Requirement of N-glycan on GPI-anchored proteins for efficient binding of aerolysin but not Clostridium septicum alpha-toxin

Aerolysin of the Gram-negative bacterium Aeromonas hydrophila consists of small (SL) and large (LL) lobes. The alpha-toxin of Gram-positive Clostridium septicum has a single lobe homologous to LL. These toxins bind to glycosylphosphatidylinositol (GPI)-anchored proteins and generate pores in the cell's plasma membrane. We isolated CHO cells resistant to aerolysin, with the aim of obtaining GPI biosynthesis mutants. One mutant unexpectedly expressed GPI-anchored proteins, but nevertheless bound aerolysin poorly and was 10-fold less sensitive than wild-type cells. A cDNA of N-acetylglucosamine transferase I (GnTI) restored the binding of aerolysin to this mutant. Therefore, N-glycan is involved in the binding. Removal of mannoses by alpha-mannosidase II was important for the binding of aerolysin. In contrast, the alpha-toxin killed GnTI-deficient and wild-type CHO cells equally, indicating that its binding to GPI-anchored proteins is independent of N-glycan. Because SL bound to wild-type but not to GnTI-deficient cells, and because a hybrid toxin consisting of SL and the alpha-toxin killed wild-type cells 10-fold more efficiently than GnTI- deficient cells, SL with its binding site for N-glycan contributes to the high binding affinity of aerolysin.     

The glycan core of GPI-anchored proteins modulates aerolysin binding but is not sufficient: the polypeptide moiety is required for the toxin-receptor interaction

Sensitivity of mammalian cells to the bacterial toxin aerolysin is due to the presence at their surface of glycosylphosphatidyl inositol (GPI)-anchored proteins which act as receptors. Using a panel of mutants that are affected in the GPI biosynthetic pathway and Trypanosoma brucei variant surface glycoproteins, we show that addition of an ethanolamine phosphate residue on the first mannose of the glycan core does not affect binding. In contrast, the addition of a side chain of up to four galactose residues at position 3 of this same mannose leads to an increase in binding. However, protein free GPIs, which accumulate in mutant cells deficient in the transamidase that transfers the protein to the pre-formed GPI-anchor, were unable to bind the toxin indicating a requirement for the polypeptide moiety, the nature and size of which seem of little importance although two exceptions have been identified.     

TbGPI16 is an essential component of GPI transamidase in Trypanosoma brucei

Glycosylphosphatidylinositol (GPI) is widely used by eukaryotic cell surface proteins for membrane attachment. De novo synthesized GPI precursors are attached to proteins post-translationally by the enzyme complex, GPI transamidase. TbGPI16, a component of the trypanosome transamidase, shares similarity with human PIG-T. Here, we show that TbGPI16 is the orthologue of PIG-T and an essential component of GPI transamidase by creating a TbGPI16 knockout. TbGPI16 forms a disulfide-linked complex with TbGPI8. A cysteine to serine mutant of TbGPI16 was unable to fully restore the surface expression of GPI-anchored proteins upon transfection into the knockout cells, indicating that its disulfide linkage with TbGPI8 is important for the full transamidase activity.     

Two subunits of glycosylphosphatidylinositol transamidase,GPI8 and PIG-T,form a functionally important intermolecular disulfide bridge

Many eukaryotic proteins are tethered to the plasma membrane via glycosylphosphatidylinositol (GPI). GPI transamidase is localized in the endoplasmic reticulum and mediates post-translational transfer of preformed GPI to proteins bearing a carboxyl-terminal GPI attachment signal. Mammalian GPI transamidase is a multimeric complex consisting of at least five subunits. Here we report that two subunits of mammalian GPI transamidase, GPI8 and PIG-T, form a functionally important disulfide bond between conserved cysteine residues. GPI8 and PIG-T mutants in which relevant cysteines were replaced with serines were unable to fully restore the surface expression of GPI-anchored proteins upon transfection into their respective mutant cells. Microsomal membranes of these transfectants had markedly decreased activities in an in vitro transamidase assay. The formation of this disulfide bond is not essential but required for full transamidase activity. Antibodies against GPI8 and PIG-T revealed that endogenous as well as exogenous proteins formed a disulfide bond. Furthermore trypanosome GPI8 forms a similar intermolecular disulfide bond via its conserved cysteine residue, suggesting that the trypanosome GPI transamidase is also a multimeric complex likely containing the orthologue of PIG-T. We also demonstrate that an inactive human GPI transamidase complex that consists of non-functional GPI8 and four other components was co-purified with the proform of substrate proteins, indicating that these five components are sufficient to hold the substrate proteins.     

