Unusual Glycosylation Research Articles

The melanoma tumor glyco-code impacts human dendritic cells' functionality and dictates clinical outcomes.

Subversion of immunity is a hallmark of cancer development. Dendritic cells (DCs) are strategic immune cells triggering anti-tumor immune responses, but tumor cells exploit their versatility to subvert their functions. Tumor cells harbor unusual glycosylation patterns, which can be sensed through glycan-binding receptors (lectins) expressed by immune cells that are crucial for DCs to shape and orientate antitumor immunity. Yet, the global tumor glyco-code and its impact on immunity has not been explored in melanoma. To decrypt the potential link between aberrant glycosylation patterns and immune evasion in melanoma, we investigated the melanoma tumor glyco-code through the GLYcoPROFILE™ methodology (lectin arrays), and depicted its impact on patients' clinical outcome and DC subsets' functionality. Specific glycan patterns correlated with clinical outcome of melanoma patients, GlcNAc, NeuAc, TF-Ag and Fuc motifs being associated with poor outcome, whereas Man and Glc residues elicited better survival. Strikingly, tumor cells differentially impacting cytokine production by DCs harbored distinct glyco-profiles. GlcNAc exhibited a negative influence on cDC2s, whereas Fuc and Gal displayed inhibitory impacts on cDC1s and pDCs. We further identified potential booster glycans for cDC1s and pDCs. Targeting specific glycans on melanoma tumor cells restored DCs' functionality. The tumor glyco-code was also linked to the nature of the immune infiltrate. This study unveils the impact of melanoma glycan patterns on immunity, and paves the way for innovative therapeutic options. Glycans/lectins interactions arise as promising immune checkpoints to rescue DCs from tumor' hijacking to reshape antitumor immunity and inhibit immunosuppressive circuits triggered by aberrant tumor glycosylation.

Unique CLR expression patterns on circulating and tumor-infiltrating DC subsets correlated with clinical outcome in melanoma patients.

Subversion of immunity by tumors is a crucial step for their development. Dendritic cells (DCs) are strategic immune cells that orchestrate anti-tumor immune responses but display altered functions in cancer. The bases for such DCs' hijacking are not fully understood. Tumor cells harbor unusual glycosylation patterns of surface glycoproteins and glycolipids. DCs express glycan-binding receptors, named C-type lectin receptors (CLR), allowing them to sense changes in glycan signature of their environment, and subsequently trigger a response. Recognition of tumor glycans by CLRs is crucial for DCs to shape antitumor immunity, and decisive in the orientation of the response. Yet the status of the CLR machinery on DCs in cancer, especially melanoma, remained largely unknown. We explored CLR expression patterns on circulating and tumor-infiltrating cDC1s, cDC2s, and pDCs of melanoma patients, assessed their clinical relevance, and further depicted the correlations between CLR expression profiles and DCs' features. For the first time, we highlighted that the CLR repertoire of circulating and tumor-infiltrating cDC1s, cDC2s, and pDCs was strongly perturbed in melanoma patients, with modulation of DCIR, CLEC-12α and NKp44 on circulating DCs, and perturbation of Dectin-1, CD206, DEC205, DC-SIGN and CLEC-9α on tumor-infiltrating DCs. Furthermore, melanoma tumor cells directly altered CLR expression profiles of healthy DC subsets, and this was associated with specific glycan patterns (Man, Fuc, GlcNAc) that may interact with DCs through CLR molecules. Notably, specific CLR expression profiles on DC subsets correlated with unique DCs' activation status and functionality and were associated with clinical outcome of melanoma patients. Higher proportions of DCIR-, DEC205-, CLEC-12α-expressing cDCs were linked with a better survival, whereas elevated proportions of CD206-, Dectin1-expressing cDCs and NKp44-expressing pDCs were associated with a poor outcome. Thus, melanoma tumor may shape DCs' features by exploiting the plasticity of the CLR machinery. Our study revealed that melanoma manipulates CLR pathways to hijack DC subsets and escape from immune control. It further paved the way to exploit glycan-lectin interactions for the design of innovative therapeutic strategies, which exploit DCs' potentialities while avoiding hijacking by tumor, to properly reshape anti-tumor immunity by manipulating the CLR machinery.

