O-linked Chains Research Articles

Boosting Type-I and Type-II ROS Production of Water-Soluble Porphyrin for Efficient Hypoxic Tumor Therapy.

As the most successful clinically approved photosensitizers, porphyrins have been extensively employed in the photodynamic therapy (PDT) of cancers. However, their poor water solubility, aggregation-induced self-quenching on ROS generation, and a low tolerance for a hypoxic condition usually result in unsatisfied therapeutic outcomes. Therefore, great efforts have been dedicated to improving the PDT efficacy of porphyrin-type photosensitizers in treating hypoxic tumors, including combination with additional active components or therapies, which can significantly complicate the therapeutic process. Herein, we report a novel water-soluble porphyrin with O-linked cationic side chains, which exhibits good water solubility, high photostability, and significantly enhanced ROS generation efficacy in both type-I and type-II photodynamic pathways. We have also found that the end charges of side chains can dramatically affect the ROS generation of the porphyrin. The cationic porphyrin exhibited high in vitro PDT efficacy with low IC50 values both in normoxia and hypoxia. Hence, during in vivo PDT study, the cationic porphyrin displayed highly effective tumor ablation capability. This study demonstrates the power of side-chain chemistry in tuning the photodynamic property of porphyrin, which offers a new effective strategy to enhance the anticancer performance of photosensitizers for fulfilling the increasing demands for cancer therapy in clinics.

The story of the Sda antigen and of its cognate enzyme B4GALNT2: What is new?

The structure Siaα2,3(GalNAcβ1,4)Gal- is the epitope of the Sda antigen, which is expressed on the erythrocytes and secretions of the vast majority of Caucasians, carried by N- and O-linked chains of glycoproteins, as well as by glycolipids. Sda is very similar, but not identical, to ganglioside GM2 [Siaα2,3(GalNAcβ1,4)Galβ1,4Glc-Cer]. The Sda synthase β1,4N-acetylgalactosaminyl transferase 2 (B4GALNT2) exists in a short and a long form, diverging in the aminoterminal domain. The latter has a very long cytoplasmic tail and displays a Golgi- as well as a post-Golgi localization. The biosynthesis of Sda is mutually exclusive with that of the cancer-associated sialyl Lewis antigens, whose structure is Siaα2,3Galβ1,3/4(Fucα1,4/3)GlcNAc-. B4GALNT2 is down-regulated in colon cancer but patients with higher expression survive longer. In experimental systems, B4GALNT2 inhibits colon cancer progression,not only through inhibition of sialyl Lewis antigen biosynthesis. By contrast, in breast cancer B4GALNT2 is associated with malignancy. In colon cancer, the B4GALNT2 gene is regulated by multiple mechanisms, which include miRNA and transcription factor expression, as well as CpG methylation. In addition, Sda/B4GALNT2 regulates the susceptibility to infectious agents, the protection from muscle dystrophy, the activity of immune system in pregnancy and the immune rejection in xenotransplantation.

An Antigen Capture Assay for the Detection of Mycolactone, the Polyketide Toxin of Mycobacterium ulcerans.

Mycolactone is a cytotoxin responsible for most of the chronic necrotizing pathology of Mycobacterium ulcerans disease (Buruli ulcer). The polyketide toxin consists of a 12-membered lactone ring with a lower O-linked polyunsaturated acyl side chain and an upper C-linked side chain. Mycolactone is unique to M. ulcerans and an immunological Ag capture assay would represent an important tool for the study of Buruli ulcer pathogenesis and for laboratory diagnosis. When testing sets of mycolactone-specific mouse mAbs, we found that Abs against the hydrophobic lower side chain only bind mycolactone immobilized on a solid support but not when present in solution. This observation supports previous findings that mycolactone forms micellar structures in aqueous solution with the hydrophobic region sequestered into the inner core of the aggregates. Although an Ag capture assay typically requires two Abs that recognize nonoverlapping epitopes, our search for matching pairs of mAbs showed that the same mAb could be used both as capture and as detecting reagent for the detection of the mycolactone aggregates. However, the combination of a core-specific and a core/upper side chain-specific mAb constituted the most sensitive ELISA with a sensitivity in the low nanogram range. The results of a pilot experiment showed that the sensitivity of the assay is sufficient to detect mycolactone in swab samples from Buruli ulcer lesions. Although the described capture ELISA can serve as a tool for research on the biology of mycolactone, the assay system will have to be adapted for use as a diagnostic tool.