Use of Clostridium septicum alpha toxins for isolation of various glycosylphosphatidylinositol-deficient cells

In eukaryotic cells, various proteins are anchored to the plasma membrane through glycosylphosphatidylinositol (GPI). To study the biosynthetic pathways and modifications of GPI, various mutant cells have been isolated from the cells of Chinese hamster ovaries (CHO) supplemented with several exogenous genes involved in GPI biosynthesis using aerolysin, a toxin secreted from gram-negative bacterium Aeromonas hydrophila. Alpha toxin from Gram-positive bacterium Clostridium septicum is homologous to large lobes (LL) of aerolysin, binds GPI-anchored proteins and possesses a cell-destroying mechanism similar to aerolysin. Here, to determine whether alpha toxins can be used as an isolation tool of GPI-mutants, like aerolysin, CHO cells stably transfected with several exogenous genes involved in GPI biosynthesis were chemically mutagenized and cultured in a medium containing alpha toxins. We isolated six mutants highly resistant to alpha toxins and deficient in GPI biosynthesis. By genetic complementation, we determined that one mutant cell was defective of the second subunit of dolichol phosphate mannose synthase (DPM2) and other five cells were of a putative catalytic subunit of inositol acyltransferase (PIG-W). Therefore, C. septicum alpha toxins are a useful screening probe for the isolation of various GPI-mutant cells.     

Human PIG-U and yeast Cdc91p are the fifth subunit of GPI transamidase that attaches GPI-anchors to proteins

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.     

Yeast Gaa1p is required for attachment of a completed GPI anchor onto proteins

Anchoring of proteins to membranes by glycosylphosphatidylinositols (GPIs) is ubiquitous among all eukaryotes and heavily used by parasitic protozoa. GPI is synthesized and transferred en bloc to form GPI- anchored proteins. The key enzyme in this process is a putative GPI:protein transamidase that would cleave a peptide bond near the COOH terminus of the protein and attach the GPI by an amide linkage. We have identified a gene, GAA1, encoding an essential ER protein required for GPI anchoring. gaal mutant cells synthesize the complete GPI anchor precursor at nonpermissive temperatures, but do not attach it to proteins. Overexpression of GAA1 improves the ability of cells to attach anchors to a GPI-anchored protein with a mutant anchor attachment site. Therefore, Gaa1p is required for a terminal step of GPI anchor attachment and could be part of the putative GPI:protein transamidase.     

In vitro biosynthesis of glycosylphosphatidylinositol in Aspergillus fumigatus

Glycosylphosphatidylinositol (GPI) represents a mechanism for the attachment of proteins to the plasma membrane found in all eukaryotic cells. GPI biosynthesis has been mainly studied in parasites, yeast, and mammalian cells. Aspergillus fumigatus, a filamentous fungus, produces GPI-anchored molecules, some of them being essential in the construction of the cell wall. An in vitro assay was used to study the GPI biosynthesis in the mycelium form of this organism. In the presence of UDP-GlcNAc and coenzyme A, the cell-free system produces the initial intermediates of the GPI biosynthesis: GlcNAc-PI, GlcN-PI, and GlcN-(acyl)PI. Using GDP-Man, two types of mannosylation are observed. First, one or two mannose residues are added to GlcN-PI. This mannosylation, never described in fungi, does not require dolichol phosphomannoside (Dol-P-Man) as the monosaccharide donor. Second, one to five mannose residues are added to GlcN-(acyl)PI using Dol-P-Man as the mannose donor. The addition of ethanolamine phosphate groups to the first, second, and third mannose residue is also observed. This latter series of GPI intermediates identified in the A. fumigatus cell-free system indicates that GPI biosynthesis in this filamentous fungus is similar to the mammalian or yeast systems. Thus, these biochemical data are in agreement with a comparative genome analysis that shows that all but 3 of the 21 genes described in the Saccharomyces cerevisiae GPI pathways are found in A. fumigatus.     

