O-sulfotransferases (OSTs) are critical enzymes in the cellular biosynthesis of the biologically and pharmacologically important heparan sulfate and heparin. Recently, these enzymes have been cloned and expressed in bacteria for application in the chemoenzymatic synthesis of glycosaminoglycan-based drugs. OST activity assays have largely relied on the use of radioisotopic methods using [35S] 3'-phosphoadenosine-5'-phosphosulfate and scintillation counting. Herein, we examine alternative assays that are more compatible with a biomanufacturing environment. A high throughput microtiter-based approach is reported that relies on a coupled bienzymic colorimetric assay for heparan sulfate and heparin OSTs acting on polysaccharide substrates using arylsulfotransferase-IV and p-nitrophenylsulfate as a sacrificial sulfogroup donor. A second liquid chromatography-mass spectrometric assay, for heparan sulfate and heparin OSTs acting on structurally defined oligosaccharide substrates, is also reported that provides additional information on the number and positions of the transferred sulfo groups within the product. Together, these assays allow quantitative and mechanistic information to be obtained on OSTs that act on heparan sulfate and heparin precursors.
enzymes; mass spectrometry; bioassays; sulfotransferases; coupled assay; heparin; heparan sulfate
Glycosaminoglycans (GAGS) are anionic, linear, polysaccharides involved in cell signaling. The GAG content, composition and structure of human tissue have been suggested to play a role in cancer and might provide useful diagnostic or prognostic markers. The current study examines 17 stomach tissue biopsy samples taken from normal individuals and from patients with gastric cancers. An ultrasensitive liquid chromatography (LC) – mass spectrometry assay was applied to individual biopsy samples as small 250 µg providing GAG content and disaccharide composition. The results of these analyses show a significant increase in non-sulfated chondroitin/dermatan sulfate concentration in all cancer samples when compared to normal tissues. In addition in advanced gastric cancer, a significant decrease is observed in hyaluronan.
Stomach cancer; Glycosaminoglycan; Chondroitin sulfate; Hyaluronan; Disaccharide analysis
Testosteronan, an unusual glycosaminoglycan first isolated from the microbe Comamonas testosteroni, was enzymatically synthesized in vitro by transferring uridine diphosphate sugars on β-p-nitrophenyl glucuronide acceptor. After chemically converting testosteronan to N-sulfotestosteronan it was tested as a substrate for sulfotransferases involved in the biosynthesis of the glycosaminoglycan, heparan sulfate. Studies using 35S-labeled 3′-phosphodenosine-5′-phosphosulfate (PAPS) showed that only 6-O-sulfotransferases acted on N-sulfotestosteronan. An oxidative depolymerization reaction was explored to generate oligosaccharides from 34S-labeled 6-O-sulfo-N-sulfotestosteroran using 34S-labeled PAPS because testosteronan was resistant to all of the tested glycosaminoglycan-degrading enzymes. Liquid chromotography-mass spectrometric analysis of the oxidatively depolymerized polysaccharides confirmed the incorporation of 34S into ~14% of the glucosamine residues. Nuclear magnetic resonance spectroscopy also showed that the sulfo groups were transferred to ~20% of the 6-hydroxyl groups in the glucosamine residue of N-sulfotestosteronan. The bioactivity of 6-O–sulfo-N-sulfotestosteronan was examined by performing protein-binding studies with fibroblast growth factors and antithrombin III using a surface plasmon resonance competition assay. The introduction of 6-O-sulfo groups enhanced N-sulfotestosteronan binding to the fibroblast growth factors, but not to antithrombin III.
bacterial polysaccharide; sulfotransferases; protein-binding; sulfonation; heparan sulfate
On the basis of suggested clinical efficacy in an uncontrolled study in ninety-seven patients with unstable angina, an animal study was conducted to investigate antithrombotic synergy between orally administered heparin and arginine. A rat venous thrombosis model tested the difference in thrombus formation when heparin (7.5 mg/kg) and arginine (113 mg/kg) were administered, alone or in combination, by stomach tube with a minimum of 20 rats/group. Oral heparin, arginine, and heparin plus arginine reduced thrombus formation by 50%, 75%, and 90%, respectively, when compared to saline administration. Heparin was recovered from endothelium, yet there was little or no observable plasma anticoagulant activity. An orally administered low-molecular- weight anticoagulant glycosaminoglycan mixture, sulodexide (7.5 mg/kg), showed an 88% reduction in stable thrombus formation when administered alone but showed no synergy with oral arginine. A 28-day study with oral sulodexide (2.9 mg/ kg) and arginine (43.9 mg/kg), 20 rats/group, showed antithrombotic activity with minimal anticoagulant activity indicating suitability for long term treatment. These findings suggest the endothelial localization of heparin and a synergistic antithrombotic effect for orally administered heparin and arginine.
