Study On The Expression Mechanism Of N-glycolylneuraminic Acid In Humans Based On Reactive Oxygen Species Levels

N-glycolylneuraminic acid（Neu5Gc）,a non-human sialic acid found in cancer patients,plays a critical role in cancer progression by participating in humoral inflammatory responses.However,in the process of evolution,due to the deletion of the human encoding cytidine 5’-monophosphate-N-acetylneuraminic acid（CMP-N-acetylneuraminic acid,CMP-Neu5Ac）hydroxylase gene CMAH,the human body cannot synthesize Neu5Gc through the metabolic pathways.The question regarding how Neu5Gc produced in cancer patients has become a question of great concern in the field of cancer research.One theory that is commonly understood in current research on the mechanism by which Neu5Gc exists in human cells is through exogenous ingestion,such as consumption of Neu5Gc-enriched red meat followed by metabolic sialyltransferase-catalyzed transfer to human glycoconjugates.Therefore,high consumption of red meat has been shown to associate with increased cancer risk.However,there is no evidence showed that vegetarian cancer patients do not express Neu5Gc,so there must be other mechanisms that lead to the Neu5Gc expression in vegan cancer patients.Existing research reports have not addressed whether Neu5Gc can be produced in the disease microenvironment rather than metabolic pathways,such as in the presence of high levels of reactive oxygen species（ROS）.This study demonstrated that Neu5Ac can be hydroxylated to Neu5Gc through chemical reactions rather than metabolic pathways in the environment contains high ROS levels,which provides a new direction for the study of the expression mechanism of Neu5Gc in human cells.The contents of this study include:（1）Establishment of a modern liquid chromatography-tandem mass spectrometry（LC-MRM）method for quantitative analysis of Neu5Gc based on multiple reaction monitoring,and verified by a standard methodology.（2）In vitro conversion reaction of Neu5Ac to Neu5Gc was designed according to the actual physiological conditions.（3）Isotopically labeled[2,3-¹³C₂]Neu5Ac was used to further validate the in vitro conversion reaction of Neu5Ac to Neu5Gc;（4）To demonstrate the hydroxylation of Neu5Ac can occur in serum biological matrix.（5）To study the conversion of endogenous Neu5Ac to Neu5Gc by stimulating A549 human lung cancer cells to produce ROS;（6）ROS and Neu5Gc immunofluorescence assay was used to verify the endogenous Neu5Ac-Neu5Gc transformation reaction and the correlation between ROS level and Neu5Gc expression.The experimental results show that:（1）In the buffer reaction in vitro,the retention time of the chromatographic peaks of the control group and the experimental group was within 1%（RE%）and the mass spectrometry showed very similar parent and daughter ions.The abundance ratio of fragment ions to molecular ions was within 20%of that of the Neu5Gc standard.The results indicate that Neu5Ac reacts with H₂O₂and iron（II）（Fe（II））to form Neu5Gc with the conversion rate of 0.01%-0.025%.（2）Isotopically labeled Neu5Ac was utilized to further proved that Neu5Gc could be converted from Neu5Ac in the presence of oxidation factors.（3）Neu5Ac was converted to Neu5Gc in the human serum in the presence of H₂O₂and Fe（II）,and the concentration rate is related to the concentrations of H₂O₂and Fe（II）.（4）Cell culture experiments further proved that endogenous Neu5Ac could be converted into Neu5Gc in cell culture,and correlated with the concentration of ROS inducing agent.The study found that not only the free stand Neu5Ac,but also the glycosidic bond linked Neu5Ac on the cell surface can be converted to Neu5Gc due to the increase of ROS.（5）Immunofluorescence assay verified that cells could endogenously produce Neu5Gc under the condition of high ROS level,and there was a positive correlation between ROS level and Neu5Gc expression.In conclusion,this study suggests that ROS is involved in the chemical pathway of Neu5Ac acetyl hydroxylation,and this could be another mechanism for Neu5Gc expression in inflammatory and tumor environments.The results provide a new framework for the expression mechanism of Neu5Gc in various diseases,elucidate the correlation between high levels of ROS and Neu5Ac hydroxylation in cancer cells,and also provide a new reference for the mechanism of similar transformation of other biomolecules.