A beta-N-acetylglucosaminyl phosphate diester residue is attached to the glycosylphosphatidylinositol anchor of human placental alkaline phosphatase: a target of the channel-forming toxin aerolysin

Glycosylphosphatidylinositol (GPI)-anchored proteins are ubiquitous in eukaryotes. The minimum conserved GPI core structure of all GPI-anchored glycans has been determined as EtN-PO4-6Manalpha1-2Manalpha1-6Manalpha1-4GlcN-myo-inositol-PO3H. Human placental alkaline phosphatase (AP) has been reported to be a GPI-anchored membrane protein. AP carries one N-glycan, (NeuAcalpha2-->3)2Gal2GlcNAc2Man3GlcNAc(+/-Fuc)GlcNAc, and a GPI anchor, which contains an ethanolamine phosphate diester group, as a side chain. However, we found that both sialidase-treated soluble AP (sAP) and its GPI-anchored glycan bound to a Psathyrella velutina lectin (PVL)-Sepharose column, which binds beta-GlcNAc residues. PVL binding of asialo-sAP and its GPI-anchored glycan was diminished by digestion with diplococcal beta-N-acetylhexosaminidase or by mild acid treatment. After sequential digestion of asialo-sAP with beta-N-acetylhexosaminidase and acid phosphatase, the elution patterns on chromatofocusing gels were changed in accordance with the negative charges of phosphate residues. Trypsin-digested sAP was analyzed by liquid chromatography/electrospray ionization mass spectrometry, and the structures of two glycopeptides with GPI-anchored glycans were confirmed as peptide-EtN-PO4-6Manalpha1-->2(GlcNAcbeta1-PO4-->6)Manalpha1-6(+/-EtN-PO4-->)Manalpha1-->4GlcN, which may be produced by endo-alpha-glucosaminidase. In addition to AP, GPI-anchored carcinoembryonic antigen, cholinesterase, and Tamm-Horsfall glycoprotein also bound to a PVL-Sepharose column, suggesting that the beta-N-acetylglucosaminyl phosphate diester residue is widely distributed in human GPI-anchored glycans. Furthermore, we found that the beta-N-acetylglucosaminyl phosphate diester residue is important for GPI anchor recognition of aerolysin, a channel-forming toxin derived from Aeromonas hydrophila.     

Requirement of PIG-F and PIG-O for transferring phosphoethanolamine to the third mannose in glycosylphosphatidylinositol

Many eukaryotic proteins are anchored by glycosylphosphatidylinositol (GPI) to the cell surface membrane. The GPI anchor is linked to proteins by an amide bond formed between the carboxyl terminus and phosphoethanolamine attached to the third mannose. Here, we report the roles of two mammalian genes involved in transfer of phosphoethanolamine to the third mannose in GPI. We cloned a mouse gene termed Pig-o that encodes a 1101-amino acid PIG-O protein bearing regions conserved in various phosphodiesterases. Pig-o knockout F9 embryonal carcinoma cells expressed very little GPI-anchored proteins and accumulated the same major GPI intermediate as the mouse class F mutant cell, which is defective in transferring phosphoethanolamine to the third mannose due to mutant Pig-f gene. PIG-O and PIG-F proteins associate with each other, and the stability of PIG-O was dependent upon PIG-F. However, the class F cell is completely deficient in the surface expression of GPI-anchored proteins. A minor GPI intermediate seen in Pig-o knockout but not class F cells had more than three mannoses with phosphoethanolamines on the first and third mannoses, suggesting that this GPI may account for the low expression of GPI-anchored proteins. Therefore, mammalian cells have redundant activities in transferring phosphoethanolamine to the third mannose, both of which require PIG-F.     

Mechanism for release of alkaline phosphatase caused by glycosylphosphatidylinositol deficiency in patients with hyperphosphatasia mental retardation syndrome

