Abstract
This study reports a global glycoproteomic analysis of pancreatic cancer cells that describes how flux through the sialic acid biosynthetic pathway selectively modulates a subset of N-glycosylation sites found within cellular proteins. These results provide evidence that sialoglycoprotein patterns are not determined exclusively by the transcription of biosynthetic enzymes or the availability of N-glycan sequons; instead, bulk metabolic flux through the sialic acid pathway has a remarkable ability to increase the abundance of certain sialoglycoproteins while having a minimal impact on others. Specifically, of 82 glycoproteins identified through a mass spectrometry and bioinformatics approach, ≈ 31% showed no change in sialylation, ≈ 29% exhibited a modest increase, whereas ≈ 40% experienced an increase of greater than twofold. Increased sialylation of specific glycoproteins resulted in changes to the adhesive properties of SW1990 pancreatic cancer cells (e.g. increased CD44-mediated adhesion to selectins under physiological flow and enhanced integrin-mediated cell mobility on collagen and fibronectin). These results indicate that cancer cells can become more aggressively malignant by controlling the sialylation of proteins implicated in metastatic transformation via metabolic flux.
Highlights
The surfaces of mammalian cells are covered with a dense layer of carbohydrates, collectively known as the glycocalyx, that influence many aspects of the interaction between a cell and its microenvironment
These findings expand the role of metabolic flux in controlling glycosylation beyond the HBP and provide a foundation to explore the hypothesis that cancer cells modulate their metastatic potential and malignant progression via changes to bulk metabolic flux through the sialic acid biosynthetic pathway
Flow-based Adhesion Assays—To simulate the physiological shear environment of the vasculature, pancreatic carcinoma cells in D-PBS containing Caϩ2/Mgϩ2, 0.1% bovine serum albumin were perfused over immobilized E, or L-selectin-coated dishes at prescribed wall shear stresses using a parallel plate flow chamber (250-m channel depth, 5.0-mm channel width)
Summary
Materials: Adhesion Molecules, Antibodies, and Reagents—E-selectin-IgG Fc (E-selectin) L-selectin-IgG Fc (L-selectin), P-selectin-IgG Fc (P-selectin), and unlabeled anti-CD44 mAbs (2C5) were purchased from R & D Systems (Minneapolis, MN). Monoclonal antibody binding to determine LeX, sLeX, and sLeA expression was performed by washing 500,000 cells with D-PBS and incubating them with antihuman CD15 FITC (eBioscience Inc., San Diego, CA), 10 g/ml of mAb PE-labeled HECA 452 (PharMingen, San Diego, CA) in 100 l D-PBS with 0.1% BSA, or anti-sialyl Lewis A (Millipore, Billerica, MA), respectively. The blots were placed under a parallel plate flow chamber CHO transfectants expressing P- or E-selectin, resuspended at 1.0 ϫ 106 cells/ml in 90% D-PBS/10% glycerol, and perfused at the shear stress of 0.5 dynes/ cm2 [16]. Cells were plated and 4.0 l from a 50 mM stock solution of 1,3,4-O-Bu3ManNAc was added to treated samples (to give a final analog concentration of 100 M) and 4.0 l of 100% ethanol was added to the controls. Statistical significance of differences between means was determined by analysis of variance
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
