Integrated Approach to Identify Heparan Sulfate Ligand Requirements of Robo1

Abstract

An integrated methodology is described to establish ligand requirements for heparan sulfate (HS) binding proteins based on a workflow in which HS octasaccharides are produced by partial enzymatic degradation of natural HS followed by size exclusion purification, affinity enrichment using an immobilized HS-binding protein of interest, putative structure determination of isolated compounds by a hydrophilic interaction chromatography-high-resolution mass spectrometry platform, and chemical synthesis of well-defined HS oligosaccharides for structure-activity relationship studies. The methodology was used to establish the ligand requirements of human Roundabout receptor 1 (Robo1), which is involved in a number of developmental processes. Mass spectrometric analysis of the starting octasaccharide mixture and the Robo1-bound fraction indicated that Robo1 has a preference for a specific set of structures. Further analysis was performed by sequential permethylation, desulfation, and pertrideuteroacetylation followed by online separation and structural analysis by MS/MS. Sequences of tetrasaccharides could be deduced from the data, and by combining the compositional and sequence data, a putative octasaccharide ligand could be proposed (GlA-GlcNS6S-IdoA-GlcNS-IdoA2S-GlcNS6S-IdoA-GlcNAc6S). A modular synthetic approach was employed to prepare the target compound, and binding studies by surface plasmon resonance (SPR) confirmed it to be a high affinity ligand for Robo1. Further studies with a number of tetrasaccharides confirmed that sulfate esters at C-6 are critical for binding, whereas such functionalities at C-2 substantially reduce binding. High affinity ligands were able to reverse a reduction in endothelial cell migration induced by Slit2-Robo1 signaling.

Figure 1

Composition analysis of the octasaccharide mixture, the GFP-bound fraction, and the Robo1-bound fraction. Each composition is given as follows: [ΔHexA, HexA, GlcN, Ac, SO₃]. Abundances are relative to the overall amount of HS detected in that sample and are not directly comparable between samples. Inset: An enlarged view of the region with the most abundant compositions; the four compositions only observed significantly in the Robo1-bound fraction are indicated by red arrows, with the largest amount of specific binding being detected for the composition [1,3,4,1,7].

Figure 3

SPR sensorgram representing the concentration-dependent kinetic analysis of the binding of 1 with immobilized Robo1 on a CM5 chip. Concentration of compound 1 (from top to bottom): 100, 80, 40, 20, 10, 5, and 2.5 μM, fitted with a Langmuir 1:1 binding model (black lines).

Figure 4

Robo1-binding octa- and tetrasaccharides reverted the inhibition of Slit2–Robo1 signaling on mouse diaphragm endothelial cell migration. Serum-starved immortalized mouse lung endothelial cells in serum-free medium were seeded into the upper chamber of a RTCA CIM-16 plate (ACEA Bioscences). Slit2 (1 μg/mL) without or with oligosaccharide (50 μg/mL) was supplemented in serum-free medium in the lower chamber. Bovine serum albumin (BSA) was included as a negative control of Slit2. The cell migration to the lower side of the chamber membrane was monitored by a RTCA DP plate reader (ACEA Biosciences). Statistical analysis was carried out using two-tailed Student t test.

Scheme 1

Octasaccharide Synthesis^a