Abstract P5-08-01: Globo H: A Globo series glycosphingolipid target antigen in breast cancer

Abstract Cancer cells may be differentiated from normal cells by the surface expression of unusual glycosylation patterns: tumor-associated carbohydrate antigens (TACAs) may be expressed as membrane-bound glycoproteins or glycolipids. Differential expression of TACAs in multiple cancer types represents a potential therapeutic target for anticancer therapies. At least 16 different types of epithelial cancers have been found to be Globo H–positive, with 60% to 65% of patients with breast cancer having Globo H–positive tumors. It has been shown that tumor-infiltrating lymphocytes in breast cancer tissues could take up Globo H ceramide (GHCer) released by tumor cells masking cancer cells from immune surveillance through the suppression of Notch 1 signaling. Globo series antigens, including Globo H and stage-specific embryonic antigen 4 could associate with the FAK/CAV1/AKT/RIP complex to trigger the downstream β-catenin signaling pathway to promote tumor cell survival. Moreover, exogenous addition of GHCer promoted tumor cell migration. Thus, treatment-mediated depletion of GHCer may overcome the immunosuppressive mechanism of cancer cells, with resulting decreases in tumor cell survival and migration. OBI Pharma, Inc., in collaboration with NeoGenomics, has developed a validated immunohistochemistry assay to measure the expression of Globo H in clinical specimens. The intensity and extent of Globo H expression are determined microscopically using the histochemical scoring system (H-score), defined as the sum of the products of the staining intensity (score of 0-3) multiplied by the percentage of cells (0-100) stained at a given intensity. To date, 420 patients with triple-negative breast cancer (TNBC) have been screened across 4 separate clinical trials. Of those patients, 173 (41%) had an H-score of ≥15, with a median H-score of 70. Of the 173 patients with an H-score of ≥15, 69 (40%) had an H-score of ≥100. Adagloxad simolenin (AS/OBI-822) is a Globo H conjugate vaccine that when administered in combination with the adjuvant OBI-821 results in anti–Globo H IgM and IgG humoral immune responses. These responses have been assessed in all clinical trials of AS/OBI-821 to date. In the early trials that administered up to 9 doses, early and robust IgM responses were noted but with delayed and lower-titer IgG responses. When the number of doses was increased to 9-12 doses over an extended time (up to either 37 or 68 weeks), it became apparent that the peak IgG titers occurred at around the time of the last AS/OBI-821 dose. The time course for the development of anti–Globo H IgM and IgG humoral responses showed that following injection, anti–Globo H IgM and IgG antibody titers demonstrated a geometric mean ratio (GMR) increase of 10.26 and 12.47, respectively, from almost nondetectable levels at baseline. The GMR in the placebo group remained low in comparison. Mean time to peak titer was approximately 4 weeks for IgM and 40 weeks for IgG. These data were employed in the design of the phase 3, randomized, open-label study of the anti–Globo H vaccine AS/OBI-821 in the adjuvant treatment of patients with high-risk, early-stage Globo H–positive TNBC (The GLORIA Study). To enter the GLORIA study, patients must have a centrally confirmed Globo H IHC H-score of ≥15, and it is anticipated that approximately 40% of patients screened will have this level of expression. The GLORIA study is presently enrolling patients in 7 countries, with another 9 countries anticipated to begin recruiting in 2021. Citation Format: Darren S. Sigal, Apostolia Maria Tsimberidou, I-Ju Chen, Pei Hsu, Tillman Pearce. Globo H: A Globo series glycosphingolipid target antigen in breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-08-01.

Opportunities and Challenges of Bacterial Glycosylation for the Development of Novel Antibacterial Strategies.