Aspergillus fumigatus Mnn9 is responsible for mannan synthesis and required for covalent linkage of mannoprotein to the cell wall

Owing to the essential role in protection of the Aspergillus fumigatus cell against human defense reactions, its cell wall has long been taken as a promising antifungal target. The cell wall of A. fumigatus composed of chitin, glucan and galactomannan and mannoproteins. Although galactomannan has been used as a diagnostic target for a long time, its biosynthesis remains unknown in A. fumigatus. In this study, a putative α1,6-mannosyltransferase gene mnn9 was identified in A. fumigatus. Deletion of the mnn9 gene resulted in an increased sensitivity to calcofluor white, Congo red, or hygromycin B as well as in reduced cell wall components and abnormal polarity. Although there was no major effect on N-glycan synthesis, covalently-linked cell wall mannoprotein Mp1 was significantly reduced in the mutant. Based on our results, we propose that Mnn9p is a mannosyltransferase responsible for the formation of the α-mannan in cell wall mannoproteins, potentially via elongation of O-linked mannose chains.

Plant Defensins NaD1 and NaD2 Induce Different Stress Response Pathways in Fungi.

Nicotiana alata defensins 1 and 2 (NaD1 and NaD2) are plant defensins from the ornamental tobacco that have antifungal activity against a variety of fungal pathogens. Some plant defensins interact with fungal cell wall O-glycosylated proteins. Therefore, we investigated if this was the case for NaD1 and NaD2, by assessing the sensitivity of the three Aspergillus nidulans (An) O-mannosyltransferase (pmt) knockout (KO) mutants (An∆pmtA, An∆pmtB, and An∆pmtC). An∆pmtA was resistant to both defensins, while An∆pmtC was resistant to NaD2 only, suggesting NaD1 and NaD2 are unlikely to have a general interaction with O-linked side chains. Further evidence of this difference in the antifungal mechanism was provided by the dissimilarity of the NaD1 and NaD2 sensitivities of the Fusarium oxysporum f. sp. lycopersici (Fol) signalling knockout mutants from the cell wall integrity (CWI) and high osmolarity glycerol (HOG) mitogen-activated protein kinase (MAPK) pathways. HOG pathway mutants were sensitive to both NaD1 and NaD2, while CWI pathway mutants only displayed sensitivity to NaD2.

Expanding the knowledge of the chemical structure of glycoconjugates from Trypanosoma cruzi TcI genotype. Contribution to taxonomic studies.

One of the main obstacles to the treatment of Chagas disease is the genetic and phenotypical variance displayed by T. cruzi strains, resulting in differences in morphology, virulence, pathogenicity and drug susceptibility. To better understand the role of glycoconjungates in Chagas disease, we performed the molecular characterization of the O-linked chains from mucins and glycoinositolphospholipids (GIPLs) of the Silvio X10 clone 1 strain. We demonstrated the presence of a β-galactofuranose (β-Galf) unity linked to the O-4 position of the α-N-acetylglucosamine (α-GlcNAc)O-4 in Tc-mucins. GIPLs analysis showed that the lipidic portion is exclusively composed of ceramide and the PI-oligossacharidic portion contains the Man4(AEP)GlcN-Ins-PO4 core, substituted by ethanolamine-phosphate (EtNP) on the third distal mannose from inositol, which may or may not have a terminal β Galf unity. These results confirm the classification of the Silvio X10/1 strain in group T. cruzi I. Again, it is noted that the study of T. cruzi surface glycoconjugates confirm the molecular results and the hypothesis that surface glycoconjugates may be interesting biomarker for the differentiation of trypanosomatid strains.

Function of Membrane-Associated Proteoglycans in the Regulation of Satellite Cell Growth.

