Heparan Sulfate

Heparan sulfate is a sulfated polysaccharide that is found on the surface of most cells as part of proteoglycans. Heparan sulfate is also present in the extracellular matrix. The polysaccharide mediates the interactions between a number of different proteins.

In Vitro-Generated Heparan Sulfate Libraries

In vitro-generated libraries are important research tools in protein and nucleic acid biochemistry. For example, enzyme libraries can be created by synthesizing random segments of DNA and then ligate them into an existing gene. Also, the whole coding region can be subjected to random mutagenesis or DNA shuffling. The mutated genes are then heterologously expressed in different systems and variant enzymes or other proteins with desired properties are screened or selected for. Libraries of RNA, obtained by a combination of organic synthesis and RNA polymerase-catalyzed reactions, are used to develop new catalytic RNA molecules. Peptide libraries are screened to find protease inhibitors.

A large class of biopolymers that may also be investigated by use of in vitro-generated libraries is the polysaccharides. Heparan sulfate is a complex polysaccharide that is suitable to explore using a library approach.

To address this we set out to make "biosynthetic" in vitro-generated libraries consisting of enzymatically sulfated heparan sulfate oligomers. These will be used as a complement to tissue-derived heparan sulfate in the search for heparan sulfate structures that bind to different proteins or function as substrates for enzymes involved in heparan sulfate metabolism.

Briefly, our strategy was to use size-defined heparan sulfate-related oligomers devoid of either O-sulfate groups or both O-sulfate and N-sulfate groups, and to sulfate these enzymatically in the test tube. This was achieved by incubating terminally ³H-labeled fragments with an extract of solubilized sulfotransferases together with PAPS as sulfate donor. To prove the functionality of the heparan sulfate-mimicing oligosaccharides obtained, products expressing the structural features required for protein binding are affinity-captured and subjected to sequence analysis.

A detailed description of how to generate these libraries, and their application as a tool to identify structure-function relationships for heparan sulfate protein interactions are presented in:

Jemth, P., Kreuger, J., Kusche-Gullberg, M., Sturiale, L., GimÈnez-Gallego, G., and Lindahl, U. (2002) Biosynthetic oligosaccharide libraries for identification of protein-binding heparan sulfate motifs. Exploring the structural diversity by screening for fibroblast growth factor (FGF) 1 and FGF2 binding. J. Biol. Chem. 277, 30567-30573 [abstract]

Heparan Sulfate - Protein interactions

All the possible modifications of heparan sulfate give rise to a heparan sulfate "sequence". Thus, there are several different motifs along the heparan sulfate chain, which may or may not interact with different proteins. It is therefore likely that the physiological effect of these different proteins, which are often growth factors, are in some way regulated by the polysaccharide.

Except for the antithrombin binding motif, detailed structures of heparan sulfate molecules that interact with distinct proteins have only recently been elucidated with the advent of heparan sulfate sequencing methodology.

Attempts to identify protein binding heparan sulfate motifs previously relied on saccharide isolated from different tissues. Because of the high variability among heparan sulfate molecules from different cells there is a risk that the epitope that interacts with a protein in vivo is not present in the sample used in the in vitro screening, and the epitope could escape discovery. Therefore we created heparan sulfate libraries in vitro.

Thus, we tried to find out whether or not there are indeed messages "encoded" by heparan sulfate. We did this by screening heparan sulfate oligosaccharide libraries for binders toward different Fibroblast Growth Factors, FGFs. Isolated oligosaccharides were then subjected to sequence analysis. We also used a panel of purified and sequenced octasaccharides to probe the specificity of FGFs.

Our results showed that the specificities of FGFs 1, 2, 4, 7 and 8a towards heparan sulfate oligosaccharides were overlapping. We suggest that FGF signalling cannot be regulated by the specificity of the FGF-heparan sulfate interaction alone. It is possible that the ternary FGF-heparan sulfate-FGF receptor complex involves specific interactions but more work is needed to validate this hypothesis.

The results are described in:
Kreuger, J., Jemth, P., Sander-Lindberg, E., Eliahu, L., Ron, D., Basilico, C., Salmivirta, M., and Lindahl, U. (2005) Fibroblast growth factors share binding sites in heparan sulfate. Biochem. J. 389, 145-150 [abstract]