Glycosylation is a ubiquitous process that is universally conserved in nature. The various products of glycosylation, such as polysaccharides, glycoproteins, and glycolipids, perform a myriad of intra- and extracellular functions. The multitude of roles performed by these molecules is reflected in the significant diversity of glycan structures and linkages found in eukaryotes and prokaryotes. Importantly, glycosylation is highly relevant for the virulence of many bacterial pathogens. Various surface-associated glycoconjugates have been identified in bacteria that promote infectious behavior and survival in the host through motility, adhesion, molecular mimicry, and immune system manipulation. Interestingly, bacterial glycosylation systems that produce these virulence factors frequently feature rare monosaccharides and unusual glycosylation mechanisms. Owing to their marked difference from human glycosylation, bacterial glycosylation systems constitute promising antibacterial targets. With the rise of antibiotic resistance and depletion of the antibiotic pipeline, novel drug targets are urgently needed. Bacteria-specific glycosylation systems are especially promising for antivirulence therapies that do not eliminate a bacterial population, but rather alleviate its pathogenesis. In this review, we describe a selection of unique glycosylation systems in bacterial pathogens and their role in bacterial homeostasis and infection, with a focus on virulence factors. In addition, recent advances to inhibit the enzymes involved in these glycosylation systems and target the bacterial glycan structures directly will be highlighted. Together, this review provides an overview of the current status and promise for the future of using bacterial glycosylation to develop novel antibacterial strategies.

Mass spectrometric characterisation of the major peptides of the male ejaculatory duct, including a glycopeptide with an unusual zwitterionic glycosylation

Peptides present in the seminal fluid of Drosophila melanogaster can function as antimicrobial agents, enzyme inhibitors and as pheromones that elicit physiological and behavioural responses in the post-mated female. Understanding the molecular interactions by which these peptides influence reproduction requires detailed knowledge of their molecular structures. However, this information is often lacking and cannot be gleaned from just gene sequences and standard proteomic data. We now report the native structures of four seminal fluid peptides (andropin, CG42782, Met75C and Acp54A1) from the ejaculatory duct of male D. melanogaster. The mature CG42782, Met75C and Acp54A1 peptides each have a cyclic structure formed by a disulfide bond, which will reduce conformational freedom and enhance metabolic stability. In addition, the presence of a penultimate Pro in CG42782 and Met75C will help prevent degradation by carboxypeptidases. Met75C has undergone more extensive post-translational modifications with the formation of an N-terminal pyroglutamyl residue and the attachment of a mucin-like O-glycan to the side chain of Thr4. Both of these modifications are expected to further enhance the stability of the secreted peptide. The glycan has a rare zwitterionic structure comprising an O-linked N-acetyl hexosamine, a hexose and, unusually, phosphoethanolamine. A survey of various genomes showed that andropin, CG42782, and Acp54A1 are relatively recent genes and are restricted to the melanogaster subgroup. Met75C, however, was also found in members of the obscura species groups and in Scaptodrosophila lebanonensis. Andropin is related to the cecropin gene family and probably arose by tandem gene duplication, whereas CG42782, Met75C and Acp54A1 possibly emerged de novo. We speculate that the post-translational modifications that we report for these gene products will be important not only for a biological function, but also for metabolic stability and might also facilitate transport across tissue barriers, such as the blood-brain barrier of the female insect. Biological significanceSeminal fluid peptides of D. melanogaster function as antimicrobials, enzyme inhibitors and as pheromones, eliciting physiological and behavioural responses in the post-mated female. A fuller understanding of how these peptides influence reproduction requires knowledge not only of their primary structure, but also of their post-translational modification. However, this information is often lacking and difficult to glean from standard proteomic data. The reported modifications, including the unusual glycosylation, adds much to our knowledge of this important class of peptides in this model organism, par excellence.

