Glycans are complex carbohydrate molecules attached to various other structures, including peptides, proteins, lipids, or aminoglycans. Two common types of glycans, N-linked and O-linked, have been extensively analyzed in the past, and N-linked protein glycosylation is one of the most crucial, prevalent and complicated co- and post-translational protein modifications. In Creative Biolabs, we always focus on glycoengineering studies and are skilled at identifying N-linked glycans of glycoproteins to determine the changes of glycans. With the help of us, the mechanisms of N-linked glycans-associated signal transduction and protein function would be solved and understood clearly.
The N-linked and O-linked glycans are the two most common glycoforms in protein glycosylation. The N-linked glycan has a common core structure of GlcNAc2Man3 that conjugates to asparagine (Asn) residues in the consensus amino-acid motif of Asn-X-Ser/Thr, in which X can be any amino acid except proline (Pro). The structure of glycans is complex due to its non-template biosynthesis. The complexity stems predominantly from its variable monosaccharides, linkages, branches, and isomers.
Fig.1 Types of N-linked glycans. (Stanley, 2017)
N-linked glycans are covalently attached to the protein by an N-glycosidic bond. Although diverse sugars adhere to Asn residues in prokaryotes, all eukaryotic N-glycans begin with GlcNAcβ1-Asn. N-linked glycans at Asn-X-Ser/Thr sequons in eukaryote glycoproteins are of three general types: oligomannose, complex, and hybrid. Each N-linked glycan contains the common core structure. Complex N-linked glycans have up to six branches initiated by GlcNAc and each can be elongated with Galβ1-4GlcNAc (LacNAc) repeats.
The synthesis of N-linked glycans begins on a lipid-like polyisoprenoid molecule termed dolichol-phosphate (Dol-P) in eukaryotes. The whole process occurs in two phases and two compartments of eukaryotic cells, the endoplasmic reticulum (ER) and the Golgi apparatus. The first phase is a highly conserved that proceeds at the ER membrane on the lipid carrier Dol-P. The second phase starts at processing of N-linked glycans by glycosidases and glycosyltransferases in the lumen of the ER and continues in the Golgi in a species-, cell type-, protein-, and site-specific manner. This synthetic pathway is conserved in all metazoans, plants, and yeasts. And bacteria can utilize related mechanisms to synthesize cell walls.
Fig.2 Typical N-glycan structures are shown. (Kizuka, 2017)
The attachment of N-linked glycan onto a protein takes place in the ER lumen, and the process is mainly co-translational. N-linked glycan precursor is biosynthesized at the ER membranes, and the common N-glycan structure comprising 14 sugars is transferred to proteins with the consensus sequence. After the transfer of 14 saccharides, the N-linked glycan is gradually trimmed by glucosidases and mannosidases in the ER and early Golgi during protein folding. Subsequent glycan maturation occurs in the Golgi apparatus by the action of glycosyltransferases, which generates a wide variety of glycan structures in Fig.2.
The functions of N-glycans are quite diverse and regulate various molecular behaviors. Defects in N-linked glycan structures of a particular glycoprotein are profoundly involved in different human diseases including type 1 diabetes, rheumatoid arthritis, Crohn's disease, and cancers, which is a rationale for glycol-targeted biomarkers and therapy. Indeed, glycoproteins with specific N-glycan epitopes are clinically used as disease markers, especially in the cancer context.
Fig.3 Quantitative MALDI-TOF MS spectra of IgG1 glycan profile in the presence of stable isotope labeled internal N-glycan standards (marked with an asterisk). (Etxebarria, 2016)
Glycan biomarkers, like N-glycans, have a huge potential for personalized medicine. The application of stable isotope labeled glycans as internal standards and heavy-isotope labeling approaches will provide the necessary method precision and robustness acceptable for clinical use. If you're interested in this field, please contact us for more details.
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