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Sulfation pattern and molecular weight (MW) play a key role in the biological actions of sulfated glycans. Besides anticoagulant effects, certain sulfated glycans can also exhibit anti-SARS-CoV-2 properties. To develop a more selective antiviral carbohydrate, an efficient strategy to separate these two actions is required. In this work, low MW fractions derived from the red alga Botryocladia occidentalis sulfated galactan (BoSG) were generated, structurally characterized, and tested for activity against SARS-CoV-2 and blood coagulation. The lowest MW fraction was found to be primarily composed of octasaccharides of monosulfated monosaccharides. Unlike heparin or native BoSG, we found that hydrolyzed BoSG products had weak anticoagulant activities as seen by aPTT and inhibitory assays using purified cofactors. In contrast, lower MW BoSG-derivatives retained anti-SARS-CoV-2 activity using SARS-CoV-2 spike (S)-protein pseudotyped lentivirus vector in HEK-293T-hACE2 cells monitored by GFP. Surface plasmon resonance confirmed that longer chains are necessary for BoSG to interact with coagulation cofactors but is not required for interactions with certain S-protein variants. We observed distinct affinities of BoSG derivatives for the S-proteins of different SARS-CoV-2 strains, including WT, N501Y (Alpha), K417T/E484K/N501Y (Gamma), and L542R (Delta) mutants, and stronger affinity for the N501Y-containing variants. Docking of the four possible monosulfated BoSG disaccharides in interactions with the N501Y mutant S-protein predicted potential binding poses of the BoSG constructs and favorable binding in close proximity to the 501Y residue. Our results demonstrate that depolymerization and fractionation of BoSG are an effective strategy to segregate its anticoagulant property from its anti-SARS-CoV-2 action.

Certain sulfated glycans, including those from marine sources, can show potential effects against SARS-CoV-2. Here, a new fucosylated chondroitin sulfate (FucCS) from the sea cucumber Pentacta pygmaea (PpFucCS) (MW ∼10–60 kDa) was isolated and structurally characterized by NMR. PpFucCS is composed of {→3)-β-GalNAcX-(1→4)-β-GlcA-[(3→1)Y]-(1→}, where X = 4S (80%), 6S (10%) or nonsulfated (10%), Y = α-Fuc2,4S (40%), α-Fuc2,4S-(1→4)-α-Fuc (30%), or α-Fuc4S (30%), and S = SO3−. The anti-SARS-CoV-2 activity of PpFucCS and those of the FucCS and sulfated fucan isolated from Isostichopus badionotus (IbFucCS and IbSF) were compared with that of heparin. IC50 values demonstrated the activity of the three holothurian sulfated glycans to be ∼12 times more efficient than heparin, with no cytotoxic effects. The dissociation constant (KD) values obtained by surface plasmon resonance of the wildtype SARS-CoV-2 spike (S)-protein receptor-binding domain (RBD) and N501Y mutant RBD in interactions with the heparin-immobilized sensor chip were 94 and 1.8 × 103 nM, respectively. Competitive surface plasmon resonance inhibition analysis of PpFucCS, IbFucCS, and IbSF against heparin binding to wildtype S-protein showed IC50 values (in the nanomolar range) 6, 25, and 6 times more efficient than heparin, respectively. Data from computational simulations suggest an influence of the sulfation patterns of the Fuc units on hydrogen bonding with GlcA and that conformational change of some of the oligosaccharide structures occurs upon S-protein RBD binding. Compared with heparin, negligible anticoagulant action was observed for IbSF. Our results suggest that IbSF may represent a promising molecule for future investigations against SARS-CoV-2.

Among the earliest deuterostomes, the echinoderms are an evolutionary important group of ancient marine animals. Within this phylum, the holothuroids (sea cucumbers) are known to produce a wide range of glycoconjugate biopolymers with apparent benefits to health; therefore, they are of economic and culinary interest throughout the world. Other than their highly modified glycosaminoglycans (e.g. fucosylated chondroitin sulfate and fucoidan), nothing is known about their protein-linked glycosylation. Here we used multistep N-glycan fractionation to efficiently separate anionic and neutral N-glycans before analyzing the N-glycans of the black sea cucumber (Holothuria atra) by MS in combination with enzymatic and chemical treatments. These analyses showed the presence of various fucosylated, phosphorylated, sialylated, and multiply sulfated moieties as modifications of oligomannosidic, hybrid, and complex-type N-glycans. The high degree of sulfation and fucosylation parallels the modifications observed previously on holothuroid glycosaminoglycans. Compatible with its phylogenetic position, H. atra not only expresses vertebrate motifs such as sulfo– and sialyl–Lewis A epitopes but displays a high degree of anionic substitution of its glycans, as observed in other marine invertebrates. Thus, as for other echinoderms, the phylum- and order-specific aspects of this species' N-glycosylation reveal both invertebrate- and vertebrate-like features.

Sulfur is present in the amino acids cysteine and methionine and in a large range of essential coenzymes and cofactors and is therefore essential for all organisms. It is also a constituent of sulfate esters in proteins, carbohydrates, and numerous cellular metabolites. The sulfation and desulfation reactions modifying a variety of different substrates are commonly known as sulfation pathways. Although relatively little is known about the function of most sulfated metabolites, the synthesis of activated sulfate used in sulfation pathways is essential in both animal and plant kingdoms. In humans, mutations in the genes encoding the sulfation pathway enzymes underlie a number of developmental aberrations, and in flies and worms, their loss-of-function is fatal. In plants, a lower capacity for synthesizing activated sulfate for sulfation reactions results in dwarfism, and a complete loss of activated sulfate synthesis is also lethal. Here, we review the similarities and differences in sulfation pathways and associated processes in animals and plants, and we point out how they diverge from bacteria and yeast. We highlight the open questions concerning localization, regulation, and importance of sulfation pathways in both kingdoms and the ways in which findings from these "red" and "green" experimental systems may help reciprocally address questions specific to each of the systems.

Background: ACKR2 is an atypical chemokine receptor that is biologically distinct from conventional chemokine receptors. Results: An N-terminal sulfated tyrosine motif is essential for ligand binding by ACKR2. Conclusion: The structure/function basis for ligand binding by ACKR2 is similar to that of conventional chemokine receptors. Significance: ACKR2 N-terminal peptides may be therapeutically useful as pan-chemokine blockers in inflammatory pathologies., The atypical chemokine receptor, ACKR2 is a pivotal regulator of chemokine-driven inflammatory responses and works by binding, internalizing, and degrading inflammatory CC-chemokines. ACKR2 displays promiscuity of ligand binding and is capable of interacting with up to 14 different inflammatory CC-chemokines. Despite its prominent biological role, little is known about the structure/function relationship within ACKR2, which regulates ligand binding. Here we demonstrate that a conserved tyrosine motif at the N terminus of ACKR2 is essential for ligand binding, internalization, and scavenging. In addition we demonstrate that sulfation of this motif contributes to ligand internalization. Furthermore, a peptide derived from this region is capable of binding inflammatory chemokines and inhibits their interaction with their cognate signaling receptors. Importantly, the peptide is only active in the sulfated form, further confirming the importance of the sulfated tyrosines for function. Finally, we demonstrate that the bacterial protease, staphopain A, can cleave the N terminus of ACKR2 and suppress its ligand internalization activity. Overall, these results shed new light on the nature of the structural motifs in ACKR2 that are responsible for ligand binding. The study also highlights ACKR2-derived N-terminal peptides as being of potential therapeutic significance.