An integrated strategy for the construction of a species-specific glycan library for mass spectrometry-based intact glycopeptide analyses

Mass spectrometry (MS)-based strategies and related software tools using glycan mass lists have greatly facilitated the analysis of intact glycopeptides. Most glycan mass lists are derived from normal glycans of mammals and contain limited monosaccharides, which has significantly hindered high throughput studies of unusual glycosylation events observed in other species. In this work, an integrated strategy was developed for the construction of a species-specific glycan mass list from glycan structure databases and published papers. We developed a computational tool called LibGlycan, which could process the different formats of glycans. Then, the software tool generated a glycan library that contained the monoisotope mass, average mass, isotope distribution, and glycan mass list for input into Byonic software. This strategy was applied to analyze the N-glycosylation of rice roots and O-glycosylation of Acinetobacter baumannii ATCC17978, leading to the identification of 296 and 145 intact glycopeptides respectively. Combined, these results show that this strategy is a robust computational approach for the determination of glycan diversity within different complex biological systems.

S-glycosyltransferase UGT74B1 can glycosylate both S- and O-acceptors: mechanistic insights through substrate specificity

UGT74B1 from Arabidopsis thaliana is one of the few characterized glycosyltransferases able to generate a thioglycosidic linkage in vivo, using the sulfur atom of thiohydroximate as the nucleophile in the glycosylation reaction. This critical biosynthetic step in glucosinolate production has been documented. Yet little is known about the molecular mechanism that enables this rare and unusual glycosylation at the sulfur atom. To identify the role of this atom in the glycosylation reaction and unravel the mechanism of UGT74B1, we used a range of substrates containing either sulfur or oxygen. We first demonstrated that the enzyme could catalyze the glycosylation of thiohydroximates but also of O-containing hydroximates analogs. If KM values were shown to be close between analogs, the reaction catalytic rate kcat was 50–100 lower in the case of hydroximates. The glycosylation reaction is catalyzed through deprotonation of the acceptor, which was confirmed by the removal of catalytic carboxylic residues by site-directed mutagenesis. Moreover, using a range of simple phenols and thiophenols as UGT74B1 substrate acceptors for glycosylation, we concluded that the glycosylation reaction rate is correlated to the acceptor atom acidity, and not to the nature of this nucleophilic atom (oxygen or sulfur).

ChemInform Abstract: An Unusual Glycosylation Product from a Partially Protected Fucosyl Donor under Silver Triflate Activation Conditions.

AbstractThe partially protected fucosyl donor (I) is activated in the presence of primary alcohols (II) to give unexpected difucoside products (III) exclusively.

Futile Protein Folding Cycles in the ER Are Terminated by the Unfolded Protein O-Mannosylation Pathway

Newly synthesized polypeptides fold and assemble with assistance from protein chaperones. Full maturation can take multiple attempts, exchanging chaperones at each round. Improperly folded molecules must exit folding cycles and be degraded. In the endoplasmic reticulum (ER), prolonged substrate cycling is detrimental because it expends chaperone and energy resources and increases toxic reactive oxygen species. In budding yeast, we found that unfolded protein O-mannosylation terminated failed folding attempts through the Pmt1/Pmt2 complex. O-mannosylation incapacitated target molecule folding and removed them from folding cycles by reducing engagement with the Kar2 chaperone. In an in vitro protein refolding assay, the modification intrinsically and irreversibly disabled the folding potential of the substrate. Thus, protein folding termination can involve a covalent glycosylation event.

An unusual glycosylation product from a partially protected fucosyl donor under silver triflate activation conditions

Partially protected glycosyl donors are extremely useful reagents for oligosaccharide synthesis allowing more facile deprotection and enhanced activity due to lower steric restraints. A partially protected fucosyl donor containing tert-butyldimethylsilyl (TBDMS) protecting groups was activated under bromine-silver triflate conditions in the presence of primary alcohols and found to give difucoside products exclusively, in good yield with excellent diastereoselectivity. The dimerisation reaction appears to require a conformational relaxation of steric crowding, induced upon activation of the glycosyl donor. The scope and limitations of this unusual glycosylation methodology are reported.

