C₅‑Epimerase and 2‑<i>O</i>‑Sulfotransferase Associate <i>in Vitro</i> to Generate Contiguous Epimerized and 2‑<i>O</i>‑Sulfated Heparan Sulfate Domains

Heparan sulfate (HS), a complex polysaccharide of the cell surface, is endowed with the remarkable ability to bind numerous proteins and, as such, regulates a large variety of biological processes. Protein binding depends on HS structure; however, in the absence of a template driving its biosynthesis, the mechanism by which protein binding sequences are assembled remains poorly known. Here, we developed a chemically defined ¹³C-labeled substrate and NMR based experiments to simultaneously follow in real time the activity of HS biosynthetic enzymes and characterize the reaction products. Using this new approach, we report that the association of C₅-epimerase and 2-<i>O</i>-sulfotransferase, which catalyze the production of iduronic acid and its 2-<i>O</i>-sulfation, respectively, is necessary to processively generate extended sequences of contiguous IdoA2S-containing disaccharides, whereas modifications are randomly introduced when the enzymes are uncoupled. These data shed light on the mechanisms by which HS motifs are generated during biosynthesis. They support the view that HS structure assembly is controlled not only by the availability of the biosynthetic enzymes but also by their physical association, which in the case of the C₅-epimerase and 2-<i>O</i>-sulfotransferase was characterized by an affinity of 80 nM as demonstrated by surface plasmon resonance experiments

Characterization of the LcrV/IFNγ interaction by intrinsic fluorescence studies.

<p>(A) Effects of NBS modification on LcrV spectral properties. LcrV was incubated with an 80-fold molar excess of NBS, and the resulting fluorescence (grey curve) was compared to that of wild-type LcrV (black curve). The fluorescence emission spectrum was recorded over the range 310–370 nm after excitation at 295 nm. (B) Evolution of the relative fluorescence intensities over injection of increasing concentrations of oxidized LcrV (LcrVox). Fitting of the data was performed as described in the experimental section. The LcrV curve was not shown for clarity. F_IFNγ; intensity of the IFNγ spectrum; F_NATA, intensity of the NATA spectrum.</p

Inhibition of LcrV/IFNγ interaction by different synthetic oligosaccharides.

<p>(A) Formula of the different oligosaccharides used in this study. m, number of disaccharide repeats, n = number of ethylene glycol repeats. Adapted from ref 51. (B) Each inhibitor tested was incubated with IFNγ, and complexes were injected over immobilized LcrV (2000 RU) at 10 µL/min. Non-specific binding to the sensor chip was subtracted for each injection. The percentage of inhibition is represented for three independent experiments, and the standard error of the mean is indicated (bars). The mean response in the absence of inhibitor (0% of inhibition) was equal to 270 RU. (C) IFNγ was pre-incubated with a range of concentrations of 2O₃₂. Each reaction mixture was injected over immobilized LcrV (1150 RU) at 10 µL/min. Non-specific binding to the sensor chip was subtracted for each injection.</p

Free IFNγ interacts with LcrV, but not with PcrV, in a surface plasmon resonance assay.

<p>(A) Scatchard analysis of IFNγ binding to LcrV immobilized on a CM3 sensorchip (1150 RU). R_eq, steady state value at equilibrium; C, concentration of injected IFNγ. (B) 5.0 µg/mL IFNγ was injected over LcrV (2700 RU, black curve) and PcrV (1200 RU, grey curve) immobilized on two different lanes of the same sensorchip. (C) Injection over immobilized LcrV (1150 RU) of 2 µg/mL IFNγ (black curve) or of 2 µg/mL IFNγ and an equimolar amount of IFNγR (grey curve). RU =  resonance units. (D) Injection of 1 µg/mL IFNγ alone (black curve) or in combination with LcrV (1 µg/mL of each, grey curve) over immobilized IFNγR (1600 RU). For all experiments, non-specific binding to the sensor chip was subtracted from the raw data.</p

CXCL12γ has an unstructured C-terminal domain but is identical to CXCL12α in the 1-68 region.

<p>(A) Sequences of the wild type and mutant CXCL12α, β and γ isoforms produced and used in this study (mutated residues are underlined). The secondary structures of CXCL12γ 1–68 domain and CXCL12α are almost identical (black boxes: α helices, white arrows: β strands, E: extended conformation). (B) ¹⁵N-HSQC spectrum of CXCL12γ (1 mM, 30°C), on which only non overlapping amide protons were indicated for clarity. Residues from the γ extension are clustered between 8–8.5 ppm ¹H frequency. (C) CXCL12γ 1–68 domain and CXCL12α fold similarly. A good correlation (Chi² = 74) is observed between N-H^N RDCs (CXCL12γ 10–64) with RDCs backcalculated from the CXCL12α structure. (D) ¹⁵N-¹H heteronuclear NOes, longitudinal (R1; square) and transversal (R2; triangle) relaxation rates on CXCL12γ. CXCL12γ is folded between residues 10 and 64 and the γ extension is disordered with low NOe, R2 and R1 values.</p

Flow cytometry analysis of CXCL12 interaction with cell surface GAGs.

<p>CHO-K1 parental cells (squares) or HS-deficient CHO-pgsD677 cells (triangles) were incubated with the indicated concentrations of CXCL12 α (open symbols) or γ (close symbols) and, after extensive washing to remove free chemokine, were labelled with K15C mAb and analyzed by flow cytometrey.</p

Comparative analysis of the CXCR4 binding and signaling properties of CXCL12α and γ.

<p>(A) ¹²⁵I-CXCL12α (0.25 nM) was bound to CXCR4⁺ CEM cells in the presence of cold CXCL12α (squares), γ (triangle) or γ-m1 (circle). (B) Intracellular calcium mobilization induced by CXCL12α (squares), γ (triangles) isoforms or CXCL12α P2G (line) in A3.01 cells. CXCL12α P2G is a non signaling mutant of CXCL12α. Data are representative of three independent experiments.</p

Analysis of CXCL12 binding to HP, HS and DS.

<p>SPR sensorgrams measured when CXCL12 were injected over HP, HS or DS activated sensorchips. The response in RU was recorded as a function of time for CXCL12α (26 to 300 nM), β (13 to 150 nM) and γ (2.6 to 30 nM).</p

Analysis of wild type and mutant CXCL12 binding to immobilized GAGs.

<p>Binding of wild type and mutant CXCL12 were recorded as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001110#pone-0001110-g002" target="_blank">Fig. 2</a>. CXCL12α (26 to 300 nM), β, β-m1, β-m2 (13 to 150 nM), γ, γ-m1, γ-m2 (2.6 to 30 nM) were injected over GAG activated sensorchips and the response in RU was recorded as a function of time.</p

Association and dissociation rate constant of the CXCL12-GAG interaction.

<p>(A) Graphical summary of the data generated from the sensorgrams of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001110#pone-0001110-g004" target="_blank">Fig. 4</a>, in which association (k_on) and dissociation (k_off) rate constants of CXCL12α (open circle), β (grey circle), β-m1 (grey square), γ (black circle) and γ-m1 (black square) for HP were determined as described. Differences were essentially observed along the k_off axis. (B) Dissociative half live of the different CXCL12/HP complexes.</p