Genetic analysis of the heparan modification network in Caenorhabditis elegans

Heparan sulfates (HS) are highly modified sugar polymers in multicellular organisms that function in cell adhesion and cellular responses to protein signaling. Functionally distinct, cell type-dependent HS modification patterns arise as the result of a conserved network of enzymes that catalyze deacetylations, sulfations, and epimerizations in specific positions of the sugar residues. To understand the genetic interactions of the enzymes during the HS modification process, we have measured the composition of HS purified from mutant strains of Caenorhabditis elegans. From these measurements we have developed a genetic network model of HS modification. We find the interactions to be highly recursive positive feed-forward and negative feedback loops. Our genetic analyses show that the HS C-5 epimerase hse-5, the HS 2-O-sulfotransferase hst-2, or the HS 6-O-sulfotransferase hst-6 inhibit N-sulfation. In contrast, hse-5 stimulates both 2-O- and 6-O-sulfation and, hst-2 and hst-6 inhibit 6-O- and 2-O-sulfation, respectively. The effects of hst-2 and hst-6 on N-sulfation, 6-O-sulfation, and 2-O-sulfation appear largely dependent on hse-5 function. This core of regulatory interactions is further modulated by 6-O-endosulfatase activity (sul-1). 47% of all 6-O-sulfates get removed from HS and this editing process is dependent on hst-2, thereby providing additional negative feedback between 2-O- and 6-O-sulfation. These findings suggest that the modification patterns are highly sensitive to the relative composition of the HS modification enzymes. Our comprehensive genetic analysis forms the basis of understanding the HS modification network in metazoans.     

Dual roles of the Cardin-Weintraub motif in multimeric Sonic hedgehog

The fly morphogen Hedgehog (Hh) and its mammalian orthologs, Sonic, Indian, and Desert hedgehog, are secreted signaling molecules that mediate tissue patterning during embryogenesis and function in tissue homeostasis and regeneration in the adult. The function of all Hh family members is regulated at the levels of morphogen multimerization on the surface of producing cells, multimer release, multimer diffusion to target cells, and signal reception. These mechanisms are all known to depend on interactions of positively charged Hh amino acids (the Cardin-Weintraub (CW) motif) with negatively charged heparan sulfate (HS) glycosaminoglycan chains. However, a precise mechanistic understanding of these interactions is still lacking. In this work, we characterized ionic HS interactions of multimeric Sonic hedgehog (called ShhNp) as well as mutant forms lacking one or more CW residues. We found that deletion of all five CW residues as well as site-directed mutagenesis of CW residues Lys(33), Arg(35), and Lys(39) (mouse nomenclature) abolished HS binding. In contrast, CW residues Arg(34) and Lys(38) did not contribute to HS binding. Analysis and validation of Shh crystal lattice contacts provided an explanation for this finding. We demonstrate that CW residues Arg(34) and Lys(38) make contact with an acidic groove on the adjacent molecule in the multimer, suggesting a new function of these residues in ShhNp multimerization rather than HS binding. Therefore, the recombinant monomeric morphogen (called ShhN) differs in CW-dependent HS binding and biological activity from physiologically relevant ShhNp multimers, providing new explanations for functional differences observed between ShhN and ShhNp.     

Heparan sulfate proteoglycans and their binding proteins in embryo implantation and placentation

Complex interactions occur among embryonic, placental and maternal tissues during embryo implantation. Many of these interactions are controlled by growth factors, extracellular matrix and cell surface components that share the ability to bind heparan sulfate (HS) polysaccharides. HS is carried by several classes of cell surface and secreted proteins called HS proteoglycan that are expressed in restricted patterns during implantation and placentation. This review will discuss the expression of HS proteoglycans and various HS binding growth factors as well as extracellular matrix components and HS-modifying enzymes that can release HS-bound proteins in the context of implantation and placentation.     

In vivo manipulation of heparan sulfate structure and its effect on Drosophila development

Heparan sulfate proteoglycans (HSPGs) participate in a wide range of biological processes through interactions with a number of ligand proteins. The nature of these interactions largely depends on the heparan sulfate (HS) moiety of HSPGs, which undergoes a series of modifications by various HS-modifying enzymes (HSMEs). Although the effects of alterations in a single HSME on physiological processes have started to be studied, it remains elusive how a combination of these molecules control the structure and function of HS. Here we systematically manipulated the HS structures and analyzed their effect on morphogenesis and signaling, using the genetically tractable model organism, Drosophila. We generated transgenic fly strains overexpressing HSMEs alone or in combination. Unsaturated disaccharide analyses of HS showed that expression of various HSMEs generates distinct HS structures, and the enzymatic activities of HSMEs are influenced by coexpression of other HSMEs. Furthermore, these transgenic HSME animals showed a different extent of lethality, and a subset of HSMEs caused specific morphological defects due to defective activities of Wnt and bone morphogenetic protein signaling. There is no obvious relationship between HS unsaturated disaccharide composition and developmental defects in HSME animals, suggesting that other structural factors, such as domain organization or sulfation sequence, might regulate the function of HS.     

