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Organization of the gene encoding common acute lymphoblastic leukemia antigen (neutral endopeptidase 24.11): multiple miniexons and separate 5' untranslated regions.

ABSTRACT: The common acute lymphoblastic leukemia antigen (CALLA) is a 749-amino acid type II integral membrane protein that has been identified recently as the neutral endopeptidase 24.11 [NEP (EC 3.4.24.11)]. Herein, we characterize the organization of the human CALLA/NEP gene and show that it spans more than 80 kilobases (kb) and is composed of 24 exons. Exons 1 and 2 encode 5' untranslated sequences; exon 3 [170 base pairs (bp)] encodes the initiation codon and transmembrane and cytoplasmic domain; 20 short exons (exons 4-23), ranging in size from 36 to 162 bp, encode most of the extracellular portion of the enzyme; and exon 24 (approximately 3400 bp) encodes the COOH-terminal 32 amino acids of the protein and contains the entire 3' untranslated region (UTR). Of note, the pentapeptide sequence (His-Glu-Ile-Thr-His) associated with metalloprotease zinc binding and substrate catalysis is encoded within a single exon (exon 19). Three types of CALLA/NEP cDNAs have been identified: these clones contain 5' UTR sequences differing from one another upstream of exon 3. These human 5' sequences are homologous to those found in rat brain and rabbit kidney NEP cDNAs. The three human CALLA cDNA types result from alternative splicing of exons 1, 2a, or 2b to the common exon 3. Moreover, exons 2a and 2b share the same 5' sequence but differ from each other by the use of two distinct donor splice sites 171 bp apart in the gene. The substantial conservation of 5' untranslated sequences among species and the existence of 5' alternative splicing suggest that CALLA gene expression may be differentially controlled in a tissue-specific and/or developmentally regulated fashion.

SUBMITTER: D'Adamio L

PROVIDER: S-EPMC298003 | biostudies-other | 1989 Sep

REPOSITORIES: biostudies-other

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Organization of the gene encoding common acute lymphoblastic leukemia antigen (neutral endopeptidase 24.11): multiple miniexons and separate 5' untranslated regions.

D'Adamio L L D'Adamio L L Shipp M A MA Masteller E L EL Reinherz E L EL

Proceedings of the National Academy of Sciences of the United States of America 19890901 18

The common acute lymphoblastic leukemia antigen (CALLA) is a 749-amino acid type II integral membrane protein that has been identified recently as the neutral endopeptidase 24.11 [NEP (EC 3.4.24.11)]. Herein, we characterize the organization of the human CALLA/NEP gene and show that it spans more than 80 kilobases (kb) and is composed of 24 exons. Exons 1 and 2 encode 5' untranslated sequences; exon 3 [170 base pairs (bp)] encodes the initiation codon and transmembrane and cytoplasmic domain; 20 ...[more]

PMID: 2528730

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Project description:The human colon cancer cell line Caco-2 undergoes spontaneous enterocytic differentiation during growth, and expresses a number of brush-border-membrane-associated hydrolases typical of a differentiated phenotype. Among these are alkaline phosphatase, dipeptidyl peptidase IV and sucrase-isomaltase (sucrase, EC 3.2.1.48). Neutral endopeptidase 24.11 [EC 3.4.24.11, neprilysin (NEP)] is another abundant protease of normal enterocytes but its presence in Caco-2 cells has not been fully documented yet. In this paper, we show that Caco-2 cell extracts hydrolyse tritiated [D-Ala2Leu5]enkephalin with a Km of 180 microM, very close to the value obtained for the NEP present in the rabbit kidney (118 microM). Western-blot analysis of brush-border membranes purified from post-confluent cells revealed a protein with an apparent molecular mass of 94000 Da similar to that of the rabbit kidney NEP. The amount of enzyme in cell extracts increased as a function of the age of the culture, indicating that NEP expression is correlated with the degree of cell differentiation as is also the case for sucrase and dipeptidylpeptidase IV (DPP-IV). Binding of a radiolabelled antibody to Caco-2 cell monolayers grown on semi-permeable filters indicated that 95% of NEP molecules present at the cell surface are on the apical side. Immunocytochemical and flow cytometric analysis of intact and permeabilized cells were also used to investigate the presence of NEP and DPP-IV at the surface of Caco-2 cells. Whereas DPP-IV staining appeared to be homogeneous throughout the entire cell population, NEP-related fluorescence exhibited a bimodal distribution which indicates an uneven expression of the protein at the cell surface. Permeabilization of monolayers with saponin before staining restored a labelling pattern for NEP similar to the one obtained for DPP-IV. This suggests that although DPP-IV and NEP follow similar patterns of expression when enzymic activities are measured on whole-cell extracts, targeting of these brush-border proteins to the cell surface appears to be regulated in different ways.

Dataset Information

Organization of the gene encoding common acute lymphoblastic leukemia antigen (neutral endopeptidase 24.11): multiple miniexons and separate 5' untranslated regions.

Publications

Organization of the gene encoding common acute lymphoblastic leukemia antigen (neutral endopeptidase 24.11): multiple miniexons and separate 5' untranslated regions.

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