Muscle growth can be divided into embryonic and postnatal periods. During the embryonic period, mesenchymal stem cells proliferate and differentiate to form muscle fibers. Postnatal muscle growth (hypertrophy) is characterized by the enlargement of existing muscle fiber size. Satellite cells (also known as adult myoblasts) are responsible for hypertrophy. The activity of satellite cells can be regulated by their extracellular matrix (ECM). The ECM is composed of collagens, proteoglycans, non-collagenous glycoproteins, cytokines and growth factors. Proteoglycans contain a central core protein with covalently attached glycosaminoglycans (GAGs: chondroitin sulfate, keratan sulfate, dermatan sulfate, and heparan sulfate) and N- or O-linked glycosylation chains. Membrane-associated proteoglycans attach to the cell membrane either through a glycosylphosphatidylinositol anchor or transmembrane domain. The GAGs can bind proteins including cytokines and growth factors. Both cytokines and growth factors play important roles in regulating satellite cell growth and development. Cytokines are generally associated with immune cells. However, cytokines can also affect muscle cell development. For instance, interleukin-6, tumor necrosis factor-α, and leukemia inhibitory factor have been reported to affect the proliferation and differentiation of satellite cells and myoblasts. Growth factors are potent stimulators or inhibitors of satellite cell proliferation and differentiation. The proper function of some cytokines and growth factors requires an interaction with the cell membrane-associated proteoglycans to enhance the affinity to bind to their primary receptors to initiate downstream signal transduction. This chapter is focused on the interaction of membrane-associated proteoglycans with cytokines and growth factors, and their role in satellite cell growth and development.

O-glycosylation in cell wall proteins in Scedosporium prolificans is critical for phagocytosis and inflammatory cytokines production by macrophages.

In this study, we analyze the importance of O-linked oligosaccharides present in peptidorhamnomannan (PRM) from the cell wall of the fungus Scedosporium prolificans for recognition and phagocytosis of conidia by macrophages. Adding PRM led to a dose-dependent inhibition of conidia phagocytosis, whereas de-O-glycosylated PRM did not show any effect. PRM induced the release of macrophage-derived antimicrobial compounds. However, O-linked oligosaccharides do not appear to be required for such induction. The effect of PRM on conidia-induced macrophage killing was examined using latex beads coated with PRM or de-O-glycosylated PRM. A decrease in macrophage viability similar to that caused by conidia was detected. However, macrophage killing was unaffected when beads coated with de-O-glycosylated PRM were used, indicating the toxic effect of O-linked oligosaccharides on macrophages. In addition, PRM triggered TNF-α release by macrophages. Chemical removal of O-linked oligosaccharides from PRM abolished cytokine induction, suggesting that the O-linked oligosaccharidic chains are important moieties involved in inflammatory responses through the induction of TNF-α secretion. In summary, we show that O-glycosylation plays a role in the recognition and uptake of S. prolificans by macrophages, killing of macrophages and production of pro- inflammatory cytokines.

A practical approach for O-linked mannose removal: the use of recombinant lysosomal mannosidase.

The methylotrophic yeast Pichia pastoris is an attractive expression system due to its ability to secrete large amounts of recombinant protein, with the potential for glycosylation. Advances in glycoengineering of P. pastoris have successfully demonstrated the humanization of both the N- and O-linked glycosylation pathways in this organism. However, in certain cases, the presence of O-linked glycans on a therapeutic protein may not be desirable. Recently, we have reported the in vitro utility of jack bean α-1,2/3/6-mannosidase to remove O-linked mannose from intact undenatured glycoproteins produced in glycoengineered P. pastoris. However, one caveat of this strategy is that jack bean mannosidase has yet to be cloned and as such is only available as crude cellular extracts. This raises several concerns for using this reagent to treat large preparations of therapeutic proteins generated in P. pastoris. Therefore, we postulated that lysosomal mannosidases which have been cloned and demonstrated to have similar activities to jack bean mannosidase on N-linked glycans would also process O-linked glycans in a similar fashion. To this end, we screened a panel of recombinant lysosomal mannosidases from different organisms and identified several which cannot only reduce extended O-linked mannose chains but which can also hydrolyze the Man-α-O-Ser/Thr glycosidic bond on intact glycoproteins. As such, not only do we show for the first time the utility of lysosomal mannosidase for O-linked mannose processing, but since this is a recombinant enzyme, it has several benefits over the use of crude jack bean mannosidase extracts.

