Effects of macromolecular crowding on the folding and aggregation of glycosylated MUC5AC.

OBJECTIVE: To investigate the effects of macromolecular crowding on the folding and aggregation of MUC5AC with different levels of glycosylation during refolding. METHODS: Part 1:An in vitro catalytic reaction comprising the ppGalNAc T2 enzyme, uridine-5'-diphospho-N-galactosamine (UDP-GalNAc) and an 11-amino acid peptide substrate, was used to assess the enzyme activity of the ppGalNAc T2 enzyme in macromolecular crowding environment respectively with bovine serum albumin (BSA), polyethylene glycol (PEG2000), Dextran70 and Ficoll70 at different concentration and temperature. Part 2: The recombinant MUC5AC was expressed in HEK293 cells and purified by nickel column chromatography. The purified protein was treated with PNGase F, and the degree of glycosylation was analyzed by SDS-PAGE. Macromolecular crowding was simulated using PEG2000 at the concentrations of 50, 100, and 200 g/L. Deglycosylated-MUC5AC (d-MUC5AC) and glycosylated MUC5AC (g-MUC5AC) were denatured by GdnHCl and renatured by dilution in a refolding buffer. Protein aggregation was monitored continuously by absorbance reading at 488 nm using a UV spectrophotometer at 25 °C. The refolded proteins were centrifuged, the protein concentration of the supernatant was measured, and refolding yield in different refolding buffers was determined. RESULTS: Enzyme activityof ppGalNAc T2 was observed to increase with increasing crowding agent concentration, with highest enzyme activity at 200 g/L. Compared with the group in the absence of crowding reagent, the refolding yield of g-MUC5AC and d-MUC5AC were reduced significantly in the presence of different concentrations of PEG2000 (200, 100, and 50 g/L). Compared with the dilute solution, aggregation increased significantly in the presence of PEG2000, especially at 200 g/L. Moreover, in the crowded reagent with the same concentration, the refolding yield of d-MUC5AC was higher than that of g-MUC5AC, whereas the degree of aggregation of d-MUC5AC was lower than that of g-MUC5AC. CONCLUSION: The crowded intracellular environment reduces the refolding rate of MUC5AC and strongly induces the misfolding and aggregation of glycosylated MUC5AC.

O-GlcNAcylation as a Therapeutic Target for Alzheimer's Disease.

Alzheimer's disease (AD) is the most common cause of dementia and the number of elderly patients suffering from AD has been steadily increasing. Despite worldwide efforts to cope with this disease, little progress has been achieved with regard to identification of effective therapeutics. Thus, active research focusing on identification of new therapeutic targets of AD is ongoing. Among the new targets, post-translational modifications which modify the properties of mature proteins have gained attention. O-GlcNAcylation, a type of PTM that attaches O-linked ß-N-acetylglucosamine (O-GlcNAc) to a protein, is being sought as a new target to treat AD pathologies. O-GlcNAcylation has been known to modify the two important components of AD pathological hallmarks, amyloid precursor protein, and tau protein. In addition, elevating O-GlcNAcylation levels in AD animal models has been shown to be effective in alleviating AD-associated pathology. Although studies investigating the precise mechanism of reversal of AD pathologies by targeting O-GlcNAcylation are not yet complete, it is clearly important to examine O-GlcNAcylation regulation as a target of AD therapeutics. This review highlights the mechanisms of O-GlcNAcylation and its role as a potential therapeutic target under physiological and pathological AD conditions.

Identification of the O-GalNAcylation site(s) on FOXA1 catalyzed by ppGalNAc-T2 enzyme in vitro.

