Human follicular fluid heparan sulfate contains abundant 3-O-sulfated chains with anticoagulant activity

Abstract

Anticoagulant heparan sulfate proteoglycans bind and activate antithrombin by virtue of a specific 3-O-sulfated pentasaccharide. They not only occur in the vascular wall but also in extravascular tissues, such as the ovary, where their functions remain unknown. The rupture of the ovarian follicle at ovulation is one of the most striking examples of tissue remodeling in adult mammals. It involves tightly controlled inflammation, proteolysis, and fibrin deposition. We hypothesized that ovarian heparan sulfates may modulate these processes through interactions with effector proteins. Our previous work has shown that anticoagulant heparan sulfates are synthesized by rodent ovarian granulosa cells, and we now have set out to characterize heparan sulfates from human follicular fluid. Here we report the first anticoagulant heparan sulfate purified from a natural human extravascular source. Heparan sulfate chains were fractionated according to their affinity for antithrombin, and their structure was analyzed by 1H NMR and MS/MS. We find that human follicular fluid is a rich source of anticoagulant heparan sulfate, comprising 50.4% of total heparan sulfate. These antithrombin-binding chains contain more than 6% 3-O-sulfated glucosamine residues, convey an anticoagulant activity of 2.5 IU/ml to human follicular fluid, and have an anti-Factor Xa specific activity of 167 IU/mg. The heparan sulfate chains that do not bind antithrombin surprisingly exhibit an extremely high content in 3-O-sulfated glucosamine residues, which suggest that they may exhibit biological activities through interactions with other proteins.

FIGURE 1.

Purification of hFF HSPG by ion exchange and gel filtration chromatography. A, fractionation of hFF PG by Mono Q chromatography. The column was loaded and washed with PBS buffer containing 0.15 m NaCl and eluted by a NaCl gradient. Proteins were followed by A₂₈₀; the aHSPG was followed by the ¹²⁵I-AT ligand-binding assay and the GAG by Alcian blue. Positive fractions were detected between 0.96 and 1.76 m NaCl, and the fractions eluting between 0.96 and 1.38 m NaCl were pooled for further purification (bar). B, gel filtration of hFF HSPG on Sepharose CL4B. HSPG (followed by Alcian blue) eluted as a major high molecular weight peak containing all the aHSPG (followed by ¹²⁵I-AT ligand binding). Fractions eluting at K_av 0–0.1 were pooled for further analysis (bar). Minor HS peaks of lower molecular weight representing free GAG were discarded. For comparison, heparin (M_r(av)16,400) eluted at K_av 0.6 (arrow) clearly separated from the HSPG peak.

FIGURE 2.

aHS·AT complexes have retarded migration on affinity coelectrophoresis. The hFF aHS and iHS (10 μg/lane) were subjected to electrophoresis in an agarose gel containing the indicated concentrations of AT. Densitometry scanning of the Azure A-stained gel shows that aHS is retarded in the presence of 30 nm AT and further retarded with 500 nm AT. In contrast, iHS migration is not affected by the presence of AT. The figure shows the data for one representative experiment. Similar results were obtained in three independent experiments.

FIGURE 3.

Molecular size distribution of hFF aHS and iHS. A representative example of molecular weight determination for aHS (a) and iHS (b). Azure A-stained PAGE shows similar size distribution of aHS and iHS. The migration position of standard GAG is indicated by arrows, heparin (M_r(av)16,400), chondroitin sulfate A (M_r(av) 21,600), and HS I (M_r(av)15,500). The modal molecular mass of aHS and iHS is about 30,000 Da.

FIGURE 4.

¹H NMR spectra of standard HS and hFF derived aHS and iHS. Shown are ¹H NMR spectra of a commercial HS standard (A), hFF aHS (B), and hFF iHS (C). The presence of H-3 of GlcNS3S and GlcNS3S6S (peak e′) in B is highlighted in D by expanding the indicated spectral regions and by showing the difference spectrum of B minus C. Peak a, H-1 GlcNAc; peak b, H-1 IdoUA2S; peak c, H-1 IdoUA; peak d, H-5 IdoUA2S; peak e, H-1 GlcA; peak f, H-2 IdoUA2S; peak g, H-3 IdoUA2S; peak h, H-6 GlcNS6S or GlcNAc6S; peak i, H-4,5,6 GlcNAc; peak j, H-2 GlcNS.

FIGURE 5.

TIC and oligosaccharide composition of hFF aHS and iHS following enzymatic digestion. A, TIC of hFF aHS; B, TIC of hFF iHS; C, composition table of each fraction. MS and MS/MS analyses confirmed the structures of the indicate disaccharides (data not shown) and oligosaccharides (Fig. 6).

FIGURE 6.

Structure determination of hFF aHS and iHS tetrasaccharides by MS/MS. Shown are representative MS/MS spectra (left) and deduced structures of aHS and iHS tetrasaccharides. A, glycosidic bond cleavage fragment ions (Y₂, Y₃, Z₃) identify peak 5 as ΔUA2S-GlcNS-UA-GlcNAc. B, glycosidic bond cleavage ions (C₂, C₃) and a cross-ring cleavage fragment ion (^0,4A₄) reveal peak 6 as ΔUA-GlcNS-UA-GlcNS6S. C, glycosidic bond cleavage ions (B₁, C₁, B₃, C₃) and a cross-ring cleavage fragment ion (^0,4A₄) show peak 7 to be ΔUA2S-GlcNAc-UA-GlcNS6S. D, glycosidic bond cleavage fragment ions (C₁, B₃, C₃) identify peak 8-1 as ΔUA2S-GlcNAc-UA-GlcNS6S3S.; E, MS/MS spectrum of hexasaccharide in peak 8-2.

FIGURE 7.

aHSPG in human GC. A, human GCs produce aHSPG. GCs from four different patients were cultured. Cell-bound and soluble aHSPG were detected by ¹²⁵I-AT cell binding assay (left) and ¹²⁵I-AT ligand-binding assay (center), respectively. The amount of aHSPG was variable in different patients but was comparable with the reference cell line LTA (right). The amount of cell-bound aHSPG was correlated to the estradiol secreted by the cells, reflecting the extent to the cells response to FSH. B,3-O-sulfotransferase isoforms expression in human granulosa cells. 3-OST transcript copies per 10 ng of RNA. Mean values from five different patients are shown.