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

Abstract

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.

Oligosaccharide Library-based Assessment of Heparan Sulfate 6-O-Sulfotransferase Substrate Specificity

“…Thus, differential 2-O-sulfation of the IdoUA residues can give rise to 16 distinct octasaccharides; one without any 2-O-sulfates, four with one, six with two, four with three, and one with four 2-O-sulfate groups. Accordingly, anion exchange HPLC revealed five oligosaccharide families recognized through sequence analysis and previous analyses of HS and modified heparin octasaccharides (18,24,25) (Fig. 1A).…”

“…Lysates (corresponding to approximately 20 g of protein) from 6-OST2-or 6-OST3-transfected cells were incubated with 0.1-2 M octasaccharide, 5 mM PAPS, 10 mM MnCl 2 , 5 mM CaCl 2 , 10 mM MgCl 2 , 3.5 M NaF, 0.3% (v/v) Triton X-100, and 50 mM Hepes, pH 7.4, in a total volume of 25 l. The reactions were incubated at 37°C for various periods of time (1 min to overnight), depending on the desired degree of 6-O-sulfation and the enzymatic activity. Incubations with microsomal OSTs from a mouse mastocytoma were performed as described (18). The reactions were heat inactivated (90°C for 1 min) and centrifuged, and the supernatants analyzed by anion exchange chromatography using the ProPac PA1 column.…”

“…Elution profiles were monitored by scintillation counting and the central fractions from distinct peaks were pooled and desalted on PD-10 gel filtration columns (Sephadex G-25) (Amersham Biosciences). The resulting purified octasaccharide preparations were subjected to sequence analysis using a combination of chemical (HNO 2 ) and enzymatic (iduronate 2-sulfatase) cleavage as described previously (18,(23)(24)(25).…”

Isolation and Characterization of Low Sulfated Heparan Sulfate Sequences with Affinity for Lipoprotein Lipase

“…Individual charge homogeneous peaks were pooled and desalted on PD-10 columns before re-analysis on the anion-exchange column and partial deamination at pH 1.5. The deamination products were sequentially treated with iduronate-2-sulfatase and iduronidase, and the intermediate products of the sequencing reactions were separated on the anionexchange column and their elution profiles compared with profiles of characterized structures (6,34).…”

“…Sequencing of Affinity Isolated NS Fragments-Partial sequences of NS domains were determined essentially as described (6,34). Affinity-purified 8-mer N-sulfated [1-3 H]aMan R end group-labeled fragments were fractionated by anionexchange chromatography on a Propac column with a gradient of [NaCl] from 0 to 1.5 M at pH 3.0 with an increment of 10 mM/min.…”