An Attenuated Targeted-TNF Localizes to Tumors In Vivo and Regains Activity at the Site of Disease.

Antibody-cytokine fusion proteins (immunocytokines) are gaining importance for cancer therapy, but those products are often limited by systemic toxicity related to the activity of the cytokine payload in circulation and in secondary lymphoid organs. Tumor necrosis factor (TNF) is used as a pro-inflammatory payload to trigger haemorrhagic necrosis and boost anti-cancer immunity at the tumor site. Here we describe a depotentiated version of TNF (carrying the single point mutation I97A), which displayed reduced binding affinity to its cognate receptor tumor necrosis factor receptor 1 (TNFR-1) and lower biocidal activity. The fusion of the TNF(I97A) mutant to the L19 antibody promoted restoration of anti-tumor activity upon accumulation on the cognate antigen, the alternatively spliced EDB domain of fibronectin. In vivo administration of high doses (375 µg/Kg) of the fusion protein showed a potent anti-tumor effect without apparent toxicity compared with the wild type protein. L19-TNFI97A holds promise for the targeted delivery of TNF activity to neoplastic lesions, helping spare normal tissues.

X-ray structure of a bacterial oligosaccharyltransferase.

Asparagine-linked glycosylation is a post-translational modification of proteins containing the conserved sequence motif Asn-X-Ser/Thr. The attachment of oligosaccharides is implicated in diverse processes such as protein folding and quality control, organism development or host-pathogen interactions. The reaction is catalysed by oligosaccharyltransferase (OST), a membrane protein complex located in the endoplasmic reticulum. The central, catalytic enzyme of OST is the STT3 subunit, which has homologues in bacteria and archaea. Here we report the X-ray structure of a bacterial OST, the PglB protein of Campylobacter lari, in complex with an acceptor peptide. The structure defines the fold of STT3 proteins and provides insight into glycosylation sequon recognition and amide nitrogen activation, both of which are prerequisites for the formation of the N-glycosidic linkage. We also identified and validated catalytically important, acidic amino acid residues. Our results provide the molecular basis for understanding the mechanism of N-linked glycosylation.

Substrate specificity of cytoplasmic N-glycosyltransferase.

N-Linked protein glycosylation is a very common post-translational modification that can be found in all kingdoms of life. The classical, highly conserved pathway entails the assembly of a lipid-linked oligosaccharide and its transfer to an asparagine residue in the sequon NX(S/T) of a secreted protein by the integral membrane protein oligosaccharyltransferase. A few species in the class of γ-proteobacteria encode a cytoplasmic N-glycosylation system mediated by a soluble N-glycosyltransferase (NGT). This enzyme uses nucleotide-activated sugars to modify asparagine residues with single monosaccharides. As these enzymes are not related to oligosaccharyltransferase, NGTs constitute a novel class of N-glycosylation catalyzing enzymes. To characterize the NGT-catalyzed reaction, we developed a sensitive and quantitative in vitro assay based on HPLC separation and quantification of fluorescently labeled substrate peptides. With this assay we were able to directly quantify glycopeptide formation by Actinobacillus pleuropneumoniae NGT and determine its substrate specificities: NGT turns over a number of different sugar donor substrates and allows for activation by both UDP and GDP. Quantitative analysis of peptide substrate turnover demonstrated a strikingly similar specificity as the classical, oligosaccharyltransferase-catalyzed N-glycosylation, with NX(S/T) sequons being the optimal NGT substrates.

A catalytically essential motif in external loop 5 of the bacterial oligosaccharyltransferase PglB.

Asparagine-linked glycosylation is a post-translational protein modification that is conserved in all domains of life. The initial transfer of a lipid-linked oligosaccharide (LLO) onto acceptor asparagines is catalyzed by the integral membrane protein oligosaccharyltransferase (OST). The previously reported structure of a single-subunit OST enzyme, the Campylobacter lari protein PglB, revealed a partially disordered external loop (EL5), whose role in catalysis was unclear. We identified a new and functionally important sequence motif in EL5 containing a conserved tyrosine residue (Tyr293) whose aromatic side chain is essential for catalysis. A synthetic peptide containing the conserved motif can partially but specifically rescue in vitro activity of mutated PglB lacking Tyr293. Using site-directed disulfide cross-linking, we show that disengagement of the structurally ordered part of EL5 is an essential step of the glycosylation reaction, probably by allowing sequon binding or glyco-product release. Our findings define two distinct mechanistic roles of EL5 in OST-catalyzed glycosylation. These functions, exerted by the two halves of EL5, are independent, because the loop can be cleaved by specific proteolysis with only slight reduction in activity.

