A novel family of sugar-specific phosphodiesterases that remove zwitterionic modifications of GlcNAc.

The zwitterions phosphorylcholine (PC) and phosphoethanolamine (PE) are often found esterified to certain sugars in polysaccharides and glycoconjugates in a wide range of biological species. One such modification involves PC attachment to the 6-carbon of N-acetylglucosamine (GlcNAc-6-PC) in N-glycans and glycosphingolipids (GSLs) of parasitic nematodes, a modification that helps the parasite evade host immunity. Knowledge of enzymes involved in the synthesis and degradation of PC and PE modifications is limited. More detailed studies on such enzymes would contribute to a better understanding of the function of PC modifications and have potential application in the structural analysis of zwitterion-modified glycans. In this study, we used functional metagenomic screening to identify phosphodiesterases encoded in a human fecal DNA fosmid library that remove PC from GlcNAc-6-PC. A novel bacterial phosphodiesterase was identified and biochemically characterized. This enzyme (termed GlcNAc-PDase) shows remarkable substrate preference for GlcNAc-6-PC and GlcNAc-6-PE, with little or no activity on other zwitterion-modified hexoses. The identified GlcNAc-PDase protein sequence is a member of the large endonuclease/exonuclease/phosphatase superfamily where it defines a distinct subfamily of related sequences of previously unknown function, mostly from Clostridium bacteria species. Finally, we demonstrate use of GlcNAc-PDase to confirm the presence of GlcNAc-6-PC in N-glycans and GSLs of the parasitic nematode Brugia malayi in a glycoanalytical workflow.

Ultraviolet Photodissociation Permits Comprehensive Characterization of O-Glycopeptides Cleaved with O-Glycoprotease IMPa.

Complete O-glycosite characterization, including identification of the peptides, localization of the glycosites, and mapping of the glycans, has been a persistent challenge in O-glycoproteomics owing to the technical challenges surrounding O-glycan analysis. Multi-glycosylated peptides pose an even greater challenge owing to their potential heterogeneity. Ultraviolet photodissociation (UVPD) can localize multiple post-translational modifications and is well-suited for the characterization of glycans. Three glycoproteins were assessed based on a strategy combining the use of O-glycoprotease IMPa and HCD-triggered UVPD for the complete characterization of O-glycopeptides. This approach localized multiple adjacent or proximal O-glycosites on individual glycopeptides and identified a previously unknown glycosite on etanercept at S218. Nine different glycoforms were characterized as a multi-glycosylated peptide from etanercept. The performance of UVPD was compared to that of HCD and EThcD for the localization of O-glycosites and the characterization of the constituent peptides and glycans.

A Broad-Specificity O-Glycoprotease That Enables Improved Analysis of Glycoproteins and Glycopeptides Containing Intact Complex O-Glycans.

Characterization of mucin-type O-glycans linked to serine/threonine of glycoproteins is technically challenging, in part, due to a lack of effective enzymatic tools that enable their analysis. Recently, several O-glycan-specific endoproteases that can cleave the protein adjacent to the appended glycan have been described. Despite significant progress in understanding the biochemistry of these enzymes, known O-glycoproteases have specificity constraints, such as inefficient cleavage of glycoproteins bearing sialylated O-glycans, high selectivity for certain types of glycoproteins, or protein sequence bias. These factors limit their analytical application. In this study, we examined the capabilities of an immunomodulating metalloprotease (IMPa) from Pseudomonas aeruginosa. Peptide sequence selectivity and its impact on IMPa activity were interrogated using an array of synthetic peptides and their glycoforms. We show that IMPa has no specific P1 residue preference and can tolerate most amino acids at the P1 position, except aspartic acid. The enzyme does not cleave between two adjacent O-glycosites, indicating that O-glycosylated serine/threonine is not allowed at position P1. Glycopeptides with as few as two amino acids on either side of an O-glycosite were cleaved by IMPa. Finally, IMPa efficiently cleaved peptides and proteins carrying sialylated and asialylated O-glycans of varying complexity. We present the use of IMPa in a one-step O-glycoproteomic workflow for glycoprofiling of the purified glycoproteins granulocyte colony-stimulating factor and receptor-type tyrosine-protein phosphatase C without the need for glycopeptide enrichment. In these examples, IMPa enabled both the identification of O-glycosites and the range of complex O-glycan structures at each site.

