Cleavage without anchor addition accompanies the processing of a nascent protein to its glycosylphosphatidylinositol-anchored form.

Abstract

Rough microsomal membranes from most mammalian cells, in the presence of a translation system, process nascent proteins with appropriate COOH-terminal signal peptides to their mature glycosylphosphatidylinositol (GPI)-linked forms. The present study, using preprominiplacental alkaline phosphatase as substrate, shows that as much as 10%Yo of the mature product is cleaved correctly but is not linked to GPI. Some of the factors that influence the relative proportions of GPI linked to free mini-placental alkaline phosphatase are the amounts ofGPI in the cells and the amino acid substituent at the o site of the nascent protein. A mechanism for explaining cleavage both with and without GPI addition is presented, which supports a transamidase type of enzyme as the catalyst.A nascent protein (preproprotein), prior to processing to a glycosylphosphatidylinositol (GPI) form, contains both NH2-and COOH-terminal signal peptides that are cleaved sequentially to yield the mature form. Cleavage of the NH2-terminal peptide follows translocation of the nascent protein into the endoplasmic reticulum and results in the proprotein. Cleavage of the COOH terminus from the latter, accompanied by attachment of the GPI moiety, yields the mature protein. We have investigated processing in cell-free systems using a shortened, engineered form of nascent placental alkaline phosphatase (PLAP) to which we have given the name preprominiPLAP (see Fig. 1). In studies with rough microsomal membranes (RMs) from normal and GPI-deficient cells, we presented evidence that GPI is an obligatory cosubstrate for COOH-terminal processing of nascent proteins (1). During those studies we noticed that, in addition to the major product of the reaction, mature GPI-linked miniPLAP, a minor product is also formed. From its migration on gels, we suggested that this could represent mature miniPLAP that was not GPI-linked. In this report, we present clear evidence that processing of preprominiPLAP in the endoplasmic reticulum to its mature GPI-linked form is accompanied by some cleavage at the w site (the amino acid in a nascent protein that accepts the GPI moiety and, after cleavage, becomes the COOH-terminal residue of the mature protein) without GPI addition. The significance of this finding with respect to the mechanism of cleavage/GPI addition is discussed.

Leishmania mexicanaMutants Lacking Glycosylphosphatidylinositol (GPI):Protein Transamidase Provide Insights into the Biosynthesis and Functions of GPI-anchored Proteins

“…The nucleophilic ethanolamine component of the GPI anchor reacts with the active carbonyl, and the resultant GPI-anchored protein is then trafficked to the cell surface. This proposed reaction mechanism is supported by the finding that nucleophiles such as hydrazine can replace the GPI anchor in a cell-free assay system for GPI anchoring (Maxwell et al, 1995b;Sharma et al, 1999) and that the process is ATP and GTP independent (Mayor et al, 1991). Interestingly, the GPI signal sequence differs between species, and that of Trypanosoma brucei variable surface glycoprotein functioned poorly when expressed in mammalian cells (Moran and Caras, 1994).…”

“…This signal is recognized by the GPI:protein transamidase complex, which proteolytically cleaves the precursor protein at the amino acid and links the newly generated C terminus to the terminal ethanolamine phosphate (EtN-P) of a preassembled GPI anchor precursor having the structure EtN-PMan 3 GlcN-phosphatidylinositol (PI) (Udenfriend and Kodukula, 1995). The transamidase is thought to form an intermediate with the precursor protein, resulting in an activated carbonyl group at the site (Maxwell et al, 1995b;Sharma et al, 1999). The nucleophilic ethanolamine component of the GPI anchor reacts with the active carbonyl, and the resultant GPI-anchored protein is then trafficked to the cell surface.…”

A defect in glycosylphosphatidylinositol (GPI) transamidase activity in mutant K cells is responsible for their inability to display GPI surface proteins.

“…The use of this method permits analysis of the effects of another nucleophilic agent, hydroxylamine, which can serve as second alternative donor to the GPI anchor that otherwise would interfere with translation (17). To determine whether COOH-terminal processing of prominiPLAP by RM from mutant K cells could be achieved with hydroxylamine or otherwise forced under experimental conditions used in preloading, we compared posttranslational studies of this type using RM from HeLa, K562 parental, Thy-i-lym- phoma mutant F, and mutant K cells.…”

“…To further assess the nature of the block in COOH-terminal processing in mutant K cells, we conducted experiments with hydrazine, which can function as an alternate nucleophile (17) in the transamidation reaction that couples GPI anchor structures to polypeptides. As can be seen in Fig.…”

“…1). We exploited recent findings that prominiPLAP, in the presence of the GPI transamidase can react with the nucleophiles hydrazine or hydroxylamine in place of GPI to yield the hydrazide or hydroxamate of miniPLAP (17).…”