Quail Sulf1 Function Requires Asparagine-linked Glycosylation

Ambasta, Rashmi K.; Ai, Xingbin; Emerson, Charles P.

doi:10.1074/jbc.m706744200

Cited by 27 publications

(17 citation statements)

References 21 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…For example, in common with other sulfatases (37,38), the activity of both Sulfs relies on the conversion of a highly conserved cysteine within the catalytic domain into C␣-formylglycine, the site at which sulfate is directly bound (7,8,33). In addition, N-linked glycosylation is required for QSulf-1 activity (39). In contrast to this information about post-translational modifications, little has been reported concerning the proteolytic processing of these enzymes.…”

Section: Discussionmentioning

confidence: 82%

Functional Consequences of the Subdomain Organization of the Sulfs

Tang

Rosen

2009

Journal of Biological Chemistry

View full text Add to dashboard Cite

Sulf-1 and Sulf-2 are novel extracellular sulfatases that act on internal glucosamine 6-O-sulfate modifications within heparan sulfate proteoglycans and regulate their interactions with various signaling molecules, including Wnt ligands. Although the Sulfs are multidomain proteins, there is limited information available about how the subdomains contribute to their enzymatic and signaling activities. In this study, we found that both human Sulfs were synthesized as prepro-enzymes and cleaved by a furin-type proteinase to form disulfide-bond linked heterodimers of 75-and 50-kDa subunits. The mature Sulfs were secreted into conditioned medium, as well as retained on the cell membrane. Although the catalytic center resides in the N-terminal 75-kDa subunit, the C-terminal 50-kDa subunit was indispensable for both arylsufatase and glucosamine 6-O-sulfate-endosulfatase activity. We found that the hydrophilic regions of the Sulfs were essential for endosulfatase activity but not for arylsulfatase activity. Using Edman sequencing, we identified furin-type proteinase cleavage sites in Sulf-1 and Sulf-2. Deletion of these sequences resulted in uncleavable forms of Sulfs. The uncleavable Sulfs retained enzymatic activity. However, they were unable to potentiate Wnt signaling, which may be due to their defective localization into lipid rafts on the plasma membrane.Heparan sulfate proteoglycans (HSPGs) 2 are major components of the extracellular matrix/cell surface and regulate a variety of biological phenomena, including cell proliferation, cell migration, and differentiation (1). These effects are mediated through the ability of HSPGs to bind to a diverse repertoire of protein ligands. Among these are morphogens, growth factors, chemokines, and other classes of molecules (2, 3).HSPGs consist of multiple heparan sulfate (HS) chains covalently linked to a limited set of core proteins. The HS chains contain repeating uronic acid and glucosamine disaccharide units. The binding functions of HSPGs depend on the fine structure of the attached heparan sulfate chains where sulfation modifications occur in four positions (N-, 3-O, and 6-O of glucosamine and 2-O of uronic acid) in highly variegated, yet highly regulated patterns (3, 4). 6-O-Sulfation of glucosamine is established to be critical for certain HSPG functions in organisms from Drosophila through mammals (5, 6).Several years ago, we cloned cDNAs encoding two novel extracellular sulfatases (Sulf-1 and Sulf-2) in human and mouse (7), initiated by the identification of the Sulf-1 ortholog in the quail embryo (QSulf-1) (8). We and others showed that both Sulfs are neutral pH endosulfatases, which remove glucosamine-6-O-sulfate from internal glucosamine residues of highly sulfated subregions within heparin/HSPGs (7, 9, 10). The ability of these enzymes to modulate the heparin/HSPG interactions of a number of growth factors, morphogens, and chemokines has been confirmed in direct binding assays (9, 11-13). In some cellular contexts, the Sulfs act to promote signaling pathways (...

show abstract

Section: Discussionmentioning

confidence: 82%

Functional Consequences of the Subdomain Organization of the Sulfs

Tang

Rosen

2009

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Both SULFs are modified by asparagine-linked glycans before secretion, perhaps at 6-12 sites, and various varieties have been discovered in cell medium[14]. N -linked glycosylation of quail SULF1 appears essential for secretion, membrane targeting, and enzyme activity[17]. Little conservation exists among potential glycosylation sites between species.…”

Section: Sulf1 and Sulf2 Domain Structure Substrate Specificity And mentioning

confidence: 99%

Heparin-Degrading Sulfatases in Hepatocellular Carcinoma: Roles in Pathogenesis and Therapy Targets

Lai

Thompson²,

Sandhu³

et al. 2008

Future Oncol.