FOXA1 functions as a pioneer factor of transcriptional regulation that binds to specific sites in the chromatin and recruits other transcription factors, promoting the initiation of gene transcription and mediating the regulation of downstream target gene expression. FOXA1 was reported to facilitate or reprogram ERα binding, thus playing a key function in breast cancer progression. Our previous results indicated that the O-linked N-acetylgalactosamine (O-GalNAc) modification of FOXA1 plays a potentially significant role in the ERα transcription network. However, further investigations are needed to identify the specific mechanism of modification and the specific glycosylation sites on FOXA1. In this study, we first suggested that FOXA1 could be O-GalNAcylated by ppGalNAc-T2 in vitro. By dividing and expressing recombinant FOXA1 as three segments, two O-GalNAcylation sites were found on FOXA1, both located at the C-terminal of the protein. Then, synthesized peptides, including the predicted O-GalNAc sites in the C-terminus of FOXA1, were used in a vitro reaction, and peptides mutated at the predicted O-GalNAc sites were employed as controls. Through an ESI-MS assay, S354 and S355 were identified as probable O-GalNAcylation sites on FOXA1. Additionally, we performed ESI-ETD-MS/MS analysis of the full-length O-GalNAcylated FOXA1 protein and identified S355 as the O-GalNAc modification site on FOXA1, consistent with the peptide reaction. In conclusion, our results demonstrated that FOXA1 can be O-GalNAcylated by ppGalNAc-T2 at S355 in vitro. These results will provide new insights for studying the role of O-GalNAcylation in the development of breast cancer.

Molecular cloning, expression, and characterization of UDP N-acetyl-α-d-galactosamine: Polypeptide N-acetylgalactosaminyltransferase 4 from Cryptosporidium parvum.

Cryptosporidium spp. are the causative agents of diarrheal disease worldwide, but effective treatments are lacking. Cryptosporidium employs mucin-like glycoproteins with O-glycans to attach to and infect host intestinal epithelial cells. The Tn antigen (GalNAcα1-Ser/Thr) is an O-glycan essential for these processes, as Tn-specific lectins and a Tn-specific monoclonal antibody block attachment to and infection of host cells in vitro. The enzymes in Cryptosporidium catalyzing their synthesis, however, have not been studied. Previously, we identified four genes encoding putative UDP N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts) in the genomes of three Cryptosporidium spp. Here we report the in silico analysis, cloning, expression, purification, and characterization of one of the four enzymes Cryptosporidium parvum (Cp)-ppGalNAc-T4. This enzyme contains the characteristic domains and motifs conserved in ppGalNAc-Ts and is expressed at multiple time points during in vitro infection. Recombinant soluble Cp-ppGalNAc-T4 was enzymatically active against an unmodified EA2 peptide suggesting that it may function as an "initiating" ppGalNAc-T. Cp-ppGalNAc-T4 also exhibited a strong preference for UDP-GalNAc over other nucleotide sugar donors and was active against unmodified and O-glycosylated versions of the C. parvum gp40-derived peptide, with a preference for the former, suggesting it may play a role in modifying this glycoprotein in vivo. Given the importance of mucin-type O-glycosylation in Cryptosporidium spp., the enzymes that catalyze their synthesis may serve as potential therapeutic targets.

Systematic identification of the protein substrates of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase-T1/T2/T3 using a human proteome microarray.

O-GalNAc glycosylation is the initial step of the mucin-type O-glycosylation. In humans, it is catalyzed by a family of 20 homologous UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts). So far, there is very limited information on their protein substrate specificities. In this study, we developed an on-chip ppGalNAc-Ts assay that could rapidly and systematically identify the protein substrates of each ppGalNAc-T. In detail, we utilized a human proteome microarray as the protein substrates and UDP-GalNAz as the nucleotide sugar donor for click chemistry detection. From a total of 16 368 human proteins, we identified 570 potential substrates of ppGalNAc-T1, T2, and T3. Among them, 128 substrates were overlapped, while the rest were isoform specific. Further cluster analysis of these substrates showed that the substrates of ppGalNAc-T1 had a closer phylogenetic relationship with that of ppGalNAc-T3 compared with ppGalNAc-T2, which was consistent with the topology of the phylogenetic tree of these ppGalNAc-Ts. Taken together, our microarray-based enzymatic assay comprehensively reveals the substrate profile of the ppGalNAc-T1, T2, and T3, which not only provides a plausible explanation for their partial functional redundancy as reported, but clearly implies some specialized roles of each enzyme in different biological processes.

