Carlo Unverzagt

Chemical assembly of N-glycoproteins: a refined toolbox to address a ubiquitous posttranslational modification

Incremental developments in the chemistry of peptides, proteins and carbohydrates have enabled researchers to assemble entire glycoproteins with high precision. Based on sophisticated ligation chemistries pure glycoproteins bearing a single glycosylation pattern have become available. The impact of N-glycosylation on the function of glycoproteins is generally recognized but not well understood. Based on the recent advances in the synthesis of glycoproteins by chemical methods researchers can finally start to elucidate the various roles of carbohydrates in complex biomolecules in detail.

Semisynthesis of a Homogeneous Glycoprotein Enzyme: Ribonuclease C: Part 2

Active RNase glycoprotein from three pieces: The glycoprotein enzyme ribonuclease C, which contains a complex saccharide N-glycan, was synthesized by sequential native chemical ligation. An optimized ligation and isolation protocol allowed the efficient assembly and refolding of the 124 amino acid enzyme.

From structural to functional glycomics: core substitutions as molecular switches for shape and lectin affinity of N-glycans

Abstract Glycan epitopes of cellular glycoconjugates act as versatile biochemical signals (sugar coding). Here, we test the hypothesis that the common N-glycan modifications by core fucosylation and introduction of the bisecting N-acetylglucosamine moiety have long-range effects with functional consequences. Molecular dynamics simulations indicate a shift in conformational equilibria between linear extension or backfolding of the glycan antennae upon substitution. We also present a new fingerprint-like mode of presentation for this multi-parameter system. In order to delineate definite structure-function relationships, we strategically combined chemoenzymatic synthesis with bioassaying cell binding and the distribution of radioiodinated neoglycoproteins in vivo. Of clinical relevance, tailoring the core region affects serum clearance markedly, e.g., prolonging circulation time for the neoglycoprotein presenting the N-glycan with both substitutions. α2,3-Sialylation is another means toward this end, similarly seen for type II branching in triantennary N-glycans. This discovery signifies that rational glycoengineering along the given lines is an attractive perspective to optimize pharmacokinetic behavior of glycosylated pharmaproteins. Of general importance for the concept of the sugar code, the presented results teach the fundamental lesson that N-glycan core substitutions convey distinct characteristics to the concerned oligosaccharide relevant for cis and trans biorecognition processes. These modifications are thus molecular switches.

Structural Basis of Flocculin-Mediated Social Behavior in Yeast

In the budding yeast Saccharomyces cerevisiae, self-recognition and the thereby promoted aggregation of thousands of cells into protective flocs is mediated by a family of cell-surface adhesins, the flocculins (Flo). Based on this social behavior FLO genes fulfill the definition of “greenbeard” genes, which direct cooperation toward other carriers of the same gene. The process of flocculation plays an eminent role in the food industry for the production of beer and wine. However, the precise mode of flocculin-mediated surface recognition and the exact structure of cognate ligands have remained elusive. Here, we present structures of the adhesion domain of a flocculin complexed to its cognate ligands derived from yeast high-mannose oligosaccharides at resolutions up to 0.95 Å. Besides a PA14-like architecture, the Flo5A domain reveals a previously undescribed lectin fold that utilizes a unique DcisD calcium-binding motif for carbohydrate binding and that is widely spread among pro- and eukaryotes. Given the high abundance of high-mannose oligosaccharides in yeast cell walls, the Flo5A structure suggests a model for recognition, where social non-self- instead of unsocial self-interactions are favored.

