Complex sugar chains decorate the cell surface. The cell makes and organizes these complicated molecules on their surface, for reasons best known to the cells themselves. We want to understand their function of sugar chains (glycans).
We know they help cells communicate about their past and future journeys within the developing body, exactly where they go and what they become during development. Human genetic disorders where just one step in their assembly is missing, and mental retardation, seizures, blindness and poor motor skills often result.
Embryonic stem cells have a particular set of glycans on their surface and it changes as the cells develop into different cell types. We have two major goals in this project. First, to identify the human embryonic stem (hES) cell glyco-signatures and then determine what changes as they differentiate into neural precursor cells (NPC). Second, to ask whether these glycan changes are important for correct timing and normal differentiation of the cells. What happens if we change or keep the original sugar coating? Does it change the cell’s fate?
We can produce substantial amounts of hES cells that uniformly transition into NPC. Because these are early days of understanding stem cells, it was important to analyze different hES cell lines that differentiate into NPC under two different conditions. Glycan analysis can be done indirectly using sugar-binding proteins called lectins, or directly by mass spectrometry of glycans released from proteins or lipids. Another way to infer an alteration in glycan composition is by changes in the expression of genes encoding enzymes that make glycan chains. This is called transcriptional profiling. If more glycan is needed, more enzyme may be needed to produce that glycan; less glycan involves reduced expression of those genes. Sometimes more than one enzyme (gene product) carries out the very synthetic same step, but one of the enzymes may prefer a certain kind of glycan over another.
We used both approaches (lectin binding and transcriptional profiling) in parallel. Lectins bind to various types of N- and O-linked glycans and we used an instrument that detects small changes in the binding of cell fragments to a battery of 40 lectins. Carefully worked out and standardized conditions showed that only a few lectins consistently increased binding during transitions from hES cells into NPC. These lectins recognized some changes, but were in the 2-3 fold range at most. Comparison of hES cells grown on mouse feeder-layers to those grown without feeders, showed mostly similar (not identical) patterns. The changes were subtle, so instead of continuing glycan analysis by mass spectrometry, we chose to analyze transcriptional profiles. Our goal was to identify genes whose expression changed quite substantially.
Initially, we used results obtained by Dr. Terskikh to profile the entire genome when hES cells developed into NPC. Here, subtle changes in glycosylation gene expression (2-3 fold increases/decreases) were near baseline making it hard to know if they were accurate. An exception was the chondroitin sulfate core protein, decorin, which increased >30-fold during differentiation. Since many glycosylation-related genes are expressed at low levels, we went to colleagues at the University of Georgia for analysis of >700 genes using Real Time PCR. Their 5 order of magnitude dynamic range and reproducibility of triplicate samples (+/-15-20%) is impressive.
These results showed no (<2-fold) changes in sugar precursor metabolism, N-linked glycan precursor synthesis, and only a decrease in tetra-branched chains N-glycans. Considerable increases (5→100 fold) in terminal modifications common to both N- and O-linked glycans (Sda, polysialic acids, Lewisa) in the different hES cells prepared by either method. Fringe genes encoding glycans that modify Notch signaling (LFNG, especially) increased. GPI anchor biosynthesis gene that adds a final mannose (PIGZ) is decreased as is palmitoylation of the anchors. Fine differences in HS-6 sulfation changed and one especially prominent gene that appears only in neural tissue (NDST4) showed a substantial increase. These transcriptional changes are more wide-ranging and different than what we could see using lectin profiles or direct analysis of N- and O-glycans. The two approaches do not necessarily give congruent results. These transcriptional changes allow us to focus on genes more likely to be affected by the knockdown strategies we planned for aim two. In that aim, we want to prevent the up-regulation of those specific genes to see if it affects differentiation. If preventing transcriptional increase (by siRNA) does have developmental consequences then we will be in a better position to analyze which specific glycans are important. Our top knockdown candidates are: Decorin, Sd antigen (B4GALNT2), polysialic acid (STaSia1, 2, 3), NDST4 and Lewis antigen (FT3).