A Novel Regulated Hybrid Promoter That Permits Autoinduction of Heterologous Protein Expression in Kluyveromyces lactis.

The yeast Kluyveromyces lactis has been a successful host for the production of heterologous proteins for over 30 years. Currently, the galactose-/lactose-inducible and glucose-repressible LAC4 promoter (P LAC4 ) is the most widely used promoter to drive recombinant protein expression in K. lactis However, P LAC4 is not fully repressed in the presence of glucose and significant protein expression still occurs. Thus, P LAC4 is not suitable in processes where tight regulation of heterologous gene expression is required. In this study, we devised a novel K. lactis promoter system that is both strong and tightly controllable. We first tested several different endogenous K. lactis promoters for their ability to express recombinant proteins. A novel hybrid promoter (termed P350) was created by combining segments of two K. lactis promoters, namely, the strong constitutive P GAP1 promoter and the carbon source-sensitive P ICL1 promoter. We demonstrate that P350 is tightly repressed in the presence of glucose or glycerol and becomes derepressed upon depletion of these compounds by the growing cells. We further illustrate the utility of P350-controlled protein expression in shake flask and high-cell-density bioreactor cultivation strategies. The P350 hybrid promoter is a strong derepressible promoter for use in autoinduction of one-step fermentation processes for the production of heterologous proteins in K. lactisIMPORTANCE The yeast Kluyveromyces lactis is an important host for the expression of recombinant proteins at both laboratory and industrial scales. However, the system lacks a tightly regulated promoter that permits controlled expression of heterologous proteins. In this study, we report the engineering of a highly regulated strong hybrid promoter (termed P350) for use in K. lactis P350 is tightly repressed by glucose or glycerol in the medium but strongly promotes gene expression once the carbon source has been consumed by the cells. This feature permits heterologous protein expression to be "autoinduced" at any scale without the addition of a gratuitous inducer molecule or changing feed solutions.

Comprehensive Profiling of Glycosphingolipid Glycans Using a Novel Broad Specificity Endoglycoceramidase in a High-Throughput Workflow.

The biological function of glycosphingolipids (GSLs) is largely determined by their glycan headgroup moiety. This has placed a renewed emphasis on detailed GSL headgroup structural analysis. Comprehensive profiling of GSL headgroups in biological samples requires the use of endoglycoceramidases with broad substrate specificity and a robust workflow that enables their high-throughput analysis. We present here the first high-throughput glyco-analytical platform for GSL headgroup profiling. The workflow features enzymatic release of GSL glycans with a novel broad-specificity endoglycoceramidase I (EGCase I) from Rhodococcus triatomea, selective glycan capture on hydrazide beads on a robotics platform, 2AB-fluorescent glycan labeling, and analysis by UPLC-HILIC-FLD. R. triatomea EGCase I displayed a wider specificity than known EGCases and was able to efficiently hydrolyze gangliosides, globosides, (n)Lc-type GSLs, and cerebrosides. Our workflow was validated on purified GSL standard lipids and was applied to the characterization of GSLs extracted from several mammalian cell lines and human serum. This study should facilitate the analytical workflow in functional glycomics studies and biomarker discovery.

Proteomic identification of mammalian cell surface derived glycosylphosphatidylinositol-anchored proteins through selective glycan enrichment.