Amyloid propagation requires high levels of sequence specificity so that only molecules with very high sequence identity can form cross-β-sheet structures of sufficient stringency for incorporation into the amyloid fibril. This sequence specificity presents a barrier to the transmission of prions between two species with divergent sequences, termed a species barrier. Here we study the relative effects of protein sequence, seed conformation, and environment on the species barrier strength and specificity for the yeast prion protein Sup35p from three closely related species of the Saccharomyces sensu stricto group; namely, Saccharomyces cerevisiae, Saccharomyces bayanus, and Saccharomyces paradoxus. Through in vivo plasmid shuffle experiments, we show that the major characteristics of the transmission barrier and conformational fidelity are determined by the protein sequence rather than by the cellular environment. In vitro data confirm that the kinetics and structural preferences of aggregation of the S. paradoxus and S. bayanus proteins are influenced by anions in accordance with their positions in the Hofmeister series, as observed previously for S. cerevisiae. However, the specificity of the species barrier is primarily affected by the sequence and the type of anion present during the formation of the initial seed, whereas anions present during the seeded aggregation process typically influence kinetics rather than the specificity of prion conversion. Therefore, our work shows that the protein sequence and the conformation variant (strain) of the prion seed are the primary determinants of cross-species prion specificity both in vivo and in vitro.

Heparan sulfate (HS) plays a crucial role in the fibrosis associated with chronic allograft dysfunction by binding and presenting cytokines and growth factors to their receptors. These interactions critically depend on the distribution of 6-O-sulfated glucosamine residues, which is generated by glucosaminyl-6-O-sulfotransferases (HS6STs) and selectively removed by cell surface HS-6-O-endosulfatases (SULFs). Using human renal allografts we found increased expression of 6-O-sulfated HS domains in tubular epithelial cells during chronic rejection as compared with the controls. Stimulation of renal epithelial cells with TGF-β induced SULF2 expression. To examine the role of 6-O-sulfated HS in the development of fibrosis, we generated stable HS6ST1 and SULF2 overexpressing renal epithelial cells. Compared with mock transfectants, the HS6ST1 transfectants showed significantly increased binding of FGF2 (p = 0.0086) and pERK activation. HS6ST1 transfectants displayed a relative increase in mono-6-O-sulfated disaccharides accompanied by a decrease in iduronic acid 2-O-sulfated disaccharide structures. In contrast, SULF2 transfectants showed significantly reduced FGF2 binding and phosphorylation of ERK. Structural analysis of HS showed about 40% down-regulation in 6-O-sulfation with a parallel increase in iduronic acid mono-2-O-sulfated disaccharides. To assess the relevance of these data in vivo we established a murine model of fibrosis (unilateral ureteric obstruction (UUO)). HS-specific phage display antibodies (HS3A8 and RB4EA12) showed significant increase in 6-O-sulfation in fibrotic kidney compared with the control. These results suggest an important role of 6-O-sulfation in the pathogenesis of fibrosis associated with chronic rejection.

Diastrophic dysplasia (DTD) is an incurable recessive chondrodysplasia caused by mutations in the SLC26A2 transporter responsible for sulfate uptake by chondrocytes. The mutations cause undersulfation of glycosaminoglycans in cartilage. Studies of dtd mice with a knock-in Slc26a2 mutation showed an unusual progression of the disorder: net undersulfation is mild and normalizing with age, but the articular cartilage degrades with age and bones develop abnormally. To understand underlying mechanisms, we studied newborn dtd mice. We developed, verified and used high-definition infrared hyperspectral imaging of cartilage sections at physiological conditions, to quantify collagen and its orientation, noncollagenous proteins, and chondroitin chains, and their sulfation with 6-μm spatial resolution and without labeling. We found that chondroitin sulfation across the proximal femur cartilage varied dramatically in dtd, but not in the wild type. Corresponding undersulfation of dtd was mild in most regions, but strong in narrow articular and growth plate regions crucial for bone development. This undersulfation correlated with the chondroitin synthesis rate measured via radioactive sulfate incorporation, explaining the sulfation normalization with age. Collagen orientation was reduced, and the reduction correlated with chondroitin undersulfation. Such disorientation involved the layer of collagen covering the articular surface and protecting cartilage from degradation. Malformation of this layer may contribute to the degradation progression with age and to collagen and proteoglycan depletion from the articular region, which we observed in mice already at birth. The results provide clues to in vivo sulfation, DTD treatment, and cartilage growth.

Slc26a2 is a ubiquitously expressed SO(4)(2-) transporter with high expression levels in cartilage and several epithelia. Mutations in SLC26A2 are associated with diastrophic dysplasia. The mechanism by which Slc26a2 transports SO(4)(2-) and the ion gradients that mediate SO(4)(2-) uptake are poorly understood. We report here that Slc26a2 functions as an SO(4)(2-)/2OH(-), SO(4)(2-)/2Cl(-), and SO(4)(2-)/OH(-)/Cl(-) exchanger, depending on the Cl(-) and OH(-) gradients. At inward Cl(-) and outward pH gradients (high Cl(-)(o) and low pH(o)) Slc26a2 functions primarily as an SO(4)(2-)(o)/2OH(-)(i) exchanger. At low Cl(-)(o) and high pH(o) Slc26a2 functions increasingly as an SO(4)(2-)(o)/2Cl(-)(i) exchanger. The reverse is observed for SO(4)(2-)(i)/2OH(-)(o) and SO(4)(2-)(i)/2Cl(-)(o) exchange. Slc26a2 also exchanges Cl(-) for I(-), Br(-), and NO(3)(-) and Cl(-)(o) competes with SO(4)(2-) on the transport site. Interestingly, Slc26a2 is regulated by an extracellular anion site, required to activate SO(4)(2-)(i)/2OH(-)(o) exchange. Slc26a2 can transport oxalate in exchange for OH(-) and/or Cl(-) with properties similar to SO(4)(2-) transport. Modeling of the Slc26a2 transmembrane domain (TMD) structure identified a conserved extracellular sequence (367)GFXXP(371) between TMD7 and TMD8 close to the conserved Glu(417) in the permeation pathway. Mutation of Glu(417) eliminated transport by Slc26a2, whereas mutation of Phe(368) increased the affinity for SO(4)(2-)(o) 8-fold while reducing the affinity for Cl(-)(o) 2 fold, but without affecting regulation by Cl(-)(o). These findings clarify the mechanism of net SO(4)(2-) transport and describe a novel regulation of Slc26a2 by an extracellular anion binding site and should help in further understanding aberrant SLC26A2 function in diastrophic dysplasia.

Bacterial degradation of sulfoacetate, a widespread natural product, proceeds via sulfoacetaldehyde and requires a considerable initial energy input. Whereas the fate of sulfoacetaldehyde in Cupriavidus necator (Ralstonia eutropha) H16 is known, the pathway from sulfoacetate to sulfoacetaldehyde is not. The genome sequence of the organism enabled us to hypothesize that the inducible pathway, which initiates sau (sulfoacetate utilization), involved a four-gene cluster (sauRSTU; H16_A2746 to H16_A2749). The sauR gene, divergently orientated to the other three genes, probably encodes the transcriptional regulator of the presumed sauSTU operon, which is subject to inducible transcription. SauU was tentatively identified as a transporter of the major facilitator superfamily, and SauT was deduced to be a sulfoacetate-CoA ligase. SauT was a labile protein, but it could be separated and shown to generate AMP and an unknown, labile CoA-derivative from sulfoacetate, CoA, and ATP. This unknown compound, analyzed by MALDI-TOF-MS, had a relative molecular mass of 889.7, which identified it as protonated sulfoacetyl-CoA (calculated 889.6). SauS was deduced to be sulfoacetaldehyde dehydrogenase (acylating). The enzyme was purified 175-fold to homogeneity and characterized. Peptide mass fingerprinting confirmed the sauS locus (H16_A2747). SauS converted sulfoacetyl-CoA and NADPH to sulfoacetaldehyde, CoA, and NADP(+), thus confirming the hypothesis.

