Glycosylation in Health and Disease
Proteins are the executors of life activities. In the study of proteins, researchers have found that proteins have many modifications. Among these modifications, glycosylation is very common, with more than 50% of the proteins discovered so far having glycosylation modifications. Glycans are the sugar components of glycoproteins, including glycosyl and various types of branching structures composed of various sugars such as glucose, galactose, mannose, and rhamnose. Polysaccharides complete the glycosylation modification of proteins by connecting to the oxygen on specific amino acid residues.
Glycosylation in physiological processes
Glycosylation is present in many important proteins involved in life activities, including chromatin proteins, nuclear pore proteins, RNA polymerase II, transcription factors, protein translation regulators, and others, involving cellular immunity, protein translation regulation, protein degradation, and many other biological processes. For example, the MHCI (major histocompatibility complex class I) requires interaction with the chaperone calnexin in the endoplasmic reticulum through glycans linked to asparagine residues to complete binding with the thiol oxidoreductase ERp57 captured by calnexin, forming disulfide bonds. Another example is proteins modified by O-GlcNAc glycosylation, which will prevent their phosphorylation, keeping them in a relatively stable state and preventing protein degradation.
Glycosylation occurring in diseases
Abnormal glycosylation is closely related to diseases. For example, in type II diabetes, hyperglycemia can lead to abnormal O-GlcNAc modification, reducing cellular sensitivity to signals, decreasing insulin receptor substrates, and preventing insulin from consuming large amounts of glucose. The first confirmed disease related to glycosylation is inclusion-cell disease, whose pathogenic mechanism is the inability of N-glycan chains to be further modified by mannose-6-phosphate, causing protein catabolism disorders and leading to storage diseases. For diseases caused by abnormal glycosylation, some glycosylation inhibitors have been used in disease treatment research. Inhibitors such as α-glucosidase are used in clinical trials for diabetes; N-butyldeoxynojirimycin and O-butyldeoxynojirimycin are used in clinical trials for HIV.
Types of glycosylation and modification mechanisms
O-glycosylation: O-glycosylation occurs on serine or threonine residues near proline, with no specific glycosylation sequence identified yet. O-glycosylation mainly involves the gradual addition of monosaccharides to form oligosaccharides, although there are cases of only monosaccharides being attached. Glycans formed through O-glycosylation do not have glycosyl groups and may have one or no branches on the carbon skeleton.
N-glycosylation: N-glycosylation occurs on the amide nitrogen of the asparagine side chain. Almost all such glycosylation modifications in animal cells are GlcNAc and are of β configuration. N-glycosylation is present in the amino acid sequence Asn-Xaa-Ser/Thr/Cys, where Xaa can be any amino acid except Pro. Glycans formed through N-glycosylation have one glycosyl group and multiple branches.
C-glycosylation: This type of glycosylation is rare and involves the connection of a mannose molecule to the second carbon of the indole ring of tryptophan through a C-C bond. It mainly occurs on the first tryptophan residue in sequences W-X-X-W-W-X-X-C or W-X-X-F.
Research methods for glycoproteins
1. Glycan capture: Lectins can specifically recognize one or multiple sugars to agglutinate glycoproteins and can be used to obtain crude glycoproteins.
2. Fluorescent dyes: Proteins are stained with fluorescent dyes, typically used in combination with high-throughput two-dimensional gel electrophoresis, for protein discovery and identification.
3. Liquid chromatography: Glycoproteins are separated using SEC-HILIC-CapLC workflows and other techniques to construct a three-dimensional liquid-phase glycan profile to obtain glycan structural information.
4. Mass spectrometry: Mass spectrometry is used to analyze the fragmentation patterns of protein and sugar skeletons, which can be used for glycosylation site analysis.
5. Nuclear magnetic resonance: By observing changes in atomic energy transitions, information such as composition, ring size, and anomalous carbon conformations can be accurately calculated and used for precise confirmation of glycan structures.
References
1. Turroni F, et al. Glycan Utilization and Cross-Feeding Activities by Bifidobacteria. Trends in Microbiology, 2018, 26(4).
2. Lindsay C, et al. Zn-α2-glycoprotein, an MHC Class I-Related Glycoprotein Regulator of Adipose Tissues: Modification or Abrogation of Ligand Binding by Site-Directed Mutagenesis. Biochemistry, 2006, 45(7).
3. Winters M P, et al. Discovery of N-Arylpyrroles as Agonists of GPR120 for the Treatment of Type II Diabetes. Bioorganic & Medicinal Chemistry Letters, 2018, 28(5).
4. Li S, Yi L, et al. O-Glycosylation of EGF repeats: identification and initial characterization of a, UDP-glucose: protein O-glucosyltransferase. Glycobiology, 2002, 1(11).
5. Priola S A, et al. Glycosylation influences cross-species formation of protease-resistant prion protein. EMBO Journal, 2001, 20(23).
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