detectable IL-6 bioactivity. However, diluted samples contained considerable amounts of detectable IL-6 bioactivity (nanogram levels). Testing the same samples for IL-6 immunoreactivity using enzyme-linked immunoabsorbent assay revealed comparable levels of mIL-6. We conclude that IFNs, TNF-alpha, and TGF-beta and possibly other factors are potent, dominant inhibitors of IL-6-dependent plasmacytoma/hybridoma growth in vitro.

The organization of the gene (EPB72) encoding the human erythrocyte band 7 integral membrane protein (protein 7.2b).

The human gene EPB72 coding for the band 7 integral membrane protein, a major protein of the erythrocyte membrane, was isolated from a genomic DNA library and characterized. Spanning approximately 30 kb, the human EPB72 gene comprises seven exons ranging from 73 to 2331 bp; intron sizes range from 970 to approximately 11,200 bp. The first exon contains the 5'-untranslated region (61 nucleotides) and the coding sequence for the N-terminal domain; the second exon encodes the hydrophobic domain, including flanking cysteine and lysine residues. Exon 7 contains the C-terminal portion and a 2-kb 3'-untranslated region. The potential promoter region contains several consensus sequences for ubiquitous transcription factors (Sp1, AP1, AP2, CP1/2, NF kappa B, CREB, Ets-1, and CACC/GT-BF) and two imperfect sequences for erythroid factors (EKLF and GATA-1), in accordance with the ubiquitous distribution of the EPB72 mRNA in different cell types. No TATA box was apparent. An inverted Alu repeat element, flanked by nonamer direct repeats, was identified within the region -913/-620, relative to the cap site. Six additional Alu repeat elements, including one monomer and one trimer, were identified within the introns and the 3'-untranslated region. Two polyadenylation signals in the 3'-noncoding region of exon 7 enable the production of two mRNA species.