Natural Glycoforms of Human Interleukin 6 Show Atypical Plasma Clearance.

A library of glycoforms of human interleukin 6 (IL-6) comprising complex and mannosidic N-glycans was generated by semisynthesis. The three segments were connected by sequential native chemical ligation followed by two-step refolding. The central glycopeptide segments were assembled by pseudoproline-assisted Lansbury aspartylation and subsequent enzymatic elongation of complex N-glycans. Nine IL-6 glycoforms were synthesized, seven of which were evaluated for in vivo plasma clearance in rats and compared to non-glycosylated recombinant IL-6 from E. coli. Each IL-6 glycoform was tested in three animals and reproducibly showed individual serum clearances depending on the structure of the N-glycan. The clearance rates were atypical, since the 2,6-sialylated glycoforms of IL-6 cleared faster than the corresponding asialo IL-6 with terminal galactoses. Compared to non-glycosylated IL-6 the plasma clearance of IL-6 glycoforms was delayed in the presence of larger and multibranched N-glycans in most cases.

S-Glycosides: synthesis of S-linked arabinoxylan oligosaccharides.

S-Glycosides are important tools for the elucidation of specific protein-carbohydrate interactions and can significantly aid structural and functional studies of carbohydrate-active enzymes, as they are often inert or act as enzyme inhibitors. In this context, this work focuses on the introduction of an S-linkage into arabinoxylan oligosaccharides (AXs) in order to obtain a small collection of synthetic tools for the study of AXs degrading enzymes. The key step for the introduction of the S-glycosidic linkage involved anomeric thiol S-alkylation of an orthogonally protected l-arabinopyranoside triflate. The resulting S-linked disaccharide was subsequently employed in a series of glycosylation reactions to obtain a selectively protected tetrasaccharide. This could be further elaborated through chemoselective deprotection and glycosylation reactions to introduce branching l-arabinofuranosides.

Synthesis of Erythropoietins Site-Specifically Conjugated with Complex-Type N-Glycans.

The biological activity of the glycoprotein hormone erythropoietin (EPO) is dependent mainly on the structure of its N-linked glycans. We aimed to readily attach defined N-glycans to EPO through copper-catalyzed azide alkyne cycloaddition. EPO variants with an alkyne-bearing non-natural amino acid (Plk) at the N-glycosylation sites 24, 38, and 83 were obtained by amber suppression followed by protein purification and refolding. Click conjugation of the alkynyl EPOs with biantennary N-glycan azides provided biologically active site-specifically modified EPO glycoconjugates.

Substrate specificity of novel GH16 endo-ß-(1→3)-galactanases acting on linear and branched ß-(1→3)-galactooligosaccharides.

Arabinogalactan proteins are proteoglycans located in the plant cell wall. Most arabinogalactan proteins are composed of carbohydrate moieties of ß-(1→3)-galactan main chains with ß-(1→6)-galactan side chains terminated by other glycans. In this study, three novel endo-ß-(1→3)-galactanases were identified and the substrate specificity was further studied using well-defined galactan oligomers. Linear and branched ß-(1→3)-linked galactans, which resemble the carbohydrate core of the arabinogalactan protein, were used for the characterization of endo-ß-(1→3)-galactanases. The identified enzymes required at least three consecutive galactose residues for activity. Non-substituted regions were preferred, but substituents in the -2 and +2 and in some cases also -1 and +1 subsites were tolerated to some extent, depending on the branching pattern, however at a significantly lower rate/frequency.

Galectin binding to cells and glycoproteins with genetically modified glycosylation reveals galectin-glycan specificities in a natural context.