6-O-sulfation of heparan sulfate differentially regulates various fibroblast growth factor-dependent signalings in culture

Heparan sulfate (HS) interacts with diverse heparin-binding growth factors and thereby regulates their bioactivities. These interactions depend on the structures characterized by the sulfation pattern and isomer of uronic acid residues. One of the biosynthetic modifications of HS, namely 6-O-sulfation, is catalyzed by three isoforms of HS6-O-sulfotransferase. We generated HS6ST-1- and/or HS6ST-2-deficient mice (6ST1-KO, 6ST2-KO, and double knock-out (dKO)) that exhibited different phenotypes. We examined the effects of HS 6-O-sulfation in heparin-binding growth factor signaling using fibroblasts derived from these mutant mice. Mouse embryonic fibroblasts (MEF) prepared from E14.5 dKO mice produced HS with little 6-O-sulfate, whereas 2-O-sulfation in HS from dKO-MEF (dKO-HS) was increased by 1.9-fold. HS6-O-sulfotransferase activity in the dKO-MEF was hardly detected, and HS2-O-sulfotransferase activity was 1.5-fold higher than that in wild type (WT)-MEFs. The response of dKO-MEFs to fibroblast growth factors (FGFs) was distinct from that of WT-MEFs; in dKO-MEFs, FGF-4- and FGF-2-dependent signalings were reduced to approximately 30 and 60% of WT-MEFs, respectively, and FGF-1-dependent signaling was moderately reduced compared with that of WT-MEFs but only at the lower FGF-1 concentrations. Analysis with a surface plasmon resonance biosensor demonstrated that the apparent affinity of dKO-HS for FGF-4 was markedly reduced and was also reduced for FGF-1. In contrast, the affinity of dKO-HS for FGF-2 was 2.5-fold higher than that of HS from WT-MEFs. Thus, 6-O-sulfate in HS may regulate the signalings of some of HB-GFs, including FGFs, by inducing different interactions between ligands and their receptors.     

Heparin-derived heparan sulfate mimics to modulate heparan sulfate-protein interaction in inflammation and cancer

The heparan sulfate (HS) chains of heparan sulfate proteoglycans (HSPG) are “ubiquitous” components of the cell surface and the extracellular matrix (EC) and play important roles in the physiopathology of developmental and homeostatic processes. Most biological properties of HS are mediated by interactions with “heparin-binding proteins” and can be modulated by exogenous heparin species (unmodified heparin, low molecular weight heparins, shorter heparin oligosaccharides and various non-anticoagulant derivatives of different sizes). Heparin species can promote or inhibit HS activities to different extents depending, among other factors, on how closely their structure mimics the biologically active HS sequences. Heparin shares structural similarities with HS, but is richer in “fully sulfated” sequences (S domains) that are usually the strongest binders to heparin/HS-binding proteins. On the other hand, HS is usually richer in less sulfated, N-acetylated sequences (NA domains). Some of the functions of HS chains, such as that of activating proteins by favoring their dimerization, often require short S sequences separated by rather long NA sequences. The biological activities of these species cannot be simulated by heparin, unless this polysaccharide is appropriately chemically/enzymatically modified or biotechnologically engineered. This mini review covers some information and concepts concerning the interactions of HS chains with heparin-binding proteins and some of the approaches for modulating HS interactions relevant to inflammation and cancer. This is approached through a few illustrative examples, including the interaction of HS and heparin-derived species with the chemokine IL-8, the growth factors FGF1 and FGF2, and the modulation of the activity of the enzyme heparanase by these species. Progresses in sequencing HS chains and reproducing them either by chemical synthesis or semi-synthesis, and in the elucidation of the 3D structure of oligosaccharide–protein complexes, are paving the way for rational approaches to the development of HS-inspired drugs in the field of inflammation and cancer, as well in other therapeutic fields.     

