Sulf Loss Influences N-, 2-O-, and 6-O-Sulfation of Multiple Heparan Sulfate Proteoglycans and Modulates Fibroblast Growth Factor Signaling

Abstract

Sulf1 and Sulf2 are two heparan sulfate 6-O-endosulfatases that regulate the activity of multiple growth factors, such as fibroblast growth factor and Wnt, and are essential for mammalian development and survival. In this study, the mammalian Sulfs were functionally characterized using overexpressing cell lines, in vitro enzyme assays, and in vivo Sulf knock-out cell models. Analysis of subcellular Sulf localization revealed significant differences in enzyme secretion and detergent solubility between the human isoforms and their previously characterized quail orthologs. Further, the activity of the Sulfs toward their native heparan sulfate substrates was determined in vitro, demonstrating restricted specificity for S-domain-associated 6S disaccharides and an inability to modify transition zone-associated UA-GlcNAc(6S). Analysis of heparan sulfate composition from different cell surface, shed, glycosylphosphatidylinositol-anchored and extracellular matrix proteoglycan fractions of Sulf knock-out cell lines established differential effects of Sulf1 and/or Sulf2 loss on nonsubstrate N-, 2-O-, and 6-O-sulfate groups. These findings indicate a dynamic influence of Sulf deficiency on the HS biosynthetic machinery. Real time PCR analysis substantiated differential expression of the Hs2st and Hs6st heparan sulfate sulfotransferase enzymes in the Sulf knock-out cell lines. Functionally, the changes in heparan sulfate sulfation resulting from Sulf loss were shown to elicit significant effects on fibroblast growth factor signaling. Taken together, this study implicates that the Sulfs are involved in a potential cellular feed-back mechanism, in which they edit the sulfation of multiple heparan sulfate proteoglycans, thereby regulating cellular signaling and modulating the expression of heparan sulfate biosynthetic enzymes.

Dynamic expression patterns of 6‐O endosulfatases during zebrafish development suggest a subfunctionalisation event for sulf2

“…Although these sequences are fairly conserved in the zebrafish Sulfs, the additional stretch of sequences in the C-terminal region of HD of Sulf2 and Sulf2a may confer different function, such that it may alter heparin binding affinity or its localisation to the cell surface. Recently, it has been shown that human SULF2 can modify sulfation of the ECM and shed HSPGs consistent with reports of active fulllength enzyme found in the conditioned medium (Lamanna et al, 2008) thus suggesting Sulfs are more mobile than previously anticipated. It would be interesting to address the HD function of zebrafish Sulfs to observe whether this activity is conserved in all vertebrates and if Sulfs can modify 6-O sulfation on neighbouring cells.…”

“…Since double Sulf knockout mice die shortly after birth, this is a crucial biological process required for normal growth and development (Lamanna et al, 2006(Lamanna et al, , 2008. Therefore, it is not surprising we found most structures to have at least one family member expressed during early embryonic development.…”

“…This is further supported by the intracellular localisation of full-length protein in the endoplasmic reticulum (ER). Thus, in addition to their function as extracellular 6-O-sulfatases, sulfs may play a more significant role in regulating all types of sulfation inside and outside of the cell (Lamanna et al, 2008), could this imply a dual function of the Sulf enzymes? Fig.…”

Functional Consequences of the Subdomain Organization of the Sulfs

“…In contrast to this information about post-translational modifications, little has been reported concerning the proteolytic processing of these enzymes. The present study was prompted by the observation of Sulf fragments in Western blots (7,16,24,25), which suggested chain cleavage and the possible existence of a subunit organization. Here, we provide information about the maturation of these enzymes, their subunit organization, and the requirements for their enzymatic and signaling functions.…”

“…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)(12)(13). In some cellular contexts, the Sulfs act to promote signaling pathways (Wnts, bone morphogenetic protein, and glial cell-derived neurotrophic factor) (9 -11, 14), whereas in others the Sulfs are inhibitory (fibroblast growth factor-2 and transforming growth factor-␤) (15)(16)(17). The importance of the Sulfs in development has been revealed by gene knockdown (8) and knock-out studies (11, 18 -20).…”