TY - JOUR
T1 - Molecular cloning and expression of a cDNA encoding the membrane-associated rat intestinal alkaline phosphatase
AU - Lowe, Mark
AU - Strauss, Arnold W.
AU - Alpers, Ruth
AU - Seetharam, Shakuntla
AU - Alpers, David H.
N1 - Funding Information:
This work was supported in part by grants AM 10438 and AM 20407 from the National Institutes of Health. The authors are grateful to Drs. Harry Hams and Paula S. Henthorn for providing the cDNA clone encoding humar, intestinal alkaline phosphatase.
PY - 1990/2/9
Y1 - 1990/2/9
N2 - 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.
AB - 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.
KW - Alkaline phosphatase
KW - Phosphatidylinositol
UR - http://www.scopus.com/inward/record.url?scp=0025019390&partnerID=8YFLogxK
U2 - 10.1016/0167-4838(90)90164-B
DO - 10.1016/0167-4838(90)90164-B
M3 - Article
C2 - 2155025
AN - SCOPUS:0025019390
SN - 0167-4838
VL - 1037
SP - 170
EP - 177
JO - Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
JF - Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
IS - 2
ER -