A common sugar-nucleotide-mediated mechanism of inhibition of (glycosamino)glycan biosynthesis, as evidenced by 6F-GalNAc (Ac3).

Glycosaminoglycan (GAG) polysaccharides have been implicated in a variety of cellular processes, and alterations in their amount and structure have been associated with diseases such as cancer. In this study, we probed 11 sugar analogs for their capacity to interfere with GAG biosynthesis. One analog, with a modification not directly involved in the glycosidic bond formation, 6F-N-acetyl-d-galactosamine (GalNAc) (Ac3), was selected for further study on its metabolic and biologic effect. Treatment of human ovarian carcinoma cells with 50 µM 6F-GalNAc (Ac3) inhibited biosynthesis of GAGs (chondroitin/dermatan sulfate by ∼50-60%, heparan sulfate by ∼35%), N-acetyl-d-glucosamine (GlcNAc)/GalNAc containing glycans recognized by the lectins Datura stramonium and peanut agglutinin (by ∼74 and ∼43%, respectively), and O-GlcNAc protein modification. With respect to function, 6F-GalNAc (Ac3) treatment inhibited growth factor signaling and reduced in vivo angiogenesis by ∼33%. Although the analog was readily transformed in cells into the uridine 5'-diphosphate (UDP)-activated form, it was not incorporated into GAGs. Rather, it strongly reduced cellular UDP-GalNAc and UDP-GlcNAc pools. Together with data from the literature, these findings indicate that nucleotide sugar depletion without incorporation is a common mechanism of sugar analogs for inhibiting GAG/glycan biosynthesis.

Crystal structure of Helicobacter pylori pseudaminic acid biosynthesis N-acetyltransferase PseH: implications for substrate specificity and catalysis.

Helicobacter pylori infection is the common cause of gastroduodenal diseases linked to a higher risk of the development of gastric cancer. Persistent infection requires functional flagella that are heavily glycosylated with 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-nonulosonic acid (pseudaminic acid). Pseudaminic acid biosynthesis protein H (PseH) catalyzes the third step in its biosynthetic pathway, producing UDP-2,4-diacetamido-2,4,6-trideoxy-ß-L-altropyranose. It belongs to the GCN5-related N-acetyltransferase (GNAT) superfamily. The crystal structure of the PseH complex with cofactor acetyl-CoA has been determined at 2.3 Å resolution. This is the first crystal structure of the GNAT superfamily member with specificity to UDP-4-amino-4,6-dideoxy-ß-L-AltNAc. PseH is a homodimer in the crystal, each subunit of which has a central twisted ß-sheet flanked by five α-helices and is structurally homologous to those of other GNAT superfamily enzymes. Interestingly, PseH is more similar to the GNAT enzymes that utilize amino acid sulfamoyl adenosine or protein as a substrate than a different GNAT-superfamily bacterial nucleotide-sugar N-acetyltransferase of the known structure, WecD. Analysis of the complex of PseH with acetyl-CoA revealed the location of the cofactor-binding site between the splayed strands ß4 and ß5. The structure of PseH, together with the conservation of the active-site general acid among GNAT superfamily transferases, are consistent with a common catalytic mechanism for this enzyme that involves direct acetyl transfer from AcCoA without an acetylated enzyme intermediate. Based on structural homology with microcin C7 acetyltransferase MccE and WecD, the Michaelis complex can be modeled. The model suggests that the nucleotide- and 4-amino-4,6-dideoxy-ß-L-AltNAc-binding pockets form extensive interactions with the substrate and are thus the most significant determinants of substrate specificity. A hydrophobic pocket accommodating the 6'-methyl group of the altrose dictates preference to the methyl over the hydroxyl group and thus to contributes to substrate specificity of PseH.

Structures of complexes of a metal-independent glycosyltransferase GT6 from Bacteroides ovatus with UDP-N-acetylgalactosamine (UDP-GalNAc) and its hydrolysis products.