TASK-2 (KCNK5 or K(2P)5.1) is a background K(+) channel that is opened by extracellular alkalinization and plays a role in renal bicarbonate reabsorption and central chemoreception. Here, we demonstrate that in addition to its regulation by extracellular protons (pH(o)) TASK-2 is gated open by intracellular alkalinization. The following pieces of evidence suggest that the gating process controlled by intracellular pH (pH(i)) is independent from that under the command of pH(o). It was not possible to overcome closure by extracellular acidification by means of intracellular alkalinization. The mutant TASK-2-R224A that lacks sensitivity to pH(o) had normal pH(i)-dependent gating. Increasing extracellular K(+) concentration acid shifts pH(o) activity curve of TASK-2 yet did not affect pH(i) gating of TASK-2. pH(o) modulation of TASK-2 is voltage-dependent, whereas pH(i) gating was not altered by membrane potential. These results suggest that pH(o), which controls a selectivity filter external gate, and pH(i) act at different gating processes to open and close TASK-2 channels. We speculate that pH(i) regulates an inner gate. We demonstrate that neutralization of a lysine residue (Lys(245)) located at the C-terminal end of transmembrane domain 4 by mutation to alanine abolishes gating by pH(i). We postulate that this lysine acts as an intracellular pH sensor as its mutation to histidine acid-shifts the pH(i)-dependence curve of TASK-2 as expected from its lower pK(a). We conclude that intracellular pH, together with pH(o), is a critical determinant of TASK-2 activity and therefore of its physiological function.

The Zap1 transcription factor is a central player in the response of yeast to changes in zinc status. Previous studies identified over 80 genes activated by Zap1 in zinc-limited cells. In this report, we identified 36 genes repressed in a zinc- and Zap1-responsive manner. As a result, we have identified a new mechanism of Zap1-mediated gene repression whereby transcription of the MET3, MET14, and MET16 genes is repressed in zinc-limited cells. These genes encode the first three enzymes of the sulfate assimilation pathway. We found that MET30, encoding a component of the SCF(Met30) ubiquitin ligase, is a direct Zap1 target gene. MET30 expression is increased in zinc-limited cells, and this leads to degradation of Met4, a transcription factor responsible for MET3, MET14, and MET16 expression. Thus, Zap1 is responsible for a decrease in sulfate assimilation in zinc-limited cells. We further show that cells that are unable to down-regulate sulfate assimilation under zinc deficiency experience increased oxidative stress. This increased oxidative stress is associated with an increase in the NADP(+)/NADPH ratio and may result from a decrease in NADPH-dependent antioxidant activities. These studies have led to new insights into how cells adapt to nutrient-limiting growth conditions.

Matrix metalloproteinases are maintained in an inactive state by a bond between the thiol of a conserved cysteine in the prodomain and a zinc atom in the catalytic domain. Once this bond is disrupted, MMPs become active proteinases and can act on a variety of extracellular protein substrates. In vivo, matrilysin (MMP7) activates pro-alpha-defensins (procryptdins), but in vitro, processing of these peptides is slow, with about 50% conversion in 8-12 h. Similarly, autolytic activation of promatrilysin in vitro can take up to 12-24 h for 50% conversion. These inefficient reactions suggest that natural cofactors enhance the activation and activity of matrilysin. We determined that highly sulfated glycosaminoglycans (GAG), such as heparin, chondroitin-4,6-sulfate (CS-E), and dermatan sulfate, markedly enhanced (50-fold) the intermolecular autolytic activation of promatrilysin and the activity of fully active matrilysin to cleave specific physiologic substrates. In contrast, heparan sulfate and less sulfated forms of chondroitin sulfate did not augment matrilysin activation or activity. Chondroitin-2,6-sulfate (CS-D) also did not enhance matrilysin activity, suggesting that the presentation of sulfates is more important than the overall degree of sulfation. Surface plasmon resonance demonstrated that promatrilysin bound heparin (K(D), 400 nm) and CS-E (K(D), 630 nm). Active matrilysin bound heparin (K(D), 150 nm) but less so to CS-E (K(D), 60 microm). Neither form bound heparan sulfate. These observations demonstrate that sulfated GAGs regulate matrilysin activation and its activity against specific substrates.

Protein-tyrosine sulfation is mediated by two Golgi tyrosyl-protein sulfotransferases (TPST-1 and TPST-2) that are widely expressed in vivo. However, the full substrate repertoire of this enzyme system is unknown and thus, our understanding of the biological role(s) of tyrosine sulfation is limited. We reported that whereas Tpst1-/- male mice have normal fertility, Tpst2-/- males are infertile despite normal spermatogenesis. However, Tpst2-/- sperm are severely defective in their motility in viscous media and in their ability to fertilize eggs. These findings suggest that sulfation of unidentified substrate(s) is crucial for normal sperm function. We therefore sought to identify tyrosine-sulfated proteins in the male genital tract using affinity chromatography on PSG2, an anti-sulfotyrosine monoclonal antibody, followed by mass spectrometry. Among the several candidate tyrosine-sulfated proteins identified, RNase 9 and Mfge8 were examined in detail. RNase 9, a catalytically inactive RNase A family member of unknown function, is expressed only in the epididymis after onset of sexual maturity. Mfge8 is expressed on mouse sperm and Mfge8-/- male mice are subfertile. Metabolic labeling coupled with sulfoamino acid analysis confirmed that both proteins are tyrosine-sulfated and both proteins are expressed at comparable levels in wild type, Tpst1-/-, and Tpst2-/- epididymides. However, we demonstrate that RNase 9 and Mfge8 are tyrosine-sulfated in wild type and Tpst1-/-, but not in Tpst2-/- mice. These findings suggest that lack of sulfation of one or both of these proteins may contribute mechanistically to the infertility of Tpst2-/- males.

Sulfate reduction is one of the earliest types of energy metabolism used by ancestral organisms to sustain life. Despite extensive studies, many questions remain about the way respiratory sulfate reduction is associated with energy conservation. A crucial enzyme in this process is the dissimilatory sulfite reductase (dSiR), which contains a unique siroheme-[4Fe4S] coupled cofactor. Here, we report the structure of desulfoviridin from Desulfovibrio vulgaris, in which the dSiR DsrAB (sulfite reductase) subunits are bound to the DsrC protein. The α2β2γ2 assembly contains two siroheme-[4Fe4S] cofactors bound by DsrB, two sirohydrochlorins and two [4Fe4S] centers bound by DsrA, and another four [4Fe4S] centers in the ferredoxin domains. A sulfite molecule, coordinating the siroheme, is found at the active site. The DsrC protein is bound in a cleft between DsrA and DsrB with its conserved C-terminal cysteine reaching the distal side of the siroheme. We propose a novel mechanism for the process of sulfite reduction involving DsrAB, DsrC, and the DsrMKJOP membrane complex (a membrane complex with putative disulfide/thiol reductase activity), in which two of the six electrons for reduction of sulfite derive from the membrane quinone pool. These results show that DsrC is involved in sulfite reduction, which changes the mechanism of sulfate respiration. This has important implications for models used to date ancient sulfur metabolism based on sulfur isotope fractionations.

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.

Heparin-like saccharides play an essential role in binding to both fibroblast growth factors (FGF) and their receptors at the cell surface. In this study we prepared a series of heparin oligosaccharides according to their size and sulfation level. We then investigated their affinity for FGF2 and their ability to support FGF2 mitogenesis of heparan sulfate-deficient cells expressing FGFR1c. Tetra- and hexasaccharides bound FGF2, but failed to dimerize the growth factor. Nevertheless, these saccharides promoted FGF2-mediated cell growth. Furthermore, whereas enzymatic removal of the non-reducing end 2-O-sulfate group had little effect on the 1:1 interaction with FGF2, it eliminated the mitogenic activity of these saccharides. This evidence supports the symmetric two-end model of ternary complex formation. In contrast, even at very low concentrations, octasaccharide and larger heparin fragments conferred a potent mitogenic activity that was independent of terminal 2-O-sulfation. This correlated with the ability to dimerize FGF2 in an apparently cooperative manner. This data suggests that potent mitogenic signaling results from heparin-mediated trans-dimerization of FGF2, consistent with the asymmetric model of ternary complex formation. We propose that, depending on saccharide structure, there are different architectures and modes of ternary complex assembly that differ in stability and/or efficiency of transmembrane signaling.