Hyperphosphatasia mental retardation syndrome (HPMR), an autosomal recessive disease characterized by mental retardation and elevated serum alkaline phosphatase (ALP) levels, is caused by mutations in the coding region of the phosphatidylinositol glycan anchor biosynthesis, class V (PIGV) gene, the product of which is a mannosyltransferase essential for glycosylphosphatidylinositol (GPI) biosynthesis. Mutations found in four families caused amino acid substitutions A341E, A341V, Q256K, and H385P, which drastically decreased expression of the PIGV protein. Hyperphosphatasia resulted from secretion of ALP, a GPI-anchored protein normally expressed on the cell surface, into serum due to PIGV deficiency. In contrast, a previously reported PIGM deficiency, in which there is a defect in the transfer of the first mannose, does not result in hyperphosphatasia. To provide insights into the mechanism of ALP secretion in HPMR patients, we took advantage of CHO cell mutants that are defective in various steps of GPI biosynthesis. Secretion of ALP requires GPI transamidase, which in normal cells, cleaves the C-terminal GPI attachment signal peptide and replaces it with GPI. The GPI-anchored protein was secreted substantially into medium from PIGV-, PIGB-, and PIGF-deficient CHO cells, in which incomplete GPI bearing mannose was accumulated. In contrast, ALP was degraded in PIGL-, DPM2-, or PIGX-deficient CHO cells, in which incomplete shorter GPIs that lacked mannose were accumulated. Our results suggest that GPI transamidase recognizes incomplete GPI bearing mannose and cleaves a hydrophobic signal peptide, resulting in secretion of soluble ALP. These results explain the molecular mechanism of hyperphosphatasia in HPMR.     

Gaa1p and gpi8p are components of a glycosylphosphatidylinositol (GPI) transamidase that mediates attachment of GPI to proteins

Many eukaryotic cell surface proteins are anchored to the membrane via glycosylphosphatidylinositol (GPI). The GPI is attached to proteins that have a GPI attachment signal peptide at the carboxyl terminus. The GPI attachment signal peptide is replaced by a preassembled GPI in the endoplasmic reticulum by a transamidation reaction through the formation of a carbonyl intermediate. GPI transamidase is a key enzyme of this posttranslational modification. Here we report that Gaa1p and Gpi8p are components of a GPI transamidase. To determine a role of Gaa1p we disrupted a GAA1/GPAA1 gene in mouse F9 cells by homologous recombination. GAA1 knockout cells were defective in the formation of carbonyl intermediates between precursor proteins and transamidase as determined by an in vitro GPI-anchoring assay. We also show that cysteine and histidine residues of Gpi8p, which are conserved in members of a cysteine protease family, are essential for generation of a carbonyl intermediate. This result suggests that Gpi8p is a catalytic component that cleaves the GPI attachment signal peptide. Moreover, Gaa1p and Gpi8p are associated with each other. Therefore, Gaa1p and Gpi8p constitute a GPI transamidase and cooperate in generating a carbonyl intermediate, a prerequisite for GPI attachment.     

GPI7 is the second partner of PIG-F and involved in modification of glycosylphosphatidylinositol

Many eukaryotic cell surface proteins are anchored to the membrane via glycosylphosphatidylinositol (GPI). GPI is synthesized from phosphatidylinositol by stepwise reactions and attached en bloc to nascent proteins. In mammalian cells, the major GPI species transferred to proteins is termed H7. By attachment of an additional ethanolamine phosphate (EtNP) to the second mannose, H7 can be converted to H8, which acts as a minor type of protein-linked GPI and also exists as a free GPI on the cell surface. Yeast GPI7 is involved in the transfer of EtNP to the second mannose, but the corresponding mammalian enzyme has not yet been clarified. Here, we report that the human homolog of Gpi7p (hGPI7) forms a protein complex with PIG-F and is involved in the H7-to-H8 conversion. We knocked down hGPI7 by RNA interference and found that H7 accumulated with little production of H8. Immunoprecipitation experiments revealed that hGPI7 was associated with and stabilized by PIG-F, which is known to bind to and stabilize PIG-O, a protein homologous to hGPI7. PIG-O is a transferase that adds EtNP to the third mannose, rendering GPI capable of attaching to proteins. We further found that the overexpression of hGPI7 decreased the level of PIG-O and, therefore, decreased the level of EtNP transferred to the third mannose. Finally, we propose a mechanism for the regulation of GPI biosynthesis through competition between the two independent enzymes, PIG-O and hGPI7, for the common stabilizer, PIG-F.     