Mechanism of bacterial oligosaccharyltransferase: in vitro quantification of sequon binding and catalysis.

N-Linked glycosylation is an essential post-translational protein modification in the eukaryotic cell. The initial transfer of an oligosaccharide from a lipid carrier onto asparagine residues within a consensus sequon is catalyzed by oligosaccharyltransferase (OST). The first X-ray structure of a complete bacterial OST enzyme, Campylobacter lari PglB, was recently determined. To understand the mechanism of PglB, we have quantified sequon binding and glycosylation turnover in vitro using purified enzyme and fluorescently labeled, synthetic peptide substrates. Using fluorescence anisotropy, we determined a dissociation constant of 1.0 µm and a strict requirement for divalent metal ions for consensus (DQNAT) sequon binding. Using in-gel fluorescence detection, we quantified exceedingly low glycosylation rates that remained undetected using in vivo assays. We found that an alanine in the -2 sequon position, converting the bacterial sequon to a eukaryotic one, resulted in strongly lowered sequon binding, with in vitro turnover reduced 50,000-fold. A threonine is preferred over serine in the +2 sequon position, reflected by a 4-fold higher affinity and a 1.2-fold higher glycosylation rate. The interaction of the +2 sequon position with PglB is modulated by isoleucine 572. Our study demonstrates an intricate interplay of peptide and metal binding as the first step of protein N-glycosylation.

High-mass matrix-assisted laser desorption ionization-mass spectrometry of integral membrane proteins and their complexes.

Analyzing purified membrane proteins and membrane protein complexes by mass spectrometry has been notoriously challenging and required highly specialized buffer conditions, sample preparation methods, and apparatus. Here we show that a standard matrix-assisted laser desorption/ionization (MALDI) protocol, if used in combination with a high-mass detector, allows straightforward mass spectrometric measurements of integral membrane proteins and their complexes, directly following purification in detergent solution. Molecular weights can be determined precisely (mass error ≤ 0.1%) such that high-mass MALDI-MS was able to identify the site for N-linked glycosylation of the eukaryotic multidrug ABC transporter Cdr1p without special purification steps, which is impossible by any other current approach. After chemical cross-linking with glutaraldehyde in the presence of detergent micelles, the subunit stoichiometries of a series of integral membrane protein complexes, including the homomeric PglK and the heteromeric BtuCD as well as BtuCDF, were unambiguously resolved. This thus adds a valuable tool for biophysical characterization of integral membrane proteins.

A combined method for producing homogeneous glycoproteins with eukaryotic N-glycosylation.

We describe a new method for producing homogeneous eukaryotic N-glycoproteins. The method involves the engineering and functional transfer of the Campylobacter jejuni glycosylation machinery in Escherichia coli to express glycosylated proteins with the key GlcNAc-Asn linkage. The bacterial glycans were then trimmed and remodeled in vitro by enzymatic transglycosylation to fulfill a eukaryotic N-glycosylation. It provides a potentially general platform for producing eukaryotic N-glycoproteins.

Relaxed acceptor site specificity of bacterial oligosaccharyltransferase in vivo.

A number of proteobacteria carry the genetic information to perform N-linked glycosylation, but only the protein glycosylation (pgl) pathway of Campylobacter jejuni has been studied to date. Here, we report that the pgl gene cluster of Campylobacter lari encodes for a functional glycosylation machinery that can be reconstituted in Escherichia coli. We determined that the N-glycan produced in this system consisted of a linear hexasaccharide. We found that the oligosaccharyltransferase (OST) of C. lari conserved a predominant specificity for the primary sequence D/E-X(-1)-N-X(+1)-S/T (where X(-1) and X(+1) can be any amino acid but proline). At the same time, we observed that this enzyme exhibited a relaxed specificity toward the acceptor site and modified asparagine residues of a protein at sequences DANSG and NNNST. Moreover, C. lari pgl glycosylated a native E. coli protein. Bacterial N-glycosylation appears as a useful tool to establish a molecular description of how single-subunit OSTs perform selection of glycosyl acceptor sites.

N-Linked glycosylation of antibody fragments in Escherichia coli.

