COOH-terminal processing of nascent polypeptides by the glycosylphosphatidylinositol transamidase in the presence of hydrazine is governed by the same parameters as glycosylphosphatidylinositol addition.

Abstract

Proteins anchored to the cell membrane via a glycosylphosphatidylinositol (GPI) moiety are found in all eukaryotes. After NH2-terminal peptide cleavage of the nascent protein by the signal peptidase, a second COOH-terminal signal peptide is cleaved with the concomitant addition of the GPI unit. The proposed mechanism of the GPI transfer is a transamidation reaction that involves the formation of an activated carbonyl intermediate (enzyme-substrate complex) with the ethanolamine moiety of the preassembled GPI unit serving as a nucleophile. Other nucleophilic acceptors like hydrazine (HDZ) and hydroxylamine have been shown to be possible alternate substrates for GPI. Since GPI has yet to be purified, the use of readily available nucleophilic substitutes such as HDZ and hydroxylamine is a viable alternative to study COOH-terminal processing by the putative transamidase. As a first step in developing a soluble system to study this process, we have examined the amino acid requirements at the COOH terminus for the transamidation reaction using HDZ as the nucleophilic acceptor instead of GPI. The hydrazideforming reaction shows identical amino acid requirement profiles to that of GPI anchor addition. Additionally, we have studied other parameters relating to the kinetics of the transamidation reaction in the context of rough microsomal membranes. The findings with HDZ provide further evidence for the transamidase nature of the enzyme and also provide a starting point for development of a soluble assay.Glycosylphosphatidylinositol (GPI) anchoring is a mechanism used by all eukaryotic cells to tether proteins to the outer cell membrane (1-7). A nascent protein (preproprotein) that is destined to be GPI anchored contains an NH2-terminal signal sequence and a COOH-terminal signal sequence, both of which are cleaved sequentially first by the NH2-terminal signal peptidase and then by a putative transamidase to yield the mature GPI-linked protein. Cleavage at the COOH-terminal signal proceeds in a concerted manner in which the GPI moiety is incorporated at the newly exposed COOH terminus of the protein.The amino acid residue that condenses with the GPI moiety is designated the X residue (8,9). Analogous to the -1, -3 rule that has been described for NH2-terminal signal peptidase activity, an wc,w+2 rule for COOH-terminal processing has been described (8). (8) (Fig. 1A).In previous work with an in vitro translation system employing rough microsomal membranes (RM) (8), we demonstrated that the proform of an engineered version of human placental alkaline phosphatase (PLAP), miniPLAP, is processed to the mature COOH-terminal cleaved form. Processing of nascent preprominiPLAP (28 kDa) gives rise to prominiPLAP (27 kDa), in which the NH2-terminal signal sequence is removed. This proform is subsequently converted to mature miniPLAP (24.7 kDa), in which a 29-residue COOH-terminal signal sequence is cleaved and replaced by GPI. In addition to the formation of this anchor-containing product (Fig. 1B), cleavage of prominiPLAP...

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

“…In contrast, depletion of intracellular levels of GPI protein anchor precursors by ectopic expression of T. brucei GPI-PLC in L. major resulted in the secretion of a hydrophilic form of gp63 (Mensa-Wilmot et al, 1994). Similarly, cleavage of the signal peptide without anchor addition has been reported in both mammalian and trypanosomal in vitro systems and resulted in the release of processed, but GPI-free, protein (Maxwell et al, 1995a;Ramalingam et al, 1996;Sharma et al, 1999). However, in the absence of the proteolytic processing event (as would be the situation in ⌬GPI8), the protein is targeted for degradation.…”

Addition of a Glycophosphatidylinositol to Acetylcholinesterase

“…A third possibility is that the lytic secreted molecules may be generated by an aborted transamidation, in which cleavage of the polypeptide chain would be followed by reaction with a water molecule instead of the GPI anchor (17,44). Such a reaction was observed with the nucleophilic reagent hydrazine: by reacting with an intermediate of the transamidation reaction, hydrazine prevents the attachment of the GPI anchor and releases a cleaved, soluble protein.…”

“…The structural requirements of the C-terminal signal peptides that induce GPI addition have been investigated extensively by the groups of Caras and Udenfriend, by mutagenesis of the "decay-accelerating factor" (9 -12) and of placental alkaline phosphatase (13)(14)(15)(16)(17). They showed that glypiation requires a C-terminal hydrophobic sequence and an upstream cleavage/addition site (2,13).…”

The GPI Transamidase Complex ofSaccharomyces cerevisiaeContains Gaa1p, Gpi8p, and Gpi16p

“…The transfer of preformed GPIs onto proteins has been studied in microsomal translation/translocation/GPI-anchoring systems (Ramalingam et al, 1996;Sharma et al, 1999) or translocation/GPI-anchoring systems (Doering and Schekman, 1997) in several organisms, and these studies allowed a preliminary biochemical characterization of the GPI transfer reaction. The GPI transferase is believed to act as a transamidase, i.e., to jointly remove the GPI-anchoring signal and transfer the preformed GPI (Ramalingam et al, 1996;Sharma et al, 1999).…”

“…The GPI transferase is believed to act as a transamidase, i.e., to jointly remove the GPI-anchoring signal and transfer the preformed GPI (Ramalingam et al, 1996;Sharma et al, 1999). Genetic approaches have identified genes required for the addition of GPI anchors.…”