In vitro enzymatic treatment to remove O-linked mannose from intact glycoproteins

The methylotrophic yeast Pichia pastoris is an attractive expression system for heterologous protein production due to its ability to perform posttranslational modifications, such as glycosylation, and secrete large amounts of recombinant protein. However, the structures of N- and O-linked oligosaccharide chains in yeast differ significantly from those of mammalian cells. The most common O-linked glycan structures added by P. pastoris are typically polymers of between one and four α-linked mannose residues, with a subset of glycans being capped by a β-1,2-mannose disaccharide or phosphomannose residue. Such mannosylation of recombinant proteins is considered a key factor in immunomodulation, with mannose-specific receptors binding and promoting enhanced immune responses. As a result of engineering the N-linked glycosylation pathway of P. pastoris, the recombinant proteins expressed in this system are devoid of phospho- and β-mannose on O-linked glycans, leaving only α-mannose polymers. Here we screen a library of α-mannosidases for their ability to decrease the extent of O-mannosylation on glycoproteins secreted from this expression system. In doing so, we demonstrate the utility of the α-1,2/3/6-mannosidase from Jack bean in not only reducing extended O-linked mannose chains but also in specifically hydrolyzing the Man-α-O-Ser/Thr glycosidic bond on intact glycoproteins. As such, this presents for the first time a strategy to remove O-linked glycosylation from intact glycoproteins expressed in P. pastoris. We additionally show that this strategy can be used to significantly decrease the extent of O-mannosylation on commercial products produced in other similar expression systems.

Antimicrobial Glycopeptides: Synthesis and Antibacterial Activity of N-linked and O-linked Smaller Chain Glycopeptides

Our objective is to synthesize the smaller chain N-linked and O-linked glycopeptides using sugars belong to mono, di and polysaccharides, with the efforts mainly directed towards the identification of antibacterial compounds. 7 glycopeptides, viz., Arg-Asn-Mannose, Arg-Asn-Lactose, His-Asn-Mannose, His-Asn-Lactose (N-glycopeptides), Arg-Ser-Lactose, Arg-Thr-Lactose, Arg-Thr-Starch (O-glycopeptides) were prepared by dicyclohexyl carbodimide (DCC) coupling for amino acids using microwave oven (50W power; 15 min) and activated and coupled with respective sugar moieties using microwave oven at 120 W for 20-25 min. The column eluted compounds were tested for disc diffusion assay using 3 gram positive S. aureus, B. subtilis, S. caprae and 3 gram negative E. coli, P. aeruginosa and S. sonnei strains at different concentrations predicted by pH and inhibitory concentrations. One of the test glycopeptide, His-Asn-Lactose was found to be very effective against all the microbial strains tested and 3 other Test Compounds, viz., His-Asn-Mannose, Arg-Thr-Lactose and Arg-Thr-Starch are also proved to be effective against 2 gram positive and 2 gram negative strains tested. Maximum activity was observed at a concentration of 450 μg/ml (747.51 µM) for the N-glycopeptide His-Asn-Lactose with the corresponding zonal inhibition diameters (15 mm; 19 mm; 14 mm; 18 mm; 16 mm; 17 mm) against S. aureus, B. subtilis, S. caprae, E. coli, P. aeruginosa, S. sonnei. This is the first evidence based report that our N-glycopeptide, His-Asn-Lactose tested, has shown antibacterial action against both gram positive and gram negative strains. Further in vivo testing and clinical studies will make sure to position our potent small chain N-glycopeptide in the arsenal of new broad spectrum anti-gram positive and negative agent.

Challenges and Opportunities in Tuberculosis Drug Discovery: An Industry Perspective

Future Medicinal ChemistryVol. 5, No. 5 EditorialChallenges and opportunities in tuberculosis drug discovery: an industry perspectivePravin S Shirude, Sreekanth Ramachandran & Vinayak HosagraharaPravin S Shirude* Author for correspondenceAstraZeneca India Pvt. Ltd., Bellary Road, Hebbal, Bangalore 560024, India. Search for more papers by this authorEmail the corresponding author at pravin.shirude@astrazeneca.com, Sreekanth RamachandranAstraZeneca India Pvt. Ltd., Bellary Road, Hebbal, Bangalore 560024, IndiaSearch for more papers by this author & Vinayak HosagraharaAstraZeneca India Pvt. Ltd., Bellary Road, Hebbal, Bangalore 560024, IndiaSearch for more papers by this authorPublished Online:10 Apr 2013https://doi.org/10.4155/fmc.13.26AboutSectionsView ArticleView Full TextPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInRedditEmail View articleKeywords: alternative approachcell-based approachtarget-based approachTB drug discoveryReferences1 World Health Organization. Global tuberculosis control report. WHO Press, Geneva, Switzerland (2011).Google Scholar2 Dye C, Williams BG. The population dynamics and control of tuberculosis. Science328,856–861 (2010).Crossref, Medline, CAS, Google Scholar3 Zulma A, Hafner R, Leinhardt C, Hoelscher M, Nunn A. Advancing the development of tuberculosis therapy. Nat. Rev. Drug Discov.11,171–172 (2012).Crossref, Medline, Google Scholar4 Ginsberg A. The TB alliance: overcoming challenges to chart the future course of TB drug development. Future Med. 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Situro FDA advanced grant accelerated approval. www.investor.jnj.com/releaseDetail.cfm?ReleaseID=730893&year=2013Google Scholar104 Phase 2a EBA Trial of AZD5847. www.clinicaltrials.gov/ct2/show/NCT01516203Google ScholarFiguresReferencesRelatedDetailsCited ByInhA, the enoyl-thioester reductase from Mycobacterium tuberculosis forms a covalent adduct during catalysisJournal of Biological Chemistry, Vol. 293, No. 44Perspective: Challenges and opportunities in TB drug discovery from phenotypic screeningBioorganic & Medicinal Chemistry, Vol. 23, No. 16 Vol. 5, No. 5 Follow us on social media for the latest updates Metrics Downloaded 70 times History Published online 10 April 2013 Published in print April 2013 Information© Future Science LtdKeywordsalternative approachcell-based approachtarget-based approachTB drug discoveryFinancial & competing interests disclosureThe authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.No writing assistance was utilized in the production of this manuscript.PDF download