Atherosclerosis is the underlying pathological process of most cardiovascular disease. A critical component of the "response to retention" hypothesis of atherogenesis is proteoglycan/low density lipoprotein (LDL) binding. Transforming growth factor beta (TGF-beta) is present in atherosclerotic lesions, regulates vascular smooth muscle cell (VSMC) proteoglycan synthesis via an unknown signaling pathway, and increases proteoglycan/LDL binding. This pathway was investigated using the activin receptor-like kinase 5 (ALK5) inhibitor SB431542 and inhibitors of p38 MAP kinase as a possible downstream or alternative mediator. TGF-beta stimulated and SB431542 inhibited the phosphorylation of Smad2/3. In human VSMC, TGF-beta increased [(35)S]sulfate incorporation into proteoglycans associated with a 19% increase in glycosaminoglycan (GAG) chain size by size exclusion chromatography. SB431542 caused a concentration-dependent decrease in TGF-beta-mediated [(35)S]sulfate incorporation with 92% inhibition at 3 mum. Two different p38 MAP kinase inhibitors, SB203580 and SB202190, but not the inactive analogue SB202474, concentration-dependently blocked TGF-beta-mediated [(35)S]sulfate incorporation. TGF-beta increased [(3)H]glucosamine incorporation into glycosaminoglycans by 180% and [(35)S]Met/Cys incorporation into proteoglycan core proteins by 35% with both effects completely inhibited by SB431542. Blocking both Smad2/3 and p38 MAP kinase pathways prevented the effect of TGF-beta to increase proteoglycan to LDL binding. TGF-beta mediates its effects on proteoglycan synthesis in VSMCs via the ALK5/Smad2/3 phosphorylation pathway as well as via the p38 MAP kinase signaling cascade. Further studies of downstream pathways controlling proteoglycan synthesis may identify potential therapeutic targets for the prevention of atherosclerosis and cardiovascular disease.

Many of the biological functions of heparan sulfate (HS) proteoglycans can be attributed to specialized structures within HS moieties, which are thought to modulate binding and function of various effector proteins. Cyclophilin B (CyPB), which was initially identified as a cyclosporin A-binding protein, triggers migration and integrin-mediated adhesion of peripheral blood T lymphocytes by a mechanism dependent on interaction with cell surface HS. Here we determined the structural features of HS that are responsible for the specific binding of CyPB. In addition to the involvement of 2-O,6-O, and N-sulfate groups, we also demonstrated that binding of CyPB was dependent on the presence of N-unsubstituted glucosamine residues (GlcNH(2)), which have been reported to be precursors for sulfation by 3-O-sulfotransferases-3 (3-OST-3). Interestingly, 3-OST-3B isoform was found to be the main 3-OST isoenzyme expressed in peripheral blood T lymphocytes and Jurkat T cells. Moreover, down-regulation of the expression of 3-OST-3 by RNA interference potently reduced CyPB binding and consequent activation of p44/42 mitogen-activated protein kinases. Altogether, our results strongly support the hypothesis that 3-O-sulfation of GlcNH(2) residues could be a key modification that provides specialized HS structures for CyPB binding to responsive cells. Given that 3-O-sulfation of GlcNH(2)-containing HS by 3-OST-3 also provides binding sites for glycoprotein gD of herpes simplex virus type I, these findings suggest an intriguing structural linkage between the HS sequences involved in CyPB binding and viral infection.

Fxralpha is known to regulate a variety of metabolic processes, including bile acid, cholesterol, and carbohydrate metabolism. In this study, we show direct evidence that Fxralpha is a key player in maintaining sulfate homeostasis. We identified and characterized the sodium/sulfate co-transporter (NaS-1; Slc13a1) as an Fxralpha target gene expressed in the kidney and intestine. Electromobility shift assays, chromatin immunoprecipitation, and promoter reporter studies identified a single functional Fxralpha response element in the second intron of the mouse Slc13a1 gene. Treatment of wild-type mice with GW4064, a synthetic Fxralpha agonist, induced Slc13a1 mRNA in the intestine and kidney. Slc13a1 mRNA was also induced in the kidney and intestine of wild-type, but not Fxralpha-/- mice, after treatment with the hepatotoxin alpha-naphthylisothiocyanate, which is known to result in elevated blood bile acid levels. Finally, we observed a decrease in Slc13a1 mRNA in the kidney and intestine of Fxralpha-/- mice and a corresponding increase in urinary excretion of free sulfates as compared with wild-type mice. These results demonstrate that mouse Slc13a1 is a novel Fxralpha target gene expressed in the kidney and intestine and that in the absence of Fxralpha, mice waste sulfate into the urine. Thus, Fxralpha is necessary for normal sulfate homeostasis in vivo.

Sulfate assimilation provides reduced sulfur for synthesis of the amino acids cysteine and methionine and for a range of other metabolites. Sulfate has to be activated prior to reduction by adenylation to adenosine 5'-phosphosulfate (APS). In plants, algae, and many bacteria, this compound is reduced to sulfite by APS reductase (APR); in fungi and some cyanobacteria and gamma-proteobacteria, a second activation step, phosphorylation to 3'-phosphoadenosine 5'-phosphosulfate (PAPS), is necessary before reduction to sulfite by PAPS reductase (PAPR). We found previously that the moss Physcomitrella patens is unique among these organisms in possessing orthologs of both APR and PAPR genes (Koprivova, A., Meyer, A. J., Schween, G., Herschbach, C., Reski, R., and Kopriva, S. (2002) J. Biol. Chem. 277, 32195-32201). To assess the function of the two enzymes, we compared their biochemical properties by analysis of purified recombinant proteins. APR from Physcomitrella is very similar to the well characterized APRs from seed plants. On the other hand, we found that the putative PAPR preferentially reduces APS. Sequence analysis, analysis of UV-visible spectra, and determination of iron revealed that this new APR, named PpAPR-B, does not contain the FeS cluster, which was previously believed to determine the substrate specificity of the otherwise relatively similar enzymes. The lack of the FeS cluster in PpAPR-B catalysis is connected with a lower turnover rate but higher stability of the protein. These findings show that APS reduction without the FeS cluster is possible and that plant sulfate assimilation is predominantly dependent on reduction of APS.

Human leukocyte elastase (HLE) and cathepsin G (CG) are expressed at high levels on the surface of activated human neutrophils (PMN) in catalytically active but inhibitor-resistant forms having the potential to contribute to tissue injury. Herein we have investigated the mechanisms by which HLE and CG bind to PMN plasma membranes. (125)I-Labeled HLE and CG bind to PMN at 0 degrees C in a saturable and reversible manner (K(D) = 5.38 and 4.36 x 10(-7) m and 11.5 and 8.1 x 10(6) binding sites/cell, respectively). Incubation of PMN with radiolabeled HLE and CG in the presence of a 200-fold molar excess of unlabeled HLE, CG, myeloperoxidase, lactoferrin, proteinase 3, phenylmethylsulfonyl fluoride (PMSF)-inactivated HLE, or PMSF-inactivated CG inhibited binding of radiolabeled ligands. This indicates that these PMN granule proteins share binding sites on PMN and that functional active sites of HLE and CG are not required for their binding to PMN. The sulfate groups of heparan sulfate- and chondroitin sulfate-containing proteoglycans are the PMN binding sites for HLE and CG since binding of HLE and CG to PMN was inhibited by incubating PMN with 1) trypsin, chondroitinase ABC, and heparitinases, but not other glycanases, and 2) purified chondroitin sulfates, heparan sulfate, and other sulfated molecules, but not with non-sulfated glycans. Thus, heparan sulfate- and chondroitin sulfate-containing proteoglycans are low affinity, high volume PMN surface binding sites for HLE and CG, which are well suited to bind high concentrations of active serine proteinases released from degranulating PMN.