Two new triterpenoid saponins from Pittosporum senacia Putterlick (Pittosporaceae)

From the branches of Pittosporum senacia Putterlick (Pittosporaceae), two new triterpenoid saponins, senaciapittosides A and B (1, 2), were isolated. Their structures were elucidated by extensive analysis of one- and two-dimensional nuclear magnetic resonance spectroscopy, high-resolution electrospray ionization mass spectrometry (HR-ESIMS) and chemical evidence as 3-O-[β-D-glucopyranosyl-(1 → 2)]-[α-L-arabinopyranosyl-(1 → 3)]-[α-L-arabinofuranosyl-(1 → 4)]-β-D-glucuronopyranosyl oleanolic acid 28-O-β-D-glucopyranosyl ester (1) and 3-O-[β-D-glucopyranosyl-(1 → 2)]-[α-L-arabinopyranosyl-(1 → 3)]-[α-L-arabinofuranosyl-(1 → 4)]-β-D-glucuronopyranosyl-22-O-α-L-arabinopyranosyl-21-acetoxy R1-barrigenol (2). Compound 2 presents an unusual glycosylation at C-22 of its aglycone.

Angucyclines: Biosynthesis, mode-of-action, new natural products, and synthesis.

Covering: 1997 to 2010. The angucycline group is the largest group of type II PKS-engineered natural products, rich in biological activities and chemical scaffolds. This stimulated synthetic creativity and biosynthetic inquisitiveness. The synthetic studies used five different strategies, involving Diels-Alder reactions, nucleophilic additions, electrophilic additions, transition-metal mediated cross-couplings and intramolecular cyclizations to generate the angucycline frames. Biosynthetic studies were particularly intriguing when unusual framework rearrangements by post-PKS tailoring oxidoreductases occurred, or when unusual glycosylation reactions were involved in decorating the benz[a]anthracene-derived cores. This review follows our previous reviews, which were published in 1992 and 1997, and covers new angucycline group antibiotics published between 1997 and 2010. However, in contrast to the previous reviews, the main focus of this article is on new synthetic approaches and biosynthetic investigations, most of which were published between 1997 and 2010, but go beyond, e.g. for some biosyntheses all the way back to the 1980s, to provide the necessary context of information.

7-Deazapurine (Pyrrolo[2,3-d]pyrimidine) 2-Deoxyribonucleosides: Syntheses and Transformations

This review reports on the synthesis of 7-deazapurine (pyrrolo[2,3-d]pyrimidine) 2-deoxyribonucleosides, including β-D- and β-L-enantiomers, fluoro derivatives, and 2,3-dideoxyribonucleosides. It covers the various aspects of convergent nucleoside synthesis. Stereochemically defined α-D and α-L 2-deoxyribonucleosides as well as sugar derivatives were prepared by nucleobase anion glycosylation. This glycosylation reaction is regioselective for the pyrrole nitrogen and stereoselective for β-nucleoside formation. Common glycosylation protocols lead to 7-deazapurine 2-deoxyribonucleosides with unusual glycosylation sites. 7-Deazapurine 2,3- dideoxyribonucleosides were also obtained from 2-deoxy- or 3-deoxyribonucleosides by Barton-McCombie deoxygenation, by elimination of sugar hydroxyl groups or by anion glycosylation. Another aspect of the review is the functionalization of pyrrolo[2,3-d]pyrimidine nucleosides. A broad range of reporter groups were introduced by the Sonogashira cross coupling or the copper(I)-catalyzed Huisgen- Meldal-Sharpless “click” reaction. The application of 7-deazapurine nucleosides as antiviral or anticancer agents, and the use of 7- deazapurine nucleoside triphosphates in the Sanger dideoxy DNA-sequencing are also reported.

Rare and unusual glycosylation of peptides and proteins

Glycosylation represents the most complex co- and post-translational modification of proteins. In addition to N- and O-glycans, almost all combinations, including the nature of the carbohydrate moiety and the amino-acid involved, but also the type of the chemical linkage, can be isolated from natural glycoconjugates. This diversity correlates with the importance and the variety of the biological processes (and consequently the diseases) glycosides are involved in. This review focuses on rare and unusual glycosylation of peptides and proteins.

