engleski

High-throughput N-glycosylation alpha-1-acid glycoprotein analysis could aid in the diagnosis of diabetes mellitus

Alpha-1-acid glycoprotein (AGP) is an acute phase glycoprotein in blood, which is primarily synthetized in the liver. Although many possible functions of AGP have been described, the detailed knowledge about its exact role in pathophysiological processes is still elusive. A large part of AGP (around 45%) is modified by glycans, so they certainly play a very important role in its biological activity. Recently, we have developed a cost-effective method for a high-throughput and site-specific N-glycosylation LC-MS analysis of alpha-1-acid glycoprotein (AGP) which can be applied on large cohorts, aid in search for novel disease biomarkers, and enable better understanding of AGP’s role and function in health and disease (Keser et al, 2021). The method does not require isolation of AGP with antibodies and affinity chromatography, but AGP is enriched by acid precipitation from 5 μl of blood plasma in a 96-well format. After trypsinization, AGP glycopeptides are purified using a hydrophilic interaction chromatography-based solid-phase extraction and analyzed by reversed-phase-liquid chromatography-electrospray ionization-MS. We used our method to show for the first time that AGP N-glycan profile is stable in healthy individuals, but it is variable between the individuals of the same population. Furthermore, we tested our method on a population including individuals with registered hyperglycemia in critical illness (59 cases and 49 controls), which represents a significantly increased risk of developing type 2 diabetes. Individuals at higher risk of diabetes presented increased N-glycan branching on AGP’s second glycosylation site and lower sialylation of N- glycans on AGP’s third and AGP1’s fourth glycosylation site. We also analyzed fucosylation of AGP in subjects with HNF1A-MODY (most common type of monogenic diabetes in adults) and other types of diabetes aiming to evaluate its diagnostic potential in 564 individuals (Tijardović et al, 2022). The results showed significant reduction in AGP fucosylation associated to HNF1A-MODY when compared to other diabetes subtypes. Additionally, ROC curve analysis confirmed significant discriminatory potential of individual fucosylated AGP glycopeptides, where the best performing glycopeptide had an AUC of 0.94 (95% CI 0.90–0.99). Altogether, our results indicate that AGP glycosylation could have a significant role in diagnosis and understanding of diabetes mellitus.

Diabetes ; high-throughput ; N-glycosylation ; alpha-1-acid glycoprotein

nije evidentirano