One of the major complicating factors in insulin-dependent diabetes mellitus is nephropathy. Several investigators have linked heparan sulfate (HS) alterations in the glomerular basement membrane (GBM) with albuminuria as a marker of abnormal blood filtration and the subsequent progression to renal failure. In this study, we examined the fine structure of HS in the glomerulus and the GBM isolated from the kidneys of rats injected with streptozotocin. Using fluorophore-assisted carbohydrate electrophoresis, we obtained disaccharide composition analyses for HS. In a time course study, we observed that normal rat HS isolated from the GBM becomes more N-sulfated as the glomeruli mature over a period of 8 weeks. Diabetic rats injected with streptozotocin at the beginning of this period showed a reversal of this trend. Using a graded sieve technique, we found that two different sizes of glomeruli could be isolated from the rat kidneys and that there was a significant difference in the HS disaccharide content between these two pools of glomeruli. Only the larger sized glomeruli had less N-sulfation of HS as a result of insulin-dependent diabetes mellitus. This change in the fine structure of HS was localized to the GBM and was not associated with cell surface HS. We also generated oligosaccharides of HS that portray fine structural alterations in the diabetic rats indicative of a loss of the sulfation of N-acetylglucosamine.

Human DHRS6 is a previously uncharacterized member of the short chain dehydrogenases/reductase family and displays significant homologies to bacterial hydroxybutyrate dehydrogenases. Substrate screening reveals sole NAD(+)-dependent conversion of (R)-hydroxybutyrate to acetoacetate with K(m) values of about 10 mm, consistent with plasma levels of circulating ketone bodies in situations of starvation or ketoacidosis. The structure of human DHRS6 was determined at a resolution of 1.8 A in complex with NAD(H) and reveals a tetrameric organization with a short chain dehydrogenases/reductase-typical folding pattern. A highly conserved triad of Arg residues ("triple R" motif consisting of Arg(144), Arg(188), and Arg(205)) was found to bind a sulfate molecule at the active site. Docking analysis of R-beta-hydroxybutyrate into the active site reveals an experimentally consistent model of substrate carboxylate binding and catalytically competent orientation. GFP reporter gene analysis reveals a cytosolic localization upon transfection into mammalian cells. These data establish DHRS6 as a novel, cytosolic type 2 (R)-hydroxybutyrate dehydrogenase, distinct from its well characterized mitochondrial type 1 counterpart. The properties determined for DHRS6 suggest a possible physiological role in cytosolic ketone body utilization, either as a secondary system for energy supply in starvation or to generate precursors for lipid and sterol synthesis.

We have shown previously that a highly sulfated sequence, GalNAc(4,6-SO(4))-GlcA(2SO(4))-GalNAc(6SO(4)), is present at the nonreducing terminal of chondroitin sulfate (CS), and this structure was synthesized from a unique sequence, GalNAc(4SO(4))-GlcA(2SO(4))-GalNAc(6SO(4)), by sulfation with N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase. Uronosyl 2-O-sulfotrasferase (2OST), which transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to position 2 of the GlcA residue of CS, is expected to be involved in synthesis of these structures; however, the specificity of 2OST concerning recognition of the sulfation pattern of the acceptor has largely remained unclear. In the present study, we examined the specificity of 2OST in terms of recognition of the sulfation pattern around the targeting GlcA residue. The recombinant 2OST could sulfate CS-A, CS-C, and desulfated dermatan sulfate. When [(35)S]glycosaminoglycans formed from CS-A after the reaction with the recombinant 2OST and [(35)S]PAPS were subjected to limited digestion with chondroitinase ACII, a radioactive tetrasaccharide (Tetra A) was obtained as a sole intermediate product. The sequence of Tetra A was found to be DeltaHexA-GalNAc(4SO(4))-GlcA(2SO(4))-GalNAc(6SO(4)) by enzymatic and chemical reactions. These observations indicate that 2OST transfers sulfate preferentially to the GlcA residue located in a unique sequence, -GalNAc(4SO(4))-GlcA-GalNAc(6SO(4))-. When oligosaccharides with different sulfation patterns were used as the acceptor, GalNAc(4SO(4))-GlcA-GalNAc(6SO(4)) and GlcA-GalNAc(4SO(4))-GlcA-GalNAc(6SO(4)) were the best acceptors for 2OST among trisaccharides and tetrasaccharides, respectively. These results suggest that 2OST may be involved in the synthesis of the highly sulfated structure found in CS-A.

We have grown polarized epithelial Madin-Darby canine kidney II (MDCK II) cells on filters in the presence of [(35)S]sulfate, [(3)H]glucosamine, or [(35)S]cysteine/[(35)S]methionine to study proteoglycan (PG) synthesis, sorting, and secretion to the apical and basolateral media. Whereas most of the [(35)S]sulfate label was recovered in basolateral PGs, the [(3)H]glucosamine label was predominantly incorporated into the glycosaminoglycan chains of apical PGs, indicating that basolateral PGs are more intensely sulfated than their apical counterparts. Expression of the PG serglycin with a green fluorescent protein tag (SG-GFP) in MDCK II cells produced a protein core secreted 85% apically, which was largely modified by chondroitin sulfate chains. Surprisingly, the 15% of secreted SG-GFP molecules recovered basolaterally were more heavily sulfated and displayed a different sulfation pattern than the apical counterpart. More detailed studies of the differential modification of apically and basolaterally secreted SG-GFP indicate that the protein cores have been designated to apical and basolateral transport platforms before pathway-specific, post-translational modifications have been completed.

Metabolomics is considered as an emerging new tool for functional proteomics in the identification of new protein function or in projects aiming at modeling whole cell metabolism. When combined with proteome studies, metabolite-profiling analyses revealed unanticipated insights into the yeast sulfur pathway. In response to cadmium, the observed overproduction of glutathione, essential for the detoxification of the metal, can be entirely accounted for by a marked drop in sulfur-containing protein synthesis and a redirection of sulfur metabolite fluxes to the glutathione pathway. A kinetic analysis showed sequential and dramatic changes in intermediate sulfur metabolite pools within the first hours of the treatment. Strikingly, whereas proteome and metabolic data were positively correlated under cadmium conditions, proteome and metabolic data were negatively correlated during other growth conditions, i.e. methionine supplementation or sulfate starvation. These differences can be explained by alternative mechanisms in the regulation of Met4, the activator of the sulfur pathway. Whereas Met4 activity is controlled by the cellular cysteine content in response to sulfur source and availability, the present study suggests that Met4 activation under cadmium conditions is cysteine-independent. The results clearly indicate that the metabolic state of a cell cannot be safely predicted based solely on proteomic and/or gene expression data. Combined metabolome and proteome studies are necessary to draw a comprehensive and integrated view of cell metabolism.

The molecular interaction of secreted granzyme B-serglycin complexes with target cells remains undefined. Targets exposed to double-labeled granzyme B-serglycin complexes show solely the uptake of granzyme B. An in vitro model demonstrates the exchange of the granzyme from serglycin to immobilized, sulfated glycosaminoglycans. Using a combination of cell binding and internalization assays, granzyme B was found to exchange to sulfated glycosaminoglycans and, depending on the cell type, to higher affinity sites. Apoptosis induced by purified granzyme B and cytotoxic T-cells was diminished in targets with reduced cell surface glycosaminoglycan content. A mechanism of delivery is proposed entailing electrostatic transfer of granzyme B from serglycin to cell surface proteins.

Hydroxyquinol 1,2-dioxygenase (1,2-HQD) catalyzes the ring cleavage of hydroxyquinol (1,2,4-trihydroxybenzene), a central intermediate in the degradation of aromatic compounds including a variety of particularly recalcitrant polychloro- and nitroaromatic pollutants. We report here the primary sequence determination and the analysis of the crystal structure of the 1,2-HQD from Nocardioides simplex 3E solved at 1.75 A resolution using the multiple wavelength anomalous dispersion of the two catalytic irons (1 Fe/293 amino acids). The catalytic Fe(III) coordination polyhedron composed by the side chains of Tyr164, Tyr197, His221, and His223 resembles that of the other known intradiol-cleaving dioxygenases, but several of the tertiary structure features are notably different. One of the most distinctive characteristics of the present structure is the extensive openings and consequent exposure to solvent of the upper part of the catalytic cavity arranged to favor the binding of hydroxyquinols but not catechols. A co-crystallized benzoate-like molecule is also found bound to the metal center forming a distinctive hydrogen bond network as observed previously also in 4-chlorocatechol 1,2-dioxygenase from Rhodococcus opacus 1CP. This is the first structure of an intradiol dioxygenase specialized in hydroxyquinol ring cleavage to be investigated in detail.

