SULF1
Sulfatase 1, also known as SULF1, is an enzyme which in humans is encoded by the SULF1 gene.
Heparan sulfate proteoglycans act as co-receptors for numerous heparin-binding growth factors and cytokines and are involved in cell signaling. Heparan sulfate 6-O-endo-sulfatases, such as SULF1, selectively remove 6-O-sulfate groups from heparan sulfate. This activity modulates the effects of heparan sulfate by altering binding sites for signaling molecules.
Function
Heparan sulfate proteoglycans are widely expressed throughout most tissues of nearly all multicellular species. The function of HSPGs extends beyond providing an extracellular matrix structure and scaffold for cells. They are integral regulators of essential cell signaling pathways affecting cell growth, proliferation, differentiation, and migration. Although the core protein is important, the large heparan sulfate chains extending from the core are responsible for most receptor signaling. HS chains are heterogeneous structures that differ in specific and conditional cell contexts. Of particular importance is the HS sulfation pattern, which was once thought to be static after HS biosynthesis in the Golgi. However, this paradigm changed after the discovery of two extracellular 6-O-S glucosamine arylsulfatases, Sulf1 and Sulf2. These two enzymes allow rapid extracellular modification of sulfate content in HSPGs, impacting signaling involving Shh, Wnt, BMP, FGF, VEGF, HB-EGF, GDNF, and HGF. In addition, Sulfs may exercise another level of regulation over HS composition by down or upregulating HS biosynthetic enzymes present in the Golgi through the very same signaling pathways they modify.Discovery
Before the cloning and characterization of Sulf1 and Sulf2, HS composition was thought to be unchanging after localization to the cell surface. However, this changed when the quail orthologue of Sulf1, QSulf1, was identified in a screen for Sonic hedgehog response genes activated during somite formation in quail embryos. Sequence alignment analysis indicates QSsulf1 is homologous with lysosomal N-acetyl glucosamine sulfatases that catalyze the hydrolysis of 6-O sulfates from N-acetyl glucosamines of heparan sulfate during the degradation of HSPGs. In contrast to lysosomal active sulfatases, QSulf1 localizes exclusively to the cell surface by interacting hydrophilically with a non-heparan sulfate outer membrane component, and is enzymatically active at a neutral pH. By mutating the catalytically active cysteines to alanine, thereby blocking N-formylglycine formation, they found QSulf1 was responsible for Wingless release from HS chains to activate the Frizzled receptor; this was the first evidence that an extracellular sulf was capable of modifying HS and therefore cell signaling. The overall structure of QSulf is followed closely by its orthologues and paralogues, including human and mouse. The human and murine orthologues of QSulf1, HSulf1 and MSulf1, respectively, were cloned and characterized after the discovery of QSulf1. In addition, a paralogue, Sulf2, sharing 63-65% identity with Sulf1 also was discovered through BLAST sequence analysis. The HSulf1 gene has an open reading frame of 2616 bp, encoding a protein of 871 amino acid, and HSulf2 has an open reading frame of 2613 bp, encoding a protein of 870 aa. The HSulf1 and 2 genes localize to 8q13.2-13.3 and 20q13.12, respectively. They contain putative Asn-linked glycosylation sites, and furin cleavage sites responsible for proteolytic processing in the Golgi. The function or substrate specificity these cleavage sites impart has yet to be determined.Validation of the predicted N-linked glycosylation sites on QSulf1 were performed using tunicamycin and QSulf1 variants missing the N-terminal domain or HD, which contain predicted N-linked glycosylation sites. The N- and C-terminal showed unbranched N-linked glycosylation, but was absent in the hydrophilic domain even though it contains two putative sites. In addition, O-linked or sialylated glycosylation were not present in QSulf1. Importantly, proper glycosylation is necessary to localize to the cell surface, possibly to bind HS moieties, and was required for enzymatic activity.
Structure and mechanism
Sulf1 and Sulf2 are new members of a superfamily of arylsulfatases, being closely related to arylsulfatase A, B and glucosamine 6-sulfatase. The x-ray crystal structure of neither Sulf1 or Sulf2 has been attempted, but ARSA active site crystal structure was deciphered. In ARSA, the conserved cysteine, which is posttranslationally modified to a C alpha formylglycine is critical for catalytic activity. In the first step, one of the two oxygens of the aldehyde hydrate attacks the sulfur of the sulfate ester. This leads to a transesterification of the sulfate group onto the aldehyde hydrate. Simultaneously the substrate alcohol is released. In the second step, sulfate is eliminated from the enzyme-sulfate intermediate by an intramolecular rearrangement. The “intramolecular hydrolysis” allows the aldehyde group to be regenerated. The active site of ARSA contains nine conserved residues that were found to be critical for catalytic activity. Some residues, such as Lys123 and Lys302, bind the substrate while others either participate in catalysis directly, such as His125 and Asp281, or indirectly. In addition a magnesium ion is needed to coordinate the oxygen that attacks the sulfur in the first step of sulfate cleavage. The crystal structure and residue mutations need to be performed in Sulf1 and Sulf2 to determine if any differences exist from lysosomal sulfatases.Enzymatic specificity