Mammalian members of glycosyltransferase family 6 (GT6) of the CAZy database have a GT-A fold containing a conserved Asp-X-Asp (DXD) sequence that binds an essential metal cofactor. Bacteroides ovatus GT6a represents a GT6 clade found in more than 30 Gram-negative bacteria that is similar in sequence to the catalytic domains of mammalian GT6, but has an Asn(95)-Ala-Asn(97) (NXN) sequence substituted for the DXD motif and metal-independent catalytic activity. Co-crystals of a low activity mutant of BoGT6a (E192Q) with UDP-GalNAc contained protein complexes with intact UDP-GalNAc and two forms with hydrolysis products (UDP plus GalNAc) representing an initial closed complex and later open form primed for product release. Two cationic residues near the C terminus of BoGT6a, Lys(231) and Arg(243), interact with the diphosphate moiety of UDP-GalNAc, but only Lys(231) interacts with the UDP product and may function in leaving group stabilization. The amide group of Asn(95), the first Asn of the NXN motif, interacts with the ribose moiety of the substrate. This metal-independent GT6 resembles its metal-dependent homologs in undergoing conformational changes on binding UDP-GalNAc that arise from structuring the C terminus to cover this substrate. It appears that in the GT6 family, the metal cofactor functions specifically in binding the UDP moiety in the donor substrate and transition state, actions that can be efficiently performed by components of the polypeptide chain.

Hyper-O-GlcNAcylation is anti-apoptotic and maintains constitutive NF-κB activity in pancreatic cancer cells.

Cancer cell metabolic reprogramming includes a shift in energy production from oxidative phosphorylation to less efficient glycolysis even in the presence of oxygen (Warburg effect) and use of glutamine for increased biosynthetic needs. This necessitates greatly increased glucose and glutamine uptake, both of which enter the hexosamine biosynthetic pathway (HBP). The HBP end product UDP-N-acetylglucosamine (UDP-GlcNAc) is used in enzymatic post-translational modification of many cytosolic and nuclear proteins by O-linked ß-N-acetylglucosamine (O-GlcNAc). Here, we observed increased HBP flux and hyper-O-GlcNAcylation in human pancreatic ductal adenocarcinoma (PDAC). PDAC hyper-O-GlcNAcylation was associated with elevation of OGT and reduction of the enzyme that removes O-GlcNAc (OGA). Reducing hyper-O-GlcNAcylation had no effect on non-transformed pancreatic epithelial cell growth, but inhibited PDAC cell proliferation, anchorage-independent growth, orthotopic tumor growth, and triggered apoptosis. PDAC is supported by oncogenic NF-κB transcriptional activity. The NF-κB p65 subunit and upstream kinases IKKα/IKKß were O-GlcNAcylated in PDAC. Reducing hyper-O-GlcNAcylation decreased PDAC cell p65 activating phosphorylation (S536), nuclear translocation, NF-κB transcriptional activity, and target gene expression. Conversely, mimicking PDAC hyper-O-GlcNAcylation through pharmacological inhibition of OGA suppressed suspension culture-induced apoptosis and increased IKKα and p65 O-GlcNAcylation, accompanied by activation of NF-κB signaling. Finally, reducing p65 O-GlcNAcylation specifically by mutating two p65 O-GlcNAc sites (T322A and T352A) attenuated the induction of PDAC cell anchorage-independent growth. Our data indicate that hyper-O-GlcNAcylation is anti-apoptotic and contributes to NF-κB oncogenic activation in PDAC.

Glycosylation of α-dystroglycan: O-mannosylation influences the subsequent addition of GalNAc by UDP-GalNAc polypeptide N-acetylgalactosaminyltransferases.