Galectins compose a protein family defined by a conserved sequence motif conferring affinity for ß-galactose-containing glycans. Moreover, galectins gain higher affinity and fine-tune specificity by glycan interactions at sites adjacent to their ß-galactoside-binding site, as revealed by extensive testing against panels of purified glycans. However, in cells, galectins bind glycans on glycoproteins and glycolipids in the context of other cellular components, such as at the cell surface. Because of difficulties in characterizing natural cellular environments, we currently lack a detailed understanding of galectin-binding specificities in the cellular context. To address this challenge, we used a panel of genetically stable glycosylation mutated CHO cells that express defined glycans to evaluate the binding affinities of 10 different carbohydrate-recognition domains in galectins to N-glycans and mucin-type O-glycans. Using flow cytometry, we measured the cell-surface binding of the galectins. Moreover, we used fluorescence anisotropy to determine the galectin affinities to recombinant erythropoietin used as a reporter glycoprotein produced by the glycoengineered cells and to synthetic N-glycans with defined branch structures. We found that all galectins, apart from galectin-8N, require complex N-glycans for high-affinity binding. Galectin-8N targeted both N- and O-linked glycans with high affinity, preferring 2,3-sialylated N-acetyllactosamine (LacNAc) structures. Furthermore, we found that 2,3-sialylation suppresses high-affinity binding of select galectins, including galectin-2, -3, -4N, and -7. Structural modeling provided a basis for interpreting the observed binding preferences. These results underscore the power of a glycoengineered platform to dissect the glycan-binding specificities of carbohydrate-binding proteins.

A Tomato Tocopherol-Binding Protein Sheds Light on Intracellular α-Tocopherol Metabolism in Plants.

Tocopherols are non-polar compounds synthesized in the plastids, which function as major antioxidants of the plant cells and are essential in the human diet. Both the intermediates and final products of the tocopherol biosynthetic pathway must cross plastid membranes to reach their sites of action. So far, no protein with tocopherol binding activity has been reported in plants. Here, we demonstrated that the tomato SlTBP protein is targeted to chloroplasts and able to bind α-tocopherol. SlTBP-knockdown tomato plants exhibited reduced levels of tocopherol in both leaves and fruits. Several tocopherol deficiency phenotypes were apparent in the transgenic lines, such as alterations in photosynthetic parameters, dramatic distortion of thylakoid membranes and significant variations in the lipid profile. These results, along with the altered expression of genes related to photosynthesis, and tetrapyrrole, lipid, isoprenoid, inositol/phosphoinositide and redox metabolism, suggest that SlTBP may act in conducting tocopherol (or its biosynthetic intermediates) between the plastid compartments and/or at the interface between chloroplast and endoplasmic reticulum membranes, affecting interorganellar lipid metabolism.

Synthesis of branched and linear 1,4-linked galactan oligosaccharides.

We report the synthesis of linear and branched (1→4)-d-galactans. Four tetrasaccharides and one pentasaccharide were accessed by adopting a procedure of regioselective ring opening of a 4,6-O-naphthylidene protecting group followed by glycosylation using phenyl thioglycoside donors. The binding of the linear pentasaccharide with galectin-3 is also investigated by the determination of a co-crystal structure. The binding of the (1→4)-linked galactan to Gal-3 highlights the oligosaccharides of pectic galactan, which is abundant in the human diet, as putative Gal-3 ligands.

Synthesis and Application of Branched Type II Arabinogalactans.

The synthesis of linear and (1 → 6)-branched ß-(1 → 3)-d-galactans, structures found in plant arabinogalactan proteins (AGPs), is described. The synthetic strategy relies on iterative couplings of monosaccharide and disaccharide thioglycoside donors, followed by a late-stage glycosylation of heptagalactan backbone acceptors to introduce branching. A key finding from the synthetic study was the need to match protective groups in order to tune reactivity and ensure selectivity during the assembly. Carbohydrate microarrays were generated to enable the detailed epitope mapping of two monoclonal antibodies known to recognize AGPs: JIM16 and JIM133.

Breaking the Limits in Analyzing Carbohydrate Recognition by NMR Spectroscopy: Resolving Branch-Selective Interaction of a Tetra-Antennary N-Glycan with Lectins.

