O-Glycosylation of glycine-serine linkers in recombinant Fc-fusion proteins

A xylose-based glycosaminoglycan (GAG) core was recently identified at a Ser residue in the linker sequence of a recombinant Fc fusion protein. The linker sequence, G-S-G-G-G-G, and an upstream acidic residue were serving as a substrate for O-xylosyltransferase, resulting in a major glycan composed of Xyl-Gal-Gal-GlcA and other minor intermediates. In this paper, a portion of an unrelated protein was fused to the C-terminus of an IgG Fc domain using the common (G4S)₄ linker repeat. This linker resulted in a heterogenous population of xylose-based glycans all containing at least a core Xyl. Commonly observed glycan structures include GAG-related di-, tri-, tetra-, and penta-saccharides (e.g., Xyl-Gal, Xyl-Gal-Gal, Xyl-Gal-Gal-GlcA, and Xyl-Gal-Gal-GlcA-HexNAc), as well as Xyl-Gal-Neu5Ac. Following alkaline phosphatase or sialidase treatment combined with CID fragmentation, low-level glycans with a mass addition of 79.9 Da were confirmed to be a result of phosphorylated xylose. A minute quantity of phosphorylated GAG pentasaccharides may also be sulfated (also 79.9 Da), possibly at the HexNAc moiety due to non-reactivity to alkaline phosphatase. The xylose moiety may be randomly incorporated in one of the three G-S-G sequence motifs; and the linker peptide shows evidence for multiple additions of xylose at very low levels.     

Antibody–drug conjugates (ADCs) for cancer therapy: Strategies,challenges, and successes

Targeted delivery of therapeutic molecules into cancer cells is considered as a promising strategy to tackle cancer. Antibody–drug conjugates (ADCs), in which a monoclonal antibody (mAb) is conjugated to biologically active drugs through chemical linkers, have emerged as a promising class of anticancer treatment agents, being one of the fastest growing fields in cancer therapy. The failure of early ADCs led researchers to explore strategies to develop more effective and improved ADCs with lower levels of unconjugated mAbs and more-stable linkers between the drug and the antibody, which show improved pharmacokinetic properties, therapeutic indexes, and safety profiles. Such improvements resulted in the US Food and Drug Administration approvals of brentuximab vedotin, trastuzumab emtansine, and, more recently, inotuzumab ozogamicin. In addition, recent clinical outcomes have sparked additional interest, which leads to the dramatically increased number of ADCs in clinical development. The present review explores ADCs, their main characteristics, and new research developments, as well as discusses strategies for the selection of the most appropriate target antigens, mAbs, cytotoxic drugs, linkers, and conjugation chemistries.     

Small photoactivatable molecules for controlled fluorescence activation in living cells

The search for chemical probes which allow a controlled fluorescence activation in living cells represent a major challenge in chemical biology. To be useful, such probes have to be specifically targeted to cellular proteins allowing thereof the analysis of dynamic aspects of this protein in its cellular environment. The present paper describes different methods which have been developed to control cellular fluorescence activation emphasizing the photochemical activation methods known to be orthogonal to most cellular components and, in addition, allowing a spatio-temporal controlled triggering of the fluorescent signal.     

A new method for modeling large-scale rearrangements of protein domains

A method for modeling large-scale rearrangements of protein domains connected by a single- or a double-stranded linker is proposed. Multidomain proteins may undergo substantial domain displacements, while their intradomain structure remains essentially unchanged. The method allows automatic identification of an interdomain linker and builds an all-atom model of a protein structure in internal coordinates. Torsion angles belonging to the interdomain linkers and side chains potentially able to form domain interfaces are set free while all remaining torsions, bond lengths, and bond angles are fixed. Large-scale sampling of the reduced torsion conformational subspace is effected with the “biased probability Monte Carlo-minimization” method [Abagyan, R.A., Totrov, M.M. (1994): J. Mol. Biol. 235, 983–1002]. Solvation and side-chain entropic contributions are added to the energy function. A special procedure has been developed to generate concerted deformations of a double-stranded interdomain linker in such a way that the polypeptide chain continuity is preserved. The method was tested on Bence-Jones protein with a single-stranded linker and lysine/arginine/ornithine-binding (LAO) protein with a double-stranded linker. For each protein, structurally diverse low-energy conformations with ideal covalent geometry were generated, and an overlap between two sets of conformations generated starting from the crystallographically determined “closed” and “open” forms was found. One of the low-energy conformations generated in a run starting from the LAO “closed” form was only 2.2 Å away from the structure of the “open” form. The method can be useful in predicting the scope of possible domain rearrangements of a multidomain protein. Proteins 27:410–424, 1997. © 1997 Wiley-Liss, Inc.     

Linkers of secondary structures in proteins.

Linkers that connect repeating secondary structures fall into conformational classes based on distance and main-chain torsion clustering. A data set of 300 unique protein chains with low pairwise sequence identity was clustered into only a few groups representing the preferred motifs. The linkers of two to eight residues for the nonredundant data set are designated H-Ln-H, H-Ln-E, E-Ln-H, E-Ln-E, where n is the length, H stands for alpha-helices, and E for beta-strands. Most of the clusters identified here corroborate earlier findings. However, 19 new clusters are identified in this paper, with many of them having seven and eight residue linkers. In our first analysis, the secondary structures flanking the linkers are both interacting and noninteracting and there is no precise angle of orientation between them. A second analysis was performed on a set of proteins with restricted orientations for the flanking elements, namely, mainly alpha class of proteins with orthogonal architecture. Two definite clusters are identified, one corresponding to linkers of orthogonal helices and the other to linkers of antiparallel helices. Loops forming binding sites or involved in catalytic activity are important determinants of the function of proteins. Although the structural conservation of the residues around the catalytic triad of serine proteases has been studied widely, there has not been a systematic analysis of the conformation of the loops that contain them. Residues of the catalytic triad reside in the linkers of beta-strands, with varying lengths of more than eight residues. Here, we analyze the structural conservation of such linkers by superposition, and observe a conserved structural feature of the linkers incorporating each of the three residues of the catalytic triad.     

Reducing the renal retention of low- to moderate-molecular-weight radiopharmaceuticals

The field of nuclear imaging and therapy is rapidly progressing with the development of targeted radiopharmaceuticals that show rapid targeting and rapid clearance with minimal background. Unfortunately, they are often reabsorbed in the kidneys, leading to possible nephrotoxicity, limiting the therapeutic dose, and/or reducing imaging quality. The blocking of endocytic receptors has been extensively used as a strategy to reduce kidney radiation. Alternatively, the physicochemical properties of radiotracers can be modulated to either prevent their reuptake or promote the excretion of radiometabolites. Other interesting strategies focus on the insertion of a cleavable linker between the radiolabel and the targeting moiety or pretargeting approaches in which the targeting moiety and radiolabel are administered separately. In the context of this review, we will discuss the latest advances and insights on strategies used to reduce renal retention of low- to moderate-molecular-weight radiopharmaceuticals.     

Fmoc chemistry compatible thio-ligation assembly of proteins

We describe, and compare, two methods which enable theassembly of proteins from peptide thioester fragmentsprepared by Fmoc chemistry mediated solid phase synthesis. The first, which utilizes iso-thiouronium salts,allows formation of thiophenyl esters directly frompartially protected peptides, either in solution or onresin. The second uses sulfonamide based safety-catchresins. Data on yields, generality and potential for side-reactions are provided.