Heparin; thrombosis; endothelium; arginine; oral
Heparin and its low molecular weight heparin derivatives, widely used as clinical anticoagulants, are acidic polysaccharide members of a family of biomacromolecules called glycosaminoglycans (GAGs). Heparin and the related heparan sulfate are biosynthesized in the Golgi apparatus of eukaryotic cells. Heparin is a polycomponent drug that currently is prepared for clinical use by extraction from animal tissues. A heparin pentasaccharide, fondaparinux, has also been prepared through chemical synthesis for use as a homogenous anticoagulant drug. Recent enabling technologies suggest that it may now be possible to synthesize heparin and its derivatives enzymatically. Moreover, new technologies including advances in synthetic carbohydrate synthesis, enzyme-based GAG synthesis, micro- and nano-display of GAGs, rapid on-line structural analysis, and microarray/microfluidic technologies might be applied to the enzymatic synthesis of heparins with defined structures and exhibiting selected activities. The advent of these new technologies also makes it possible to consider the construction of an artificial Golgi to increase our understanding of the cellular control of GAG biosyntheses in this organelle.
Heparin; enzymes; biosynthesis; Golgi; microfluidics
Heparin is a major anticoagulant with activity mediated primarily through its interaction with antithrombin (AT). Heparan sulfate (HS), structurally related to heparin, binds a wide range of proteins of different functionality, taking part in various physiological and pathological processes. The heparin–AT complex, the most well understood facet of anticoagulation, serves as a prototypical example of the important role of heparin/HS in vascular biology. Extensive studies have identified common structural features in heparin/HS–binding sites of proteins. These include the elucidation of consensus sequences in proteins, patterns of clusters of basic and nonbasic residues, and common spatial arrangements of basic amino acids in the heparin-binding sites. Although these studies have provided valuable information, heparin/HS–binding proteins differ widely in structure. The prediction of heparin/HS–binding proteins from sequence information is not currently possible, and elucidation of protein-binding sites requires the individual study of each glycosaminoglycan–protein complex. Thus, x-ray crystallography and site-directed mutagenesis experiments are among the most powerful tools, providing accurate structural information, facilitating the characterization of heparin–protein complexes.
heparin-binding domains; glycosaminoglycan; basic amino acids; consensus sequence; antithrombin
Pluripotent and multipotent cells become increasingly lineage restricted through differentiation. Alterations to the cellular proteoglycan composition and structure should accompany these changes to influence cell proliferation, delineation of tissues and acquisition of cell migration capabilities. Retinoic acid plays an important role in pre-patterning of the early embryo. Retinoic acid can be used in vitro to induce differentiation, causing pluripotent and multipotent cells to become increasingly lineage restricted. We examined retinoic acid-induced changes in the cellular proteoglycan composition of the well-characterized teratocarcinoma line NCCIT. Our analysis revealed changes in the abundance of transcripts for genes encoding core proteins, enzymes that are responsible for early and late linkage region biosynthesis, as well as enzymes for GAG chain extension and modification. Transcript levels for genes encoding core proteins used as backbones for polysaccharide synthesis revealed highly significant increases in expression of lumican and decorin, 1500-fold and 2800-fold, respectively. Similarly, glypican 3, glypican 5, versican and glypican 6 showed increases between 5 and 70-fold. Significant decreases in biglycan, serglycin, glypican 4, aggrecan, neurocan, CD74 and glypican 1 were observed. Disaccharide analysis of the glycans in heparin/heparan sulfate and chondroitin/dermatan sulfate revealed retinoic acid-induced changes restricted to chondroitin/dermatan sulfate glycans. Our study provides the first detailed analysis of changes in the glycosaminoglycan profile of human pluripotent cells upon treatment with the retinoic acid morphogen.
