An Active Carbonyl Formed during Glycosylphosphatidylinositol Addition to a Protein Is Evidence of Catalysis by a Transamidase

Maxwell, Stephen E.; Ramalingam, Sandhya; Gerber, Louise; Brink, Larry; Udenfriend, Sidney

doi:10.1074/jbc.270.33.19576

Cited by 78 publications

(85 citation statements)

References 39 publications

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“…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.…”

Section: Discussionmentioning

confidence: 94%

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

Hilley

Zawadzki

McConville

et al. 2000

MBoC

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The major surface proteins of the parasitic protozoon Leishmania mexicana are anchored to the plasma membrane by glycosylphosphatidylinositol (GPI) anchors. We have cloned the L. mexicana GPI8 gene that encodes the catalytic component of the GPI:protein transamidase complex that adds GPI anchors to nascent cell surface proteins in the endoplasmic reticulum. Mutants lacking GPI8 (⌬GPI8) do not express detectable levels of GPI-anchored proteins and accumulate two putative protein-anchor precursors. However, the synthesis and cellular levels of other nonprotein-linked GPIs, including lipophosphoglycan and a major class of free GPIs, are not affected in the ⌬GPI8 mutant. Significantly, the ⌬GPI8 mutant displays normal growth in liquid culture, is capable of differentiating into replicating amastigotes within macrophages in vitro, and is infective to mice. These data suggest that GPI-anchored surface proteins are not essential to L. mexicana for its entry into and survival within mammalian host cells in vitro or in vivo and provide further support for the notion that free GPIs are essential for parasite growth. INTRODUCTIONLeishmania are protozoan parasites that cause a spectrum of diseases in humans. These parasites alternate between a flagellated promastigote stage that proliferates within the midgut of the sandfly vector and a nonmotile amastigote stage that invades mammalian macrophages, where they occupy the phagolysosome compartment. The cell surface of the promastigote stage is coated by a protective glycocalyx that comprises a number of glycosylphosphatidylinositol (GPI)-anchored glycoproteins, a complex GPI-anchored lipophosphoglycan (LPG), and a family of free GPIs (termed glycoinositolphospholipids [GIPLs]) Beverley and Turco, 1998; Figure 1). Components in this glycoclayx are thought to be essential for parasite survival and infectivity in the diverse host Beverley and Turco, 1998). The GPIanchored glycoproteins include an abundant metalloproteinase, termed gp63 or leishmanolysin, and the promastigote surface antigen (PSA2)/gp46 family of glycoproteins Murray et al., 1989;Frommel et al., 1990;Lohman et al., 1990). Gp63 has been shown to be proteolytically active against a wide variety of different peptide substrates and has been reported to act as a ligand for macrophage receptors, either directly or after opsonization with complement, to protect the parasites from complementmediated lysis and also to contribute to the pathology of lesion development (see Alexander and Russell, 1992;Joshi et al., 1998). However, the fact that these proteins are encoded by multicopy polymorphic genes (Button et al., 1989;Lohman et al., 1990;Symons et al., 1994) has hindered elucidation of their function by genetic analysis (Joshi et al., 1998). Moreover, surface expression of gp63 and PSA2 is dramatically down-regulated in the amastigote stage of some Leishmania species and variable within particular parasite populations of others (Bahr et al., 1993;Handman et al., 1995) such that the precise function of these parasite pro...

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Section: Discussionmentioning

confidence: 94%

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

Hilley

Zawadzki

McConville

et al. 2000

MBoC

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“…14, May 2003the GPI-anchored form (lane 2) as described previously (Maxwell et al, 1995). Microsomes of class L CHO mutant cells that were deficient in the second step of GPI biosynthesis did not generate the GPI-anchored form (lane 3).…”

Section: Fifth Subunit Of Gpi Transamidasementioning

confidence: 99%

Human PIG-U and Yeast Cdc91p Are the Fifth Subunit of GPI Transamidase That Attaches GPI-Anchors to Proteins