Glycomic alterations in the highly-abundant and lesser-abundant blood serum protein fractions for patients diagnosed with hepatocellular carcinoma

Hepatocellular cancer (HCC) is a serious human disease with an unfortunately low survival rate. It further poses a significant epidemic threat to our society through its viral vectors associated with cirrhosis conditions preceding the cancer. A search for biomarkers of these diseases enlists analytical glycobiology, in general, and quantitative biomolecular mass spectrometry (MS), in particular, as valuable approaches to cancer research. The recent advances in quantitative glycan permethylation prior to MALDI-MS oligosaccharide profiling has enabled us to compare the glycan quantitative proportions in the small serum samples of cancer and cirrhotic patients against control individuals. In this investigation, reasoning that some of the observed glycomic changes could be at least partly explained by acute-phase or immune responses, we further fractionated the major serum proteins from the minor components and compared statistically their differential glycosylation, elucidating some causes of quantitatively unusual glycosylation events. Numerous glycan structures were identified and tentatively connected with their originating proteins, with a particular emphasis on sialylated and fucosylated glycans. In particular, for the highly-abundant protein fraction, several smaller, neutral glycans were observed to be different between disease-free individuals and those diagnosed with either HCC or cirrhosis. Further, these types of glycans were also different between the two diseases. In the lesser-abundant protein fraction, a number of sialylated glycans were different between each state-of-health.

Improved Mass Spectrometric Characterization of Protein Glycosylation Reveals Unusual Glycosylation of Maize-Derived Bovine Trypsin

Although bottom-up proteomics using tryptic digests is widely used to locate post-translational modifications (PTM) in proteins, there are cases where the protein has several potential modification sites within a tryptic fragment and MS(2) strategies fail to pinpoint the location. We report here a method using two proteolytic enzymes, trypsin and pepsin, in combination followed by tandem mass spectrometric analysis to provide fragments that allow one to locate the modification sites. We used this strategy to find a glycosylation site on bovine trypsin expressed in maize (TrypZean). Several glycans are present, and all are attached to a nonconsensus N-glycosylation site on the protein.

Glycosylation Profile of Differently Charged IgA1 and Their Binding Characteristics to Cultured Mesangial Cells in IgA Nephropathy

Background: IgA nephropathy (IgAN) is characterized by mesangial deposition of polymeric IgA1 (pIgA1), yet the pathogeneic mechanism remains unresolved. In the present study, we examined the glycosylation profile of differently charged IgA1 from IgAN patients. The binding characteristics of these IgA1 fractions to cultured human mesangial cells (HMC) and hepatoma cell lines (HepG2) were studied. Methods: Differently charged IgA1 were isolated by ion exchange chromatography. The glycosylation profile in the carbohydrate moieties of these differently charged IgA1 was analyzed by galactose (Gal)-, galactose-acetylgalactosamine (Gal-GalNAc)-, or sialic acid-specific enzyme-linked lectin binding assays (ELLA). The binding characteristic of these IgA1 to HMC was examined by flow cytometry and competitive binding assay. Results: Anionic pIgA from IgAN patients showed less reactivity in (Gal)- and (Gal-GalNAc)-specific ELLA (p < 0.01). There was higher reactivity for anionic pIgA1 in α(2,6)-linked sialic acid-specific ELLA (p < 0.01). Anionic pIgA1 from IgAN patients exhibited increased binding to cultured HMC and the binding was significantly reduced after neuraminidase treatment (p < 0.05). In contrast, anionic pIgA1 from IgAN patients bound less to cultured HepG2 cells and the binding was enhanced following neuraminidase treatment (p < 0.05). Conclusions: We demonstrated an unusual glycosylation and sialylation pattern of anionic pIgA1 in IgAN which may have an important effect on its pathogenesis.

The Sandfish's Skin: Morphology, Chemistry and Reconstruction

The sandfish is a lizard having the remarkable ability to move in desert sand in a swimming-like fashion. The most outstanding adaptations to this mode of life are the low friction behaviour and the extensive abrasion resistance of the sandfish skin against sand, outperforming even steel. We investigated the topography, the composition and the mechanical properties of sandfish scales. These consist of glycosylated keratins with high amount of sulphur but no hard inorganic material, such as silicates or lime. Remarkably, atomic force microscopy shows an almost complete absence of attractive forces between the scale surface and a silicon tip, suggesting that this is responsible for the unusual tribological properties. The unusual glycosylation of the keratins was found to be absolutely necessary for the described phenomenon. The scales were dissolved and reconstituted on a polymer surface resulting in properties similar to the original scale. Thus, we provide a pathway towards exploitation of the reconstituted scale material for future engineering applications.