The biological recognition of complex-type N-glycans is part of many key physiological and pathological events. Despite their importance, the structural characterization of these events remains unsolved. The inherent flexibility of N-glycans hampers crystallization and the chemical equivalence of individual branches precludes their NMR characterization. By using a chemoenzymatically synthesized tetra-antennary N-glycan conjugated to a lanthanide binding tag, the NMR signals under paramagnetic conditions discriminated all four N-acetyl lactosamine antennae with unprecedented resolution. The NMR data revealed the conformation of the N-glycan and permitted for the first time the direct identification of individual branches involved in the recognition by two N-acetyllactosamine-binding lectins, Datura stramonium seed lectin (DSL) and Ricinus Communis agglutinin (RCA120).

A Synthetic Glycan Microarray Enables Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies.

In the last three decades, more than 200 monoclonal antibodies have been raised against most classes of plant cell wall polysaccharides by different laboratories worldwide. These antibodies are widely used to identify differences in plant cell wall components in mutants, organ and tissue types, and developmental stages. Despite their importance and broad use, the precise binding epitope has been determined for only a few of these antibodies. Here, we use a plant glycan microarray equipped with 88 synthetic oligosaccharides to comprehensively map the epitopes of plant cell wall glycan-directed antibodies. Our results reveal the binding epitopes for 78 arabinogalactan-, rhamnogalacturonan-, xylan-, and xyloglucan-directed antibodies. We demonstrate that, with knowledge of the exact epitopes recognized by individual antibodies, specific glycosyl hydrolases can be implemented into immunological cell wall analyses, providing a framework to obtain structural information on plant cell wall glycans with unprecedented molecular precision.

Synthetic Glycoforms Reveal Carbohydrate-Dependent Bioactivity of Human Saposin†D.

The main glycoforms of the hydrophobic lysosomal glycoprotein saposin D (SapD) were synthesized by native chemical ligation. An approach for the challenging solid-phase synthesis of the fragments was developed. Three SapD glycoforms were obtained following a general and robust refolding and purification protocol. A crystal structure of one glycoform confirmed its native structure and disulfide pattern. Functional assays revealed that the lipid-binding properties of three SapD glycoforms are highly affected by the single sugar moiety of SapD showing a dependency of the size and the type of N-glycan.

Convergent strategy for the synthesis of S-linked oligoxylans.

Arabinoxylans (AX) are a major class of hemicellulose and an important polysaccharide component of lignocellulosic biomass. To utilize the glycan polymer effectively, it is desirable to learn more about the enzymatic hydrolysis of AXs. Well-defined glycans can help to elucidate these processes. Here, we report the efficient synthesis of a mixed O- and S-linked tetraxylan. This thio-oligosaccharide has been developed as a putative inhibitor of arabinoxylan degrading enzymes used for the saccharification of biomass. Two common approaches for the synthesis of thio-oligosaccharides, either involving 1-thioglycoside donors or thioacceptors, are presented and compared regarding byproduct formation and yields. Both methods have shown to be useful for the synthesis of thiolinkages in oligoxylans assembly. However, the success of the reaction is highly dependent on the "match" between donors and acceptors.

Characterization of the LM5 pectic galactan epitope with synthetic analogues of ß-1,4-d-galactotetraose.

Plant cell wall glycans are important polymers that are crucial to plant development and serve as an important source of sustainable biomass. The study of polysaccharides in the plant cell wall relies heavily on monoclonal antibodies (mAbs) for localization and visualization of glycans, using e.g. immunofluorescent microscopy. Here, we describe the detailed epitope mapping of the mAb LM5 that is shown to bind to a minimum of three sugar residues at the non-reducing end of linear beta-1,4-linked galactan. The study uses de novo synthetic analogues of galactans combined with carbohydrate microarray and competitive inhibition ELISA for analysis of antibody-carbohydrate interactions.

A recombinant fungal lectin for labeling truncated glycans on human cancer cells.

