Glycosylation modification serves as a pivotal quality attribute in glycoprotein-based therapeutics, emphasizing its role in drug safety and efficacy. Prior studies have underscored the potential immunogenic risks posed by the presence of galactose-α-1,3-galactose (α-Gal) and N-glycolylneuraminic acid (NeuGc) in glycoprotein formulations. This accentuates the imperative for developing robust qualitative and quantitative analytical methods to monitor these immunogenic epitopes, thereby ensuring drug safety. In the present investigation, α-Gal and NeuGc were accurately quantified using UPLC-FLR-MS/MS at the oligosaccharide level. Remarkably, α-Gal could be identified when the ion intensity ratio or the mass-to-charge ratio (m/z) of 528.19 to 366.14 exceeded 1. Concurrently, NeuGc and N-acetylneuraminic acid (NeuAc) could be unambiguously identified through their respective fragment ions at m/z 673.23 and m/z 657.23. Furthermore, relative quantification of α-Gal and NeuGc was achieved using fluorescence signals. This study introduces a sensitive and reliable analytical approach for the qualitative and quantitative assessment of α-Gal and NeuGc in glycoprotein pharmaceuticals. The methodology offers significant potential for application in process control and optimization of glycoprotein production, aimed at minimizing immunogenicity and enhancing product quality.
Obesity has become a common health concern around the world. Maternal obesity could cause poor reproductive outcomes due to chronic ovarian inflammation and decreased oocyte quality. However, the strategies to improve the poor reproductive outcomes of obese females have not been fully studied. In this study, we aimed to explore the effects and underlying mechanisms of nicotinamide mononucleotide (NMN) on oocyte quality and reproductive performance of obese mice. The obese mouse model was established by feeding high-fat diet which was confirmed by body weight record, fasting blood glucose test and oral glucose tolerance test. The expression of ovary development related genes and inflammation related genes, including Lhx8, Bmp4, Adgre1, Ccl2, TNF-α, Gal-3, Clec10a and IL-10 in ovaries and the expression of Bax and Sod1 in oocytes were detected using quantitative reverse transcription PCR (RT-qPCR). The adipose size of abdominal fat tissue was determined with haematoxylin and eosin (H&E) staining. Immunofluorescence staining was performed to measure the ROS level, spindle/chromosome structure, mitochondrial function, actin dynamics and DNA damage of oocytes. The administration of NMN restored ovarian weight and reduced the adipose size of abdominal fat tissue and ovarian inflammation in high fat diet (HFD) mice. Furthermore, NMN treatment improved the oocytes quality partially by restoring the mitochondrial function and actin dynamics, reducing meiotic defects, DNA damage and ROS level and lipid droplet distribution of oocytes in HFD mice. On the long-term effect, NMN restored offspring body weight of HFD mice. NMN could improve the oocyte quality of HFD-induced obese mice.
The enzyme α-galactosidase (α-GAL), a member of glycoside hydrolase family 27, catalyzes the removal of a nonreducing terminal α-galactose residue from polysaccharides, glycolipids, and glycopeptides. α-GAL is believed to have the double displacement retaining reaction mechanism. In this work, the glycosylation step catalyzed by human α-GAL was computationally simulated with quantum mechanics/molecular mechanics metadynamics. Our simulations show that the overall catalytic mechanism follows a D(N)*A(N)-like mechanism, and the transition state has a oxocarbenium ion like character with a partially formed double bond between the ring oxygen and C5' carbon atoms. In addition, the galactosyl ring of the substrate follows a conformational itinerary of (4)C(1) → [E(3)/(4)H(3)](++) → (1)S(3) along the reaction coordinate.