Heparan sulfate proteoglycans as key regulators of the mesenchymal niche of hematopoietic stem cells

The complex microenvironment that surrounds hematopoietic stem cells (HSCs) in the bone marrow niche involves different coordinated signaling pathways. The stem cells establish permanent interactions with distinct cell types such as mesenchymal stromal cells, osteoblasts, osteoclasts or endothelial cells and with secreted regulators such as growth factors, cytokines, chemokines and their receptors. These interactions are mediated through adhesion to extracellular matrix compounds also. All these signaling pathways are important for stem cell fates such as self-renewal, proliferation or differentiation, homing and mobilization, as well as for remodeling of the niche. Among these complex molecular cues, this review focuses on heparan sulfate (HS) structures and functions and on the role of enzymes involved in their biosynthesis and turnover. HS associated to core protein, constitute the superfamily of heparan sulfate proteoglycans (HSPGs) present on the cell surface and in the extracellular matrix of all tissues. The key regulatory effects of major medullar HSPGs are described, focusing on their roles in the interactions between hematopoietic stem cells and their endosteal niche, and on their ability to interact with Heparin Binding Proteins (HBPs). Finally, according to the relevance of HS moieties effects on this complex medullar niche, we describe recent data that identify HS mimetics or sulfated HS signatures as new glycanic tools and targets, respectively, for hematopoietic and mesenchymal stem cell based therapeutic applications.     

Tinkering with heparan sulfate sulfation to steer development

Heparan sulfate (HS) proteoglycans, at the cell surface and extracellular matrix, facilitate ligand-receptor interactions crucial to many physiological processes. The distinct sulfation patterns of HS sugar chains presented by their protein core are key to HS proteoglycan activity. Tight regulation of several Golgi complex enzyme families is crucial to produce complex tissue-specific HS sequences. Several in vivo models deficient in HS biosynthesis enzymes demonstrate that developmental abnormalities result from modified HS structure. This review will discuss the plasticity of sulfation requirements on HS for activating protein ligands, which might reflect a flexible HS biosynthetic mechanism. In addition, the latest discovery of HS acting enzymes, the Sulfs, responsible for extracellular tweaking of HS sulfation levels subsequent to biosynthesis will be considered.     

Sequence analysis of heparan sulfate epitopes with graded affinities for fibroblast growth factors 1 and 2.

Proteins that belong to the fibroblast growth factor (FGF) family regulate proliferation, migration, and differentiation of many cell types. Several FGFs, including the prototype factors FGF-1 and FGF-2, depend on interactions with heparan sulfate (HS) proteoglycans for activity. We have assessed tissue-derived HS fragments for binding to FGF-1 and FGF-2 to identify the authentic saccharide motifs required for interactions. Sequence information on a range of N-sulfated HS octasaccharides spanning from low to high affinity for FGF-1 was obtained. All octasaccharides with high affinity for FGF-1 (> or =0.5 m NaCl required for elution) contained an internal IdoUA(2-OSO(3))-GlcNSO(3)(6-OSO(3))-IdoUA(2-OSO(3))-trisaccharide motif. Octasaccharides with a higher overall degree of sulfation but lacking the specific trisaccharide motif showed lower affinity for FGF-1. FGF-2 was shown to bind to a mono-O-sulfated HS 6-mer carrying a single internal IdoUA(2-OSO(3))-unit. However, a di-O-sulfated -IdoUA(2-OSO(3))-GlcNSO(3)-IdoUA(2-OSO(3))-trisaccharide sequence within a HS 8-mer gave stronger binding. These findings show that not only the number but also the positions of individual sulfate groups determine affinity of HS for FGFs. Our findings support the notion that FGF-dependent processes can be modulated in vivo by regulated expression of distinct HS sequences.     

Biological implications of glycosaminoglycan interactions with haemopoietic cytokines

Heparan sulphate (HS) glycosaminoglycans (GAGs) are an integral part of the signalling complex of fibroblast derived growth factor (FGF) family members, HS being regarded as a coreceptor. FGFs are also retained in the tissues by binding to HS structures. Early studies on the contribution of the bone marrow stroma to haemopoiesis suggested that cytokines with a role in haemopoiesis were similarly retained in the stroma through interactions with HS. However, the functional outcomes of these cytokines binding HS were poorly understood. Here the GAG-binding properties of cytokines of the four alpha-helical bundle family and the biological consequences of such binding are reviewed. From this analysis it is apparent that although many of these cytokines do bind GAGs, GAG binding is not a consistent feature, nor is the site of GAG binding conserved among these cytokines. The biological outcome of GAG binding depends, in part, on the location of the GAG-binding site on the cytokine. In some cases GAG binding appears to block signalling, whereas in others signalling is likely to be facilitated by binding. It is postulated that the interactions of these cytokines with their receptor complexes evolved independently of GAG binding, with GAG binding being an additional feature for a subset of this cytokine family. Nevertheless, because GAG binding localizes cytokines to sites within tissues, these interactions are likely to be critically important for the biology of these cytokines.     

