A strategy for rapid sequencing of heparan sulfate and heparin saccharides

Heparin and heparan sulfate (HS) are structurally diverse glycosaminoglycans (GAG) that are known to interact, via unique structural motifs, with a wide range of functionally distinct proteins and modulate their biological activity. To define the GAG motifs that interact with proteins, we assessed the ability of 15 totally synthetic HS mimetics to interact with 10 functionally diverse proteins that bind heparin/HS. The HS mimetics consisted of cyclitol-based pseudo-sugars coupled by linkers of variable chain length, flexibility, orientation, and hydrophobicity, with variations in sulfation also being introduced into some molecules. Three of the proteins tested, namely hepatocyte growth factor, eotaxin, and elastase, failed to interact with any of the sulfated linked cyclitols. In contrast, each of the remaining seven proteins tested exhibited a unique reactivity pattern with the panel of HS mimetics, with tetrameric cyclitols linked by different length alkyl chains being particularly informative. Thus, compounds with short alkyl spacers (2-3 carbon atoms) effectively blocked the interaction of fibroblast growth factor-1 (FGF-1) and lipoprotein lipase with heparin/HS, whereas longer chain spacers (7-10 carbon atoms) were required for optimal inhibition of FGF-2 and vascular endothelial growth factor binding. This effect was most pronounced with the chemokine, interleukin-8, where alkyl-linked tetrameric cyclitols were essentially inactive unless a spacer of >7 carbon atoms was used. The heparin-inhibitable enzymes heparanase and cathepsin G also displayed characteristic inhibition patterns, cathepsin G interacting promiscuously with most of the sulfated cyclitols but heparanase activity being inhibited most effectively by HS mimetics that structurally resemble a sulfated pentasaccharide. These data indicate that a simple panel of HS mimetics can be used to probe the HS binding specificity of proteins, with the position of anionic groups in the HS mimetics being critical. Heparan sulfate (HS)1 is a glycosaminoglycan that is ubiquitously expressed as a proteoglycan on cell surfaces and throughout the extracellular matrix (ECM) in most multicellular animals (1-5). The polysaccharide is composed of alternating glucuronic acid and N-acetylglucosamine units, which, during biosynthesis, are subjected to a range of modifications such as O-sulfation at various positions, N-deacetylation, and Nsulfation of N-acetylglucosamine residues as well as C-5 epimerization of glucuronic acid to iduronic acid. Theoretically up to 48 different disaccharides could occur in HS due to these modifications, although so far only 23 have been identified (2). Nevertheless, as a result of the presence of these different disaccharides, HS chains exhibit remarkable structural heterogeneity. Such diversity is further enhanced by the considerable variation in chain length of the glycosaminoglycan. Heparin, a glycosaminoglycan with a much more restricted cellular distribution than HS, is structurally closely related to HS but has ϳ80% of its disacc...

Section: Discussionmentioning

confidence: 99%

Use of Sulfated Linked Cyclitols as Heparan Sulfate Mimetics to Probe the Heparin/Heparan Sulfate Binding Specificity of Proteins

Freeman¹,

Liu

Banwell

et al. 2005

“…Great technical progress has been made in HS sequencing and structural determination in recent years (21,24,26,(31)(32)(33), but HS whole chain sequencing has not been attempted yet. The identification of the NRE of HS may open a door to this avenue.…”