glycomics; teratocarcinoma; pluriotent; glycosaminoglycans
The sensitive detection of highly
toxic botulinum neurotoxin (BoNT)
from Clostridium botulinum is of critical importance
because it causes human illnesses if foodborne or introduced in wounds
and as an iatrogenic substance. Moreover, it has been recently considered
a possible biological warfare agent. Over the past decade, significant
progress has been made in BoNT detection technologies, including mouse
lethality assays, enzyme-linked immunosorbent assays, and endopeptidase
assays and by mass spectrometry. Critical assay requirements, including
rapid assay, active toxin detection, sensitive and accurate detection,
still remain challenging. Here, we present a novel method to detect
active BoNTs using a Glyco-quantitative polymerase chain-reaction
(qPCR) approach. Sialyllactose, which interacts with the binding-domain
of BoNTs, is incorporated into a sialyllactose-DNA conjugate as a
binding-probe for active BoNT and recovered through BoNT-immunoprecipitation.
Glyco-qPCR analysis of the bound sialyllactose-DNA is then used to
detect low attomolar concentrations of BoNT and attomolar to femtomolar
concentrations of BoNT in honey, the most common foodborne source
of infant botulism.
Heparin is a critically important anticoagulant drug that was contaminated with a persulfonated polysaccharide in 2008, resulting in a number of severe adverse reactions, some leading to death. Controversy remains as to the precise composition of the 2008 contaminant and new information suggests that heparin may now be subject to adulteration with a new, difficult to detect, contaminant, N-sulfo oversulfated chondroitin sulfate. This study synthesizes this new potential contaminant and describes the use of radical depolymerization followed by liquid chromatography-mass spectrometry to detect N-sulfo oversulfated chondroitin sulfate and to confirm the structure of the 2008 contaminant as oversulfated chondroitin sulfate and not oversulfated heparan sulfate.
heparin; oversulfated chondroitin sulfate; oversulfated heparan sulfate; N-sulfated oversulfated chondroitin sulfate; contaminant; radical depolymerization; mass spectrometry
Although most pharmaceutical heparin used today is obtained from porcine intestine, heparin has historically been prepared from bovine lung and ovine intestine. There is some regulatory concern about establishing the species origin of heparin. This concern began with the outbreak of mad cow disease in the 1990s and was exacerbated during the heparin shortage in the 2000s and the heparin contamination crisis of 2007–2008. Three heparins from porcine, ovine, and bovine were characterized through state-of-the-art carbohydrate analysis methods with a view profiling their physicochemical properties. Differences in molecular weight, monosaccharide and disaccharide composition, oligosaccharide sequence, and antithrombin III-binding affinity were observed. These data provide some insight into the variability of heparins obtained from these three species and suggest some analytical approaches that may be useful in confirming the species origin of a heparin active pharmaceutical ingredient.
bioanalysis; glycosaminoglycans; heparin; LC–MS; NMR; mass spectomery; surface plasmon resonance; antithrombin III
Fibroblast growth factor (FGF) signals cell growth through its interaction with a fibroblast growth factor receptor (FGFR) and a glycosaminoglycn (GAG) co-receptor. Here we examine the signaling of five different FGFs (FGF1, FGF2, FGF6, FGF8 and FGF8b) through FGFR3c. A small library of GAG and GAG-derivative co-receptors are screened to better understand the structure-activity relationship of these co-receptors on signaling. Initially, data were collected in a microtiter well-based cell proliferation assay. In an effort to reduce reagent requirements and improve assay throughput a cell-based microarray platform was developed. In this cell-based microarray, FGFR3c expressing cells were printed in alginate hydrogel droplets of ~30 nL and incubated with FGF and GAG. Heparin was the most effective GAG co-receptor for all FGFs studied. Other GAGs, such as 2-O-desulfated heparin and chondroitin sulfate B, were also effective co-receptors. Signaling by FGF8 and FGF8b showed the widest tolerance for co-receptor structure. Finally, this on-chip cell-based microarray provides comparable data to a microtiter well-based assay, demonstrating that the co-receptor assay can be converted into a high throughput assay.
