Two cDNAs encode rat intestinal alkaline phosphatases having completely different carboxyl-terminal peptides; one is hydrophobic and fulfills the consensus requirements for glycan phosphatidylinositol linkage, and the other is neither hydrophobic nor hydrophilic, but contains a small amino acid domain (-NSASS-) just distal to a region of 17 threonine residues. Constructs were created using 80% of the amino-terminal portion of one alkaline phosphatase and the carboxyl-terminal portions of each of the isoforms. Both of the carboxyl-terminal peptides supported glycan phosphatidylinositol linkage as demonstrated by the following criteria: 1) plasma membrane targeting in transfected COS-1 cells, 2) release of transfected alkaline phosphatase by phosphatidylinositol-specific phospholipase C, 3) appearance of the trypanosome variable glycoprotein cross-reacting determinant after phospholipase C treatment, 4) ethanolamine incorporation into newly synthesized enzyme, 5) loss of phospholipase C release after mutation of the ω and ω + 2 positions in the putative linkage site, -NSA-, and 6) evidence of surface membrane localization by immunofluorescence using antibody against rat intestinal alkaline phosphatase. These data demonstrate that a predicted hydrophobic carboxyl-terminal sequence is not essential for glycan phosphatidylinositol linkage. Moreover, because both isomers are membrane-bound, the origin of soluble enzyme in the serum is likely to arise from the action of serum phosphatidylinositol-specific phospholipase D.
CITATION STYLE
Engle, M. J., Mahmood, A., & Alpers, D. H. (1995). Two rat intestinal alkaline phosphatase isoforms with different carboxyl-terminal peptides are both membrane-bound by a glycan phosphatidylinositol linkage. Journal of Biological Chemistry, 270(20), 11935–11940. https://doi.org/10.1074/jbc.270.20.11935
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