Nat. of biomolecules that are involved in a wide variety of physiological and disease processes.1 Rabbit Polyclonal to PLCG1 The biological functions of protein glycosylation are truly multifaceted. 1 It is well documented that glycosylation can profoundly affect a proteins intrinsic properties such as the conformations, protease Phensuximide stability, antigenicity, and immunoge-nicity.2 The glycans of glycoproteins can also directly participate in a number of biological recognition processes including intracellular trafficking, cell adhesion, signaling, development, host-pathogen interactions, and immune responses, to name Phensuximide a few.13C5 For example, N-glycosylation plays an important role in the lectin (calnexin/calreticulin)-mediated protein folding and the ER-associated degradation pathways in quality control;6,7 the mannose-6-phosphate (M6P)-tagged glycosylation of lysosomal enzymes is critical for successfully targeting the enzymes to lysosomes for degrading various dysfunctional biomolecules; ,9 cell surface glycans often serve as ligands for glycan-binding protein mediated host-pathogen interactions such as bacterial and viral infections; 10C14 and aberrant glycosylation is often associated with disease development and progression such as cancer and autoimmune disorders.15C17 A majority of therapeutic proteins, including monoclonal antibodies, are glycosylated, and the presence as well as the fine structures of the sugar chains are critical for Phensuximide the stability and biological functions. 8,19 Thus, understanding the structure and functions, as well as the control of the glycosylation status, is essential for the development and production of efficient protein-based therapeutics.20,21 A major challenging in dealing with glycoproteins comes from the structural heterogeneity of natural and recombinant glycoproteins usually aroused from the variations of glycan components Phensuximide and/or the sites of glycosylation. In fact, recombinant glycoproteins such as therapeutic antibodies are usually produced as mixtures of glycoforms that have the same protein backbone but differ in the pendent oligosaccharides, from which pure glycoforms are difficult to isolate using current chromatographic techniques. This situation significantly hampers a detailed understanding of the structure-function relationships and slows down the therapeutic and diagnostic applications of glycoproteins.18,21C27 Thus, efficient methods that allow controlling glycosylation during expression or permit in construction of glycan-defined glycoproteins are urgently needed. Tremendous progress has been made in recent years for producing structurally well-defined, homogeneous glycoproteins, including tailor-made glycoforms of intact antibodies.28C31 These include total chemical synthesis,32C45 tag and modify approaches for site-selective protein-glycan conjugation,46C48 chemoenzymatic synthesis using enzymes for key modification andligation,28C31,49C62 and glycosylation engineering by manipulating the biosynthetic pathways in different host expression systems.63C69 The present review provides a survey of the chemoenzymatic methods that have been developed for the synthesis of glycopeptides and glycoproteins carrying defined oligosaccharides, with a focus on the advances in the past decade. Particular attention was turned to the following areas: the generation of various glycosynthases from glycosidases for synthetic purposes, the endoglycosidase-catalyzed glycoprotein synthesis and glycan remodeling of intact glycoproteins, and the direct enzymatic glycosylation of polypeptides and proteins using oligosacchar-yltransferase, N-glycosyltransferase, O-GlcNAc transferase and O-GalNAc transferase. Selected examples are presented to discuss the concept of the respective methods, while more indepth technical points can be found in the cited primary literature. 2.?GENERAL ASPECTS OF THE STRUCTURE, FUNCTION, AND SYNTHESIS OF GLYCOPROTEINS 2.1. Structural Features of Glycans and Glycoproteins The covalent attachment of mono- or oligosaccharide moieties to proteins, collectively called protein glycosylation, is one of the most prevalent posttranslational modifications (PTMs). Protein glycosylation was once considered as the event only reserved in eukaryotic systems. However, recent discoveries have shown that protein glycosylation is also a common trend in some microorganisms, including bacteria, archaea, and fungi.70,71 In contrast to most other PTMs, such as phosphorylation, that usually involve a simple functional group transfer to one or a handful of amino acid residues, glycosylation can be much more complex and structurally and functionally varied.1,72 So far, over 40 different types of the sugar-amino acid junctions have been identified that involve at least 8 different amino acid residues and 13 different proximal monosaccharides.73,74 While microorganisms are found to have diverse rare monosaccharide devices in secondary metabolites, surprisingly only a dozen or so common monosaccharides are present in typical eukaryotic glycoproteins (Number 1). However, the limited numbers of building blocks can still form incredibly diverse constructions due to the huge possibilities of linkage types, anomeric stereochemistry, and/or additional noncarbohydrate decorations of the sugars chains. Open in a separate window Number 1. Constructions and symbols of common monosaccharide devices found in eukaryotic systems. For most glycoproteins found in eukaryotic.