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. 1998 Aug 4;95(16):9512-7.
doi: 10.1073/pnas.95.16.9512.

Mammalian glycophosphatidylinositol anchor transfer to proteins and posttransfer deacylation

R Chen  1 , E I Walter, G Parker, J P Lapurga, J L Millan, Y Ikehara, S Udenfriend, M E Medof
Affiliations

Mammalian glycophosphatidylinositol anchor transfer to proteins and posttransfer deacylation

R Chen et al. Proc Natl Acad Sci U S A. .

Abstract

The glycophosphatidylinositol (GPI) anchors of proteins expressed on human erythrocytes and nucleated cells differ with respect to acylation of an inositol hydroxyl group, a structural feature that modulates their cleavability by PI-specific phospholipase C (PI-PLC). To determine how this GPI anchor modification is regulated, the precursor and protein-associated GPIs in two K562 cell transfectants (ATCC and .48) exhibiting alternatively PI-PLC-sensitive and resistant surface proteins were analyzed and the temporal relationship between GPI protein transfer and acquisition of PI-PLC sensitivity was determined. Nondenaturing PAGE analyses demonstrated that, whereas in .48 transfectants the GPI anchors in decay accelerating factor (DAF) and placental alkaline phosphatase (PLAP) were >95% acylated, in ATCC transfectants, they were 60 and 33% unsubstituted, respectively. In contrast, TLC analyses revealed that putative GPI donors in the two lines were identical and were >/=95% acylated. Studies of de novo DAF biosynthesis in HeLa cells bearing proteins with >90% unacylated anchors showed that within 5 min at 37 degreesC (or at 18 degreesC, which does not permit endoplasmic reticilum exit), >50% of the anchor in nascent 44-kDa proDAF protein exhibited PI-PLC sensitivity. In vitro analyses of the microsomal processing of miniPLAP, a truncated PLAP reporter protein, demonstrated that the anchor donor initially transferred to prominiPLAP was acylated and then progressively was deacylated. These findings indicate that (i) the anchor moiety that initially transfers to nascent proteins is acylated, (ii) inositol acylation in mature surface proteins is regulated via posttransfer deacylation, which in general is cell-specific but also can be protein-dependent, and (iii) deacylation occurs in the endoplasmic reticulum immediately after GPI transfer.

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Figures

Figure 1
Figure 1
(A) In situ cleavage by PI-PLC of endogenous DAF in K562 ATCC and K562.48 cells. Cells were treated with PI-PLC and then stained with anti-DAF or nonrelevant mAbs and analyzed by flow cytometry. Percent DAF released equals the difference between the fluorescence of buffer- and PI-PLC-treated cells. (B) ND-PAGE analyses of in situ PLAP protein. Samples in lanes 1 and 2 were treated with buffer at 0°C overnight, and in lanes 3 and 4 treated with hydroxylamine, pH 10.7 at 0°C overnight. Samples in lanes 1 and 3 then were incubated with buffer and samples in lanes 2 and 4 with PI-PLC (1:50) at 37°C for 2 hr. F, protein freed from detergent micelles; B, protein bound to detergent micelles; O, origin.
Figure 2
Figure 2
TLC analyses of GPI products deriving from [3H]mannose labeling of K562 ATCC and .48 lines. [3H]mannose-labeled GPI intermediates from tunicamycin-pretreated HeLa cells (A), K562 ATCC cells (B), or K562.48 cells (C) were separated on TLC plates developed in chloroform:methanol:water (10:10:3). The positions of previously characterized HeLa cell bands H6, H7, and H8 and of dolichol-phosphoryl-mannose are indicated. As control (D) [3H]mannose-labeled Tryp GPIs were prepared from Tryp lysate (1 × 107 cell equivalent) incubated with 10 μCi GDP-[3H]mannose. Aliquots of GPI products deriving from each source alternatively were incubated with buffer or PI-PLC. The radioactivity (cpm) falling within the peak (H8 and H7 for the K562 cell lines and A′ for Tryp) was electronically measured.
Figure 3
Figure 3
GPI anchor acylation in newly synthesized DAF polypeptides. HeLa cells were labeled with [35S]C for 5 or 15 min at 37°C, [35S]-labeled surface proteins affinity purified, and products eluted from the anti-DAF beads were incubated with PI-PLC or corresponding buffer and partitioned in TX-114 detergent. [35S]-labeled proteins in the aqueous and organic phases were then repurified with IA10-Sepharose.
Figure 4
Figure 4
Microsomal processing of preprominiPLAP. miniPLAP RNA was added in cotranslational assays with [35S]M and RM. (A) Products deriving from processing of preprominiPLAP by K562.ATCC (lanes 1–4) and .48 RM (lanes 5–8), treated with buffer alone (lanes 1 and 5), buffer and PI-PLC (lanes 2 and 6), alkaline/hydroxylamine alone (lanes 3 and 7), or alkaline/hydroxylamine and PI-PLC (lanes 4 and 8). (B) Products deriving from HeLa cell RM were treated with buffer or hydroxylamine and PI-PLC and then partitioned in TX-114 detergent. A, aqueous phase; D, detergent phase. (C and D) Cotranslational processing of preprominiPLAP in the presence of HeLa RM was stopped after the indicated times, the mixture treated with buffer (lanes 1–6) or PI-PLC (lanes 7–12) and the products analyzed. (D) Densitometric data.
Figure 5
Figure 5
Proposed relationship between GPI anchor transfer and deacylation. The anchor initially transfers in an acylated form. Inositol deacylation then occurs either(i) as a subsequent reaction physically unassociated with transamidation mediated by separate factor (deacylase) or (ii) as a concerted reaction carried out by a multiprotein transamidase deacylation complex. Hatched bar, C-terminal signal peptide; solid bar, EthN-P-containing glycan of GPI donor; striped bar, N-terminal signal peptide.
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