Dayoung Park; Narine Arabyan; Cynthia Williams; Ting Song; Bart Weimer; Carlito Lebrilla
University of California, Davis, Davis, CA
Methodology to analyze thousands of membrane glycan structures simultaneously with linkage and isomer differentiation and identify glycans involved in infection.
Cell membranes consist of proteins that contain short saccharide chains called glycans, which mediate nearly all interactions. In the gastrointestinal tract, a single layer of epithelial cells lines the inner layer as a protective interface between the embedded tissues and microorganisms that continually contact the internal organs. Regulation of these bacterial-host interactions is critical for host health. Using high resolution mass spectrometric techniques, a glycomic analysis was performed to characterize glycosylation changes on epithelial cell surfaces upon prolonged contact with foreign and resident bacteria of the gut. This new technique allows for rapid profiling and quantitation of membrane glycans based on retention time and accurate mass and was used to understand which glycans are involved in bacterial colonization and infection.
Epithelial colorectal adenocarcinoma Caco-2 cells were cultured in vitro. Selected bacterial genes that express glycosidases were knocked out. Upon infection with wildtype bacteria and the knockouts, cells were lysed using probe sonication and the membrane fraction was extracted from the resulting lysate by ultracentrifugation. Glycans were enzymatically released from isolated cell membrane glycoproteins and analyzed by microfluidic chip-based nano-LC quadrupole time-of-flight mass spectrometry. The total glycan profile of detected membrane glycans were constructed using a theoretical, retrosynthetic mass library, providing glycan compositions based on known synthetic pathways. Changes in relative abundances of individual N-glycans were characterized statistically. To elucidate exact structures, tandem MS was employed and reduced glycans were digested with exoglycosidases for linkage information and isomeric differentiation.
The cell sample set was optimized using different cell amounts varying from one to four million cells. Samples with at least 2 million cells showed the highest glycan signal and reproducibility.
Caco-2 cell surface glycosylation is dominated by sialylated and fucosylated complex and hybrid glycans, comprising almost three fourths of the total number of glycans. When considering the relative intensities, however, high mannose glycans are among the most abundant. These results suggest that Caco-2 membranes have a large amount of terminal mannose residues, which may have functional significance in epithelial cells during infection.
Bacterial infection times were varied to observe changes in Caco-2 membrane glycosylation. From 0 min to 45 min, only slight variations in glycan profiles were observed. At the 1 hr time point, there was a significant change in glycan compositions and relative abundances. Notably, nondecorated and high mannose glycans increased in signal after 1 hr of infection but later decreased past 2 hrs. By the third hour of infection, the overall profile recovered and resembled that of the uninfected set.
During the course of infection, levels of bisecting and triantennary complex glycans were significantly altered. The most abundant glycan in the uninfected sample, a bisecting, monofucosylated, bisialylated complex glycan, decreased dramatically in signal post-infection, becoming suppressed by other high abundant glycans. An isomer of this glycan, which eluted at a later time, increased in abundance fifteen fold after infection. Terminal fucose and sialic acid residues on a glycan with more than two antennas may act as receptors for bacteria and utilized as a source of energy, carbon, and nitrogen. Deficient glycan degrading enzyme activity of the bacteria led to an accumulation of certain oligosaccharide substrates on the cell surface. On average, 176 glycan compositions were identified in the uninfected sample and 166 compositions for the infected sample.