Selectivity of the cleavage/attachment site of phosphatidylinositol-glycan-anchored membrane proteins determined by site-specific mutagenesis at Asp-484 of placental alkaline phosphatase.

Abstract

Many proteins are now known to be anchored to the plasma membrane by a phosphatidylinositol-glycan (PI-G) moiety that is attached to their COOH termini. Placental alkaline phosphatase (PLAP) has been used as a model for investigating mechanisms involved in the COOH-terminal processing of PI-G-tailed proteins. The COOH-terminal domain of pre-pro-PLAP provides a signal for processing during which a largely hydrophobic 29-residue COOH-terminal peptide is removed, and the PI-G moiety is added to the newly exposed Asp484 terminus. This cleavage/attachment site was subjected to an almost saturation mutagenesis, and the enzymatic activities, COOH-terminal processing, and cellular localizations of the various mutant PLAP forms were determined. Substitution of Asp-484 by glycine, alanine, cysteine, asparagine, or serine (category I) resulted in PI-G-tailed and enzymatically active proteins. However, not all category I mutant proteins were PI-G tailed to the same extent. Prepro-PLAP with other substituents at position 484

Biosynthesis of glycosylphosphatidylinositol (GPI)-anchored membrane proteins in intact cells: specific amino acid requirements adjacent to the site of cleavage and GPI attachment.

“…For example, when mutating alanine at co + 1, the wild-type amino acids at ~0 and ~ + 2 (aspartic acid and aianine, respectively) were preserved, thus the ~0 + I mutants w~d have a sequence Asp-X-AIa and the ~0 + 2 mutants ha~e an Asp-Ala-X motif in the small amino acid domain. Similarly, the site mutations at the ~0 position had an X-AlaAla motif as reported earlier (18,19).…”

“…Processing of the COOH terminus is a prerequisite for PLAP to be expressed on the cell surface (18,19). Transfected cells, both w + 1 and w + 2 mutants, were homogenized and total PLAP enzyme activity was assayed.…”

“…Mutational studies carried out at the co site of the nascent forms of placental alkaline phosphatase (PLAP) in intact cells (18,19) and of the engineered protein miniPLAP in a cell-free system (14) indicated that only glycine, alanine, serine, cysteine, aspartic acid, and asparagine are allowed. These same six amino acids are the only ones that have been reported to be present at the co sites of the GPI-anchored proreins that have been characterized thus far (8, I0, 11).…”

Cellular function and molecular structure of ecto-nucleotidases

“…The N-terminal signal peptide is specifically removed and the C-terminal of the nascent pre-pro-PLAP provides the signal for processing, during which a largely hydrophobic 29-residue C-terminal peptide is removed. The phosphatidylinositol-glycan moiety is then added to the newly exposed Asp-484 terminus [555,556]. Phosphatidylinositol-glycan tailing is a prerequisite for transport from the ER and for PLAP enzyme activity.…”

“…Phosphatidylinositol-glycan tailing is a prerequisite for transport from the ER and for PLAP enzyme activity. Otherwise, proteins are retained in the ER in an inactive conformation [555]. Conversely, interruption of the C-terminal 29-amino acid hydrophobic stretch by a charged residue resulted in protein secretion into the medium [557].…”