Kinetic and structural studies on interactions between heparin or heparan sulfate and proteins of the hedgehog signaling pathway

Heparan sulfate (HS) proteoglycans (PGs) interact with a number of extracellular signaling proteins, thereby playing an essential role in the regulation of many physiological processes. These interactions are important for both normal signal transduction and regulation of the tissue distribution of signaling molecules. In this study, we use surface plasmon resonance (SPR) to study interactions of HS and structurally related heparin with proteins in the Hedgehog signaling pathway. SPR analysis shows that heparin binds with different affinities to active fragments of the proteins Hedgehog (Hh), Interference Hedgehog (Ihog), Cam-related/Down-regulated by Oncogenes (CDO), and Sonic Hedgehog (Shh). Solution competition studies show that the minimum size of a heparin oligosaccharide capable of interacting with Ihog is larger than a tetrasaccharide and for interacting with Shh is larger than an octasaccharide. In comparison with heparin, Ihog and Shh exhibited a lower affinity for HS than for heparin, and CDO and Hh exhibit negligible binding to HS. This study clearly demonstrates Shh and Ihog are heparin and HS binding proteins and that both molecules preferentially bind heparin or HS having a high level of sulfation.     

Isolation and characterization of heparan sulfate from various murine tissues

Heparan sulfate (HS), is a proteoglycan (PG) found both in the extracellular matrix and on cell surface. It may represent one of the most biologically important glycoconjugates, playing an essential role in a variety of different events at molecular level. The publication of the mouse genome, and the intensive investigations aimed at understanding the proteome it encodes, has motivated us to initiate studies in mouse glycomics focused on HS. The current study is aimed at determining the quantitative and qualitative organ distribution of HS in mice. HS from brain, eyes, heart, lung, liver, kidney, spleen, intestine and skin was purified from 6–8 week old male and female mice. The recovered yield of HS from these organs is compared with the recovered whole body yield of HS. Structural characterization of the resulting HS relied on disaccharide analysis and ¹H-NMR spectroscopy. Different organs revealed a characteristic HS structure. These data begin to provide a structural understanding of the role of HS in cell-cell interactions, cell signaling and sub-cellular protein trafficking as well as a fundamental understanding of certain aspects of protein-carbohydrate interactions.     

Dissolved humic substances – ecological driving forces from the individual to the ecosystem level?

1. This review focuses on direct and indirect interactions between dissolved humic substances (HS) and freshwater organisms and presents novel opinions and hypotheses on their ecological significance. Despite their abundance in freshwaters, the role of HS is still inadequately understood. These substances have been considered too large to be taken up by freshwater organisms. On the contrary, here we present evidence that dissolved HS are indeed taken up and interact directly and/or indirectly with freshwater organisms. 2. We show that dissolved HS exert a mild chemical stress upon aquatic organisms in many ways; they induce molecular chaperones (stress shock proteins), induce and modulate biotransformation enzymes and modulate (mainly inhibiting) the photosynthetic release of oxygen by freshwater plants. Furthermore, they produce an oxidative stress, which may lead to membrane oxidation. HS modulate the multixenobiotic resistance activity and probably other membrane‐bound pumps. This property may lead to the increased bioaccumulation of xenobiotic chemicals. Furthermore, they can modulate the numbers of offspring in a nematode and feminise fish and amphibians. The ecological consequences of this potential remain obscure at present. HS also have the potential to act as chemical attractants (as shown with a nematode). 3. In some macrophytes and algae we show that HS interfere with photosynthesis and growth. For instance, the presence of HS suppresses cyanobacteria more than eukaryotic algae. By applying a quantitative structure activity relationship approach, we show that quinones in the HS interfere with photosynthetic electron transport. We show that even Phragmites leachate can act as a kind of phytotoxin. HS also have the potential to suppress fungal growth, as shown with the water mould Saprolegnia parasitica and force the fungus to respond by spore production. 4. In very soft, humic freshwaters, such as the Rio Negro, Brazil, HS stimulate the uptake of essential ions, such as Na and Ca, at extremely low pH (3.5–4.0) and prevent the ionoregulatory disturbance induced by acid waters, thereby enabling fish to survive in these environments. 5. We discuss whether or not HS are directly utilised by aquatic microorganisms or via exoenzymes, which may be washed in from the terrestrial catchment. There is accumulating evidence that the quality of the HS controls microbial growth. In total, net‐heterotrophy may result from HS‐mediated suppression of primary production by the quinone structures and/or from HS‐mediated support of microbial growth. As there is also evidence that HS have the potential to support photoautotrophic growth and suppress microbial growth, the opposite community effect could result. Consequently, dissolved organic carbon (DOC) has to be chemically characterised, rather than simply measuring bulk DOC concentration. 6. In sum, dissolved HS interact with freshwater organisms in a variety of ways in unenriched humic lakes. In addition to the well known effects of HS on light regime, for example, and the direct and indirect supply with carbon (energy), other interactions may be much more subtle. For instance, HS may induce internal biochemical stress defence systems and have the potential to cause acclimatisation and even adaptation. We are just at the beginning of understanding these interactions between dissolved HS and freshwater organisms.     