Nuclear mRNA retention and aberrant doppel protein expression in human astrocytic tumor cells

Doppel (Dpl) is a paralogue of the mammalian Prion (PrP) protein. It is abundant in testis and, unlike PrP, it is expressed at low levels in the adult central nervous system (CNS). Besides, Dpl overexpression correlates with some prion-disease pathological features, such as ataxia and death of cerebellar neurons. Recently, ectopic expression of doppel was found in two different tumor types, specifically in glial and haematological cancers. In this study the doppel gene (PRND) mRNA and protein expression in PRT-HU2 and IPDDC-A2 astrocytoma-derived cell lines was investigated. Northern blot analysis revealed two equally abundant PRND mRNA isoforms, while real-time PCR, on nuclear and cytoplasmic RNA fractions, and cRNA in situ hybridization, on astrocytoma cells and bioptical specimens, showed a nuclear retention of PRND transcripts. Western blot analysis showed that the amount of protein expressed is low compared to the level of mRNA. Moreover deglycosylation studies indicated that Dpl undergoes unusual glycosylation processes. Immunohistochemistry experiments demonstrated that Dpl was mainly localised in the cytoplasm of the astrocytic tumor cells, and that it failed to be GPI-anchored to the cell membrane. This unusual cellular localization was also confirmed through EGFP-Dpl expression in astrocytomas; on the contrary, HeLa cells exhibited the expected Dpl membrane localization. Our findings suggest an aberrant doppel gene expression pattern, characterized by a substantial nuclear retention of the transcript, an altered post-translational modification of the protein and an unusual cytoplasmic localization.

Molecular cloning and characterization of a novel human 1,3-glucosyltransferase, which is localized at the endoplasmic reticulum and glucosylates O-linked fucosylglycan on thrombospondin type 1 repeat domain

Protein O-linked fucosylation is an unusual glycosylation associated with many important biological functions such as Notch signaling. Two fucosylation pathways synthesizing O-fucosylglycans have been reported on cystein-knotted proteins, that is, on epidermal growth factor-like (EGF-like) domains and on thrombospondin Type 1 repeat (TSR) domains. We report here the molecular cloning and characterization of a novel beta1,3-glucosyltransferase (beta3Glc-T) that synthesizes a Glcbeta1,3Fucalpha- structure on the TSR domain. We found a novel glycosyltransferase gene with beta1,3-glycosyltransferase (beta3GT) motifs in databases. The recombinant enzyme expressed in human embryonic kidney 293T (HEK293T) cells exhibited glucosyltransferase activity toward fucose-alpha-para-nitrophenyl (Fucalpha-pNp). Thin-layer chromatography (TLC) analysis revealed that the product of the recombinant enzyme migrated to the same position as did the product of endogenous beta3Glc-T of Chinese hamster ovary (CHO) cells. The two products could be digested by beta-glucosidase from almond and by exo-1,3-beta-glucanase from Trichoderma sp. These results strongly suggested that the product has the structure of Glcbeta1-3Fuc. Therefore, we named this novel enzyme beta3Glc-T. Immunostaining revealed that FLAG-tagged beta3Glc-T is an enzyme residing in the endoplasmic reticulum (ER) via retention signal, "REEL," which is a KDEL-like sequence, at the C-terminus. The TSR domain expressed in Escherichia coli was first fucosylated by the recombinant protein O-fucosyltransferase 2 (POFUT2), after which it became an acceptor substrate for the recombinant beta3Glc-T, which could apparently transfer Glc to the fucosylated TSR domain. Our results suggest that a novel glycosyltransferase, beta3Glc-T, contributes to the elongation of O-fucosylglycan and that this occurs specifically on TSR domains.