Cell surface glycoconjugates present alterations of their structures in chronic diseases and distinct oligosaccharide epitopes have been associated with cancer. Among them, truncated glycans present terminal non-reducing ß-N-acetylglucosamine (GlcNAc) residues that are rare on healthy tissues. Lectins from unconventional sources such as fungi or algi provide novel markers that bind specifically to such epitopes, but their availability may be challenging. A GlcNAc-binding lectin from the fruiting body of the fungus Psathyrella velutina (PVL) has been produced in good yield in bacterial culture. A strong specificity for terminal GlcNAc residues was evidenced by glycan array. Affinity values obtained by microcalorimetry and surface plasmon resonance demonstrated a micromolar affinity for GlcNAcß1-3Gal epitopes and for biantennary N-glycans with GlcNAcß1-2Man capped branches. Crystal structure of PVL complexed with GlcNAcß1-3Gal established the structural basis of the specificity. Labeling of several types of cancer cells and use of inhibitors of glycan metabolism indicated that rPVL binds to terminal GlcNAc but also to sialic acid (Neu5Ac). Analysis of glycosyltransferase expression confirmed the higher amount of GlcNAc present on cancer cells. rPVL binding is specific to cancer tissue and weak or no labeling is observed for healthy ones, except for stomach glands that present unique αGlcNAc-presenting mucins. In lung, breast and colon carcinomas, a clear delineation could be observed between cancer regions and surrounding healthy tissues. PVL is therefore a useful tool for labeling agalacto-glycans in cancer or other diseases.

Semisynthesis of biologically active glycoforms of the human cytokine interleukin†6.

Human interleukin 6 (IL-6) is a potent cytokine with immunomodulatory properties. As the influence of N-glycosylation on the in vivo activities of IL-6 could not be elucidated so far, a semisynthesis of homogeneous glycoforms of IL-6 was established by sequential native chemical ligation. The four cysteines of IL-6 are convenient for ligations and require only the short synthetic glycopeptide 43-48. The Cys-peptide 49-183 could be obtained recombinantly by cleavage of a SUMO tag. The fragment 1-42 was accessible by the simultaneous cleavage of two inteins, leading to the 1-42 thioester with the native N-terminus. Ligation and refolding studies showed that the inherently labile Asp-Pro bond 139-140 was detrimental for the sequential C- to N-terminal ligation. A reversed ligation sequence using glycopeptide hydrazides gave full-length IL-6 glycoproteins, which showed full bioactivity after efficient refolding and purification.

Breaking pseudo-symmetry in multiantennary complex N-glycans using lanthanide-binding tags and NMR pseudo-contact shifts.

Controlling NMR shifts by lanthanides tagged to a "symmetrical" N-glycan reveals individual resonances for the residues of the otherwise identical A and B arms. This method provides a global perspective of conformational features and interactions in solution.

Three year results of endovascular therapy with a new generation nitinol stent for femoro-popliteal artery lesions - a single-center outcome analysis of a subcohort of MISAGO 2 study.

BACKGROUND: Advances in stent technology have widened the field of indications for stent treatment of femoro-popliteal artery lesions, however the use of stents in bending arterial segments is restricted because some first- and second-generation nitinol stent designs did not respond well to the mechanical forces of femoro-popliteal segments in motion which pose a substantial risk of stent fracture inducing in-stent-stenosis. New generation nitinol stents are supposed to overcome these limitations but long-term results are rare. PATIENTS AND METHODS: In forty-five patients (mean age 68 y, range 50 - 85) with peripheral arterial disease (TASC II A-C, Rutherford category 2 - 5) forty-six lesions of the superficial femoral artery (37) or popliteal artery (9) were treated [25 high-grade stenoses, mean length 53 mm (range 30 - 145 mm); 21 chronic total occlusions, mean length 74 mm (range 30 - 180 mm)]. 74 % of lesions were located in the mobile bending arterial segments in the distal femoral or the popliteal segment. Clinical reevaluation performed at discharge, at 6, 12, 24, and 36 months included at least the measurement of ankle-brachial index (ABI) and duplex sonography. RESULTS: Procedural success rate was 100 %. At 6, 12, 24, and 36 months, cumulative primary patency rate was 93.5 %, 84.8 %, 80.5 %, and 74.3 % (SE<10); freedom from target lesion revascularization rate was 95.7 %, 89.2 %, 84.9 %, and 79.3 % (SE<10); Rutherford category and ABI improved in all patients and clinical success was maintained in more than 85 % of patients. CONCLUSIONS: Sustained technical and clinical success and good clinical long-term results were achieved with Misago™ nitinol stent implantation in femoro-popliteal lesions with moderate risk for in-stent-stenosis, and in the distal femoral and popliteal mobile segment.