O-Linked glycosylation is a functionally and structurally diverse type of protein modification present in many tissues and across many species. α-Dystroglycan (α-DG), a protein linked to the extracellular matrix, whose glycosylation status is associated with human muscular dystrophies, displays two predominant types of O-glycosylation, O-linked mannose (O-Man) and O-linked N-acetylgalactosamine (O-GalNAc), in its highly conserved mucin-like domain. The O-Man is installed by an enzyme complex present in the endoplasmic reticulum. O-GalNAc modifications are initiated subsequently in the Golgi apparatus by the UDP-GalNAc polypeptide N-acetylgalactosaminyltransferase (ppGalNAc-T) enzymes. How the presence and position of O-Man influences the action of the ppGalNAc-Ts on α-DG and the distribution of the two forms of glycosylation in this domain is not known. Here, we investigated the interplay between O-Man and the addition of O-GalNAc by examining the activity of the ppGalNAc-Ts on peptides and O-Man-containing glycopeptides mimicking those found in native α-DG. These synthetic glycopeptides emulate intermediate structures, not otherwise readily available from natural sources. Through enzymatic and mass spectrometric methods, we demonstrate that the presence and specific location of O-Man can impact either the regional exclusion or the site of O-GalNAc addition on α-DG, elucidating the factors contributing to the glycosylation patterns observed in vivo. These results provide evidence that one form of glycosylation can influence another form of glycosylation in α-DG and suggest that in the absence of proper O-mannosylation, as is associated with certain forms of muscular dystrophy, aberrant O-GalNAc modifications may occur and could play a role in disease presentation.

UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferases: completion of the family tree.

The formation of mucin-type O-glycans is initiated by an evolutionarily conserved family of enzymes, the UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts). The human genome encodes 20 transferases; 17 of which have been characterized functionally. The complexity of the GalNAc-T family reflects the differential patterns of expression among the individual enzyme isoforms and the unique substrate specificities which are required to form the dense arrays of glycans that are essential for mucin function. We report the expression patterns and enzymatic activity of the remaining three members of the family and the further characterization of a recently reported isoform, GalNAc-T17. One isoform, GalNAcT-16 that is most homologous to GalNAc-T14, is widely expressed (abundantly in the heart) and has robust polypeptide transferase activity. The second isoform GalNAc-T18, most similar to GalNAc-T8, -T9 and -T19, completes a discrete subfamily of GalNAc-Ts. It is widely expressed and has low, albeit detectable, activity. The final isoform, GalNAc-T20, is most homologous to GalNAc-T11 but lacks a lectin domain and has no detectable transferase activity with the panel of substrates tested. We have also identified and characterized enzymatically active splice variants of GalNAc-T13 that differ in the sequence of their lectin domain. The variants differ in their affinities for glycopeptide substrates. Our findings provide a comprehensive view of the complexities of mucin-type O-glycan formation and provide insight into the underlying mechanisms employed to heavily decorate mucins and mucin-like domains with carbohydrate.

In vitro metabonomic study detects increases in UDP-GlcNAc and UDP-GalNAc, as early phase markers of cisplatin treatment response in brain tumor cells.

O-linked ß-N-acetylglucosamine glycosylation (O-GlcNAcylation) is important in a number of biological processes and diseases including transcription, cell stress, diabetes, and neurodegeneration and may be a marker of tumor metastasis. Uridine diphospho-N-acetylglucosamine (UDP-GlcNAc), the donor molecule in O-GlcNAcylation, can be detected by (1)H nuclear magnetic resonance spectroscopy ((1)H NMR), giving the potential to measure its level noninvasively, providing a novel biomarker of prognosis and treatment monitoring. In this in vitro metabonomic study, four brain cancer cell lines were exposed to cisplatin and studied for metabolic responses using (1)H NMR. The Alamar blue assay and DAPI staining were used to assess cell sensitivity to cisplatin treatment and to confirm cell death. It is shown that in the cisplatin responding cells, UDP-GlcNAc and uridine diphospho-N-acetylgalactosamine (UDP-GalNAc), in parallel with (1)H NMR detected lipids, increased with cisplatin exposure before or at the onset of the microscopic signs of evolving cell death. The changes in UDP-GlcNAc and UDP-GalNAc were not detected in the nonresponders. These glycosylated UDP compounds, the key substrates for glycosylation of proteins and lipids, are commonly implicated in cancer proliferation and malignant transformation. However, the present study mechanistically links UDP-GlcNAc and UDP-GalNAc to cancer cell death following chemotherapeutic treatment.