HS enzymatic specificity of QSulf1 was first analyzed. QSulf1 enzymatic specificity on 6-O sulfates was linked to the trisulfated disaccharides in S domains of HS and not NA/NS domains. Sulf1 and 2 null murine embryonic fibroblasts were generated to test the HS specificity of mammalian Sulf as opposed to avian Sulf. Investigators found mSulf1−/−;mSulf2−/− HS showed overall large increases in all 6S disaccharides. Cooperativity between mSulf1/2 was found because a 2-fold increase in S-domain-associated disaccharides and UA–GlcNS) was observed in double knock-out HS as compared with either single knock-out HS alone. However, one difference from mSulf1 is that mSulf2−/− HS shows an increase in 6S almost exclusively within the non-sulfated and transition zones. This sulfation effect on non-sulfated and transition zones is also different from QSulfs, which catalyze desulfation exclusively in S-domains. Although 6S changes were dominant, other small changes in NS and 2S sulfation do occur in the Sulf knock out MEFs, which may be a compensatory mechanism. Further biochemical studies elucidated specificity and localization of human Sulfs 1 and 2. Sulf1 and 2 hydrophilic domains associate with the cell membrane components through electrostatic interactions and not by integration with into the lipid bilayer. In addition to cell membrane association, Sulfs also secreted freely into the media, which contrasts the findings with QSulf1 and 2. Biochemical analysis of HSPGs in Sulf 1 and 2 knockout MEFS reveal enzyme specificities to disulfated and, primarily, trisulfated 6S disaccharide units UA-GlcNS and UA-GlcNS within the HS chain, with specific exclusion of monosulfated disaccharide units. In vivo studies, however, demonstrate that loss of Sulf1 and Sulf2 result in sulfation changes of nonsubstrates, indicating Sulf modulates HS biosynthetic machinery. This was further demonstrated by PCR analysis, showing dynamic changes in HS biosynthesis enzymes after Sulf1 and 2 loss. Also, the authors showed in an MEF model system, that Sulf1 and Sulf2 definitively and differentially modify HS proteoglycan fractions including cell surface, GPI-anchored, shed, and ECM-associated proteoglycans.Role in cancer
The next section gives a detailed description of Sulf1 and Sulf2’s involvement in cancer. Much of what is known about signaling pathways mediated by Sulfs has been determined through investigating extracellular Sulf role and function in cancer. Therefore, they will be described in tandem. Additionally, this emphasizes how small changes in HS sulfation patterns have major impacts in health and disease.Ovarian Cancer
The first signs of Sulf1 dysregulation were found in ovarian cancer. The expression of Sulf1 mRNA was found to be downregulated or absent in a majority of ovarian cancer specimens. The same investigators also found lowered mRNA expression in breast, pancreatic, and hepatic malignant cell lines. This absent or hypomorhic Sulf1 expression results in highly sulfated HSPGs. The lack of Sulf1 expression also augments heparin binding-epidermal growth factor response by way of greater EGF Receptor and extracellular signal-regulated kinase signaling, which are common signatures of ovarian cancer. Even further, Sulf1 N-terminal sulfatase actitivity was specifically required for cisplatin-induced apoptosis of the ovarian cancer cell line, OV207. The mechanism by which Sulf1 is downregulated in ovarian cancer was investigated. Epigenetic silencing of CpG sites within Sulf1 exon 1A by methylation is associated with ovarian cancer cells and primary ovarian cancer tissues lacking Sulf1 expression. Furthermore, CpG sites showed increased levels of histone H3 K9 methylation in Sulf1 negative ovarian cancer cell lines.Breast Cancer
Breast cancer expression of Sulf1 at the mRNA level was shown to be downregulated. Investigations into this relationship revealed that angiogenesis in breast cancer was shown to be regulated in part by Sulf1. Breast cancer xenografts overexpressing Sulf1 in athymic mice showed marked decreases in angiogenesis. Specifically, Sulf1 inhibited the ability of vascular endothelial cell heparan sulfate to participate in complex formation with FGF-2, thereby abolishing growth signaling. FGF-2 is a HB-GF, requiring the formation of a ternary complex with HS and the FGF Receptor to cause receptor dimerization, activation, and autophosphorylation, which then leads to induction of the mitogen-activated protein kinase pathway. This results in several responses including cell proliferation and angiogenesis. Importantly, this response is dependent upon the degree and signature of HS-GAG sulfation. To further validate the response in breast cancer, human umbilical vein endothelial cells, overexpressing Sulf1 inhibited vascular endothelial growth factor 165 signaling which is dependent upon HS, but not HS-independent VEGF121. Sulf2 also was implicated in breast cancer. In contrast to Sulf1, Sulf2 was upregulated at both the mRNA and protein levels in tumor tissue in two mammary carcinoma mouse models.Sulf1 displays regulation of amphiregulin and HB-EGF-mediated autocrine and paracrine signaling in breast cancer. Loss of Sulf1 in a breast cancer cell line, MDA-MB-468, shows increased ERK1/2 and EGFR activation, which was shown to be mediated by HB-EGF and amphiregulin, which require complexes with specifically sulfated HS. Breast cancer samples show loss of Sulf1 expression in invasive lobular carcinomas. These carcinomas are predominantly, estrogen receptor and progesterone receptor -positive, and HER-2, p53, and EGFR-negative, but do not confer an increased survival. The authors suggest that enhanced amphiregulin and HB-EGF signaling due to a lack of Sulf1, and therefore oversulfation of HS, may make lobular carcinomas more aggressive than expected. The mechanism by which Sulf1 is downregulated in breast cancer was further investigated. The authors found aberrant hypermethylation of the Sulf1 promoter in both breast cancer and gastric cancer cell lines and patient samples, leading to a reduction of Sulf1 expression, which is similar to ovarian cancer.
Despite this evidence, disagreements are found in the literature regarding the role of Sulf in breast cancer. In contrast to previous reports, Sulf1 transcript expression was highly upregulated in invasive ductal carcinoma with respect to confined ductal carcinoma in situ. The authors, therefore, propose that Sulf1 is involved in the acquisition of the capacity to invade adjacent tissues in ductal carcinoma in situ.