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are an important class of glycoproteins that are tethered to the surface of mammalian cells via the lipid GPI. GPI-APs have been implicated in many important cellular functions including cell adhesion, cell signaling, and immune regulation. Proteomic identification of mammalian GPI-APs en masse has been limited technically by poor sensitivity for these low abundance proteins and the use of methods that destroy cell integrity. Here, we present methodology that permits identification of GPI-APs liberated directly from the surface of intact mammalian cells through exploitation of their appended glycans to enrich for these proteins ahead of LC-MS/MS analyses. We validate our approach in HeLa cells, identifying a greater number of GPI-APs from intact cells than has been previously identified from isolated HeLa membranes and a lipid raft preparation. We further apply our approach to define the cohort of endogenous GPI-APs that populate the distinct apical and basolateral membrane surfaces of polarized epithelial cell monolayers. Our approach provides a new method to achieve greater sensitivity in the identification of low abundance GPI-APs from the surface of live cells and the nondestructive nature of the method provides new opportunities for the temporal or spatial analysis of cellular GPI-AP expression and dynamics.

A set of aspartyl protease-deficient strains for improved expression of heterologous proteins in Kluyveromyces lactis.

Secretion of recombinant proteins is a common strategy for heterologous protein expression using the yeast Kluyveromyces lactis. However, a common problem is degradation of a target recombinant protein by secretory pathway aspartyl proteases. In this study, we identified five putative pfam00026 aspartyl proteases encoded by the K. lactis genome. A set of selectable marker-free protease deletion mutants was constructed in the prototrophic K. lactis GG799 industrial expression strain background using a PCR-based dominant marker recycling method based on the Aspergillus nidulans acetamidase gene (amdS). Each mutant was assessed for its secretion of protease activity, its health and growth characteristics, and its ability to efficiently produce heterologous proteins. In particular, despite having a longer lag phase and slower growth compared with the other mutants, a Δyps1 mutant demonstrated marked improvement in both the yield and the quality of Gaussia princeps luciferase and the human chimeric interferon Hy3, two proteins that experienced significant proteolysis when secreted from the wild-type parent strain.

A bi-specific lectin from the mushroom Boletopsis grisea and its application in glycoanalytical workflows.

The BLL lectin from the edible Japanese "Kurokawa" mushroom (Boletopsis leucomelaena) was previously reported to bind to N-glycans harboring terminal N-acetylglucosamine (GlcNAc) and to induce apoptosis in a leukemia cell line. However, its gene has not been reported. In this study, we used a transcriptomics-based workflow to identify a full-length transcript of a BLL functional ortholog (termed BGL) from Boletopsis grisea, a close North American relative of B. leucomelaena. The deduced amino acid sequence of BGL was an obvious member of fungal fruit body lectin family (Pfam PF07367), a highly conserved group of mushroom lectins with a preference for binding O-glycans harboring the Thomsen-Friedenreich antigen (TF-antigen; Galß1,3GalNAc-α-) and having two ligand binding sites. Functional characterization of recombinant BGL using glycan microarray analysis and surface plasmon resonance confirmed its ability to bind both the TF-antigen and ß-GlcNAc-terminated N-glycans. Structure-guided mutagenesis of BGL's two ligand binding clefts showed that one site is responsible for binding TF-antigen structures associated with O-glycans, whereas the second site specifically recognizes N-glycans with terminal ß-GlcNAc. Additionally, the two sites show no evidence of allosteric communication. Finally, mutant BGL proteins having single functional bindings site were used to enrich GlcNAc-capped N-glycans or mucin type O-glycopeptides from complex samples in glycomics and glycoproteomics analytical workflows.

Production and сharacterization of the exopolysaccharide from strain Paenibacillus polymyxa 2020.

Paenibacillus spp. exopolysaccharides (EPSs) have become a growing interest recently as a source of biomaterials. In this study, we characterized Paenibacillus polymyxa 2020 strain, which produces a large quantity of EPS (up to 68 g/L),and was isolated from wasp honeycombs. Here we report its complete genome sequence and full methylome analysis detected by Pacific Biosciences SMRT sequencing. Moreover, bioinformatic analysis identified a putative levan synthetic operon. SacC and sacB genes have been cloned and their products identified as glycoside hydrolase and levansucrase respectively. The Fourier transform infrared (FT-IR) and nuclear magnetic resonance (NMR) spectra demonstrated that the EPS is a linear ß-(2→6)-linked fructan (levan). The structure and properties of levan polymer produced from sucrose and molasses were analyzed by FT-IR, NMR, scanning electron microscopy (SEM), high performance size exclusion chromatography (HPSEC), thermogravimetric analysis (TGA), cytotoxicity tests and showed low toxicity and high biocompatibility. Thus, P. polymyxa 2020 could be an exceptional cost-effective source for the industrial production of levan-type EPSs and to obtain functional biomaterials based on it for a broad range of applications, including bioengineering.