Discovery and Structural Characterization of Fucosylated Oligomannosidic N-Glycans in Mushrooms

L-fucose is a common constituent of Asn-linked glycans in vertebrates, invertebrates, and plants, but in fungal glycoproteins, fucose has not been found so far. However, by mass spectrometry we detected N-glycans and O-glycans containing one to six deoxyhexose residues in fruit bodies of several basidiomycetes. The N-glycans of chanterelles (Cantharellus cibarius) contained a deoxyhexose chromatographically identical to fucose and sensitive to α-L-fucosidase. Analysis of individual glycan species by tandem MS, glycosidase digestion, and finally (1)H NMR revealed the presence of L-fucose in α1,6-linkage to an α1,6-mannose of oligomannosidic N-glycans. The substitution by α1,6-mannose of α1,2-mannosyl residues of the canonical precursor structure was yet another hitherto unknown modification. No indication for the occurrence of yet other modifications, e.g. bisecting N-acetylglucosamine, was seen. Besides fucosylated N-glycans, short O-linked mannan chains substituted with fucose were present on chanterelle proteins. Although undiscovered so far, L-fucose appears to represent a prominent feature of protein-linked glycans in the fungal kingdom.

The Pathogenic Fungus Paracoccidioides brasiliensis Exports Extracellular Vesicles Containing Highly Immunogenic α-Galactosyl Epitopes

Exosome-like vesicles containing virulence factors, enzymes, and antigens have recently been characterized in fungal pathogens, such as Cryptococcus neoformans and Histoplasma capsulatum. Here, we describe extracellular vesicles carrying highly immunogenic α-linked galactopyranosyl (α-Gal) epitopes in Paracoccidioides brasiliensis. P. brasiliensis is a dimorphic fungus that causes human paracoccidioidomycosis (PCM). For vesicle preparations, cell-free supernatant fluids from yeast cells cultivated in Ham's defined medium-glucose were concentrated in an Amicon ultrafiltration system and ultracentrifuged at 100,000 × g. P. brasiliensis antigens were present in preparations from phylogenetically distinct isolates Pb18 and Pb3, as observed in immunoblots revealed with sera from PCM patients. In an enzyme-linked immunosorbent assay (ELISA), vesicle components containing α-Gal epitopes reacted strongly with anti-α-Gal antibodies isolated from both Chagas' disease and PCM patients, with Marasmius oreades agglutinin (MOA) (a lectin that recognizes terminal α-Gal), but only faintly with natural anti-α-Gal. Reactivity was inhibited after treatment with α-galactosidase. Vesicle preparations analyzed by electron microscopy showed vesicular structures of 20 to 200 nm that were labeled both on the surface and in the lumen with MOA. In P. brasiliensis cells, components carrying α-Gal epitopes were found distributed on the cell wall, following a punctuated confocal pattern, and inside large intracellular vacuoles. Lipid-free vesicle fractions reacted with anti-α-Gal in ELISA only when not digested with α-galactosidase, while reactivity with glycoproteins was reduced after β-elimination, which is indicative of partial O-linked chain localization. Our findings open new areas to explore in terms of host-parasite relationships in PCM and the role played in vivo by vesicle components and α-galactosyl epitopes.