Phosphoenolpyruvate carboxylases (PEPC, EC 4.1.1.31) from higher plants are regulated by both allosteric effects and reversible phosphorylation. Previous x-ray crystallographic analysis of Zea mays PEPC has revealed a binding site for sulfate ion, speculated to be the site for an allosteric activator, glucose 6-phosphate (Glc-6-P) (Matsumura, H., Xie, Y., Shirakata, S., Inoue, T., Yoshinaga, T., Ueno, Y., Izui, K., and Kai, Y. (2002) Structure (Lond.) 10, 1721-1730). Because kinetic experiments have also supported this notion, each of the four basic residues (Arg-183, -184, -231, and -372' on the adjacent subunit) located at or near the binding site was replaced by Gln, and the kinetic properties of recombinant mutant enzymes were investigated. Complete desensitization to Glc-6-P was observed for R183Q, R184Q, R183Q/R184Q (double mutant), and R372Q, as was a marked decrease in the sensitivity for R231Q. The heterotropic effect of Glc-6-P on an allosteric inhibitor, l-malate, was also abolished, but sensitivity to Gly, another allosteric activator of monocot PEPC, was essentially not affected, suggesting the distinctness of their binding sites. Considering the kinetic and structural data, Arg-183 and Arg-231 were suggested to be involved directly in the binding with phosphate group of Glc-6-P, and the residues Arg-184 and Arg-372 were thought to be involved in making up the site for Glc-6-P and/or in the transmission of an allosteric regulatory signal. Most unexpectedly, the mutant enzymes had almost lost responsiveness to regulatory phosphorylation at Ser-15. An apparent lack of kinetic competition between the phosphate groups of Glc-6-P and of phospho-Ser at 15 suggested the distinctness of their binding sites. The possible roles of these Arg residues are discussed.

Multiple ligand co-recognition of 3'-sulfogalactosylceramide (SGC) and sulfotyrosine initiated the comparison of SGC and sulfotyrosine and, subsequently, phosphotyrosine (pY) binding. SGC is a receptor for ligands involved in cell adhesion/microbial pathology. pY forms a Src homology domain 2 recognition motif in intracellular signaling. Using hsp70, anti-SGC, and anti-pY antibodies, ligand binding is retained following phosphate/sulfate and tyrosine/galactose substitution in SGC and sulfate/phosphate exchange in pY. Remarkable lipid-dependent binding to phosphatidylethanolamine-conjugated sulfotyrosine suggests "microenvironmental" modulation of sulfotyrosine-containing receptors, similar to glycosphingolipids. Based on an aryl substrate-bound co-crystal of arylsulfatase A, a sulfogalactose and phosphotyrosine esterase, modeling provides a solvation basis for co-recognition. c-Src/Src homology domain 2:SGC/phosphogalactosylceramide binding confirms our hypothesis, heralding a carbohydrate-based approach to regulation of phosphotyrosine-mediated recognition.

Sialic acid-binding immunoglobulin-like lectin 8 (Siglec-8) is selectively expressed on human eosinophils, basophils, and mast cells, where it regulates their function and survival. Previous studies demonstrated sialic acid-dependent binding of Siglec-8 but failed to reveal significant substructure specificity or high affinity of that binding. To test a broader range of potential ligands, a Siglec-8-Ig chimeric protein was tested for binding to 172 different glycan structures immobilized as biotinylated glycosides on a 384-well streptavidin-coated plate. Of these, approximately 40 structures were sialylated. Among these, avid binding was detected to a single defined glycan, NeuAcalpha2-3(6-O-sulfo)Galbeta1-4[Fucalpha1-3]GlcNAc, also referred to in the literature as 6'-sulfo-sLex. Notably, neither unsulfated sLex (NeuAcalpha2-3Galbeta1-4[Fucalpha1-3]GlcNAc) nor an isomer with the sulfate on the 6-position of the GlcNAc residue (6-sulfo-sLex, NeuAcalpha2-3Galbeta1-4[Fucalpha1-3](6-O-sulfo)GlcNAc) supported detectable binding. Subsequent secondary screening was performed using surface plasmon resonance. Biotin glycosides immobilized on streptavidin biosensor chips were exposed to Siglec-8-Ig in solution. Whereas surfaces derivatized with sLex and 6-sulfo-sLex failed to support detectable Siglec-8 binding, 6'-sulfo-sLex supported significant binding with a Kd of 2.3 microm. In a separate test of binding specificity, aminopropyl glycosides were covalently immobilized at different concentrations on activated (N-hydroxysuccinimidyl) glass surfaces (Schott-Nexterion Slide H). Subsequent exposure to Siglec-8-Ig precomplexed with fluorescein isothiocyanate anti-human Fc resulted in fluorescent signals at immobilized concentrations of 6'-sulfo-sLex of5 pmol/spot. In contrast, sLex and 6-sulfo-sLex did not support any Siglec-8 binding at the highest concentration tested (300 pmol/spot). We conclude that Siglec-8 binds preferentially to the sLex structure bearing an additional sulfate ester on the galactose 6-hydroxyl.

Glycosaminoglycans have been implicated in the binding and activation of a variety of growth factors, cytokines, and chemokines. In this way, glycosaminoglycans are thought to participate in events such as development and wound repair. In particular, heparin and heparan sulfate have been well studied, and specific aspects of their structure dictate their participation in a variety of activities. In contrast, although dermatan sulfate participates in many of the same biological processes as heparin and heparan sulfate, the interactions of dermatan sulfate have been less well studied. Dermatan sulfate is abundant in the wound environment and binds and activates growth factors such as fibroblast growth factor-2 (FGF-2) and FGF-7, which are present during the wound repair process. To determine the minimum size and sulfation content of active dermatan sulfate oligosaccharides, dermatan sulfate was first digested and then separated by size exclusion high pressure liquid chromatography, and the activity to facilitate FGF-2 and FGF-7 was assayed by the cellular proliferation of cell lines expressing FGFR1 or FGFR2 IIIb. The minimum size required for the activation of FGF-2 was an octasaccharide and for FGF-7 a decasaccharide. Active fractions were rich in monosulfated, primarily 4-O-sulfated, disaccharides and iduronic acid. Increasing the sulfation to primarily 2/4-O-sulfated and 2/6-O-sulfated disaccharides did not increase activity. Cell proliferation decreased or was abolished with higher sulfated dermatan sulfate preparations. This indicated a preference for specific dermatan sulfate oligosaccharides capable of promoting FGF-2- and FGF-7-dependent cell proliferation. These data identify critical oligosaccharides that promote specific members of the FGF family that are important for wound repair and angiogenesis.