Glycosylphosphatidylinositol (GPI) proteins of Saccharomyces cerevisiae contain ethanolamine phosphate groups on the alpha1,4-linked mannose of the GPI anchor

In humans and Saccharomyces cerevisiae the free glycosylphosphatidylinositol (GPI) lipid precursor contains several ethanolamine phosphate side chains, but these side chains had been found on the protein-bound GPI anchors only in humans, not yeast. Here we confirm that the ethanolamine phosphate side chain added by Mcd4p to the first mannose is a prerequisite for the addition of the third mannose to the GPI precursor lipid and demonstrate that, contrary to an earlier report, an ethanolamine phosphate can equally be found on the majority of yeast GPI protein anchors. Curiously, the stability of this substituent during preparation of anchors is much greater in gpi7Delta sec18 double mutants than in either single mutant or wild type cells, indicating that the lack of a substituent on the second mannose (caused by the deletion of GPI7) influences the stability of the one on the first mannose. The phosphodiester-linked substituent on the second mannose, probably a further ethanolamine phosphate, is added to GPI lipids by endoplasmic reticulum-derived microsomes in vitro but cannot be detected on GPI proteins of wild type cells and undergoes spontaneous hydrolysis in saline. Genetic manipulations to increase phosphatidylethanolamine levels in gpi7Delta cells by overexpression of PSD1 restore cell growth at 37 degrees C without restoring the addition of a substituent to Man2. The three putative ethanolamine-phosphate transferases Gpi13p, Gpi7p, and Mcd4p cannot replace each other even when overexpressed. Various models trying to explain how Gpi7p, a plasma membrane protein, directs the addition of ethanolamine phosphate to mannose 2 of the GPI core have been formulated and put to the test.     

Ethanolamine phosphate linked to the first mannose residue of glycosylphosphatidylinositol (GPI) lipids is a major feature of the GPI structure that is recognized by human GPI transamidase

Glycosylphosphatidylinositol (GPI) anchoring of proteins is catalyzed by GPI transamidase (GPIT), a multisubunit, endoplasmic reticulum (ER)-localized enzyme. GPIT recognizes ER-translocated proteins that have a GPI-directing C-terminal signal sequence and replaces this sequence with a preassembled GPI anchor. Although the GPI signal sequence has been extensively characterized, little is known about the structural features of the GPI lipid substrate that enable its recognition by GPIT. In a previous study we showed that mature GPIs could be co-immunoprecipitated with GPIT complexes containing functional subunits (Vainauskas, S., and Menon, A. K. (2004) J. Biol. Chem. 279, 6540-6545). We now use this approach, as well as a method that reconstitutes the interaction between GPIs and GPIT, to define the basis of the interaction between GPI and human GPIT. We report that (i) human GPIT can interact with GPI biosynthetic intermediates, not just mature GPIs competent for transfer to protein, (ii) the ethanolamine phosphate group on the third mannose residue of the GPI glycan is not critical for GPI recognition by GPIT, (iii) the ethanolamine phosphate residue linked to the first mannose of the GPI structure is a major feature of GPIs that is recognized by human GPIT, and (iv) the simplest GPI recognized by human GPIT is EtN-P-2Manalpha1-4GlcN-(acyl)-phosphatidyl-inositol. These studies define the molecular characteristics of GPI that are recognized by GPIT and open the way to identifying GPIT subunits that are involved in this process.     

Clostridium septicum alpha toxin uses glycosylphosphatidylinositol-anchored protein receptors.

The alpha toxin produced by Clostridium septicum is a channel-forming protein that is an important contributor to the virulence of the organism. Chinese hamster ovary (CHO) cells are sensitive to low concentrations of the toxin, indicating that they contain toxin receptors. Using retroviral mutagenesis, a mutant CHO line (BAG15) was generated that is resistant to alpha toxin. FACS analysis showed that the mutant cells have lost the ability to bind the toxin, indicating that they lack an alpha toxin receptor. The mutant cells are also resistant to aerolysin, a channel-forming protein secreted by Aeromonas spp., which is structurally and functionally related to alpha toxin and which is known to bind to glycosylphosphatidylinositol (GPI)-anchored proteins, such as Thy-1. We obtained evidence that the BAG15 cells lack N-acetylglucosaminyl-phosphatidylinositol deacetylase-L, needed for the second step in GPI anchor biosynthesis. Several lymphocyte cell lines lacking GPI-anchored proteins were also shown to be less sensitive to alpha toxin. On the other hand, the sensitivity of CHO cells to alpha toxin was increased when the cells were transfected with the GPI-anchored folate receptor. We conclude that alpha toxin, like aerolysin, binds to GPI-anchored protein receptors. Evidence is also presented that the two toxins bind to different subsets of GPI-anchored proteins.     