A novel broad specificity fucosidase capable of core α1-6 fucose release from N-glycans labeled with urea-linked fluorescent dyes.

Exoglycosidases are often used for detailed characterization of glycan structures. Bovine kidney α-fucosidase is commonly used to determine the presence of core α1-6 fucose on N-glycans, an important modification of glycoproteins. Recently, several studies have reported that removal of core α1-6-linked fucose from N-glycans labeled with the reactive N-hydroxysuccinimide carbamate fluorescent labels 6-aminoquinolyl-N-hydroxysuccinimidylcarbamate (AQC) and RapiFluor-MS is severely impeded. We report here the cloning, expression and biochemical characterization of an α-fucosidase from Omnitrophica bacterium (termed fucosidase O). We show that fucosidase O can efficiently remove α1-6- and α1-3-linked core fucose from N-glycans. Additionally, we demonstrate that fucosidase O is able to efficiently hydrolyze core α1-6-linked fucose from N-glycans labeled with any of the existing NHS-carbamate activated fluorescent dyes.

Profiling of core fucosylated N-glycans using a novel bacterial lectin that specifically recognizes α1,6 fucosylated chitobiose.

A novel fucose-binding lectin (SL2-1) from the bacterium Streptomyces rapamycinicus was identified by analysis of metagenomic DNA sequences. SL2-1 belongs to a new group of bacterial fucose-specific lectins that have no similarity to known bacterial fucose-binding proteins, but are related to certain eukaryotic fucose-binding lectins. The 17 kDa protein was expressed recombinantly in E. coli and purified by affinity chromatography. Glycan microarray analysis with fluorescently labeled recombinant SL2-1 demonstrated its ability to bind to core α1-6 fucosylated N-glycans, but not to core α1-3 fucosylated N-glycans, or other α1-2, α1-3 and α1-4 fucosylated oligosaccharides. The minimal high affinity binding epitope of SL2-1 was α1-6 fucosylated di-n-acetylchitobiose. The recombinant lectin was efficient in detection of N-glycan core fucosylation using lectin blotting and lectin ELISA assays. Finally, a workflow using SL2-1 for selective and quantitative profiling of core fucosylated N-glycans using UPLC-HILIC-FLR analysis was established. The approach was validated for selective capture and analysis of core fucosylated N-glycans present in complex glycan mixtures derived from mammalian serum IgG.

In vivo incorporation of an azide-labeled sugar analog to detect mammalian glycosylphosphatidylinositol molecules isolated from the cell surface.

N-Acetylgalactosamine (GalNAc) linked to the first mannose of glycosylphosphatidylinositol (GPI) core has been previously reported to be heterogeneously present on some mammalian GPI-anchored proteins. Here we present a method for profiling GalNAc-containing GPI-anchored proteins in mammalian cells by metabolic labeling with tetraacetylated N-azidoacetylgalactosamine (GalNAz) followed by biotinylation of the incorporated sugar analog. We have labeled both endogenous and recombinant GPI-anchored proteins with GalNAz, and demonstrated that the azide-activated sugar gets incorporated into the GPI glycan, likely as an unsubstituted side branch of the core structure. GalNAz was detected only on GPI molecules attached to proteins, and not on GPI precursors, indicating that GalNAc modification takes place after the GPI anchor is transferred to protein. We have highlighted the utility of this cell labeling approach by demonstrating the ability to examine specific GalNAc-containing GPI-anchored proteins isolated non-destructively from separate membrane domains (apical and basolateral) in polarized epithelial cells. This study represents the first demonstration of site-specific in vivo labeling of a GPI moiety with a synthetic sugar analog.

Trends in the incidence of occupational diseases in Lithuania between 1999 and 2008.