A UDP-HexNAc:polyprenol-P GalNAc-1-P transferase (WecP) representing a new subgroup of the enzyme family.

The Aeromonas hydrophila AH-3 WecP represents a new class of UDP-HexNAc:polyprenol-P HexNAc-1-P transferases. These enzymes use a membrane-associated polyprenol phosphate acceptor (undecaprenyl phosphate [Und-P]) and a cytoplasmic UDP-d-N-acetylhexosamine sugar nucleotide as the donor substrate. Until now, all the WecA enzymes tested were able to transfer UDP-GlcNAc to the Und-P. In this study, we present in vitro and in vivo proofs that A. hydrophila AH-3 WecP transfers GalNAc to Und-P and is unable to transfer GlcNAc to the same enzyme substrate. The molecular topology of WecP is more similar to that of WbaP (UDP-Gal polyprenol-P transferase) than to that of WecA (UDP-GlcNAc polyprenol-P transferase). WecP is the first UDP-HexNAc:polyprenol-P GalNAc-1-P transferase described.

The catalytic and lectin domains of UDP-GalNAc:polypeptide alpha-N-Acetylgalactosaminyltransferase function in concert to direct glycosylation site selection.

UDP-GalNAc:polypeptide alpha-N-Acetylgalactosaminyltransferases (ppGalNAcTs), a family (EC 2.4.1.41) of enzymes that initiate mucin-type O-glycosylation, are structurally composed of a catalytic domain and a lectin domain. Previous studies have suggested that the lectin domain modulates the glycosylation of glycopeptide substrates and may underlie the strict glycopeptide specificity of some isoforms (ppGalNAcT-7 and -10). Using a set of synthetic peptides and glycopeptides based upon the sequence of the mucin, MUC5AC, we have examined the activity and glycosylation site preference of lectin domain deletion and exchange constructs of the peptide/glycopeptide transferase ppGalNAcT-2 (hT2) and the glycopeptide transferase ppGalNAcT-10 (hT10). We demonstrate that the lectin domain of hT2 directs glycosylation site selection for glycopeptide substrates. Pre-steady-state kinetic measurements show that this effect is attributable to two mechanisms, either lectin domain-aided substrate binding or lectin domain-aided product release following glycosylation. We find that glycosylation of peptide substrates by hT10 requires binding of existing GalNAcs on the substrate to either its catalytic or lectin domain, thereby resulting in its apparent strict glycopeptide specificity. These results highlight the existence of two modes of site selection used by these ppGalNAcTs: local sequence recognition by the catalytic domain and the concerted recognition of distal sites of prior glycosylation together with local sequence binding mediated, respectively, by the lectin and catalytic domains. The latter mode may facilitate the glycosylation of serine or threonine residues, which occur in sequence contexts that would not be efficiently glycosylated by the catalytic domain alone. Local sequence recognition by the catalytic domain differs between hT2 and hT10 in that hT10 requires a pre-existing GalNAc residue while hT2 does not.

Substrate-induced conformational changes and dynamics of UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferase-2.

