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Biosynthesis of glycosylphosphatidylinositol-anchored human placental alkaline phosphatase: evidence for a phospholipase C-sensitive precursor and its post-attachment conversion into a phospholipase C-resistant form.

ABSTRACT: Previous studies have shown that some cells (e.g. SKG3a) express human placental alkaline phosphatase (AP) in a form which can be released from the membrane by bacterial PtdIns-specific phospholipase C (PI-PLC) while others (e.g. HeLa) are relatively resistant to this enzyme. Chemical and enzymic degradation studies have suggested that the PI-PLC resistance of AP is due to inositol acylation of its glycosylphosphatidylinositol (GPI) anchor. In order to identify the biosynthetic origin of PI-PLC resistance we determined the PI-PLC sensitivity of AP in 35S-labelled cells (10 min pulse; 0-60 min chase) by Triton X-114 phase separation. At the beginning of the chase period, the majority of the AP synthesized was hydrophilic, indicating that it had not acquired a GPI anchor. The concentration of hydrophilic AP species decreased with a t1/2 of 30-60 min but was not processed to an endoglycosidase H-resistant species or secreted into the medium. In both SKG3a and HeLa cells all of the hydrophobic, GPI-anchored AP detectable at the beginning of the chase was PI-PLC sensitive. PI-PLC-resistant species of AP were only observed in HeLa cells and these only appeared after about 30 min. The delayed appearance of PI-PLC resistance was unexpected as previous studies have suggested that candidate GPI-anchor precursors are PI-PLC-resistant as a result of inositol acylation. This work reveals unanticipated complexities in the biosynthesis of AP and its GPI anchor.

SUBMITTER: Wong YW

PROVIDER: S-EPMC1137163 | biostudies-other | 1994 Jul

REPOSITORIES: biostudies-other

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Project description:Placental alkaline phosphatase (PLAP) is initially synthesized as a precursor (proPLAP) with a C-terminal extension. We constructed a recombinant cDNA which encodes a chimeric protein (alpha GL-PLAP) comprising rat alpha 2u-globulin (alpha GL) and the C-terminal extension of PLAP. Two molecular species (25 kDa and 22 kDa) were expressed in the COS-1 cell transfected with the cDNA for alpha GL-PLAP. Only the 22 kDa form was labelled with both [3H]stearic acid and [3H]ethanolamine. Upon digestion with phosphatidylinositol-specific phospholipase C the 22 kDa form was released into the medium, indicating that this form is anchored on the cell surface via glycosylphosphatidylinositol (GPI). A specific IgG raised against a C-terminal nonapeptide of proPLAP precipitated the 25 kDa form but not the 22 kDa form, suggesting that the 25 kDa form is a precursor retaining the C-terminal propeptide. When a mutant alpha GL-PLAP, in which the aspartic acid residue is replaced with tryptophan at a putative cleavage/attachment site, was expressed in COS-1 cells, the 25 kDa precursor was the only form found inside the cell and retained in the endoplasmic reticulum, as judged by immunofluorescence microscopy. In vitro translation programmed with mRNAs coding for the wild-type and mutant forms of alpha GL-PLAP demonstrated that the C-terminal propeptide was cleaved from the wild-type chimeric protein, but not from the mutant one. This gave rise to the 22 kDa form attached with a GPI anchor, suggesting that GPI is covalently linked to the aspartic acid residue (Asp159) of alpha GL-PLAP. Taken together, these results indicate that the C-terminal propeptide of PLAP functions as a signal to render alpha GL a GPI-linked membrane protein in vitro and in vivo in cultured cells, and that the chimeric protein constructed in this study may be useful for elucidating the mechanism underlying the cleavage of the propeptide and attachment of GPI, which occur in the endoplasmic reticulum.