OBJECTIVES: The aim of the study was to investigate the trends in the incidence of occupational diseases in Lithuania during the period of 1999-2008. The analysis concerned both the individuals with diagnosed disease(s) and the number of diagnosed cases. MATERIAL AND METHODS: Incidence rates were calculated using data from the Republic of Lithuania National Register of Occupational Diseases and data on the employed population provided by the government Department of Statistics. The rates were age-standardized using the direct standardization method. The changes in the incidence rates throughout the study period were analyzed using segmented regression calculated with the JOINPOINT (v. 3.3.1) statistical software. We determined joinpoints in the dynamic lines of the incidence rates and calculated mean annual absolute change and mean annual relative (percentage) change for each period. RESULTS AND CONCLUSIONS: During the study period, the number of occupational diseases was, on average, 1.5 times as high as the number of individuals diagnosed with such diseases. Joinpoint positions in the dynamic lines of the incidence rates coincided for individuals with occupational diseases and for the cases of occupational diseases. The incidence was found to slightly increase during the period of 1999-2003, then to rise more rapidly during that of 2003-2006, and to decrease from 2006 to 2008.

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.

Endoplasmic reticulum localization of Gaa1 and PIG-T, subunits of the glycosylphosphatidylinositol transamidase complex.

After integration into the endoplasmic reticulum (ER) membrane, ER-resident membrane proteins must be segregated from proteins that are exported to post-ER compartments. Here we analyze how human Gaa1 and PIG-T, two of the five subunits of the ER-localized glycosylphosphatidylinositol transamidase complex, are retained in the ER. Neither protein contains a known ER localization signal. Gaa1 is a polytopic membrane glycoprotein with a cytoplasmic N terminus and a large luminal loop between its first two transmembrane spans; PIG-T is a type I membrane glycoprotein. To simplify our analyses, we studied Gaa1 and PIG-T constructs that could not interact with other subunits of the transamidase. We now show that Gaa1(282), a truncated protein consisting of the first TM domain and luminal loop of Gaa1, is correctly oriented, N-glycosylated, and ER-localized. Removal of a potential ER localization signal in the form of a triple arginine cluster near the N terminus of Gaa1 or Gaa1(282) had no effect on ER localization. Fusion proteins consisting of different elements of Gaa1(282) appended to alpha2,6-sialyltransferase or transferrin receptor could exit the ER, indicating that Gaa1(282), and by implication Gaa1, does not contain any dominant ER-sorting determinants. The data suggest that Gaa1 is passively retained in the ER by a signalless mechanism. In contrast, similar analyses of PIG-T revealed that it is ER-localized because of information in its transmembrane span; fusion of the PIG-T transmembrane span to Tac antigen, a plasma membrane-localized protein, caused the fusion protein to remain in the ER. These data are discussed in the context of models that have been proposed to account for retention of ER membrane proteins.

A conserved proline in the last transmembrane segment of Gaa1 is required for glycosylphosphatidylinositol (GPI) recognition by GPI transamidase.

Glycosylphosphatidylinositol (GPI)-anchored proteins are synthesized as precursor proteins that are processed in the endoplasmic reticulum by GPI transamidase (GPIT). Human GPIT is a multisubunit membrane-bound protein complex consisting of Gaa1, Gpi8, phosphatidylinositol glycan (PIG)-S, PIG-T, and PIG-U. The enzyme recognizes a C-terminal signal sequence in the proprotein and replaces it with a preformed GPI lipid. The nature of the functional interaction of the GPIT subunits with each other and with the proprotein and GPI substrates is largely unknown. We recently analyzed the GPIT subunit Gaa1, a polytopic protein with seven transmembrane (TM) spans, to identify sequence determinants in the protein that are required for its interaction with other subunits and for function (Vainauskas, S., Maeda, Y., Kurniawan, H., Kinoshita, T., and Menon, A. K. (2002) J. Biol. Chem. 277, 30535-30542). We showed that elimination of the C-terminal TM segment of Gaa1 allows the protein to interact with Gpi8, PIG-S, and PIG-T but renders the resulting GPIT complex nonfunctional. We now show that GPIT complexes containing C-terminally truncated Gaa1 possess a full complement of subunits and are able to interact with a proprotein substrate but cannot co-immunoprecipitate GPI. We go on to show that mutation of a conserved proline residue centrally located within the C-terminal TM span of Gaa1 is sufficient to abrogate the ability of the resulting GPIT complex to co-immunoprecipitate GPI. We suggest that the putative dynamic hinge created by the proline residue provides a structural basis for the interaction of GPI with GPIT.