O-Glycan biosynthesis is initiated by the transfer of N-acetylgalactosamine (GalNAc) from a nucleotide sugar donor (UDP-GalNAc) to Ser/Thr residues of an acceptor substrate. The detailed transfer mechanism, catalyzed by the UDP-GalNAc polypeptide:N-acetyl-alpha-galactosaminyltransferases (ppGalNAcTs), remains unclear despite structural information available for several isoforms in complex with substrates at various stages along the catalytic pathway. We used all-atom molecular dynamics simulations with explicit solvent and counterions to study the conformational dynamics of ppGalNAcT-2 in several enzymatic states along the catalytic pathway. ppGalNAcT-2 is simulated both in the presence and in the absence of substrates and reaction products to examine the role of conformational changes in ligand binding. In multiple 40-ns-long simulations of more than 600 ns total run time, we studied systems ranging from 45,000 to 95,000 atoms. Our simulations accurately identified dynamically active regions of the protein, as previously revealed by the X-ray structures, and permitted a detailed, atomistic description of the conformational changes of loops near the active site and the characterization of the ensemble of structures adopted by the transferase complex on the transition pathway between the ligand-bound and ligand-free states. In particular, the conformational transition of a functional loop adjacent to the active site from closed (active) to open (inactive) is correlated with the rotameric state of the conserved residue W331. Analysis of water dynamics in the active site revealed that internal water molecules have an important role in enhancing the enzyme flexibility. We also found evidence that charged side chains in the active site rearrange during site opening to facilitate ligand binding. Our results are consistent with the single-displacement transfer mechanism previously proposed for ppGalNAcTs based on X-ray structures and mutagenesis data and provide new evidence for possible functional roles of certain amino acids conserved across several isoforms.

Congenital disorders of glycosylation: a rapidly expanding disease family.

Congenital disorders of glycosylation (CDG) are a large family of genetic diseases resulting from defects in the synthesis of glycans and in the attachment of glycans to other compounds. These disorders cause a wide range of human diseases, with examples emanating from all medical subspecialties. Since our 2001 review on CDG ( 36 ), this field has seen substantial growth: The number of N-glycosylation defects has doubled (from 6 to 12), five new O-glycosylation defects have been added to the two previously known ones, three combined N- and O-glycosylation defects have been identified, the first lipid glycosylation defects have been discovered, and a new domain, that of the hyperglycosylation defects, has been introduced. A number of CDG are due to defects in enzymes with a putative glycosyltransferase function. There is also a growing group of patients with unidentified defects (CDG-x), some with typical clinical presentations and others with presentations not seen before in CDG. This review focuses on the clinical, biochemical, and genetic characteristics of CDG and on advances expected in their future study and clinical management.

UDP-N-acetylglucosamine 4'-epimerase from the intestinal protozoan Giardia intestinalis lacks UDP-glucose 4'-epimerase activity.

The protozoan parasite Giardia intestinalis has a simple life cycle consisting of an intestinal trophozoite stage and an environmentally resistant cyst stage. The cyst is formed when a trophozoite encases itself within an external filamentous covering, the cyst wall, which is crucial to the cyst's survival outside of the host. The filaments in the cyst wall consist mainly of a beta (1-3) polymer of N-acetylgalactosamine. Its precursor, UDP-N-acetylgalactosamine, is synthesized from fructose 6-phosphate by a pathway of five inducible enzymes. The fifth, UDP-N-acetylglucosamine 4'-epimerase, epimerizes UDP-N-acetylglucosamine to UDP-N-acetylgalactosamine reversibly. The epimerase of G. intestinalis lacks UDP-glucose/UDP-galactose 4'-epimerase activity and shows characteristic amino acyl residues to allow binding of only the larger UDP-N-acetylhexosamines. While the Giardia epimerase catalyzes the reversible epimerization of UDP-N-acetylglucosamine to UDP-N-acetylgalactosamine, the reverse reaction apparently is favored. The enzyme has a higher Vmax and a smaller Km in this direction. Therefore, an excess of UDP-N-acetylglucosamine is required to drive the reaction towards the synthesis of UDP-N-acetylgalactosamine, when it is needed for cyst wall formation. This forms the ultimate regulatory step in cyst wall biosynthesis.

Dynamic association between the catalytic and lectin domains of human UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminyltransferase-2.

