The majority of biological turnover of lignocellulosic biomass in nature is conducted by fungi, which commonly use Family 1 carbohydrate-binding modules (CBMs) for targeting enzymes to cellulose. Family 1 CBMs are glycosylated, but the effects of glycosylation on CBM function remain unknown. Here, the effects of O-mannosylation are examined on the Family 1 CBM from the Trichoderma reesei Family 7 cellobiohydrolase at three glycosylation sites. To enable this work, a procedure to synthesize glycosylated Family 1 CBMs was developed. Subsequently, a library of 20 CBMs was synthesized with mono-, di-, or trisaccharides at each site for comparison of binding affinity, proteolytic stability, and thermostability. The results show that, although CBM mannosylation does not induce major conformational changes, it can increase the thermolysin cleavage resistance up to 50-fold depending on the number of mannose units on the CBM and the attachment site. O-Mannosylation also increases the thermostability of CBM glycoforms up to 16°C, and a mannose disaccharide at Ser3 seems to have the largest themostabilizing effect. Interestingly, the glycoforms with small glycans at each site displayed higher binding affinities for crystalline cellulose, and the glycoform with a single mannose at each of three positions conferred the highest affinity enhancement of 7.4-fold. Overall, by combining chemical glycoprotein synthesis and functional studies, we show that specific glycosylation events confer multiple beneficial properties on Family 1 CBMs. chemical synthesis | cellulase | biofuels | protein engineering
The importance of the glycan structure and size, amino acid residues near the glycosylation site, and glycosidic linkage in controlling the effects of CBM O-glycosylation is shown.
Diabetes is a leading cause of death worldwide and results in over 3 million annual deaths. While insulin manages the disease well, many patients fail to comply with injection schedules, and despite significant investment, a more convenient oral formulation of insulin is still unavailable. Studies suggest that glycosylation may stabilize peptides for oral delivery, but the demanding production of homogeneously glycosylated peptides has hampered transition into the clinic. We report here the first total synthesis of homogeneously glycosylated insulin. After characterizing a series of insulin glycoforms with systematically varied O-glycosylation sites and structures, we demonstrate that O-mannosylation of insulin B-chain Thr27 reduces the peptide’s susceptibility to proteases and self-association, both critical properties for oral dosing, while maintaining full activity. This work illustrates the promise of glycosylation as a general mechanism for regulating peptide activity and expanding its therapeutic use.
Protein glycosylation has been shown to have a variety of site-specific and glycan-specific effects, but so far, the molecular logic that leads to such observations has been elusive. Understanding the structural changes that occur and being able to correlate those with the physical properties of the glycopeptide are valuable steps toward being able to predict how specific glycosylation patterns will affect the stability of glycoproteins. By systematically comparing the structural features of the O-glycosylated carbohydrate-binding module of a Trichoderma reesei-derived Family 7 cellobiohydrolase, we were able to develop a better understanding of the influence of O-glycan structure on the molecule's physical stability. Our results indicate that the previously observed stabilizing effects of O-glycans come from the introduction of new bonding interactions to the structure and increased rigidity, while the decreased stability seemed to result from the impaired interactions and increased conformational flexibility. This type of knowledge provides a powerful and potentially general mechanism for improving the stability of proteins through glycoengineering.
Protein glycosylation is a diverse post-translational modification that serves myriad biological functions.
Protein glycosylation is one of the most common post-translational modifications and can influence many properties of proteins. Abnormal protein glycosylation can lead to protein malfunction and serious disease. While appreciation of glycosylation's importance is growing in the scientific community, especially in recent years, a lack of homogeneous glycoproteins with well-defined glycan structures has made it difficult to understand the correlation between the structure of glycoproteins and their properties at a quantitative level. This has been a significant limitation on rational applications of glycosylation and on optimizing glycoprotein properties. Through the extraordinary efforts of chemists, it is now feasible to use chemical synthesis to produce collections of homogeneous glycoforms with systematic variations in amino acid sequence, glycosidic linkage, anomeric configuration, and glycan structure. Such a technical advance has greatly facilitated the study and application of protein glycosylation. This Perspective highlights some representative work in this research area, with the goal of inspiring and encouraging more scientists to pursue the glycosciences.
Protein O-glycosylation is a diverse, common, and important post-translational modification of both proteins inside the cell and those that are secreted or membrane-bound. Much work has shown that O-glycosylation can alter the structure, function, and physical properties of the proteins to which it is attached. One gap remaining in our understanding of O-glycoproteins is how O-glycans might affect the folding of proteins. Here, we took advantage of synthetic, homogeneous O-glycopeptides to show that certain glycosylation patterns have an intrinsic effect, independent of any cellular folding machinery, on the folding pathway of a model O-glycoprotein, a carbohydrate binding module (CBM) derived from the Trichoderma reesei cellulase TrCel7A. The strongest effect, a 6-fold increase in overall folding rate, was observed when a single O-mannose was the glycan, and the glycosylation site was near the N-terminus of the peptide sequence. We were also able to show that glycosylation patterns affected the kinetics of each step in unique ways, which may help to explain the observations made here. This work is a first step toward quantitative understanding of how O-glycosylation might control, through intrinsic means, the folding of O-glycoproteins. Such an understanding is expected to facilitate future investigations into the effects of glycosylation on more biological processes related to protein folding.
Thesis directed by Assistant Professor Zhongping TanProtein glycosylation, the covalent attachment of carbohydrates to amino acid side chains of proteins, is a ubiquitous post-translational modification across all branches of life. Due to many factors including the vast structural complexity of glycans and the convoluted processes regulating their construction, protein glycosylation is a significantly understudied phenomenon. In particular, the study of protein O-glycosylation, where the carbohydrate moieties are attached via the oxygen of a serine or threonine residue, is lacking because there exists no well-defined consensus sequence for its occurrence and the enzymatic construction of O-glycosylated proteins in a controlled manner is very difficult. We employed chemical synthesis for the construction of homogeneous and well-defined O-glycoproteins with a large variety of structures and used these synthetic biomolecules to systematically and quantitatively investigate the effects of glycosylation on the biophysical and biological properties of proteins. Our ultimate goal is to develop a set of principles that can be widely applied to the rational engineering of enzymes and therapeutic proteins through glycosylation and other post-translational modifications. The initial focus was on examining the effects of O-glycosylation on the properties of a carbohydrate binding module (CBM) of an industrially important fungal cellulase. We used a wide range of biochemical assays to characterize a library of 51 differently-glycosylated CBM isoforms, and observed strong effects of glycosylation, in a pattern specific manner, on the folding, stability, solubility, chromatographic behavior, binding affinity and specificity of this small domain. In the long term, this project is expected to lead to fungal cellulases with optimal stability, specificity, activity required to achieve efficient saccharification of biomass for biofuels production. We then expanded our methodology to investigate the influence of Oglycosylation on two important therapeutic peptides: insulin and glucagon-like peptide-1 (GLP-1). As with the CBM system, we observed that glycosylation can significantly impact physical and/or functional properties of these molecules. We have identified specific isoforms of both insulin and GLP-1 that have iv increased stability and unchanged biological functions. It is our hope that further development of the most promising lead candidates for insulin and GLP-1 could lead to better therapies for the treatment of metabolic disorders.
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