heparan sulfate; chondroitin sulfate; ultra low molecular weight heparin; fibroblast growth factors; signaling complex; cell-based microarray
Roundabout 1 (Robo1) is the cognate receptor for secreted axon guidance molecule, Slits, which function to direct cellular migration during neuronal development and angiogenesis. The Slit2–Robo1 signaling is modulated by heparan sulfate, a sulfated linear polysaccharide that is abundantly expressed on the cell surface and in the extracellular matrix. Biochemical studies have further shown that heparan sulfate binds to both Slit2 and Robo1 facilitating the ligand–receptor interaction. The structural requirements for heparan sulfate interaction with Robo1 remain unknown. In this report, surface plasmon resonance (SPR) spectroscopy was used to examine the interaction between Robo1 and heparin and other GAGs and determined that heparin binds to Robo1 with an affinity of ~650 nM. SPR solution competition studies with chemically modified heparins further determined that although all sulfo groups on heparin are important for the Robo1–heparin interaction, the N-sulfo and 6-O-sulfo groups are essential for the Robo1–heparin binding. Examination of differently sized heparin oligosaccharides and different GAGs also demonstrated that Robo1 prefers to bind full-length heparin chains and that GAGs with higher sulfation levels show increased Robo1 binding affinities.
Heparin; Robo1; Binding; Surface plasmon resonance
Glycomics research requires the isolation of glycans from cells for structural characterization and functional studies of the glycans. A method for cell-based microscale isolation and quantification of highly sulfated, moderately sulfated, and nonsulfated glycosaminoglycans (GAGs) was developed using Chinese hamster ovary (CHO) cells. This microscale isolation relies on a mini-strong anion exchange spin column eluted stepwise with different concentrations of sodium chloride solution. Hyaluronic acid, chondroitin sulfate, and heparin were used to optimize the isolation of the endogenous glycosaminoglycans in CHO cells. This method can also be used to determine the presence of nonsulfated GAGs including heparosan, hyaluronic acid, and nonsulfated chondroitin.
Chinese hamster ovary cells; Glycosaminoglycans; Anion exchange chromatography; Heparosan; Disaccharide analysis
Rapid and highly sensitive detection of the carbohydrate components of glycoconjugates is critical for advancing glycobiology. Fluorescence (or Forster) resonance energy transfer (FRET) is commonly used in detection of DNA, in protein structural biology, and in protease assays, but is less frequently applied to glycan analysis due to difficulties in inserting two fluorescent tags into small glycan structures. We report an ultrasensitive method for the detection and quantification of a chondroitin sulfate disaccharide based on FRET, involving a CdSe-ZnS core-shell nanocrystal quantum dot (QD)-streptavidin conjugate donor and a Cy5 acceptor. The disaccharide was doubly labeled with biotin and Cy5. QDs then served to concentrate the target disaccharide, enhancing the overall energy transfer efficiency, with unlinked QDs and Cy5-hydrazide producing nearly zero background signal in capillary electrophoresis using laser-induced fluorescence detection with two different band-pass filters. This method is generally applicable to the ultrasensitive analysis of acidic glycans and offers promise for the high-throughput disaccharide analysis of glycosaminoglycans.
Fluorescence resonance energy transfer; quantum dots; glycosaminoglycans; ultrasensitive detection; capillary electrophoresis
Heparin is a critically important anticoagulant drug that is prepared from pig intestine. In 2007-2008, there was a crisis in the heparin market when the raw material was adulterated with the toxic polysaccharide, oversulfated chondroitin sulfate, which was associated with 100 deaths in the U.S. alone. As the result of this crisis, our laboratory and others have been actively pursuing alternative sources for this critical drug, including synthetic heparins and bioengineered heparin. In assessing the bioengineering processing costs it has become clear that the use of both enzyme-catalyzed cofactor recycling and enzyme immobilization will be needed for commercialization. In the current study, we examine the use of immobilization of C5-epimerase and 2-O-sulfotransferase involved in the first enzymatic step in the bioengineered heparin process, as well as arylsulfotransferase-IV involved in cofactor recycling in all three enzymatic steps. We report the successful immobilization of all three enzymes and their use in converting N-sulfo, N-acetyl heparosan into N-sulfo, N-acetyl 2-O-sulfo heparin.