Sulfated derivatives of Escherichia coli K5 polysaccharides as modulators of fibroblast growth factor signaling

Heparan sulfate (HS) proteoglycans are intimately involved in the regulation of fibroblast growth factor (FGF) signaling. HS and the related glycosaminoglycan heparin interact with FGFs and FGF receptors (FGFRs), and it is believed that both interactions are required for productive FGF signaling. Attempts to inhibit FGF activity have been made with modified heparin preparations, various heparin-like polysaccharide analogues and other polyanionic molecules, which may all act by interfering with the physiological HS-FGF-FGFR interactions on the cell surface. Here, we have studied the potential of sulfated derivatives of a bacterial polysaccharide (capsular polysaccharide from Escherichia coli K5 (K5PS)) in the modulation of FGF-heparin/HS interactions and FGF signaling. We demonstrate that O-sulfated and N,O-sulfated species of K5PS, with high degrees of sulfation, displaced FGF-1, FGF-2, and FGF-8b from heparin. However, only O-sulfated K5PS efficiently inhibited the FGF-induced proliferation of S115 mammary carcinoma cells and 3T3 fibroblasts, whereas N,O-sulfated K5PS had little or no inhibitory effect. Studies with CHO677 cells lacking endogenous HS, as well as with chlorate-treated S115 cells expressing undersulfated HS, indicated that whereas exogenously administered heparin and N,O-sulfated K5PS restored the cellular response toward FGF stimulation, O-sulfated K5PS was largely devoid of such stimulatory activity. Our data suggest that highly O-sulfated species of K5PS may be efficient inhibitors of FGF signaling.     

Spatiotemporal distribution of heparan sulfate epitopes during murine cartilage growth plate development

Heparan sulfate proteoglycans (HSPGs) are abundant in the pericellular matrix of both developing and mature cartilage. Increasing evidence suggests the action of numerous chondroregulatory molecules depends on HSPGs. In addition to specific functions attributed to their core protein, the complexity of heparan sulfate (HS) synthesis provides extraordinary structural and functional heterogeneity. Understanding the interactions of chondroregulatory molecules with HSPGs and their subsequent outcomes has been limited by the absence of a detailed analysis of HS species in cartilage. In this study, we characterize the distribution and variety of HS species in developing cartilage of normal mice. Cryo-sections of femur and tibia from normal mouse embryos were evaluated using immunostaining techniques. A panel of unique phage display antibodies specific to particular HS species were employed and visualized with secondary antibodies conjugated to Alexa-fluor dyes. Confocal microscopy demonstrates that HS species are dynamic structures within developing growth plate cartilage and the perichondrium. GlcNS6S-IdoUA2S-GlcNS6S species are down regulated and localization of GlcNS6S-IdoUA-GlcNS6S species within the hypertrophic zone of the growth plate is lost during normal development. Regional differences in HS structures are present within developing growth plates, implying that interactions with and responses to HS-binding proteins also may display regional specialization.     

