ABSTRACT
The 5' flanking region of the gene encoding the small intestinal brush-border peptidase aminopeptidase N (APN) was screened for the presence of enhancer regions. A 300 bp region with enhancer activity was identified 2.7 kb upstream of the transcriptional start site which is used in epithelial cells. The enhancer stimulated transcription from a heterologous promoter (the simian virus 40 early promoter) in a position- and orientation-independent manner. The activity of the enhancer is cell-type dependent and it is active in liver (HepG2), intestinal (Caco-2) and myeloid (K562) cells. As the epithelial APN promoter is active in the first two cell-types and the myeloid APN promoter in the last, the results may suggest that the enhancer, through a cooperation with either of the promoters, is important for the tissue-specific expression of APN. A detailed analysis of the enhancer led to the identification of four functionally important regions that are protected against DNase I digestion by Caco-2 nuclear extract. Sequence analysis suggests that two of the regions may interact with members of the Ets transcription factor family (Ets is a transformation-specific protein first discovered in the E26 avian erythroblastosis virus), one region with a CCAAT enhancer-binding protein and one region with Sp1, a transcriptional activator first described as a factor binding to the simian virus 40 early promoter.
Subject(s)
CD13 Antigens/genetics , Chromosome Mapping , Enhancer Elements, Genetic/genetics , Promoter Regions, Genetic/genetics , Base Sequence , Caco-2 Cells , Epithelium , HeLa Cells , Humans , Leukocytes , Molecular Sequence Data , Organ Specificity , Point Mutation , Sequence Analysis, DNAABSTRACT
The importance of HNF1 binding proteins for intestinal aminopeptidase N expression was investigated using the Caco-2 cell-line. Aminopeptidase N promoter activity in Caco-2 cells depends on the HNF1 element (positions -85 to -58) and co-transfection with an HNF1 alpha expression vector demonstrates a direct activation of the promoter by HNF1 alpha through this element. Electrophoretic mobility shift assays using nuclear extracts from Caco-2 cells show the presence of high amounts of HNF1 binding proteins irrespective of their state of differentiation.
Subject(s)
Aminopeptidases/genetics , DNA-Binding Proteins , Intestinal Mucosa/enzymology , Nuclear Proteins , Transcription Factors/metabolism , Animals , CD13 Antigens , Cell Nucleus/metabolism , Gene Expression Regulation, Neoplastic , HeLa Cells , Hepatocyte Nuclear Factor 1 , Hepatocyte Nuclear Factor 1-alpha , Hepatocyte Nuclear Factor 1-beta , Humans , Promoter Regions, Genetic , Protein Binding , Swine , Tumor Cells, CulturedABSTRACT
Aminopeptidase N/CD13 is a metallopeptidase found in many tissues. Aminopeptidase N activity is high in the small intestinal mucosa, moderate in the liver, and low in the spleen. Using DNase I footprinting and electrophoretic mobility shift assays with nuclear extracts from these tissues, three cis elements (DF, LF-B1, UF) were identified in the aminopeptidase N promoter. The DF region (-53 to -30) interacts with the ubiquitously expressed transcription factor Sp1. The LF-B1 region (-85 to -58) interacts with the liver transcription factor LF-B1 (HNF-1) which was detected as well in nuclei from small intestinal mucosa. The UF region (-112 to -90) interacts with nuclear factors which seem to be expressed differentially in the liver and the small intestine. Transfection of promoter deletions into HepG2 cells showed that the LF-B1 region is necessary for high expression of the aminopeptidase N gene in liver cells. LF-B1 could not be detected in spleen nuclei. In accordance with this, RNA analysis demonstrated that the aminopeptidase N promoter operating in the small intestine and in the liver is inactive in the spleen. In this tissue initiation of transcription from the aminopeptidase N gene occurs from an upstream promoter.
Subject(s)
Aminopeptidases/metabolism , Carcinoma, Hepatocellular/pathology , Liver Neoplasms/pathology , Nuclear Proteins/metabolism , Promoter Regions, Genetic , Animals , Base Sequence , CD13 Antigens , DNA Fingerprinting , Glycosylation , Humans , Intestine, Small/enzymology , Liver/enzymology , Molecular Sequence Data , Plasmids , RNA, Neoplasm/genetics , Spleen/enzymology , Swine , Transcription Factors , Transfection , Tumor Cells, CulturedABSTRACT
The complete primary structure (967 amino acids) of an intestinal human aminopeptidase N (EC 3.4.11.2) was deduced from the sequence of a cDNA clone. Aminopeptidase N is anchored to the microvillar membrane via an uncleaved signal for membrane insertion. A domain constituting amino acid 250-555 positioned within the catalytic domain shows very clear homology to E. coli aminopeptidase N and contains Zn2+ ligands. Therefore these residues are part of the active site. However, no homology of the anchor/junctional peptide domain is found suggesting that the juxta- and intra-membraneous parts of the molecule have been added/preserved during development. It is speculated that this part carries the apical address.