Structural requirements for the recruitment of Gaa1 into a functional glycosylphosphatidylinositol transamidase complex.

Glycosylphosphatidylinositol (GPI)-anchored proteins are synthesized on membrane-bound ribosomes, translocated across the endoplasmic reticulum membrane, and GPI-anchored by GPI transamidase (GPIT). GPIT is a minimally heterotetrameric membrane protein complex composed of Gaa1, Gpi8, PIG-S and PIG-T. We describe structure-function analyses of Gaa1, the most hydrophobic of the GPIT subunits, with the aim of assigning a functional role to the different sequence domains of the protein. We generated epitope-tagged Gaa1 mutants and analyzed their membrane topology, subcellular distribution, complex-forming capability, and ability to restore GPIT activity in Gaa1-deficient cells. We show that (i) detergent-extracted, Gaa1-containing GPIT complexes sediment unexpectedly rapidly at approximately 17 S, (ii) Gaa1 is an endoplasmic reticulum-localized membrane glycoprotein with a cytoplasmically oriented N terminus and a lumenally oriented C terminus, (iii) elimination of C-terminal transmembrane segments allows Gaa1 to interact with other GPIT subunits but renders the resulting GPIT complex nonfunctional, (iv) interaction between Gaa1 and other GPIT subunits occurs via the large lumenal domain of Gaa1 located between the first and second transmembrane segments, and (v) the cytoplasmic N terminus of Gaa1 is not required for formation of a functional GPIT complex but may act as a membrane-sorting determinant directing Gaa1 and associated GPIT subunits to an endoplasmic reticulum membrane domain.

Comparative importance in vivo of conserved glutamate residues in the EX7E motif retaining glycosyltransferase Gpi3p, the UDP-GlcNAc-binding subunit of the first enzyme in glycosylphosphatidylinositol assembly.

Saccharomyces cerevisiae Gpi3p is the UDP-GlcNAc-binding and presumed catalytic subunit of the enzyme that forms GlcNAc-phosphatidylinositol in glycosylphosphatidylinositol biosynthesis. It is an essential protein with an EX7E motif that is conserved in four families of retaining glycosyltransferases. All Gpi3ps contain a cysteine residue four residues C-terminal to EX7E. To test their importance for Gpi3p function in vivo, Glu289 and 297 in the EX7E motif of S. cerevisiae Gpi3p, as well as Cys301, were altered by site-specific mutagenesis, and the mutant proteins tested for their ability to complement nonviable GPI3-deleted haploids. Gpi3p-C301A supported growth but membranes from C301A-expressing cells had low in vitro N-acetylglucosaminylphosphatidylinositol (GlcNAc-PI) synthetic activity. Haploids harboring Gpi3p-E289A proved viable, although slow growing but Gpi3-E297A did not support growth. The E289D and E297D mutants both supported growth at 25 degrees C, but, whereas the E289D strain grew at 37 degrees C, the E297D mutant did not. Membranes from E289D mutants had severely reduced in vitro GlcNAc-PI synthetic activity and E297D membranes had none. The mutation of the first Glu in the EX7E motif of Schizosaccharomyces pombe Gpi3p (Glu277) to Asp complemented the lethal null mutation in gpi3+ and supported growth at 37 degrees C, but the E285D mutant was nonviable. Our results suggest that the second Glu residue of the EX7E motif in Gpi3p is of greater importance than the first for function in vivo. Further, our findings do not support previous suggestions that the first Glu of an EX7E protein is the nucleophile and that Cys301 has an important role in UDP-GlcNAc binding by Gpi3ps.