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.
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, end-group (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-O-sulfate. 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.
Jemth
Per
P
Department of Medical Biochemistry and Microbiology, Uppsala University, Biomedical Center, Box 582, SE-751 23 Uppsala, Sweden.
Kreuger
Johan
J
Kusche-Gullberg
Marion
M
Sturiale
Luisa
L
Giménez-Gallego
Guillermo
G
Lindahl
Ulf
U
D001665:Binding Sites; D002240:Carbohydrate Sequence; D016220:Fibroblast Growth Factor 1 / Q000378:metabolism*; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism*; D006497:Heparan Sulfate / Q000378:metabolism*; D006801:Humans; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D013429:Sulfatases; D013466:Sulfurtransferases / Q000378:metabolism
D016220:Fibroblast Growth Factor 1; D016222:Fibroblast Growth Factor 2; D006497:Heparan Sulfate; D009844:Oligosaccharides; D013429:Sulfatases; D013466:Sulfurtransferases; C043890:oligosaccharide 2-O-sulfatase