Section: Discussionmentioning

confidence: 99%

Characterizing the Non-reducing End Structure of Heparan Sulfate

Lech

2005

The reducing end of heparan sulfate has been known for a long time, but information on the non-reducing end has been lacking. Recent studies indicate that the non-reducing end of heparan sulfate might be the place where fibroblast growth factor signaling complex forms. The non-reducing end also changes with heparanase digestion and, thus, might serve as a marker for tumor pathology. Using high performance liquid chromatography-coupled mass spectrometry, we have identified and characterized the non-reducing end of bovine kidney heparan sulfate. We find that the nonreducing end region is highly sulfated and starts with a glucuronic acid (GlcA) residue. The likely sequence of the non-reducing end hexasaccharides is GlcA-GlcNS6S-UA؎2S-GlcNS؎6S-Ido2S-GlcNS؎6S (where GlcNS is N-sulfate-D-glucosamine, S is sulfate, UA is uronic acid, and Ido is iduronic acid). Our data suggests that the non-reducing end of bovine kidney heparan sulfate is not trimmed by heparanase and is capable of supporting fibroblast growth factor signaling complex formation. Heparan sulfate (HS)2 is a long linear polysaccharide attached to core proteins in the extracellular matrix and the surface of nearly all mammalian cells. The nascent HS chain consists of disaccharide repeats of glucuronic acid (GlcA) and N-acetyl-D-glucosamine (GlcNAc) (1-3). During the process of maturation, several modifications can occur on the HS chain, which include N-deacetylation and N-sulfation of GlcNAc, epimerization of GlcA to L-iduronic acid (IdoA), 2-O sulfation of IdoA, and 6-O and 3-O sulfation of the glucosamine (GlcN). These modifications usually focus on separate regions and result in domain structures (4 -6). It is the modified domains to which various extracellular proteins bind (2, 3). The region at the reducing end of HS is usually unmodified (5, 7-9). One major function of HS is to interact with both fibroblast growth factor (FGF) and its cognate receptor and promote the FGF signaling complex formation (10 -13). Defects in HS can cause complete losses of FGF as well as Hedgehog and Wingless signaling pathways and lead to severe abnormality in embryonic development (14,15). HS also plays important roles in cell migration and cancer cell metastasis (16).Despite these known structural information and biological functions of HS, the non-reducing end (NRE) of HS has not been studied. Assuming that each HS chain has a similar content of sulfation, the fact that the reducing end of HS is devoid of sulfation (7-9, 17) suggests that the NRE of HS might be sulfated and assume biological functions. Indeed, there are indications that HS uses its NRE to direct the FGF signaling complex formation (11,13,18). Consequently, an FGF signaling complex model where two molecules each of FGF and FGF receptor bind to the NREs of two approaching HS chains has been proposed (13). Whether the NRE of HS has the ability to support the complex formation relies on its structure.It is known that heparanase activity is closely related to the metastatic potential of tumor-derived cells ...

“…1). Partial cleavage at GlcNS residues (HNO 2 , pH 1.5) (6,8) yielded distinct peaks at positions corresponding to di-, tetra-, hexa-, and octasaccharides with the predicted number of N-sulfate groups (data not shown). Separate digestions of these oligosaccharide products with ␣-iduronidase and ␤-glucuronidase confirmed that most (Ͼ90%) of the hexuronates were IdoUA units, as expected for a heparin-derived material (data not shown).…”

Section: Methodsmentioning

confidence: 99%

Biosynthetic Oligosaccharide Libraries for Identification of Protein-binding Heparan Sulfate Motifs

Jemth

Kreuger

Kusche-Gullberg

et al. 2002

Heparan sulfate is crucial for vital reactions in the body because of its ability to bind various proteins. The identification of protein-binding heparan sulfate sequences is essential to our understanding of heparan sulfate biology and raises the possibility to develop drugs against diseases such as cancer and inflammatory conditions. We present proof-of-principle that in vitro generated heparan sulfate oligosaccharide libraries can be used to explore interactions between heparan sulfate and proteins, and that the libraries expand the available heparan sulfate sequence space. Oligosaccharide libraries mimicking highly 6-O-sulfated domains of heparan sulfate were constructed by enzymatic O-sulfation of O-desulfated, endgroup 3 H-labeled heparin octasaccharides. Acceptor oligosaccharides that were 6-O-desulfated but only partially 2-O-desulfated yielded oligosaccharide arrays with increased ratio of iduronyl 2-O-sulfate/glucosaminyl 6-Osulfate. The products were probed by affinity chromatography on immobilized growth factors, fibroblast growth factor-1 (FGF1) and FGF2, followed by sequence analysis of trapped oligosaccharides. An N-sulfated octasaccharide, devoid of 2-O-sulfate but with three 6-O-sulfate groups, was unexpectedly found to bind FGF1 as well as FGF2 at physiological ionic strength. However, a single 2-O-sulfate group in the absence of 6-O-sulfation gave higher affinity for FGF2. FGF1 binding was also augmented by 2-O-sulfation, preferentially in combination with an adjacent upstream 6-O-sulfate group. These results demonstrate the potential of the enzymatically generated oligosaccharide libraries.