Diagnosis of paroxysmal nocturnal hemoglobinuria by fluorescent clostridium septicum alpha toxin

Paroxysmal nocturnal hemoglobinuria (PNH), a hematopoietic stem cell disorder, is caused by the loss of glycosylphosphatidylinositol (GPI)-anchored proteins on the cell membrane. PNH can be simply diagnosed by flow cytometry using monoclonal antibodies against GPI-anchored proteins or fluorescent-tagged aerolysin, a bacterial toxin that binds GPI anchored proteins. Clostridium septicum alpha toxin is homologous to aerolysin and specifically binds GPI-anchored proteins. Previously, we found that an alpha toxin m45 mutant with two amino acid changes, S189C/S238C, lost cytotoxicity but still possessed binding activity for GPI-anchored proteins. To use this mutant toxin as a diagnostic probe in flow cytometry, we constructed the EGFP-AT(m45) expression vector, comprising a S189C/S238C alpha toxin mutant with EGFP and His tags at the N and C termini, respectively. The recombinant EGFP-AT(m45) was easily purified using single-step affinity chromatography against His tag from Escherichia coli. EGFP-AT(m45) bound to CHO and HeLa cells in a similar manner to monoclonal antibodies against GPI-anchored proteins or aerolysin. In whole blood from a PNH patient, GPI-deficient granulocytes could be differentiated by EGFP-AT(m45) using the same procedure as that employed with commercially available monoclonal antibodies. Therefore, nontoxic EGFP-conjugated C. septicum alpha toxin could be used clinically for PNH diagnosis.     

Cloning of murine glycosyl phosphatidylinositol anchor attachment protein, GPAA1

Glycosyl phosphatidylinositols (GPIs) are usedto anchor many proteins to the cell surface membrane and are utilizedin all eukaryotic cells. GPI anchoring units are attached to proteins via a transamidase reaction mediated by a GPI transamidase complex. Weisolated one of the components of this complex,mGPAA1 (murine GPI anchor attachment), by the signalsequence trap method. mGPAA1 cDNA is about 2 kb in lengthand encodes a putative 621 amino acid protein. The mGPAA1gene has 12 small exons and 11 small introns. mGPAA1 mRNA isubiquitously expressed in mammalian cells, and in situ hybridizationanalysis revealed that it is abundant in the choroid plexus, skeletalmuscle, osteoblasts of rib, and occipital bone in mouse embryos. Itsexpression levels and transamidation efficiency decreased withdifferentiation of embryonic stem cells. The 3T3 cell lines expressingantisense mGPAA1 failed to express GPI-anchored proteins onthe cell surface membrane.

     

A testis-specific regulator of complex and hybrid N-glycan synthesis

Database analyses identified 4933434I20Rik as a glycosyltransferase-like gene expressed mainly in testicular germ cells and regulated during spermatogenesis. Expression of a membrane-bound form of the protein resulted in a marked and specific reduction in N-acetylglucosaminyltransferase I (GlcNAcT-I) activity and complex and hybrid N-glycan synthesis. Thus, the novel activity was termed GlcNAcT-I inhibitory protein (GnT1IP). Membrane-bound GnT1IP localizes to the ER, the ER-Golgi intermediate compartment (ERGIC), and the cis-Golgi. Coexpression of membrane-anchored GnT1IP with GlcNAcT-I causes association of the two proteins, inactivation of GlcNAcT-I, and mislocalization of GlcNAcT-I from the medial-Golgi to earlier compartments. Therefore, GnT1IP is a regulator of GlcNAcT-I and complex and hybrid N-glycan production. Importantly, the formation of high mannose N-glycans resulting from inhibition of GlcNAcT-I by GnT1IP markedly increases the adhesion of CHO cells to TM4 Sertoli cells. Testicular germ cells might use GnT1IP to induce the expression of high mannose N-glycans on glycoproteins, thereby facilitating Sertoli–germ cell attachment at a particular stage of spermatogenesis.     

Leishmania mexicana mutants lacking glycosylphosphatidylinositol (GPI):protein transamidase provide insights into the biosynthesis and functions of GPI-anchored proteins

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 (DeltaGPI8) do not express detectable levels of GPI-anchored proteins and accumulate two putative protein-anchor precursors. However, the synthesis and cellular levels of other non-protein-linked GPIs, including lipophosphoglycan and a major class of free GPIs, are not affected in the DeltaGPI8 mutant. Significantly, the DeltaGPI8 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.