The family of UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminyltransferases (ppGalNAcTs) is unique among glycosyltransferases, containing both catalytic and lectin domains that we have previously shown to be closely associated. Here we describe the x-ray crystal structures of human ppGalNAcT-2 (hT2) bound to the product UDP at 2.75 A resolution and to UDP and an acceptor peptide substrate EA2 (PTTDSTTPAPTTK) at 1.64 A resolution. The conformations of both UDP and residues Arg362-Ser372 vary greatly between the two structures. In the hT2-UDP-EA2 complex, residues Arg362-Ser373 comprise a loop that forms a lid over UDP, sealing it in the active site, whereas in the hT2-UDP complex this loop is folded back, exposing UDP to bulk solvent. EA2 binds in a shallow groove with threonine 7 positioned consistent with in vitro data showing it to be the preferred site of glycosylation. The relative orientations of the hT2 catalytic and lectin domains differ dramatically from that of murine ppGalNAcT-1 and also vary considerably between the two hT2 complexes. Indeed, in the hT2-UDP-EA2 complex essentially no contact is made between the catalytic and lectin domains except for the peptide bridge between them. Thus, the hT2 structures reveal an unexpected flexibility between the catalytic and lectin domains and suggest a new mechanism used by hT2 to capture glycosylated substrates. Kinetic analysis of hT2 lacking the lectin domain confirmed the importance of this domain in acting on glycopeptide but not peptide substrates. The structure of the hT2-UDP-EA2 complex also resolves long standing questions regarding ppGalNAcT acceptor substrate specificity.

Characterization of the UDP-N-acetylgalactosamine binding domain of bovine polypeptide alphaN-acetylgalactosaminyltransferase T1.

UDP-GalNAc:polypeptide alphaN-acetylgalactosaminyltransferases (ppGaNTases) transfer GalNAc from UDP-GalNAc to Ser or Thr. Structural features underlying their enzymatic activity and their specificity are still unidentified. In order to get some insight into the donor substrate recognition, we used a molecular modelling approach on a portion of the catalytic site of the bovine ppGaNTase-T1. Fold recognition methods identified as appropriate templates the bovine alpha1,3galactosyltransferase and the human alpha1,3N-acetylgalactosaminyltransferase. A model of the ppGaNTase-T1 nucleotide-sugar binding site was built into which the UDP-GalNAc and the Mn2+ cation were docked. UDP-GalNAc fits best in a conformation where the GalNAc is folded back under the phosphates and is maintained in that special conformation through hydrogen bonds with R193. The ribose is found in van der Waals contacts with F124 and L189. The uracil is involved in a stacking interaction with W129 and forms a hydrogen bond with N126. The Mn2+ is found in coordination both with the phosphates of UDP and the DXH motif of the enzyme. Amino acids in contact with UDP-GalNAc in the model have been mutated and the corresponding soluble forms of the enzyme expressed in yeast. Their kinetic constants confirm the importance of these amino acids in donor substrate interactions.

Amino sugar phosphate levels in Giardia change during cyst wall formation.

The parasite Giardia intestinalis exists as a trophozoite (vegetative) that infects the human small intestine, and a cyst (infective) that is shed in host faeces. Cyst viability in the environment depends upon a protective cyst wall, which consists of proteins and a unique beta(1-3) GalNAc homopolymer. UDP-GalNAc, the precursor for this polysaccharide, is synthesized from glucose by an enzyme pathway that involves amino sugar phosphate intermediates. Using a novel method of microanalysis by capillary electrophoresis, the levels of amino sugar phosphate intermediates in trophozoites before encystment, during a period of active encystment and after the peak of encystment were measured. These levels were used to deduce metabolic control of amino sugar phosphates associated with encystment. Levels of amino sugar phosphate intermediates increased during encystment, and then decreased to nearly non-encysting levels. The most pronounced increase was in glucosamine 6-phosphate, which is the first substrate unique in this pathway, and which is the positive effector for the pathway's putative rate-controlling enzyme, UDP-GlcNAc pyrophosphorylase. Moreover, more UDP-GalNAc than UDP-GlcNAc, its direct precursor, was detected at 24 h. It is postulated that the enhanced UDP-GalNAc is a result of enhanced synthesis of UDP-GlcNAc by the pyrophosphorylase, and its preferential conversion to UDP-GalNAc. These results suggest that kinetics of amino sugar phosphate synthesis in encysting Giardia favours the direction that supports cyst wall synthesis. The enzymes involved in synthesis of UDP-GalNAc and its conversion to cyst wall might be potential targets for therapeutic inhibitors of Giardia infection.