immobilized enyzmes; 2-O-sulfotransferase; C5-Epimerase; Aryl sulfotransferase IV; heparosan; bioengineered heparin
Adeno-associated virus (AAV) is a key candidate in the development of gene therapy. In this report, we used surface plasmon resonance spectroscopy to study the interaction between AAV and heparin and other glycosaminoglycans. Surface plasmon resonance results revealed that heparin binds to AAV with extremely high affinity. Solution competition studies shows that AAV binding to heparin is chain length dependent. AAV prefers to bind full chain heparin. All sulfo groups (especially N-sulfo and 6-O-sulfo groups) on heparin are important for the AAV- heparin interaction. Higher levels of sulfo group substitution in GAGs enhance their binding affinities. Atomic force microscopy was also performed to image AAV-2 complexed with heparin.
Surface Plasmon Resonance; Adeno-associated virus; glycosaminoglycans; heparin
Inteins are intervening polypeptides that catalyze their own removal from flanking exteins, concomitant to the ligation of the exteins. The intein that interrupts the DP2 (large) subunit of DNA Polymerase II from Methanoculleus marisnigri (Mma) can promote protein splicing. However, protein splicing can be prevented or reduced by over-expression under non-reducing conditions, due to the formation of a disulfide bond between two internal intein Cys residues. This redox sensitivity leads to differential activity in different strains of E. coli as well as in different cell compartments. The redox-dependent control of in vivo protein splicing in an intein derived from an anaerobe that can occupy multiple environments hints at a possible physiological role for protein splicing.
(±)-Paeonilide, a novel monoterpenoid metabolite from the roots of Paeonia delavayi showing anti-platelet activating factor activity, is convergently synthesized in five steps with 59% overall yield. The application of benzoyl peroxide-promoted radical addition of unsaturated ester to aldehyde and subsequent topologically favored cyclization greatly simplified the synthesis.
Glycosaminoglycans (GAGs) are the portion of a proteoglycan that determine its final shape and function. The molecular structure of predominant GAG species in camel liver and lung is reported for the first time. The one-humped camel survives in an extreme, arid habitat and, thus, offers a good model to study the role of glycomics on homeostasis. Heparan sulfate (HS) from the lung and liver of the one-humped camel were isolated. Characterization of these newly isolated glycosaminoglycans included 1H NMR spectroscopy and disaccharide compositional analysis. The relative molecular weight of these GAGs was estimated by gradient polyacrylamide gel electrophoresis and their degree of sulfation was also assessed. Anticoagulant activity was determined using an anti-factor Xa assay and the HS from camel lung shows ~50% of heparin’s activity. The structural differences of camel liver GAGs compared to human and porcine liver heparin and HS is discussed. Camel lung heparan sulfate resembles both heparin and HS in its structure and properties suggesting that it is either a highly sulfated form of HS, a mixture of heparin and HS or an undersulfated heparin.
Heparin; Heparan sulfate; Glycosaminoglycans; One-humped camel; Mammalian; Liver; Lung; Disaccharide; Anticoagulant; Factor Xa
Saponins in Platycodi Radix (platycosides) exhibit potent biological activities in mammalian systems, including several beneficial effects such as anti-inflammatory, immunomodulatory and anti-obesity activities. In this study, we developed a new HPLC separation coupled with evaporative light scattering detector (ELSD) for the simultaneous quantitative determination of ten major saponins in Platycodi Radix. Simultaneous separation of these saponins was achieved on a C18 analytical column. The mobile phase consisted of a gradient of aqueous acetonitrile. The method was validated for linearity, precision, accuracy, limit of detection and quantification. Electrospray ionization mass spectrometry (ESI-MS) and liquid chromatography coupled with on-line mass spectrometry (LC-ESI MS/MS) were applied to identify platycosides in the purified fractions and in the crude extract. Under ESI-MS/MS conditions, the fragmentation patterns of [M – H]− ions exclusively show signals corresponding to cleavage of the glycosidic bonds, thus allowing a rapid identification of saponins in the crude extract of Platycodi Radix. The validated HPLC method provides a new basis of overall assessment on quality of Platycodi Radix, and ESI-MS/MS and LC-ESI MS/MS approaches offers analytical tools for a rapid screening of platycosides in the crude extract.
