Spin Diffusion Editing for Structural Fingerprints of Therapeutic Antibodies.

The growing importance of biologics and biosimilars as therapeutic and diagnostic agents is giving rise to new demands for analytical methodology that can quickly and accurately assess the chemical and physical state of protein-based products. A particular challenge exists in physical characterization where the proper fold and extent of disorder of a protein is a major concern. The ability of NMR to reflect structural and dynamic properties of proteins is well recognized, but sensitivity limitations and high levels of interference from excipients in typical biologic formulations have prevented widespread applications to quality assessment.

B4GAT1 is the priming enzyme for the LARGE-dependent functional glycosylation of α-dystroglycan.

Recent studies demonstrated that mutations in B3GNT1, an enzyme proposed to be involved in poly-N-acetyllactosamine synthesis, were causal for congenital muscular dystrophy with hypoglycosylation of α-dystroglycan (secondary dystroglycanopathies). Since defects in the O-mannosylation protein glycosylation pathway are primarily responsible for dystroglycanopathies and with no established O-mannose initiated structures containing a β3 linked GlcNAc known, we biochemically interrogated this human enzyme. Here we report this enzyme is not a β-1,3-N-acetylglucosaminyltransferase with catalytic activity towards β-galactose but rather a β-1,4-glucuronyltransferase, designated B4GAT1, towards both α- and β-anomers of xylose.

Synthetic, structural, and biosynthetic studies of an unusual phospho-glycopeptide derived from α-dystroglycan.

Aberrant glycosylation of α-dystroglycan (α-DG) results in loss of interactions with the extracellular matrix and is central to the pathogenesis of several disorders. To examine protein glycosylation of α-DG, a facile synthetic approach has been developed for the preparation of unusual phosphorylated O-mannosyl glycopeptides derived from α-DG by a strategy in which properly protected phospho-mannosides are coupled with a Fmoc protected threonine derivative, followed by the use of the resulting derivatives in automated solid-phase glycopeptide synthesis using hyper-acid-sensitive Sieber amide resin. Synthetic efforts also provided a reduced phospho-trisaccharide, and the NMR data of this derivative confirmed the proper structural assignment of the unusual phospho-glycan structure.

Principles of mucin architecture: structural studies on synthetic glycopeptides bearing clustered mono-, di-, tri-, and hexasaccharide glycodomains.

The structural characteristics of a mucin glycopeptide motif derived from the N-terminal fragment STTAV of the cell surface glycoprotein CD43 have been investigated by NMR. In this study, a series of molecules prepared by total synthesis were examined, consisting of the peptide itself, three glycopeptides having clustered sites of alpha-O-glycosylation on the serine and threonine side chains with the Tn, TF, and STF carbohydrate antigens, respectively, and one with the beta-O-linked TF antigen. Additionally, a glycopeptide having the sequence SSSAVAV, triglycosylated with the Le(y) epitope, was investigated.

Petrobactin, a photoreactive siderophore produced by the oil-degrading marine bacterium Marinobacter hydrocarbonoclasticus.

Petrobactin is a bis-catecholate, alpha-hydroxy acid siderophore produced by the oil-degrading marine bacterium Marinobacter hydrocarbonoclasticus. The Fe(III)-complexed form of petrobactin is photoreactive in natural sunlight, mediated by the Fe(III)-citrate moiety. The reaction results in decarboxylation of the petrobactin ligand and reduction of Fe(III) to Fe(II).