The unusually low 78% amino acid identity between the orthologous human SLC26A6 and mouse slc26a6 polypeptides prompted systematic comparison of their anion transport functions in Xenopus oocytes. Multiple human SLC26A6 variant polypeptides were also functionally compared. Transport was studied as unidirectional fluxes of (36)Cl(-), [(14)C]oxalate, and [(35)S]sulfate; as net fluxes of HCO(3)(-) by fluorescence ratio measurement of intracellular pH; as current by two-electrode voltage clamp; and as net Cl(-) flux by fluorescence intensity measurement of relative changes in extracellular and intracellular [Cl(-)]. Four human SLC26A6 polypeptide variants each exhibited rates of bidirectional [(14)C]oxalate flux, Cl(-)/HCO(3)(-) exchange, and Cl(-)/OH(-) exchange nearly equivalent to those of mouse slc26a6. Cl(-)/HCO(3)(-) exchange by both orthologs was cAMP-sensitive, further enhanced by coexpressed wild type cystic fibrosis transmembrane regulator but inhibited by cystic fibrosis transmembrane regulator DeltaF508. However, the very low rates of (36)Cl(-) and [(35)S]sulfate transport by all active human SLC26A6 isoforms contrasted with the high rates of the mouse ortholog. Human and mouse orthologs also differed in patterns of acute regulation. Studies of human-mouse chimeras revealed cosegregation of the high (36)Cl(-) transport phenotype with the transmembrane domain of mouse slc26a6. Mouse slc26a6 and human SLC26A6 each mediated electroneutral Cl(-)/HCO(3)(-) and Cl(-)/OH(-) exchange. In contrast, whereas Cl(-)/oxalate exchange by mouse slc26a6 was electrogenic, that mediated by human SLC26A6 appeared electroneutral. The increased currents observed in oocytes expressing either mouse or human ortholog were pharmacologically distinct from the accompanying monovalent anion exchange activities. The human SLC26A6 polypeptide variants SLC26A6c and SLC26A6d were inactive as transporters of oxalate, sulfate, and chloride. Thus, the orthologous mouse and human SLC26A6 proteins differ in anion selectivity, transport mechanism, and acute regulation, but both mediate electroneutral Cl(-)/HCO(3)(-) exchange.

Neuregulins are a family of growth and differentiation factors that act through activation of cell-surface erbB receptor tyrosine kinases and have essential functions both during development and on the growth of cancer cells. One alternatively spliced neuregulin-1 form has a distinct heparin-binding immunoglobulin-like domain that enables it to adhere to heparan sulfate proteoglycans at key locations during development and substantially potentiates its activity. We examined the structural specificity needed for neuregulin-1-heparin interactions using a gel mobility shift assay together with an assay that measures the ability of specific oligosaccharides to block erbB receptor phosphorylation in L6 muscle cells. Whereas the N-sulfate group of heparin was most important, the 2-O-sulfate and 6-O-sulfate groups also contributed to neuregulin-1 binding in these two assays. Optimal binding to neuregulin-1 required eight or more heparin disaccharides; however, as few as two disaccharides were still able to bind neuregulin-1 to a lesser extent. The physiological importance of this specificity was shown both by chemical and siRNA treatment of cultured muscle cells. Pretreatment of muscle cells with chlorate that blocks all sulfation or with an siRNA that selectively blocks N-sulfation significantly reduced erbB receptor activation by neuregulin-1 but had no effect on the activity of neuregulin-1 that lacks the heparin-binding domain. These results suggest that the regulation of glycosaminoglycan sulfation is an important biological mechanism that can modulate both the localization and potentiation of neuregulin-1 signaling.

Ypr118w is a non-essential, low copy number gene product from Saccharomyces cerevisiae. It belongs to the PFAM family PF01008, which contains the alpha-, beta-, and delta-subunits of eukaryotic translation initiation factor eIF2B, as well as proteins of unknown function from all three kingdoms. Recently, one of those latter proteins from Bacillus subtilis has been characterized as a 5-methylthioribose-1-phosphate isomerase, an enzyme of the methionine salvage pathway. We report here the crystal structure of Ypr118w, which reveals a dimeric protein with two domains and a putative active site cleft. The C-terminal domain resembles ribose-5-phosphate isomerase from Escherichia coli with a similar location of the active site. In vivo, Ypr118w protein is required for yeast cells to grow on methylthioadenosine in the absence of methionine, showing that Ypr118w is involved in the methionine salvage pathway. The crystal structure of Ypr118w reveals for the first time the fold of a PF01008 member and allows a deeper discussion of an enzyme of the methionine salvage pathway, which has in the past attracted interest due to tumor suppression and as a target of aniprotozoal drugs.

Sulfate transporters in plants and animals are structurally conserved and have an amino-terminal domain that functions in transport and a carboxyl-terminal region that has been designated the STAS domain. The STAS domain in sulfate transporters has significant similarity to bacterial anti-sigma factor antagonists. To determine if the STAS domain has a role in controlling the activity of sulfate transporters, their stability, or their localization to the plasma membrane, we examined the effect of deleting or modifying the STAS domain of dominant sulfate transporters in roots of Arabidopsis thaliana. The A. thaliana Sultr1;2 and Sultr1;1 sulfate transporters rescue the methionine-dependent growth phenotype of the yeast sulfate transporter mutant strain CP154-7B. Constructs of Sultr1;2 in which the STAS domain was deleted (ΔSTAS) resulted in synthesis of a truncated polypeptide that was unable to rescue the CP154-7B phenotype. The inability of these constructs to rescue the mutant phenotype probably reflected both low level cellular accumulation of the transporter and the inability of the truncated protein to localize to the plasma membrane. Fusing the STAS domain from other sulfate transporters to Sultr1;2 ΔSTAS constructs restored elevated accumulation and plasma membrane localization, although the kinetics of sulfate uptake in the transformants were markedly altered with respect to transformants synthesizing wild-type Sultr1;2 protein. These results suggest that the STAS domain is essential, either directly or indirectly, for facilitating localization of the transporters to the plasma membrane, but it also appears to influence the kinetic properties of the catalytic domain of transporters.

Post-translational modifications of the extracellular portions of receptors located in the cell membrane can contribute to modulating their biological activity. Using a mutagenesis approach in which single or multiple Tyr-to-Phe, Thr-to-Ala, Ser-to-Ala, and Asn-to-Gln substitutions were made at the appropriate positions, we analyzed the sulfation and glycosylation state of the murine CCR8 chemokine receptor, and the way in which these post-translational modifications affect CCR8 activity. A Y14Y15-to-F14F15 CCR8 mutant was less sulfated than the wild-type receptor. An N8-to-Q8 mutant was less glycosylated than wild-type, and a double T10T12-to-A10A12 mutant showed even less glycosylation. We established a flow cytometric analysis with an Fc-fused form of mouse CCL1 to determine precisely the ligand-binding activity of these mutants. Single mutants at amino acid positions 8, 10 or 12 bound CCL1-Fc similarly to wild-type CCR8, whereas the F14F15 double mutant was essentially inactive and the A10A12 double mutant showed about 65% of wild-type ligand-binding activity. Calcium flux activity assays were performed with these mutants, yielding results consistent with those from the ligand binding assays. These data indicate that sulfation at specific positions of the N-terminal domain of mouse CCR8 is critical for its biological activity, whereas glycosylation has a minor influence.

Fructose-1,6-bisphosphate (FBP) aldolase is an essential glycolytic enzyme that reversibly cleaves its ketohexose substrate into triose phosphates. Here we report the crystal structure of a metallo-dependent or class II FBP aldolase from an extreme thermophile, Thermus aquaticus (Taq). The quaternary structure reveals a tetramer composed of two dimers related by a 2-fold axis. Taq FBP aldolase subunits exhibit two distinct conformational states corresponding to loop regions that are in either open or closed position with respect to the active site. Loop closure remodels the disposition of chelating active site histidine residues. In subunits corresponding to the open conformation, the metal cofactor, Co(2+), is sequestered in the active site, whereas for subunits in the closed conformation, the metal cation exchanges between two mutually exclusive binding loci, corresponding to a site at the active site surface and an interior site vicinal to the metal-binding site in the open conformation. Cofactor site exchange is mediated by rotations of the chelating histidine side chains that are coupled to the prior conformational change of loop closure. Sulfate anions are consistent with the location of the phosphate-binding sites of the FBP substrate and determine not only the previously unknown second phosphate-binding site but also provide a mechanism that regulates loop closure during catalysis. Modeling of FBP substrate into the active site is consistent with binding by the acyclic keto form, a minor solution species, and with the metal cofactor mediating keto bond polarization. The Taq FBP aldolase structure suggests a structural basis for different metal cofactor specificity than in Escherichia coli FBP aldolase structures, and we discuss its potential role during catalysis. Comparison with the E. coli structure also indicates a structural basis for thermostability by Taq FBP aldolase.