Hong

Ohishi

Kang

et al. 2003

MBoC

110

123

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Many eukaryotic proteins are anchored to the cell surface via glycosylphosphatidylinositol (GPI), which is posttranslationally attached to the carboxyl-terminus by GPI transamidase. The mammalian GPI transamidase is a complex of at least four subunits, GPI8, GAA1, PIG-S, and PIG-T. Here, we report Chinese hamster ovary cells representing a new complementation group of GPI-anchored protein-deficient mutants, class U. The class U cells accumulated mature and immature GPI and did not have in vitro GPI transamidase activity. We cloned the gene responsible, termed PIG-U, that encoded a 435-amino-acid hydrophobic protein. The GPI transamidase complex affinity-purified from cells expressing epitope-tagged-GPI8 contained PIG-U and four other known components. Cells lacking PIG-U formed complexes of the four other components normally but had no ability to cleave the GPI attachment signal peptide. Saccharomyces cerevisiae Cdc91p, with 28% amino acid identity to PIG-U, partially restored GPI-anchored proteins on the surface of class U cells. PIG-U and Cdc91p have a functionally important short region with similarity to a region conserved in long-chain fatty acid elongases. Taken together, PIG-U and the yeast orthologue Cdc91p are the fifth component of GPI transamidase that may be involved in the recognition of either the GPI attachment signal or the lipid portion of GPI

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“…Nucleophilic attack on the activated carbonyl by the amino group of an Etn-P-capped GPI substrate (such as mammalian H7 or H8 or yeast CP2, a Man 4 -GPI with Etn-P on Man-1, -2, and -3; Fig. 2A) yields a GPI-anchored protein and regenerates GPIT (139)(140)(141) (Fig. 3B).…”

Section: Transfer Of Gpis To Proteinmentioning

confidence: 99%

“…3B). Small nucleophiles such as hydrazine and hydroxylamine can replace GPI in microsome-based assays of GPI anchoring (140,141), providing a simple test of carbonyl activation. Nucleophilic attack by water is also seen in these assays (142), raising the possibility that a small percentage of proprotein products is simply secreted without receiving a GPI anchor.…”

Section: Transfer Of Gpis To Proteinmentioning

confidence: 99%

Thematic review series: Lipid Posttranslational Modifications. GPI anchoring of protein in yeast and mammalian cells, or: how we learned to stop worrying and love glycophospholipids

Orlean

Menon

2007

Journal of Lipid Research

357

293

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Glycosylphosphatidylinositol (GPI) anchoring of cell surface proteins is the most complex and metabolically expensive of the lipid posttranslational modifications described to date. The GPI anchor is synthesized via a membrane-bound multistep pathway in the endoplasmic reticulum (ER) requiring .20 gene products. The pathway is initiated on the cytoplasmic side of the ER and completed in the ER lumen, necessitating flipping of a glycolipid intermediate across the membrane. The completed GPI anchor is attached to proteins that have been translocated across the ER membrane and that display a GPI signal anchor sequence at the C terminus. GPI proteins transit the secretory pathway to the cell surface; in yeast, many become covalently attached to the cell wall. Genes encoding proteins involved in all but one of the predicted steps in the assembly of the GPI precursor glycolipid and its transfer to protein in mammals and yeast have now been identified. Most of these genes encode polytopic membrane proteins, some of which are organized in complexes. The steps in GPI assembly, and the enzymes that carry them out, are highly conserved. GPI biosynthesis is essential for viability in yeast and for embryonic development in mammals. In this review, we describe the biosynthesis of mammalian and yeast GPIs, their transfer to protein, and their subsequent processing.-Orlean, P., and A. K. Menon. GPI anchoring of protein in yeast and mammalian cells, or: how we learned to stop worrying and love glycophospholipids.

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An Active Carbonyl Formed during Glycosylphosphatidylinositol Addition to a Protein Is Evidence of Catalysis by a Transamidase

Cited by 78 publications

References 39 publications

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

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

Human PIG-U and Yeast Cdc91p Are the Fifth Subunit of GPI Transamidase That Attaches GPI-Anchors to Proteins

Thematic review series: Lipid Posttranslational Modifications. GPI anchoring of protein in yeast and mammalian cells, or: how we learned to stop worrying and love glycophospholipids

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