Molecular cloning and expression of a cDNA encoding the membrane-associated rat intestinal alkaline phosphatase

Mark Lowe, Arnold W. Strauss, Ruth Alpers, Shakuntla Seetharam, David H. Alpers

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46 Scopus citations

Abstract

Rat intestinal alkaline phosphatase (IAP) has been purified and proteolytic fragments sequenced. A cDNA library was constructed from duodenal poly(A)+ RNA and screened for IAP positive clones by a full-length cDNA clone-encoding human IAP. A full length rat IAP clone (2237 bp) was isolated and sequenced, revealing a predicted primary sequence of 519 amino acids (61.974 kDa) with an additional signal peptide of 20 amino acids. 80% of amino acids from residues 1-474 were identical when compared with the human IAP, but there was only 31% identity in the COOH-terminal 45 amino acids. The homology diverges just before the putative binding site for the phosphatidylinositol-glycan (PI-glycan) anchor. The resulting peptide in rat AP contains five hydrophilic amino acids not present in the primary structure of human IAP. Binding of a synthetic 48-mer encoding a portion of this unique and divergent region (residues 476-491) was compared with that of the full-length clone on Northern blots of rat intestinal RNA. Two mRNAs, 3.0 and 2.7 kb, were detected by both probes, confirming earlier results, but the 48-mer bound preferentially to the 3.0 kb mRNA. The protein product of the full-length cDNA in a cell-free system was 62 kDa, corresponding with the smaller of the two IAP proteins produced by rat duodenal RNA. The cDNA transfected into COS-1 cells produced a membrane-bound IAP that was released by phosphatidylinositol-specific phospholipase (PI-PLC). These data provide definitive evidence that IAP is anchored by PI-glycan and conclusively demonstrate that the unique COOH-terminal structure encoded by this rat mRNA supports the addition of a PI-glycan anchor.

Original languageEnglish
Pages (from-to)170-177
Number of pages8
JournalBiochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
Volume1037
Issue number2
DOIs
StatePublished - Feb 9 1990

Keywords

  • Alkaline phosphatase
  • Phosphatidylinositol

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