Fibroblast growth factor receptor signalling is dictated by specific heparan sulphate saccharides

Signalling by fibroblast growth factors (FGFs) through FGF receptors (FGFRs) depends on the cell-surface polysaccharide heparan sulphate (HS) [1] [2]. HS has an ordered domain structure of highly diverse saccharide motifs that present unique displays of sulphate, carboxyl and hydroxyl groups [3]. These motifs interact with many proteins, particularly growth factors. HS binds both to FGFs [4] [5] [6] and FGFRs [7], and probably activates signalling by facilitating ligand-induced receptor dimerisation [8] [9]. Nevertheless, the extent to which specific HS saccharide sequences play a regulatory role has not been established. By screening a library of structurally diverse HS decasaccharides in bioassays of FGF signalling mediated by three different FGFR isoforms, we found that saccharides showed specificity for both ligands and receptors; some saccharides selectively activated FGF signalling through different FGFR isoforms, others acted as negative regulators. We conclude that HS saccharides play critical roles in dictating the specificity of ligand-receptor interactions in FGFR signalling. Controlled alterations in HS structures [10] would provide a mechanism for regulation of cellular responsiveness to growth factors that bind HS.     

Heparan sulphate biosynthesis and disease

Proteoglycans carrying heparan sulphate (HS) chains are ubiquitously expressed at cell surfaces and in extra-cellular matrices, and HS chains interact with numerous proteins, including growth factors, morphogens and extra-cellular-matrix proteins. These interactions form the basis of HS-related biological phenomena. Thus, the biosynthesis of HS regulates key events in embryonic development and homeostasis, and deranged HS biosynthesis could cause diseases. EXT1 and EXT2 genes encoding the polymerase responsible for HS biosynthesis are known as causative genes of hereditary multiple exostoses, a dominantly inherited genetic disorder characterized by the formation of multiple cartilaginous tumours. In this review, we will summarize HS biosynthesis in several model animals, the effects on cellular functions by alteration of HS biosynthesis, and HS-associated diseases. This review suggests that HS biosynthetic enzymes would be potential candidates for drug targets in various diseases.     

Heparan sulphate sulphotransferase expression in mice and Caenorhabditis elegans

Heparan sulphate (HS) acts as a multifunctional cell regulator, with specific sulphated saccharide sequences designed for selective interactions with many proteins. Functionally, these interactions result in regulation of the protein activities, and there is growing evidence that cells can dynamically alter the structure of HS sequences that they display. HS biosynthesis involves the action of a complex set of enzymes with polymerase, epimerase and sulphotransferase (ST) activities. In higher organisms, multiple isoforms of STs decorate the nascent HS chains with specific patterns of sulphation that confer selective biological functions. The study of HSSTs in model organisms provides a valuable opportunity to examine the expression of these enzymes in relation to the structure and activities of the HS produced. Here we describe that, in mice, there are stage-specific combinations of HSST isoenzymes that underlie the synthesis of different HS species at different times in the developing brain. This differential expression of HSSTs results in the synthesis of structurally variant HS species that form functional signalling complexes with specific fibroblast growth factors and their receptors. Regulated synthesis of specific HS species could be a mechanism for the regulation of proliferation and differentiation in the developing brain. We also describe evidence that a Caenorhabditis elegans orthologue of the mammalian 2OST enzyme, called HST-2, is essential for the normal development of this nematode. Together, these studies emphasize the importance of HSSTs in the biosynthesis of functionally variant HS proteoglycans, and demonstrate the importance of these complex regulatory molecules in developmental processes.     

Heparin/heparan sulfate biosynthesis: processive formation of N-sulfated domains

Heparan sulfate (HS) proteoglycans influence embryonic development as well as adult physiology through interactions with various proteins, including growth factors/morphogens and their receptors. The interactions depend on HS structure, which is largely determined during biosynthesis by Golgi enzymes. A key step is the initial generation of N-sulfated domains, primary sites for further polymer modification and ultimately for functional interactions with protein ligands. Such domains, generated through action of a bifunctional GlcNAc N-deacetylase/N-sulfotransferase (NDST) on a [GlcUA-GlcNAc](n) substrate, are of variable size due to regulatory mechanisms that remain poorly understood. We have studied the action of recombinant NDSTs on the [GlcUA-GlcNAc](n) precursor in the presence and absence of the sulfate donor, 3'-phosphoadenosine 5'-phosphosulfate (PAPS). In the absence of PAPS, NDST catalyzes limited and seemingly random N-deacetylation of GlcNAc residues. By contrast, access to PAPS shifts the NDST toward generation of extended N-sulfated domains that are formed through coupled N-deacetylation/N-sulfation in an apparent processive mode. Variations in N-substitution pattern could be obtained by varying PAPS concentration or by experimentally segregating the N-deacetylation and N-sulfation steps. We speculate that similar mechanisms may apply also to the regulation of HS biosynthesis in the living cell.