View full text Add to dashboard Cite

Hepatocellular carcinoma (HCC) is often diagnosed at an advanced stage for which there are limited treatment options. Two recently identified human heparin-degrading endosulfatases, named sulfatase 1 (SULF1) and sulfatase 2 (SULF2), have been found to be involved in liver carcinogenesis. SULF1 and SULF2 desulfate cell surface and extracellular matrix heparan sulfate proteoglycans (HSPGs) and modulate heparin-binding growth factor signaling in multiple cancers, including HCCs. SULF1 inhibits HCC tumor cell growth in vitro and in nude mice in vivo, partially through effects on gene expression mediated through histone H4 acetylation. While SULF1 is downregulated in the majority of HCC cell lines and approximately 30% of primary HCCs, SULF2 is up regulated in almost all HCC cell lines and in 60% of primary HCCs. In contrast to the tumor suppressor effect of SULF1, expression of SULF2 activates mitogen activated protein kinase (MAPK) and Akt pathways, promotes HCC cell growth in vitro and in vivo, and is associated with decreased survival of HCC patients. Targeting SULF2 or the interaction between SULF2 and SULF1 may lead to novel therapeutics for the treatment of HCCs.

show abstract

“…In the case of the quail ortholog, QSulf1, two N-glycosylation sites are predicted in the HD (positions 527 and 620). A systematic analysis of the N-glycosylation of QSulf1 has revealed that neither of these two sites are utilized in vivo (38). Furthermore, protein N-glycosylation was shown to be essential for enzymatic activity and membrane targeting of QSulf1.…”

Section: Generation and Characterization Of Sulf1 Deletion Mutants-mentioning

confidence: 99%

“…Based on glycosylation analysis of human Sulf1 (see above) and previous results from the quail ortholog QSulf1 (38), the absence of glycan structures within the HD enables heterologous expression of the isolated HD in E. coli. Therefore, N-terminal GST fusion constructs GST-HD, GST-HDB, and GST-HDC were generated, containing either the entire HD of human Sulf1 or parts thereof, corresponding to the above described Sulf1 deletion mutants (Fig.…”

Section: Generation and Characterization Of Sulf1 Deletion Mutants-mentioning

confidence: 99%

Characterization of the Human Sulfatase Sulf1 and Its High Affinity Heparin/Heparan Sulfate Interaction Domain

Frese

Milz

Dick

et al. 2009

Journal of Biological Chemistry

View full text Add to dashboard Cite

The extracellular sulfatases Sulf1 and Sulf2 remodel the 6O-sulfation state of heparan sulfate proteoglycans on the cell surface, thereby modulating growth factor signaling. Different from all other sulfatases, the Sulfs contain a unique, positively charged hydrophilic domain (HD) of about 320 amino acid residues. Using various HD deletion mutants and glutathione S-transferase (GST)-HD fusion proteins, this study demonstrates that the HD is required for enzymatic activity and acts as a high affinity heparin/heparan sulfate interaction domain. Association of the HD with the cell surface is sensitive to heparinase treatment, underlining specificity toward heparan sulfate chains. Correspondingly, isolated GST-HD binds strongly to both heparin and heparan sulfate in vitro and also to living cells. Surface plasmon resonance studies indicate nanomolar affinity of GST-HD toward immobilized heparin. The comparison of different mutants reveals that especially the outer regions of the HD mediate heparan sulfate binding, probably involving "tandem" interactions. Interestingly, binding to heparan sulfate depends on the presence of 6O-sulfate substrate groups, suggesting that substrate turnover facilitates release of the enzyme from its substrate. Deletion of the inner, less conserved region of the HD drastically increases Sulf1 secretion without affecting enzymatic activity or substrate specificity, thus providing a tool for the in vitro modulation of HS-dependent signaling as demonstrated here for the signal transduction of fibroblast growth factor 2. Taken together, the present study shows that specific regions of the HD influence different aspects of HS binding, cellular localization, and enzyme function.The human sulfatases represent a family of 17 enzymes responsible for the turnover and remodeling of sulfate esters and sulfamates. Their reaction mechanism relies on a special amino acid residue, C␣-formylglycine, which is generated posttranslationally via oxidation of a conserved cysteine residue in the active site (1-3). Besides the lysosomal sulfatases involved in the cellular degradation of various sulfated substrates (4), two extracellular sulfatases, Sulf1 and Sulf2 (the Sulfs), have been described (5, 6). The Sulfs are endosulfatases with restricted substrate specificity toward 6O-sulfate groups of heparan sulfate (HS), 2 an information-rich glycosaminoglycan (GAG) polymer attached to proteoglycans at the cell surface and in the extracellular matrix (6 -8). HS proteoglycans (HSPGs) act as co-receptors in cell signaling pathways and provide binding sites for growth factors and morphogens via specific sulfation patterns on their HS chains. By enzymatically removing 6O-sulfate groups from HSPGs on the cell surface, Sulf1 and Sulf2 differentially regulate the activity of FGF, vascular endothelial growth factor, Wnt, and other HS ligands, thereby modulating important processes such as development, cell growth, and differentiation (9 -12). Misregulation of the Sulfs has been linked with both tumor progression and suppr...

show abstract

Quail Sulf1 Function Requires Asparagine-linked Glycosylation

Cited by 27 publications

References 21 publications

Functional Consequences of the Subdomain Organization of the Sulfs

Functional Consequences of the Subdomain Organization of the Sulfs

Heparin-Degrading Sulfatases in Hepatocellular Carcinoma: Roles in Pathogenesis and Therapy Targets

Characterization of the Human Sulfatase Sulf1 and Its High Affinity Heparin/Heparan Sulfate Interaction Domain

Contact Info

Product

Resources

About