Platycodi Radix; Saponin; HPLC-ELSD; Quantification; ESI-MS/MS
Heparan sulfate proteoglycans (HSPGs) play important biological roles in cell–matrix adhesion processes and are essential regulators of growth actions. The expression of the different HSPGs in itself is tightly regulated providing strict controls on the activities of the bound ligands. Human liver is a target for a number of pathogens, and HSPGs have been demonstrated in several cases to play a pivotal role in infectivity. Despite HSPGs important biological functions, little is known about its cell-specific distribution patterns. Human liver HSPG was isolated, and a specific monoclonal antibody (mAb) 1E4-1C2 was produced. Distribution of HSPG reactive to this mAb was studied in normal blood cells, hematopoietic cell lines and blood cells isolated from patients with various hematologic disorders using indirect immunofluorescence. There was no expression of molecules recognized by this mAb on lymphoid (Daudi, Jurkat, SupT-1) and monocytoid (U937) cell lines. Peripheral blood cells, normal bone marrow, together with leukocytes isolated from patients with acute lymphoblastic leukemia, chronic myelocytic leukemia, Hodgkin’s disease or Non-Hodgkin’s lymphoma, were also negative. In contrast, 1E4-1C2 showed significant positive results on human myeloid cell lines HL-60 and K562. Moreover, it is interesting that this mAb also recognized epitopes on leukocytes isolated from acute myeloblastic leukemia. These results suggest that malignancies of cells in myeloid lineage may cause expression of HSPGs that are detected by this specific mAb, making it a potential co-marker for the diagnosis of acute myeloid leukemia.
Heparan sulfate; Monoclonal antibodies; Diagnosis; Acute myeloid leukemia
The interactions between cell surface receptors and sulfated glucosamineglycans serve ubiquitous roles in cell adhesion and receptor signaling. Heparin, a highly sulfated polymer of uronic acids and glucosamine, binds strongly to the integrin receptor αXβ2 (p150,95, CD11c/CD18). Here, we analyze the structural motifs within heparin that constitute high affinity binding sites for the I domain of integrin αXβ2. Heparin oligomers with chain lengths of 10 saccharide residues or higher provide strong inhibition of the binding by the αX I domain to the complement fragment iC3b. By contrast, smaller oligomers or the synthetic heparinoid fondaparinux were not able to block the binding. Semipurified heparin oligomers with 12 saccharide residues identified the fully sulfated species as the most potent antagonist of iC3b, with a 1.3 μM affinity for the αX I domain. In studies of direct binding by the αX I domain to immobilized heparin, we found that the interaction is conformationally regulated and requires Mg2+. Furthermore, the fully sulfated heparin fragment induced conformational change in the ectodomain of the αXβ2 receptor, also demonstrating allosteric linkage between heparin binding and integrin conformation.
Escherichia coli K5 heparosan was enzymatically modified by Chen and colleagues  to construct a library of heparan sulfate polysaccharides for evaluation, leading to the discovery that a 2-O-sulfo-iduronic acid residue is not essential for antithrombin-mediated anticoagulant activity in larger oligosaccharide and polysaccharide structures.
Two natural saponins 1 and 2, isolated from Solanum indicum L., and containing 2,3-branched sugar moieties, have been efficiently synthesized. Partially protected monosaccharide and disaccharide donors were used to facilitate target synthesis. Stereo factors were critical in incorporating 2,3-branched sugars on steroid aglycones. Saponin 1 was synthesized in five steps and 30% overall yield, while saponin 2 was obtained using six straightforward sequential reactions in 31% overall yield. Saponin 2 shows promising cytotoxic activity toward human hepatocellular carcinoma BEL-7402 with an IC50 of <6 μg/mL.
Glycosaminoglycan-protein interactions are an important frontier for discovering new mechanisms of cellular regulation by complex sugars. The integration of the ‘chemical glycomics’ strategies of synthetic chemistry, arrays and biological assays shows that the precise pattern of sugar sulfation dictates the specificity of a sugar’s function.