Characterizing the Non-reducing End Structure of Heparan Sulfate

We compare here the structural and functional properties of heparan sulfate (HS) chains from both male or female adult mouse liver through a combination of molecular sieving, enzymatic cleavage, and strong anion exchange-HPLC. The results demonstrated that male and female HS chains are significantly different by a number of parameters; size determination showed that HS chain lengths were ϳ100 and ϳ22 kDa, comprising 30 -40 and 6 -8 disaccharide repeats, respectively. Enzymatic depolymerization and disaccharide composition analyses also demonstrated significant differences in domain organization and fine structure. N-Unsubstituted glucosamine (⌬HexA-GlcNH 3 ؉ , ⌬HexA-GlcNH 3 ؉ (6S), ⌬HexA(2S)-GlcNH 3 ؉ , and N-acetylglucosamine (⌬HexA-GlcNAc) are the predominant disaccharides in male mouse liver HS. However, N-sulfated glucosamine (⌬HexA-GlcNSO 3 ) is the predominant disaccharide found in female liver. These structurally different male and female liver HS forms exert differential effects on human mesenchymal cell proliferation and subsequent osteogenic differentiation. The present study demonstrates the potential usefulness of gender-specific liver HS for the manipulation of human mesenchymal cell properties, including expansion, multipotentiality, and subsequent matrix mineralization. Our results suggest that HS chains show both tissue-and gender-specific differences in biochemical composition that directly reflect their biological activity.Heparan sulfate proteoglycan structure classically consists of a core protein to which one or more linear HS glycosaminoglycan (GAG) 2 chains are attached at specific serineglycine residues. Heparan sulfates have complex sulfated domain substructures that are initially synthesized as nonsulfated polysaccharides of D-glucuronic acid-N-acetyl-Dglucosamine (GlcA-GlcNAc) repeats (1-4). These linear, sulfated glycosaminoglycans have molecular masses that typically range from 10 to 100 kDa (5). Concurrent with the polymerization of an HS chain is a series of enzymatic modifications that generate the diverse sulfated domain clusters at intervals along the growing chain. The non-templatedriven diversity of HS structure is thought to be capable of giving rise to a wide range of biological functions through selective binding.Several studies have demonstrated that the binding of growth factors to HS that gives rise to mitogenic or adhesive activity occurs only when specific structural features are present within the HS chain (6). Such features include sulfation at specific positions within a given sequence of disaccharides; 6-O-sulfated N-sulfate glucosamine and 2-O-sulfated iduronic acid residues are thought to be particularly important, and minimum binding sequences are generally at least 5 or 6, and often 8 -14 disaccharides in length (7-9). The precise structures within HS sulfated domains that are involved in these interactions have remained elusive. Variations in composition and the organization of HS from different cells and tissues have confirmed the relationship between HS stru...

Section: Discussionsupporting

confidence: 82%

Comparative Assessment of the Effects of Gender-specific Heparan Sulfates on Mesenchymal Stem Cells

Murali

Leong

Lee

et al. 2011

“…Combining the present data with information contained in previous reports (27,33), we are able to propose a model for NRE NS domains. (a) HS chains that terminate in NS domains have a high probability of ending with particular N-sulfated disaccharides, and we observe enrichment of U0S0 in bovine, murine, and rat samples (present data and see Ref.…”

Section: Discussionmentioning

confidence: 70%

“…Few structural analyses of the NRE of HS have been completed. One study provided evidence that bovine kidney HS terminated in NS domains at least a hexamer in size and that these structures contained a GlcUA residue at the NRE (33). The epimerization state of the chain terminus was based on glucuronidase digestion of the only saturated disaccharide that was detected in a complete lyase depolymerization of HS.…”

Section: Discussionmentioning

confidence: 99%

Extended N-Sulfated Domains Reside at the Nonreducing End of Heparan Sulfate Chains

Staples

Shi

Zaia

2010

Heparan sulfate (HS) serves as a cell-surface co-receptor for growth factors, morphogens, and chemokines. These HS and protein binding events depend on the fine structure and distribution of domains along an HS chain. A given domain can vary in terms of uronic acid epimer, N-and O-sulfate, and N-acetate content. The most highly sulfated regions of HS chains, N-sulfated (NS) domains, play prominent roles in HS and protein binding. We have analyzed HS oligosaccharides from various mammalian sources and provide evidence that NS domains residing at the nonreducing end (NRE) are, on average, longer than those residing in the internal regions of the chain. Additionally, they are more highly sulfated than their internal counterparts. These features are independent of the sulfation pattern of the bulk HS chains. From disaccharide analysis, it is clear that NS domains do not always occupy HS NREs. However, when they do, they tend to terminate in a subset of N-sulfated disaccharides. Our observations are consistent with a significant role of NRE NS domains in HS-growth factor interactions.

“…In addition, Chinese hamster ovary cells lacking GlcNAc-TII activity still display GlcNAc-TI activity but lack HS chains (35) Moreover, this mutant accumulates a pentasaccharide intermediate with the structure GlcNAc-GlcA-Gal-Gal-Xyl (36), favoring the concept that EXTL3 catalyzes the initiation rather than the termination of HS chain elongation. It has been proposed in a recent report that a GlcA residue is present at the nonreducing end of the HS chains (37). EXTL3 harbors no GlcA-T activities (15), so it would be unlikely to catalyze chain termination.…”

Section: Discussionmentioning

confidence: 99%

Contribution of EXT1, EXT2, and EXTL3 to Heparan Sulfate Chain Elongation

Busse

Feta

Presto

et al. 2007

178

158

The exostosin (EXT) family of genes encodes glycosyltransferases involved in heparan sulfate biosynthesis. Five human members of this family have been cloned to date: EXT1, EXT2, EXTL1, EXTL2, and EXTL3. EXT1 and EXT2 are believed to form a Golgi-located hetero-oligomeric complex that catalyzes the chain elongation step in heparan sulfate biosynthesis, whereas the EXTL proteins exhibit overlapping glycosyltransferase activities in vitro, so that it is not apparent what reactions they catalyze in vivo. We used gene-silencing strategies to investigate the roles of EXT1, EXT2, and EXTL3 in heparan sulfate chain elongation. Small interfering RNAs (siRNAs) directed against the human EXT1, EXT2, or EXTL3 mRNAs were introduced into human embryonic kidney 293 cells. Compared with cells transfected with control siRNA, those transfected with EXT1 or EXT2 siRNA synthesized shorter heparan sulfate chains, and those transfected with EXTL3 siRNA synthesized longer chains. We also generated human cell lines overexpressing the EXT proteins. Overexpression of EXT1 resulted in increased HS chain length, which was even more pronounced in cells coexpressing EXT2, whereas overexpression of EXT2 alone had no detectable effect on heparan sulfate chain elongation. Mutations in either EXT1 or EXT2 are associated with hereditary multiple exostoses, a human disorder characterized by the formation of cartilage-capped bony outgrowths at the epiphyseal growth plates. To further investigate the role of EXT2, we generated human cell lines overexpressing mutant EXT2. One of the mutations, EXT2-Y419X, resulted in a truncated protein. Interestingly, the capacity of wild type EXT2 to enhance HS chain length together with EXT1 was not shared by the EXT2-Y419X mutant.