Chemical and enzymatic synthesis of multivalent sialoglycopeptides

Linear and branched glycopeptides containing multiple sialyl-N-acetyllactosamine side chains have been synthesized using a combined chemical and enzymatic approach. Peptide backbones in which beta-GlcNAc-Asn residues were incorporated were obtained in good yields by optimized solid-phase synthesis following the Boc strategy. The resulting multivalent glycopeptides were galactosylated in near-quantitative yields using bovine galactosyltransferase, UDP-galactose, and calf alkaline phosphatase that destroys the inhibiting side product UDP. Subsequent enzymatic sialylation yielded the desired glycopeptides containing asparagine-linked sialyl-N-acetyllactosamine side chains. The compounds were characterized by 1H NMR and FABMS. Recombinant sialyltransferase and CMP-sialate synthetase were used for the enzymatic synthesis of sialosides on a preparative scale. The synthetic glycopeptides were tested as inhibitors of influenza virus to cells, revealing that most of the multivalent sialoglycopeptides exhibit increased binding that depends on the spacing when compared to monovalent compounds. A possible mechanism for increased binding is proposed.

IDENTIFICATION OF CARBOHYDRATE DEFICIENT TRANSFERRIN FORMS BY MALDI-TOF MASS SPECTROMETRY AND LECTIN ELISA

Transferrin was isolated from sera of patients with severe alcohol abuse and from control sera by affinity chromatography using an immobilized polyclonal antibody from sheep, followed by gel filtration. The purified transferrin was then separated by MonoQ chromatography. Compared to the controls, sera from heavy alcohol consumers showed two additional transferrin peaks, eluting earlier than the three main transferrin forms present in all sera. Further analysis of the isolated transferrin forms by matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and enzyme linked immunosorbent assay with different digoxigenylated lectins (lectin ELISA) revealed that the main carbohydrate deficient transferrin (CDT) forms are lacking either one or both of the N-Glycan chains.

Convergent solid-phase synthesis of N-glycopeptides facilitated by pseudoprolines at consensus-sequence Ser/Thr residues.

N-Glycosylation is an important posttranslational modification of proteins. A carbohydrate is transferred to an asparagine within an Asn-X-Ser/Thr consensus sequence. The study of the biological aspects of N-glycosylation often requires the synthesis of N-glycopeptides, which are accessible by two main approaches. In the sequential mode glycosylamino acid cassettes are used for peptide elongation. After incorporation of larger oligosaccharides the solubility and reactivity of the peptide is affected and side reactions, for example, involving free OH groups complicate further elongation. In the convergent mode (Lansbury aspartylation) the sugar is connected to an aspartate after complete assembly of the peptide (Scheme 1). The main drawback of the convergent mode is the formation of cyclic aspartimides during peptide elongation and sugar coupling; this formation depends on the peptide sequence, and the coupling conditions. We found that a pseudoproline (Ypro) at the consensus-sequence Ser/Thr residue (AsnX-Ser/Thr(Ypro)) efficiently suppresses the formation of aspartimides in the convergent synthesis of N-glycopeptides on the solid phase. The lack of pure N-glycoproteins for biological studies has stimulated research into their synthesis; these syntheses were carried out mainly by ligation techniques. The required glycopeptides and their thioesters are difficult to obtain as the sugar component interferes with the peptide synthesis. The syntheses of longer glycopeptide thioesters are particularly difficult as they require, for example, additional ligation steps 10] or segment couplings. For longer N-glycopeptides the convergent approach provides advantages over the sequential approach, especially as the Lansbury aspartylation has been shown to be efficient also on the solid phase. The undesired aspartimide formation can be avoided by peptide backbone (NH) protection, however, this approach is tedious for residues other than glycine, and causes racemization when coupling backbone-protected dipeptides. Aspartimide formation during peptide elongation can be reduced by using bulky groups to protect the Asp side chain, for example, the 2-phenylisopropylester (PhiPr); however, trityl anchors are also cleaved under the reaction conditions for PhiPr removal. Dmab-protected Asp residues are compatible with trityl anchors, but the backbone protection of the neighboring amino acid is required. To provide complex N-glycopeptide thioesters by solidphase Lansbury aspartylation we compared three Asp-sidechain protecting groups for the Fmoc-SPPS of an interleukin6 (IL-6) 43–48 hexapeptide. The allyl-protected peptide 6a showed the highest percentage of aspartimide 7ai formation (15 %), followed by the Dmab peptide 6b (8%), and the PhiPr peptide 6c (< 1%; Scheme 2). As well as the protecting group many factors are known to contribute to aspartimide formation for example, the steric bulk of the neighboring amino acid, the basicity of the reaction media, and also the overall conformation of the peptide C-terminal relative to the Asp moiety. We reasoned that constraining the Scheme 1. Lansbury aspartylation leads to glycopeptide 3, Asn peptide 3Asn and aspartimide side product 4ai. PG=protecting group.