Project description:Alkaline phosphatase in a wide range of tissues has been shown to be anchored in the membrane by a specific interaction with the polar head group of phosphatidylinositol. It has previously been suggested that the production of low Mr alkaline phosphatase during the commonly used butanol extraction procedure may result from the activation of an endogenous phosphoinositide-specific phospholipase C which removes the 1,2-diacylglycerol responsible for membrane anchoring. This conversion process was investigated in greater detail with human placenta used as the source of alkaline phosphatase. Mr and hydrophobicity of the alkaline phosphatase were determined by gel filtration on TSK-250 and partitioning in Triton X-114, respectively. Alkaline phosphatase extracted from human placental particulate fraction with butanol at pH 5.4 or released by incubation with Staphylococcus aureus phosphatidylinositol-specific phospholipase C produced a form of alkaline phosphatase of Mr approx. 170,000 and relatively low hydrophobicity. By contrast, the butanol extract prepared at pH 8.3 was an aggregated form of Mr approx. 600,000 and was relatively hydrophobic. The effect of a variety of inhibitors and activators on the amount of low Mr alkaline phosphatase produced during butanol extraction revealed that it was a Ca2+- and thiol-dependent process. Proteinase inhibitors had no effect. [3H]Phosphatidylinositol hydrolysis by the particulate fraction, unlike low Mr alkaline phosphatase production, was relatively sensitive to heat inactivation, indicating that the phosphoinositide-specific phospholipases C from cytosol and lysosomes were unlikely to be responsible for conversion. A butanol-stimulated activity which removed the [3H]myristic acid from the variant surface glycoprotein ( [3H]mfVSG) of Trypanosoma brucei was detectable in the human placental particulate fraction. Since this activity was acid active, Ca2+- and thiol-dependent and relatively heat stable, it may be the same as that responsible for production of low Mr alkaline phosphatase. The only 3H-labelled product identified was phosphatidic acid, suggesting that the [3H]mfVSG-cleaving activity is a phospholipase D. These data strongly support the proposal that production of low Mr alkaline phosphatase during butanol extraction is an autolytic process occurring as the result of an endogenous phospholipase. However, they also suggest that the lysosomal and cytosolic phosphoinositide-specific phospholipases C that have previously been described in many mammalian tissues are not responsible for this process.

Project description:When fused in-frame with a C-terminal propeptide of placental alkaline phosphatase (PLAP), rat alpha 2u-globulin (alpha GL), a nonglycosylated secretory protein, was expressed on the cell surface as a glycosylphosphatidylinositol (GPI)-linked chimaeric protein (alpha GL-PLAP). In contrast with the wild-type alpha GL-PLAP, a mutant, in which Asp at the cleavage/attachment site of GPI was replaced by Trp, failed to become a GPI-linked mature form and was retained as a precursor form within the cell [Oda, Cheng, Saku, Takami, Sohda, Misumi, Ikehara and Millán (1994) Biochem. J. 301, 577-583]. To elucidate the molecular interactions involved in the retention of the proform within the cell, we examined the association of the proform with molecular chaperones in the endoplasmic reticulum (ER). Antibody against the ER retrieval motif KDEL coimmunoprecipitated a 25 kDa proform, but not a 22 kDa GPI-linked mature form. Pulse-chase experiments showed that the wild-type alpha GL-PLAP with a cleavable propeptide was converted into the mature form, whereas the mutant alpha GL-PLAP with an uncleavable propeptide remained associated with ER-resident proteins with a KDEL motif and underwent rapid degradation in a pre-Golgi compartment. Chemical cross-linking studies showed that, of the several ER-resident proteins immunoreactive with the anti-KDEL antibody, a 78 kDa protein was the only protein associated with the proform. Furthermore this 78 kDa protein was dissociated from the precursor molecule on incubation with ATP, allowing us tentatively to assign it as Bip/GRP78. Anticalnexin antibody, however, failed to coprecipitate any form of the chimaeric protein. Immunoelectron microscopy showed that the proform with the uncleavable propeptide was localized in the ER, but not detected in the Golgi apparatus or plasma membranes. Taken together, these results suggest that Bip/GRP78 is associated with pro alpha GL-PLAP and retains it within the ER until pro alpha GL-PLAP is either modified by GPI or degraded, thereby participating in the quality control of this GPI-linked chimaeric protein.

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Biosynthesis of glycosylphosphatidylinositol-anchored human placental alkaline phosphatase: evidence for a phospholipase C-sensitive precursor and its post-attachment conversion into a phospholipase C-resistant form.

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