| Bone marrow stromal cells (BMSC) are important components of hematopoietic microenvironment, providing the structural and physiological support for hematopoietic cells. Our studies are aiming at obtaining information about important molecules involved BMSC functions by focusing on the cloning of novel genes expressed in BMSC so as to understand molecular mechanisms underlying the unique biological functions of BMSC.Part I. Cloning, sequence analysis, expression pattern and cytolocalization of hPEBP4We detailed here a novel member of phosphatidylethanolamine-binding protein (PEBP) family, hPEBP4, isolated from the BMSC cDNA library by large-scale random sequencing. Full length of hPEBP4 contained 874 bp, with a methionine codon situated in a consensus Kozak sequence as start codon and a TGA as stop codon. The full ORF encoded a 227-amino acid protein. This protein contains a typical phosphatidylethanolamine-binding domain, and therefore has been designated as human phosphatidylethanolamine-binding protein 4 (hPEBP4). The gene had been deposited to GenBank/EMBL (GenBank Accession Number AY037148) and National Invention Patent of China was applied (Patent Application No. 02136524.6).Based on sequence analysis and existing PEBP structures, a number of features characteristic of the PEBP family, with likely functional importance, were identified in hPEBP4. As with all PEBP family members, hPEBP4 was predicted to contain a phosphatidylethanolamine (PE) binding domain, in this case between amino acids 84-191.Residues previously demonstrated to be conserved between all members of the family (Pro97, Asp98, Pro100, His112 and Argl49) were also found in hPEBP4. These residues are thought to be involved in determining the local structure of a biologically important ligand-binding site in PEBPs. The two main regions of high sequence conservation occurred between residues 91-117 and residues 141-153 (based on hPEBP4 numbering). Residues 91-101 with 96-98 and 100 forming an Asp-Pro-Asp-x-Pro motif that is universally conserved in all family members. The 141-153 conserved region formed part of the second side of the binding pocket. These structural features suggest that hPEBP4 might possess PE binding properties similar to those seen in other PEBPs.RT-PCR analysis revealed that hPEBP4 mRNA is expressed in a variety of tumor cells and freshly isolated cells, including Daudi (Burkitt's lymphoma), NAMALWA (Burkitt's lymphoma), MCF-7 (breast carcinoma), PC-3 (prostate carcinoma) and CaoV-3 (ovarian carcinoma) cells. Clontech human MTN blots revealed the presence of a single hPEBP4 mRNA as a strong 1.2 kb message, expressed strongly in testis, heart, skeletal muscle and thyroid, and weakly in lung, liver, spinal cord, brain, adrenal gland and bone marrow. Following TNFa stimulation, hPEBP4 expression increased in the A549 and LoVo cells, which do not normally express hPEBP4, reached a maximum at 12hr, and then decreasing. The inducible, time-dependent, hPEBP4 expression pattern suggests that hPEBP4 might play roles in cellular responses to extracellular stimuli and in self-protection from damage.The expression vectors of full-length hPEBP4 and p75PEBP4 (amino acids 1-75 of hPEBP4, lacking the highly conserved phosphatidylethanolamine-binding domain) fused to GFP protein in C-terminal fusion (hPEBP4-GFP, p75PEBP4-GFP) were transiently transfected into the HEK293 cells and examined by fluorescence confocal microscopy. hPEBP4-GFP, p75PEBP4-GFP were co-localizad with lysosomes. When transfected cells were stimulated with TNFa, hPEBP4-GFP translocated from lysosomes to the cell membrane while p75PEBP-GFP not.Glutathione S-transferase (GST) fusion expression system, an integrated system forthe expression, purification and detection of target protein fused with GST, was employed for the prokaryotic expression of hPEBP4. Analysis of the primary amino acid sequences of the varied PEBP family members revealed that the N terminus is poorly conserved in this family. The N-terminal 99aa section of hPEBP4 fused...
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