Chemoenzymatic synthesis of a sialylated diantennary N-glycan linked to asparagine

A partial structure of many glycoproteins, a glycosylated asparagine carrying a complex type undecasaccharide N-glycan (Neu5Ac(alpha 2-6)Gal(beta 1-4)GlcNAc(beta 1-2)Man alpha 1-3) [Neu5Ac(alpha 2-6)Gal(beta 1-4)GlcNAc(beta 1-2)Man(alpha 1-6)]Man(beta 1-4) GlcNAc(beta 1-4)GlcNAc-Asn) was obtained by total synthesis. As a starting material served a chemically synthesized diantennary heptasaccharide azide which was deprotected in a three-step sequence in high yield. The reduction of the anomeric azide was accomplished with propanedithiol in methanol-ethyldiisopropylamine. Coupling of the glycosyl amine to an activated aspartic acid gave the benzyl protected asparagine conjugate. After removal of the six benzyl functions the resulting free heptasaccharide asparagine was elongated enzymatically in the oligosaccharide part. The use of beta-1,4-galactosyltransferase and alpha-2,6-sialytransferase in the presence of alkaline phosphatase allowed the efficient transfer of four sugar units to the acceptor resulting in a full length N-glycan, a sialyated diantennary undecasaccharide-asparagine of the complex type.

Introduction of extended LEC14‐type branching into core‐fucosylated biantennary N‐glycan

A series of enzymatic substitutions modifies the basic structure of complex‐type biantennary N‐glycans. Among them, a β1,2‐linked N‐acetylglucosamine residue is introduced to the central mannose moiety of the core‐fucosylated oligosaccharide by N‐acetylglucosaminyltransferase VII. This so‐called LEC14 epitope can undergo galactosylation at the β1,2‐linked N‐acetylglucosamine residue. Guided by the hypothesis that structural modifications in the N‐glycan alter its capacity to serve as ligand for lectins, we prepared a neoglycoprotein with the extended LEC14 N‐glycan and tested its properties in three different assays. In order to allow comparison to previous results on other types of biantennary N‐glycans the functionalization of the glycans for coupling and assay conditions were deliberately kept constant. Compared to the core‐fucosylated N‐glycan no significant change in affinity was seen when testing three galactoside‐specific proteins. However, cell positivity in flow cytofluorimetry was enhanced in six of eight human tumor lines. Analysis of biodistribution in tumor‐bearing mice revealed an increase of blood clearance by about 40%, yielding a favorable tumor/blood ratio. Thus, the extended LEC14 motif affects binding properties to cellular lectins on cell surfaces and organs when compared to the core‐fucosylated biantennary N‐glycan. The results argue in favor of the concept of viewing substitutions as molecular switches for lectin‐binding affinity. Moreover, they have potential relevance for glycoengineering of reagents in tumor imaging.

Building blocks for glycoproteins: Synthesis of the ribonuclease B fragment 21–25 containing an undecasaccharide N-glycan

Abstract A glycosylated fragment of the bovine glycoprotein ribonuclease B ( 1 ) was synthesized corresponding to amino acids 21–25 with a complex type biantennary N-glycan linked to Asn 24 . The total synthesis of the undecasaccharide-glycopentapeptide 1 was accomplished by modern solution phase glycopeptide chemistry combined with enzymatic techniques for the removal of protective groups and a regio- and stereoselective transfer of the terminal carbohydrates using glycosyltransferases.