Glycomics of Proteoglycan Biosynthesis in Murine Embryonic Stem Cell Differentiation

Abstract

Glycosaminoglycans (GAGs) play a critical role in binding and activation of growth factors involved in cell signaling critical for developmental biology. The biosynthetic pathways for GAGs have been elucidated over the past decade and now analytical methodology makes it possible to determine GAG composition in as few as 10 million cells. A glycomics approach was used to examine GAG content, composition, and the level of transcripts encoding for GAG biosynthetic enzymes as murine embryonic stem cells (mESCs) differentiate to embryoid bodies (EBs) and to extraembryonic endodermal cells (ExE) to better understand the role of GAGs in stem cell differentiation. Hyaluronan synthesis was enhanced by 13-and 24-fold, most likely due to increased expression of hyaluronan synthase-2. Chondroitin sulfate (CS)/dermatan sulfate (DS) synthesis was enhanced by 4-and 6-fold, and heparan sulfate (HS) synthesis was enhanced by 5-and 8-fold following the transition from mESC to EB and ExE. Transcripts associated with the synthesis of the early precursors were largely unaltered, suggesting other factors account for enhanced GAG synthesis. The composition of both CS/DS and HS also changed upon differentiation. Interestingly, CS type E and highly sulfated HS both increase as mESCs differentiate to EBs and ExE. Differentiation was also accompanied by enhanced 2-sulfation in both CS/DS and HS families. Transcript levels for core proteins generally showed increases or remained constant upon mESC differentiation. Finally, transcripts encoding selected enzymes and isoforms, including GlcNAc-4,6-O-sulfotransferase, C5-epimerases, and 3-O-sulfotransferases involved in late GAG biosynthesis, were also enriched. These biosynthetic enzymes are particularly important in introducing GAG fine structure, essential for intercellular communication, cell adhesion, and outside-in signaling. Knowing the changes in GAG fine structure should improve our understanding the biological properties of differentiated stem cells.

Transcriptional Regulation of the Protocadherin β Cluster during Her-2 Protein-induced Mammary Tumorigenesis Results from Altered N-Glycan Branching

“…Primers used in the qRT-PCR analysis are listed in supplemental Table S1. Real time reactions were performed using the iQ TM SYBR Green Supermix (Bio-Rad) as reported previously (29). All PCRs were performed in triplicate samples and repeated at least two times.…”

LacdiNAc (GalNAcβ1-4GlcNAc) Contributes to Self-Renewal of Mouse Embryonic Stem Cells by Regulating Leukemia Inhibitory Factor/STAT3 Signaling

“…It has recently been demonstrated that the cell surface glycan heparan sulfate (HS) contributes to self-renewal and differentiation of mESCs by regulating BMP, Wnt, and FGF signaling [21,[27][28][29]. The expression patterns of several other cell surface glycans have now been described in undifferentiated and differentiated mESCs and hESCs [30][31][32][33][34]. However, although these glycans may be involved in the regulation of the signaling required for ESC self-renewal, with the exception of HS, their functional roles have not been demonstrated.…”

Loss of expression of N‐acetylglucosaminyltransferase Va results in altered gene expression of glycosyltransferases and galectins

“…We report here, for the first time, that the genetically targeted deletion of GnT-Va causes the altered gene expression of several glycosyltransferases and galectins in MEFs using a newly developed qRT-PCR transcript analysis platform [11]. We speculate that some of the inhibited invasiveness-related phenotypes in MEFs caused by deletion of GnT-Va may result, at least in part, from the combination of altered expression of these glycosyltansferases and/or galectins.…”