Extracellular Sulfatases SULF1 and SULF2

Sulfatases are enzymes that hydrolyze sulfate bonds in various molecules. Seventeen sulfatase genes have been identified in humans, and the majority of them localize to lysosomes. Lysosome-localized sulfatases sequentially degrade sulfated glycosaminoglycans and sulfated glycolipids under acidic conditions in concert with glycosidases. Human SULF1 and SULF2 were identified in 2002 as genes encoding proteins distinct from the group of intracellularly localized sulfatases (1). Human SULF1 and SULF2 proteins were shown to be secreted extracellularly and to utilize heparin/heparan sulfate (HS) chains as substrates, unlike the previously known group of intracellularly localized sulfatases (1). This is the first demonstration of extracellular endosulfatases that act on the sulfate groups within heparin/HS. SULF1 and SULF2 have been shown to release the sulfate group at position 6 of the IdoA2S-GlcNS6S unit in the highly sulfated domain called S-domain (IdoA, GlcNS, 2S, and 6S represent iduronic acid, N-sulfated glucosamine, 2-O-sulfate, and 6-O-sulfate, respectively) (Fig. 1) (1, 2). The neutral pH optimum for enzyme activity strongly supported that these enzymes work extracellularly (1).

Human SULF1 and SULF2 are 871 amino acids (aa) and 870 aa in length, respectively, and 64% aa sequence identical to each other, with an N-terminal signal peptide, a catalytic region of approximately 370 aa homologous to the lysosome-localized sulfatase family, a hydrophilic domain (HD) of approximately 320 aa, and an N-acetylglucosamine-6-sulfatase-related active region of 100 aa. The amino acid sequences of SULF1 and SULF2 are highly homologous with 93-94% identity between human and mouse species, respectively (1). SULF1 and SULF2 are biosynthesized as approximately 125 kDa proteins modified with N-linked glycans (Fig. 1). It is then processed within the HD region by Furin protease to form 75 kDa and 50 kDa fragments (1, 3-5). These fragments are linked by disulfide bonds. Under certain conditions, SULF1/SULF2 exhibit dimer formation (3) and SULF2 undergoes chondroitin sulfate addition within the HD region (6). The HD region of SULF1/SULF2 is also important for cell surface localization of SULF proteins (1, 3, 7, 8). Human SULF1 and SULF2 are enriched in lipid rafts, dynamic membrane microdomains that are formed in the plasma membrane (3). Active SULF2 secreted extracellularly can be recovered as a pellet by ultracentrifugation (9). Very interestingly, processing by Furin is not essential for either the activity of SULF1 and SULF2 on 4-methylumbelliferyl sulfate substrates or the endosulfatase activity on heparin/HS, but is required for localization to lipid rafts. The interaction of various proteins in lipid rafts is important for many signal transductions. Protein processing of SULF1 and SULF2 has been shown to be closely involved in the regulation of heparin-binding factor signaling, such as Wnt (3, 7, 10).

Figure 1. Structure and enzymatic reaction mode of extracellular sulfatase SULF1/SULF2 a. Human SULF1 and SULF2 have a conserved cysteine residue (Cys) that is converted to α-formylglycine. It is known that SULF2 undergoes chondroitin sulfate addition modification under certain conditions (*). b. SULF1 and SULF2 release a sulfate group at C-6 position of the IdoA2S-GlcNS6S disaccharides within the internal "S-domain" of the HS glycan chain. c. The signal peptide of SULF1/SULF2 is cleaved in the endoplasmic reticulum to form a proprotein. They undergo Furin protease cleavage at the sites within the hydrophilic domain (HD) region. Furin protease cleavage does not affect the sulfatase activity of SULF1/SULF2, but is required for its localization to lipid rafts and its ability to promote Wnt signaling.

It has been shown that SULF1 and SULF2 modulates the binding of many protein ligands to heparin or HS glycans (4, 11). In addition, SULF2 releases these ligand molecules, e.g. Vascular endothelial growth factor (VEGF) 165, C-X-C motif chemokine ligand 12 (CXCL12), C-C motif chemokine ligand 21 (CCL21), bound to immobilized-heparin from the binding complex. A mechanism has been proposed by which SULF1 or SULF2 release ligand molecules sequestered or stored in HS proteoglycans on the cell surface or in the extracellular matrix to facilitate their action on cells expressing their receptors. When Noggin, a bone morphogenetic protein (BMP) antagonist, is localized on the cell surface by binding to HS chains, BMP is tightly trapped by Noggin on the cell surface. SULF1-mediated disruption of Noggin localization at the cell surface is thought to increase in BMP bioavailability to its receptor, resulting in activation of BMP signaling. In contrast to ligand molecules that are positively regulated by SULF1/SULF2, Heparin-binding epidermal growth factor–like growth factor (HB-EGF), Fibroblast growth factor 2 (FGF2), and Hepatocyte growth factor (HGF) signaling are negatively regulated in SULF1/SULF2 expressing cells. Difficulty in formation of ligand-HS-receptor binding complex at the cell surface may induce a decrease in proliferative signaling into the cell (11-15). Phenotypic abnormalities are more severe in certain tissues during development in mice that are double deficient in Sulf1 and Sulf2 than in mice that are single deficient; the overlapping distribution of Sulf1 and Sulf2 and the ability of each Sulf to compensate for the deletion of the other has been demonstrated in mouse development (16-22). The individual functions and functional coordination of the two SULFs in different contexts, such as disease onset and progression, are topics for further study.

Elevated expression of SULF1 and SULF2 in human cancers has been widely reported; SULF1 is upregulated in hepatocellular carcinoma, pancreatic cancer, head and neck squamous cell carcinoma, gastric cancer, lung adenocarcinoma, and lung squamous cell carcinoma; SULF2 is upregulated in hepatocellular carcinoma, lung adenocarcinoma, and lung squamous cell carcinoma. Generation of SULF1 and SULF2 splicing variants have also been reported. See other literature for details (4, 11, 23-27). In human cancers, SULF1 and SULF2 may act in a tumor-promoting or inhibitory manner. Knowing which molecular signaling is dominant in the pathogenesis and progression of the disease of interest will help elucidate the involvement of SULF1 and SULF2 in pathogenesis.

Kenji UCHIMURA
(Unit of Glycobiology Structure and Functions, CNRS)

Jun. 15, 2023