Glycosylation is the predominant protein modification to diversify the functionality of proteins. In particular, N-linked protein glycosylation can increase the biophysical and pharmacokinetic properties of therapeutic proteins. However, the major challenges in studying the consequences of protein glycosylation on a molecular level are caused by glycan heterogeneities of currently used eukaryotic expression systems, but the discovery of the N-linked protein glycosylation system in the ε-proteobacterium Campylobacter jejuni and its functional transfer to Escherichia coli opened up the possibility to produce glycoproteins in bacteria. Toward this goal, we elucidated whether antibody fragments, a potential class of therapeutic proteins, are amenable to bacterial N-linked glycosylation, thereby improving their biophysical properties. We describe a new strategy for glycoengineering and production of quantitative amounts of glycosylated scFv 3D5 at high purity. The analysis revealed the presence of a homogeneous N-glycan that significantly increased the stability and the solubility of the 3D5 antibody fragment. The process of bacterial N-linked glycosylation offers the possibility to specifically address and alter the biophysical properties of proteins.

The Escherichia coli glycophage display system.

We describe a phage display technique that allows the production and selective enrichment of phages that display an N-glycoprotein (glycophages). We applied glycophage display to select functional glycosylation sequons from a pool of randomized acceptor sequences. Our system provides a genetic platform to study and engineer different steps in the pathway of bacterial N-linked protein glycosylation.

Structural Basis for Binding of Fluorescent CMP-Neu5Ac Mimetics to Enzymes of the Human ST8Sia Family.

Polysialyltransferases synthesize polysialic acid on cell surface-expressed glycoconjugates, which is crucial for developing processes and signaling pathways in eukaryotes. Recent advances in cancer research have rendered polysialyltransferases important drug targets because polysialic acid contributes to cancer cell progression, metastasis, and treatment of resistant tumors. To aid the development of high-throughput screening assays for polysialyltransferase inhibitors, we demonstrate that a previously developed class of fluorescent CMP-sialic acid mimetics for sialyltransferases has nanomolar affinities for oligo- and polysialyltransferases and can be used for the rapid screening of new polysialyltransferase inhibitors. We demonstrate that these CMP-Neu5Ac mimetics inhibit polysialylation in vitro and perform cell culture experiments, where we observe reduced polysialylation of NCAM. Furthermore, we describe the structural basis of CMP-Neu5Ac mimetics binding to the human oligosialyltransferase ST8SiaIII and extrapolate why their affinity is high for human polysialyltransferases. Our results show that this novel class of compounds is a promising tool for the development of potent and selective drugs against polysialyltransferase activity.

X-ray crystallographic structure of a bacterial polysialyltransferase provides insight into the biosynthesis of capsular polysialic acid.

Polysialic acid (polySia) is a homopolymeric saccharide that is associated with some neuroinvasive pathogens and is found on selective cell types in their eukaryotic host. The presence of a polySia capsule on these bacterial pathogens helps with resistance to phagocytosis, cationic microbial peptides and bactericidal antibody production. The biosynthesis of bacterial polySia is catalysed by a single polysialyltransferase (PST) transferring sialic acid from a nucleotide-activated donor to a lipid-linked acceptor oligosaccharide. Here we present the X-ray structure of the bacterial PST from Mannheimia haemolytica serotype A2, thereby defining the architecture of this class of enzymes representing the GT38 family. The structure reveals a prominent electropositive groove between the two Rossmann-like domains forming the GT-B fold that is suitable for binding of polySia chain products. Complex structures of PST with a sugar donor analogue and an acceptor mimetic combined with kinetic studies of PST active site mutants provide insight into the principles of substrate binding and catalysis. Our results are the basis for a molecular understanding of polySia biosynthesis in bacteria and might assist the production of polysialylated therapeutic reagents and the development of novel antibiotics.

Unexpected reactivity and mechanism of carboxamide activation in bacterial N-linked protein glycosylation.

The initial glycan transfer in asparagine-linked protein glycosylation is catalysed by the integral membrane enzyme oligosaccharyltransferase (OST). Here we study the mechanism of the bacterial PglB protein, a single-subunit OST, using chemically synthesized acceptor peptide analogues. We find that PglB can glycosylate not only asparagine but also glutamine, homoserine and the hydroxamate Asp(NHOH), although at much lower rates. In contrast, N-methylated asparagine or 2,4-diaminobutanoic acid (Dab) are not glycosylated. We find that of the various peptide analogues, only asparagine- or Dab-containing peptides bind tightly to PglB. Glycopeptide products are unable to bind, providing the driving force of product release. We find no suitably positioned residues near the active site of PglB that can activate the acceptor asparagine by deprotonation, making a general base mechanism unlikely and leaving carboxamide twisting as the most likely mechanistic proposal for asparagine activation.