Serum glycosylation characterization of osteonecrosis of the femoral head by mass spectrometry

Osteonecrosis of the femoral head is a recalcitrant and paralyzing disease often discovered in the end stage at the time of diagnosis, which is often performed by physical examination and diagnostic imaging. Osteonecrosis of the femoral head is typically caused by trauma or long-term steroid use. There are over 30 million patients in the US taking steroids, and roughly 40% will develop osteonecrosis of the femoral head. However, the exact pathophysiological process is not well understood. This study aims to examine the alteration in serum glycosylation of osteonecrosis of the femoral head using the state-of-the-art analytical tools to provide more chemical data for pathophysiology research and possibly biomarker discovery. A training set containing 27 serum samples from steroid-induced osteonecrosis of the femoral head patients and 25 from gender- and age-matched controls was collected and analyzed. Glycosylation of whole serum and site-specific glycosylation of immunoglobulins are characterized using electrospray ionization-Q-time of flight and electrospray ionization-Triple-Quadruple via multiple reaction monitoring, respectively. The whole serum glycosylation analysis yielded 14 N-glycan compositions and multiple reaction monitoring yielded eight glycopeptides that were altered between cases and controls with statistical significance. The increase of nonsialylated, nonfucosylated N-glycans and decrease of fucosylated N-glycans are associated with the development of osteonecrosis of the femoral head. Glycosylation is a posttranslational protein modification and is apparently affected by osteonecrosis of the femoral head. Future studies with a larger cohort and patients from earlier stage will be performed to assess these potential markers' value in disease onset

O-GlcNAcylation mediates metastasis of cholangiocarcinoma through FOXO3 and MAN1A1

The leading cause of death in cancer patients is metastasis, for which an effective treatment is still necessary. During metastasis, cancer cells aberrantly express several glycans that are correlated with poor patient outcome. This study was aimed toward exploring the effects of O-GlcNAcylation on membranous N-glycans that are associated with the progression of cholangiocarcinoma (CCA). Global O-GlcNAcylation in CCA cells was depleted using specific siRNA against O-GlcNAc transferase (OGT), which transfers GlcNAc to the acceptor proteins. Using an HPLC-Chip/Time-of-Flight (Chip/TOF) MS system, the N-glycans associated with O-GlcNAcylation were identified by comparing the membranous N-glycans of siOGT-treated cells with those of scramble siRNA-treated cells. In parallel, the membranous N-glycans of the parental cells (KKU-213 and KKU-214) were compared with those of the highly metastatic cells (KKU-213L5 and KKU-214L5). Together, these data revealed that high mannose (Hex9HexNAc2) and biantennary complex (Hex5HexNAc4Fuc1NeuAc1) N-linked glycans correlated positively with metastasis. We subsequently demonstrate that suppression of O-GlcNAcylation decreased the expression of these two N-glycans, suggesting that O-GlcNAcylation mediates their levels in CCA. In addition, the ability of highly metastatic cells to migrate and invade was reduced by the presence of Pisum Sativum Agglutinin (PSA), a mannose-specific lectin, further indicating the association of high mannose type N-glycans with CCA metastasis. The molecular mechanism of O-GlcNAc-mediated progression of CCA was shown to proceed via a series of signaling events, involving the activation of Akt/Erk (i), an increase in FOXO3 phosphorylation (ii), which results in the reduction of MAN1A1 expression (iii) and thus the accumulation of Hex9HexNAc2 N-glycans (iv). This study demonstrates for the first time the association between O-GlcNAcylation, high mannose type N-glycans, and the progression of CCA metastasis, suggesting a novel therapeutic target for treatment of metastatic CCA