Study of the biosynthetic pathway and the role of heparan sulfate in biological systems

Abstract

We used the teratocarcinoma line NCCIT and human embryonic stem cells as models to study changes in the cellular proteoglycan composition along differentiation towards various cell lineages. Our analysis revealed retinoic acid-induced changes in the abundance of transcripts for genes encoding core proteins, enzymes that are responsible for early and late linkage region biosynthesis, as well as enzymes for GAG chain extension and modification in NCCIT cells. Disaccharide analysis of the glycans in HS/HP and chondroitin/dermatan sulfate revealed RA-induced changes restricted to chondroitin/dermatan sulfate glycans.

Heparan sulfate (HS) is a linear, highly charged acidic glycosaminoglycan (GAG) that interacts with multiple signaling molecules. These interactions have the ability to modulate signaling pathways that determine cell fate at various stages of development. HS is a polymeric carbohydrate with repeating disaccharide units consisting of N-acetylglucosamine / N-sulfoglucosamine and uronic acid. HS is attached to core proteins and localizes to the extracellular environment, associating with either the cell-surface or the extracellular matrix. A veritable orchestra of HS chain initiation, polymerization, and chain modification enzymes works together to biosynthesize HS chains with varying length and structure, which account for the prodigious heterogeneity of HS. Some of these enzymes have isozyme forms that possess unique spatial and temporal distribution.

Although the HS biosynthetic pathway has been analyzed previously, many aspects of it remain unresolved. For example, the factors that determine which isozyme is active and how that activity affects the final structure of HS is unknown. Another subject of interest is how heterogeneity of the HS chain affects its interaction with diverse biologically active molecules at different stages of development.

The HS biosynthetic pathway is a complex yet elegant system where the cooperative effort of various components results in HS chains of various structures important for interaction with different molecules. Understanding the details of this pathway will enable us to gain better control over it with the aim to produce HS chains of the desired structure, including the one with anticoagulant activity. This knowledge will provide us with powerful tools to control and direct stem cell differentiation.

Chinese hamster ovary (CHO) cells have been used as a model to examine the effect that HS pathway modification has upon HS structure. We bioengineered CHO cell clones with the aim of producing heparin (HP), the oversulfated anticoagulant version of HS. We introduced human N-deacetylase/N-sulfotransferase (NDST2) (responsible for N-sulfonation) and mouse heparan sulfate 3-O-sulfotransferase 1 (Hs3st1) (responsible for 3-O-sulfonation) enzymes into CHO cells. Although we have observed increased N-sulfonation of the HS chain in our clones, we detect only a modest increase in 3-O-sulfonation, which is important for anticoagulation activity.

We used murine mastocytoma cells (MST), natural producers of HP, to establish expression levels and localization of HS biosynthetic enzymes, and also to investigate expression levels of core proteins required to produce HS chains similar in structure to HP. Our aim is to apply knowledge gained from these experiments to our CHO cell system in order to produce HP. Although MST cells express non-anticoagulant HP, its structure is similar to the clinically relevant form of HP. When we compared MST cells to Dual-29 cells (a clonal line of NDST2 and Hs3st1 bioengineered CHO cells), we observed that all HS biosynthetic enzymes transcripts were expressed in both cell lines, but the protein levels of these enzymes were reduced less in MST cells than in Dual-29 cells. Ext2, Hs3st1 and Hs6st1 were not detected in MST cells, whereas they were observed in Dual-29 cells. MST cells expressed only glypican-1 and syndecan-1 proteoglycans core proteins, whereas Dual-29 expressed syndecan-1 and -3 and glypican-1, -2, -3, -5 and -6. When we compared MST-10H cells (HS3ST1 transfected) with Dual-29 cells, we also observed reduced enzyme expression in MST-10H cells compared to Dual-29 cells. However the expression pattern of core proteins was the same for both MST-10H and Dual-29 cells except for glypican-6, which was detected only in Dual-29 cells. Nevertheless, not only were trisulfated disaccharide levels increased, but in MST-10H cells we also detected 3-O-sulfo group-containing structures, which are required for anticoagulation activity. The reason why we observe all biosynthetic enzymes including Hs3st1 being expressed in Dual-29 cells despite producing less sulfated HS compared to MST and MST-10H cells and how transfection of Hs3st1 affects trisulfation of the HS structure, are questions that remain to be answered.

Human embryonic stem cell line WA09 (H9) was differentiated into Isl-1 and early hepatic cells and glycomic changes accompanying these transitions were studied. Pluripotent H9 cells use the non-glycosylated form of lumican whereas Isl-1 cells use the glycosylated form. The most dramatic difference among HS biosynthetic enzymes was observed in expression of Hs3st2, which was reduced ~29-fold in Isl-1 cells. H9 cells use simple primarily non-sulfated HS chains whereas upon differentiation towards both Isl-1 and hepatic lineages N-sulfonation increases, with the greatest change being observed in the structure of HS from early hepatic cells.