The Sulfs are 6-O endosulfatases that act on the trisulfated disaccharide unit (-IdoA(2-OSO3
)-) which is the most common unit in heparin but is largely confined to the S-domains of HS [15
]. As the heterogeneous pattern of sulfation within S domains is known to dictate the binding specificity of many proteins for heparin/HS [7
], the Sulfs could potentially regulate those interactions with a dependence on the presence of trisulfated disaccharides within the binding motif. Here, we have employed Sulf-2 as a tool to explore the binding requirements of several proteins, some of which had been previously characterized and others whose binding requirements were largely unknown. Previous work has shown that QSulf-1 treatment of soluble recombinant form of an HSPG (Glypican-1) reduced its ability to bind a Wnt ligand [16
]. Also, the interaction of Noggin with cell surface HSPGs was diminished by the overexpression of QSulf-1 in the cells [17
]. In the present study we have taken advantage of two sources of soluble HSulf-2: recombinant enzyme purified from stably transfected 293 cells and a natural form of the enzyme in CM of a human breast carcinoma cell line (MCF-7). It should be noted that we have not been able to obtain expression of high level of Sulf-1 in CM of transfected 293 cells, nor have we yet identified a soluble form of this enzyme from a native source.
We have previously shown that native Sulf-2 in MCF-7 CM has arylsulfatase activity [25
]. In the present study, we have used HPLC to demonstrate that MCF-7 CM possesses endosulfatase activity against intact heparin. This result has also been established by an alternative approach using mass spectroscopy [38
]. Employing an ELISA based on binding of protein ligands to heparin-BSA conjugate, we found that rHSulf-2 strongly modulated the binding of VEGF165
, FGF-1, and SDF-1 to porcine intestinal heparin. In the case of FGF-1, the inhibitory effect of Sulf-2 was also demonstrated for heparan sulfate chains as well, using a HS-BSA conjugate. All of the effects obtained with the recombinant enzyme were observed for MCF-7 CM, and we confirmed that the activity in the CM was due to Sulf-2. The porcine intestinal heparin used in this study contained 70% trisulfated disaccharides, 18% disulfated disaccharides, 7% monosulfated disaccharides and 5% unsulfated disaccharides (data not shown). Based on our previous work [15
] and the present study, we estimate that about 80% of the total trisulfated disaccharides in heparin-BSA was converted into disulfated disaccharides by Sulf-2. Thus, only about 22% of the overall sulfate moieties were removed by the enzyme treatment, arguing against the possibility that the reduced binding of selective protein ligands to treated heparin was due to a reduction in overall charge. As heparan sulfates have limited S-domains [7
], Sulf-2 would be predicted to have an even smaller effect on the global sulfation of HSPGs. A further indication of the selectivity of Sulf-2 is that there is no relationship between the estimated Kd
for binding of each ligand to heparin-BSA (Table ) and the susceptibility of the interaction to the enzyme (Table ).
The Sulf-2 effects on FGF-1 binding were anticipated based on a number of correlative studies [28
], which have implicated the trisulfated disaccharide motif as a binding element in HS chains. Our VEGF165
results are compatible with a previous study in which chemical 6-O-desulfation of heparin strongly weakened its ability to interact with VEGF165
]. However, our findings provide the first direct evidence that 6-O-sulfation of heparin within the context of the trisulfated disaccharide motif is essential for the interaction. The majority of VEGF isoforms are able to bind to heparin/HS [40
], and it is anticipated that our results will generalize to these other forms as well. With respect to SDF-1, it has long been known that this highly basic chemokine binds relatively strongly to heparin [41
], but the fine specificity of the interaction has not previously been explored. SLC and IL-8 showed a partial sensitivity to the desulfation effects of Sulf-2 on heparin. This partial susceptibility implies that the 6-O-sulfation of the trisulfated disaccharide can contribute but is not absolutely required for a measurable binding interaction with heparin. One important avenue for future investigation is based on our observation that Sulf-2 strongly modulated the interactions of two chemokines (SDF-1 and SLC) with heparin. SDF-1 has been implicated in a diverse range of processes such as lymphocyte chemotaxis, stem cell homing and retention, tissue repair, angiogenesis, and organ-specific metastasis [42
], whereas SLC is important in lymphoid organ homeostasis and inflammation [43
]. Thus, Sulf-2 though effects on the ECM-association of chemokines could have important roles in a number of normal and pathophysiologic processes. HSulf-1 with an apparently indistinguishable enzymatic activity [15
] may exert similar effects on HSPG-bound growth factors and chemokines.
It is well established that one function of heparan sulfate proteoglycans in the ECM and basement membranes is to sequester protein ligands away from signal transduction receptors [44
]. A known mechanism to regulate the mobilization of such factors is through the action of heparanase, an endo-ß-D-glucuronidase that degrades heparan sulfate chains into relatively large fragments [46
]. This enzyme is present in a number of normal cell types (e.g., leukocytes, platelets, cytotrophoblasts) and is upregulated in several cancers. Expression of heparanase in a number of settings elicits angiogenesis, apparently through the release of HSPG-bound angiogenic factors [44
]. Our finding that Sulf-2 can reverse the association between heparin and angiogenic factors (e.g., VEGF) suggests the possibility of functional parallels between these two enzymes. Sulf-2 may mobilize VEGF or other angiogenic factors sequestered in the ECM and increase their bioavailability to endothelial cells that express the appropriate signaling receptors. The ability of rHSulf-2 to promote angiogenesis in the chick chorioallantoic membrane (CAM) assay is consistent with such a scenario [25
]. Thus, the observed upregulation of Sulf-2 in mammary carcinomas and its secretion from these cells could directly contribute to tumor angiogenesis and thus tumor growth. Sulf-2 derived from a cancer cell could also conceivably mobilize HSPG-bound growth factors that then act back on the cancer cell and thus trigger its own proliferation. The ability of QSulf-1 to promote Wnt signaling within the cells that express the enzyme provides a plausible paradigm for this type of autocrine pathway [14
]. It should be borne in mind that in the context of some other signaling pathways (e.g., FGF-2 and HGF), Sulf-2 is likely to exert a negative effect.