Cyclohexanecarboxamide

The title compound, C(7)H(13)NO, forms R(2)(2)(8) N-H...O hydrogen-bonded dimers and C4 N-H...O-linked chains, which are further stabilized by a C-H...O interaction. The combination of these interactions results in a hydrogen-bonded network parallel to (100), with a motif that can be described by the secondary graph set R(4)(6)(16). The existence of the same hydrogen-bonding motif in 1-phenylcyclopentanecarboxamide and 1-(2-bromophenyl)cyclohexanecarboxamide [Lemmerer & Michael (2008). CrystEngComm, 10, 95-102 indicates that replacing the H atom on position 1 with a more bulky group does not necessarily disrupt the observed hydrogen-bonding pattern. The presence of a C-H...O interaction to stabilize the R(4)(6)(16) network does, however, seem to be required. In addition, the title compound is isomorphous with a previously published structure of cyclopentanecarboxamide [Winter et al. (1981). Acta Cryst. B37, 2183-2185].

Structural and biological characterization of a novel acutobin‐like enzyme isolated from the venom of the sharp‐nosed pit viper (Deinagkistrodon acutus)

We previously reported the purification of a serine proteinase from the venom of the sharp-nosed pit viper (Deinagkistrodon acutus) using a combination of affinity chromatography and ion-exchange chromatography [Xin, Dong, Wang and Li, R. (2007) J. Chromatogr. B 859, 111-118]. The high fibrinogen-clotting activity [2025 NIH (National Institutes of Health) units/mg] of this protein indicated that it may have great potential as a drug for treating thrombolysis. In order to systemically determine the purified protein's structure and activity, it was characterized using the following methods: MS, isoelectric focusing, deglycosylation analysis, amino acid composition analysis, peptide mass fingerprinting, N-terminal amino acid sequencing, CD, hydrophobic-site analysis and bioactivity assays. In addition, a fluorescence probe was synthesized and conjugated to the protein in order to analyse its active site. The results indicated that the protein is a novel acutobin-like enzyme (designated acutobin II) with strong clotting and esterase activities and is composed of a 28 kDa peptide chain plus approx. 6 kDa of O-linked glycan chains. The protein contains 249 amino acids and, remarkably, no tryptophan residues. The pI of the protein is 4.8+/-0.2. The protein's secondary structure is dominated by beta-sheets (49%) and random coils (43%), and its tertiary structure does not contain any metal ions or disulfide bonds and possesses only one hydrophobic pocket. Analysis revealed that the hydrophobic pocket is most likely the enzymatic active site.

Global analysis of the glycoproteome in Saccharomyces cerevisiae reveals new roles for protein glycosylation in eukaryotes

To further understand the roles of protein glycosylation in eukaryotes, we globally identified glycan-containing proteins in yeast. A fluorescent lectin binding assay was developed and used to screen protein microarrays containing over 5000 proteins purified from yeast. A total of 534 yeast proteins were identified that bound either Concanavalin A (ConA) or Wheat-Germ Agglutinin (WGA); 406 of them were novel. Among the novel glycoproteins, 45 were validated by mobility shift upon treatment with EndoH and PNGase F, thereby extending the number of validated yeast glycoproteins to 350. In addition to many components of the secretory pathway, we identified other types of proteins, such as transcription factors and mitochondrial proteins. To further explore the role of glycosylation in mitochondrial function, the localization of four mitochondrial proteins was examined in the presence and absence of tunicamycin, an inhibitor of N-linked protein glycosylation. For two proteins, localization to the mitochondria is diminished upon tunicamycin treatment, indicating that protein glycosylation is important for protein function. Overall, our studies greatly extend our understanding of protein glycosylation in eukaryotes through the cataloguing of glycoproteins, and describe a novel role for protein glycosylation in mitochondrial protein function and localization.