Development of a new intravascular low-profile device for exclusion of aortic aneurysm: an experimental pilot study.

PURPOSE: To present a new intravascular device for the treatment of aorto-iliac aneurysms. METHODS: This new device was tested in five dogs with abdominal aortic aneurysm created experimentally by over-dilation of a balloon-expandable stent with a 16 or 18 mm wide PTA balloon catheter. The design of the device is based on a self-expanding aortic stent which consists of two stretchable circular frames filled with a textile Dacron mesh membrane that is suspended horizontally into the infrarenal abdominal aorta proximally to the aneurysm. The frames are part of a preshaped double helical structure that is introduced longitudinally through a catheter in a parallel fashion and forming the desired shape at the vessel site to be occluded. Two iliac stent-grafts are introduced in a low-profile status through the membrane sealing the aneurysm sac and holding the stent-grafts in place. After stent-graft expansion, a new bifurcation located more proximally than the natural one is created. The follow-up of the dogs was performed clinically and angiographically, and specimens were evaluated histomorphologically. RESULTS: The membrane device can be introduced through a 9 Fr vascular sheath. Technical success was achieved in four of five dogs. Nine of ten stent-grafts could be fixed securely within the membrane, thus preventing dislocation. Aneurysms were excluded immediately, and blood flow to the external iliac arteries was restored by the stent-grafts. At 6-9 months follow-up of technically successful implanted devices, there were no endoleaks, no migration, no stenoses at contact sites between the implant material and vascular wall, and no stenosis or occlusion of the stent-grafts. At microscopic evaluation, the interspace between the membranes was filled with thrombotic material, thus ensuring exclusion of the aneurysm. CONCLUSION: This new device was found to be flexible, low profile and useful in excluding abdominal aortic aneurysm in the experimental setting.

Clopidogrel plus long-term aspirin after femoro-popliteal stenting. The CLAFS project: 1- and 2-year results.

The aim of this study was to determine the patency rate after femoro-popliteal stenting followed by oral clopidogrel plus long-term aspirin. In a prospective trial, 31 patients with a total of 33 femoro-popliteal artery lesions (21 stenoses, 12 occlusions; 24 femoral, 9 popliteal) were treated with flexible tantalum stents after unsuccessful percutaneous transluminal angioplasty (PTA) preceded by local fibrinolysis in 5 of 12 patients with total occlusion. Post-interventionally, oral aspirin 100 mg was started simultaneously for the long term and was combined with an oral loading dose of 300 mg clopidogrel, followed by 75 mg clopidogrel daily for 28 days. Patients were followed for at least 12 months (maximum 34 months) by clinical examination, Doppler pressure measurement, color and duplex sonography, and angiography in case of suspicion of restenosis. In a retrospective analysis, the results were compared with those of historical groups of patients having received aspirin only (41 patients) or a long-term high-dose low molecular weight heparin (LMWH)+aspirin treatment (42 patients). Three small puncture aneurysms were treated successfully by conservative means and were categorized as minor bleeding complication. Cumulative primary patency rate (PPR) was 76 +/- 7.5% (1 year), and 70 +/- 9.6% (2 years) in the clopidogrel+aspirin group, thus being tendentiously better than in the aspirin-only group showing 75 +/- 4.6% (1 year), and 50 +/- 8.1% (2 years). Long-term high-dose LMWH+aspirin treatment showed 87 +/- 5.8% (1 year), and 72 +/- 9.1% (2 years), thus being superior to the other treatment regimes, with a statistically significant difference (p<0.05) between the LMWH+aspirin and the aspirin group. Clopidogrel plus aspirin is a safe medication regimen and may be effective in the prevention of early stent thrombosis. Mid- and long-term patency rate seems to be intermediate as compared with other therapeutic regimens. The LMWH+aspirin seems to be superior compared with CLAFS; however, randomized studies with larger patient numbers are recommended.