Integrity of cartilage fails in joint disease. The current work aimed to identify candidate active proteinases in joint diseases using an in vitro model for cartilage degradation induced by interleukin-1. A critical event in the process of cartilage destruction in joint disease is the failure of the collagen fiber network to maintain integrity. Proteins binding to the surface of the fibers are likely early points of failure. Fibromodulin, a member of the leucine-rich repeat protein family, is one predominant protein in cartilage and is known for its roles in the formation of collagen fibrils and sustained interaction with these formed fibers. Cleavage removes the tyrosine sulfate-rich region in the N terminus of fibromodulin. Whereas fibromodulin bound to collagen in tissue was digested, purified fibromodulin was not cleaved. In contrast an N-terminal 10-kDa fragment, Gln19-Lys98, of the protein generated by Lys-C digestion contains the cleavage site and was a substrate cleaved by the enzyme in medium from stimulated cultures. In solution, digestion of this substrate with matrix metalloproteinase (MMP)-2, -9, -8, and -13 demonstrated that only MMP-13 was capable to efficiently cleave it. The cleavage product obtained after MMP-13 digestion was identical to that observed in cleaved fibromodulin from cartilage explant cultures stimulated with interleukin-1. MMP-13 treatment of fresh articular cartilage also produced the fragment under study. The elucidation of the enzyme responsible for such cleavage may lead to treatment modalities involving its selective inhibition for patients suffering from arthritis. The known structure of the fragments permits the generation of neo-epitope antibodies to the cleavage site, which can be used to detect ongoing cartilage degradation in patients with arthritic disease, an important adjunct in monitoring disease progression, active disease, and efficacy of treatment.

The bacterial enzyme, glycerol-3-phosphate cytidylyltransferase (GCT), is a model for mammalian cytidylyltransferases and is a member of a large superfamily of nucleotidyltransferases. Dimeric GCT from Bacillus subtilis displays unusual negative cooperativity in substrate binding and appears to form products only when both active sites are occupied by substrates. Here we describe a complex of GCT with the product, CDP-glycerol, in a crystal structure in which bound sulfate serves as a partial mimic of the second product, pyrophosphate. Binding of sulfate to form a pseudo-ternary complex is observed in three of the four chains constituting the asymmetric unit and is accompanied by a backbone rearrangement at Asp11 and ordering of the C-terminal helix. Comparison with the CTP complex of GCT, determined previously, reveals that in the product complex the active site closes around the glycerol phosphate moiety with a concerted motion of the segment 37-47 that includes helix B. This rearrangement allows lysines 44 and 46 to interact with the glycerol and cytosine phosphates of CDP-glycerol. Binding of CDP-glycerol also induces smaller movements of residues 92-100. Roles of lysines 44 and 46 in catalysis have been confirmed by mutagenesis of these residues to alanine, which decreases Vmax(app) and has profound effects on the Km(app) for glycerol-3-phosphate.

Cartilage glycosaminoglycan (GAG) synthesis and composition, upon which its structural integrity depends, varies with age, is modified by anabolic and catabolic stimuli, and is regulated by UDP-glucuronate availability. However, how such stimuli, prototypically represented by transforming growth factor-beta1 (TGF-beta1) and IL-1alpha, modify GAG synthesis during aging of normal human articular cartilage is not known. Using explants, we show that chondroitin sulfate (CS):total GAG ratios decrease, whereas C6S:C4S ratios increase with cartilage maturation, and that chondrocytes in the cartilage mid-zone, but not the superficial or deep zones, exhibit uridine 5'-diphosphoglucose dehydrogenase (UDPGD) activity, which is also increased in mature cartilage. We also show that IL-1alpha treatment reduces both total GAG and CS synthesis, decreases C6S:C4S ratios (less C6S), but fails to modify chondrocyte UDPGD activity at all ages. On the other hand, TGF-beta1 increases total GAG synthesis in immature, but not mature, cartilage (stimulates CS but not non-CS), age-independently decreases C6S:C4S (more C4S), and increases chondrocyte UDPGD activity in a manner inversely correlated with age. Our findings show that TGF-beta1, but not IL-1alpha, modifies matrix synthesis such that its composition more closely resembles "less mature" articular cartilage. These effects of TGF-beta1, which appear to be restricted to periods of skeletal immaturity, are closely associated although not necessarily mechanistically linked with increases in chondrocyte UDPGD activity. The antianabolic effects of IL-1alpha are, on the other hand, likely to be independent of any direct modification in UDPGD activity and manifest equally in human cartilage of all ages.

The complement anaphylatoxin C3a and its cellular seven-transmembrane segment receptor, C3aR, are implicated in a variety of pathological inflammatory processes. C3aR is a G-protein-coupled receptor with an exceptionally large second extracellular loop of 172 amino acids. Previously reported deletion studies have shown that at least part of this region plays a critical role in binding C3a. Our data now demonstrate that five tyrosines in the second extracellular loop of the C3aR are posttranslationally modified by the addition of sulfate. Blocking sulfation by mutation of tyrosine to phenylalanine at positions 184, 188, 317, and/or 318 does not affect ligand binding or signal transduction. However, when tyrosine 174 is mutated to phenylalanine, binding of native C3a is completely blocked. This variant efficiently mobilizes calcium in response to synthetic C3a agonist peptides, but not to native C3a. This finding is consistent with a two-site model of ligand association typical of many peptide ligand-receptor interactions and identifies sulfotyrosine 174 as the critical C3a docking site. Tyrosine sulfation in the amino-terminal extracellular domain has been shown to be important in several other seven-transmembrane segment receptors. Our data now demonstrate that tyrosine sulfate in other extracellular domains can function for ligand interactions as well.

Fibroblast growth factors (FGFs) interact with heparan sulfate glycosaminoglycans and the extracellular domains of FGF cell surface receptors (FGFRs) to trigger receptor activation and biological responses. FGF homologous factors (FHF1-FHF4; also known as FGF11-FGF14) are related to FGFs by substantial sequence homology, yet their only documented interactions are with an intracellular kinase scaffold protein, islet brain-2 (IB2) and with voltage-gated sodium channels. In this report, we show that recombinant FHFs can bind heparin with high affinity like classical FGFs yet fail to activate any of the seven principal FGFRs. Instead, we demonstrate that FHFs bind IB2 directly, furthering the contention that FHFs and FGFs elicit their biological effects by binding to different protein partners. To understand the molecular basis for this differential target binding specificity, we elucidated the crystal structure of FHF1b to 1.7-A resolution. The FHF1b core domain assumes a beta-trefoil fold consisting of 12 antiparallel beta strands (beta 1 through beta 12). The FHF1b beta-trefoil core is remarkably similar to that of classical FGFs and exhibits an FGF-characteristic heparin-binding surface as attested to by the number of bound sulfate ions. Using molecular modeling and structure-based mutational analysis, we identified two surface residues, Arg52 in the beta 4-beta 5 loop and Val95 in the beta 9 strand of FHF1b that are required for the interaction of FHF1b with IB2. These two residues are unique to FHFs, and mutations of the corresponding residues of FGF1 to Arg and Val diminish the capacity of FGF1 to activate FGFRs, suggesting that these two FHF residues contribute to the inability of FHFs to activate FGFRs. Hence, FHFs and FGFs bear striking structural similarity but have diverged to direct related surfaces toward interaction with distinct protein targets.

The calcitic prisms of the shells of two bivalves, Pinna and Pinctada, are considered simple prisms according to some morphological and mineralogical characteristics. Scanning electron microscopic and atomic force microscopic studies show that the microstructures and nanostructures of these two shells are different. Pinna prisms are monocrystalline, whereas Pinctada prisms are not. Moreover, intraprismatic membranes are present only in the Pinctada prisms. The soluble organic matrices extracted from these prisms are acidic, but their bulk compositions differ. Ultraviolet and infrared spectrometries, fluorescence, high pressure liquid chromatography, and electrophoresis show that the sugar-protein ratios and the molecular weights are different. Sulfur is mainly associated with acidic sulfated sugars, not with amino acids, and the role of acidic sulfated sugars is still underestimated. Thus, the simple prism concept is not a relevant model for the biomineralization processes in the calcitic prismatic layer of mollusk shells.