Enzymic Remodelling of the N- and O-Linked Carbohydrate Chains of Human Chorionic Gonadotropin

The effects of altered terminal sequences in human chorionic gonadotropin (hCG) N- and O-linked glycans on receptor binding and signal transduction were analyzed using forms of hCG with remodelled carbohydrate chains. hCG derivatives were obtained by enzymic removal of the α3-linked sialic acid residues followed by α6-sialylation, α3-galactosylation or α3-fucosylation of uncovered Galβ14GlcNAc (LacNAc) termini, or α3-sialylation of Galβ13GalNAc sequences. Also a form that carried GalNAcβ14-GlcNAc units, which are typical for pituitary hormone oligosaccharides, was derived by enzymic desialylation and degalactosylation followed by β4-N-acetylgalactosaminylation. The potency to stimulate testosterone production and the binding to the lutotropin/choriogonadotropin receptor of the preparations were compared with those of native and desialylated hCG (as-hCG). The decrease in bioactivity caused by desialylation of hCG was only restored upon α6-sialylation of the Galβ14GlcNAcβ12Manα13Man branch of the N-linked glycans. This was without a major effect on receptor binding. Further α6-sialylation, occurring at the Galβ14GlcNAcβ12Manα16Man branch, resulted in a bioactivity below a level found with as-hCG, concomitant with a decreased receptor binding affinity. Similarly α3-galactosylation of the Galβ14GlcNAcβ12-Manα16Man branch yielded a hCG derivative that showed decreased bioactivity and receptor binding. α3-Fucosylation of native as well as as-hCG also led to a decreased activity. Re-α3-sialylation of the O-linked chains on as-hCG had little effect on the bioactivity and receptor binding. Hormone preparations with GalNAcβ4GlcNAc termini showed lower bioactivity and receptor affinity than as-hCG. It is concluded that the Galβ14GlcNAcβ12Manα13Man- rather than the Galβ1 4GlcNAcβ12-Manα16Man branch of the N-linked glycans on hCG plays an essential role in signal transduction, whereas the latter branch can potentially interfere with receptor binding. Furthermore attachment of sialic acid, but not of other sugars, to the first branch fulfils the requirement for the full expression of bioactivity, while sialylation of the O-linked chains is of minor importance.

Extension of microwave-accelerated residue-specific acid cleavage to proteins with carbohydrate side chains and disulfide linkages

This laboratory has introduced a chemical method for residue-specific protein cleavage and has provided a preliminary assessment of the suitability of microwave-accelerated acid cleavage as a proteomic tool. This report is a continuing assessment of the fate of common protein modifications in microwave-accelerated acid cleavage. We have examined the cleavage of ribonuclease A and the related N-linked glycoprotein ribonuclease B, and the O-linked glycoprotein alpha crystallin A chain, using MALDI-TOF and LC–ESI-MS to identify the peptide products. RNase A and B each contains four disulfide bonds, and the addition of a reducing reagent, such as dithiothreitol, was found to be required to achieve efficient acidic proteolysis. The linkage of the glycosidic group to the asparagine side chain in ribonuclease B was found not to be cleaved by brief microwave treatment in 12.5% acetic acid. The distribution of the heterogeneous carbohydrate side chain in the glycopeptide products of acid cleavage was compared to that of the glycopeptide products of tryptic digestion. Hydrolysis within the carbohydrate chain itself is minimal under the conditions used. The O-linked side chain on alpha crystalline A was found to be cleaved during acid cleavage of the protein.

Preparation and Intramolecular [2+2]-Photocycloaddition of 1,5-Dihydropyrrol-2-ones and 5,6-Dihydro-1H-pyridin-2-ones with C-, N-, and O-linked Alkenyl Side Chains at the 4-Position

The 1,5-dihydropyrrol-2-ones 2, 6, 9, and 11 were prepared from methyl tetramates (1a-c), N-Boc-protected tetramic acid (3), or N-Boc-protected tetramic acid bromide (7) in short reaction sequences and in very good overall yields. The homologous 5,6-dihydro-1H-pyridin-2-ones 16, 18, 20, 21, 23, and 27 were prepared along analogous routes starting from piperidin-2,4-dione (19) or from its N-tert-butyl derivative 15. Optimized conditions for the [2+2]-photocycloaddition include the use of dichloromethane as the solvent and an irradiation with a mercury low-pressure lamp (lambda = 254 nm). Upon applying these conditions at ambient temperature, the corresponding intramolecular photocycloaddition products 28-37 were obtained in good yields (52-79%) and with perfect diastereoselectivity. The constitution and configuration of the products was elucidated by NMR-spectroscopy. For the O-tethered substrates 2a and 20, a strong decrease of the photocycloaddition rate with temperature was observed. The effect was less pronounced for N- and C-tethered substrates 6, 9, 23, and 27. The use of a chiral complexing agent to achieve enantioselective reactions appears viable. Complexing agent (-)-38, however, is not suited because of its instability at lambda = 254 nm.