Heparinase III with High Activity and Stability: Heterologous Expression, Biochemical Characterization, and Application in Depolymerization of Heparin.

A novel heparinase III from Pedobacter schmidteae (PsHep-III) with high activity and good stability was successfully cloned, expressed, and characterized. PsHep-III displayed the highest specific activity ever reported of 192.8 U mg-1 using heparin as the substrate. It was stable at 25 °C with a half-life of 323 h in an aqueous solution. PsHep-III was employed for the depolymerization of heparin, and the enzymatic hydrolyzed products were analyzed with gel permeation chromatography and high-performance liquid chromatography. PsHep-III can break glycosidic bonds in heparin like →4]GlcNAc/GlcNAc6S/GlcNS/GlcNS6S/GlcN/GlcN6S(1 → 4)ΔUA/ΔUA2S[1 → and efficiently digest heparin into seven disaccharides including N-acetylated, N-sulfated, and N-unsubstituted modification, with molecular masses of 503, 605, 563, 563, 665, 360, and 563 Da, respectively. These results indicated that PsHep-III with broad substrate specificity could be combined with heparinase I to overcome the low selectivity at the N-acetylated modification binding sites of heparinase I. This work will contribute to the application of PsHep-III for characterizing heparin and producing low-molecular-weight heparin effectively.

A turn-on fluorescence sensor for detection of heparinase with heparin templated aggregation of tetracationic porphyrin derivative.

Heparinase is the only mammalian endoglycosidase that breaks down the commonly used blood-anticoagulant heparin into therapeutically relevant low-molecular-weight-heparin. Importantly, heparinase has been considered a malignant disease diagnostic marker. Thus, it is essential to develop detection scheme for heparinase. However, optical methods for heparinase determination are limited. In the present work, we report a turn-on fluorescence sensor for detection of heparinase that utilizes heparin-templated aggregation of a tetra-cationic porphyrin derivative, TMPyP4+, as a sensing framework. Heparinase cleaves the glycosidic linkage between hexosamine and uronic acid in the structure of heparin to destroy its polyelectrolytic nature that originally causes the aggregation of TMPyP4+. Thus, heparinase leads to dissociation of TMPyP4+ aggregates and generates an optical signal. This system leads to a sensitive and selective response towards heparinase with a Limit of Detection (LOD) of 0.3 pmol/L. Further, the same system is demonstrated to sense a trace amount of Oversulfated Chondrootin Sulphate (OSCS) in heparin, which is a heparin adulterant, by utilizing the fact that OSCS serves as an inhibitor for heparinase activity, which leads to reverse modulation in the photo-physical features of the monomer/aggregate equilibrium of the TMPyP4+-heparin-heparinase system. The sensing mechanism has been thoroughly demonstrated by ground-state absorption, steady-state emission, and time-resolved emission measurements. The selectivity of the sensor was tested using lysozyme, α-amylase, pepsin, trypsin, lipase, and glucose oxidase in the heparinase selectivity study and the method is also validated using another method reported in the literature. The study provides a new approach for the development of optical methods for the detection of heparinase and oversulfated chondroitin sulfate, which is currently limited.

Combination of engineering the substrate and Ca2+ binding domains of heparinase I to improve the catalytic activity.

Heparinase I (EC 4.2.2.7), is an enzyme that cleaves heparin, showing great potential for eco-friendly production of low molecular weight heparin (LMWH). However, owing to its poor catalytic activity and thermal stability, the industrial application of heparinase I has been severely hindered. To improve the catalytic activity, we proposed to engineer both the substrate and Ca2+ binding domains of heparinase I. Several heparinases I from different organisms were selected for multiple sequence alignment and molecular docking to screen the key residues in the binding domain. Nine single-point mutations were selected to enhance the catalytic activity of heparinase I. Among them, T250D was the most highly active one, whereas mutations around Ca2+ binding domain yielded two active mutants. Mutant D152S/R244K/T250D with significantly increased catalytic activity was obtained by combined mutation. The catalytic efficiency of the mutant was 118,875.8 min-1·µM-1, which was improved 5.26 times. Molecular modeling revealed that the improved activity and stability of the mutants were probably attributed to the formation of new hydrogen bonds. The highly active mutant had great potential applications in industry and the strategy could be used to improve the performance of other enzymes.

HighlightsImproved catalytic activity of heparinase I by engineering the binding domains of substrate and Ca²⁺.The mutant D152S/R244K/T250D showed the highest catalytic performance.The increased hydrogen bonds attribute to the increased activity.

Expression and characterization of heparinase II with MBP tag from a novel strain, Raoultella NX-TZ-3-15.

The enzymes are biological macromolecules that biocatalyze certain biochemical reactions without undergoing any modification or degradation at the end of the reaction. In this work, we constructed a recombinant novel Raoultella sp. NX-TZ-3-15 strain that produces heparinase with a maltose binding tag to enhance its production and activity. Additionally, MBP-heparinase was purified and its enzymatic capabilities are investigated to determine its industrial application. Moreover, the recombinant plasmid encoding the MBP-heparinase fusion protein was effectively generated and purified to a high purity. According to SDS-PAGE analysis, the MBP-heparinase has a molecular weight of around 70 kDa and the majority of it being soluble with a maximum activity of 5386 U/L. It has also been noted that the three ions of Ca2 + , Co2 + , and Mg2 + can have an effect on heparinase activities, with Mg2 + being the most noticeable, increasing by about 85%, while Cu2 + , Fe2 + , Zn2 + having an inhibitory effect on heparinase activities. Further investigations on the mechanistic action, structural features, and genomes of Raoultella sp. NX-TZ-3-15 heparinase synthesis are required for industrial-scale manufacturing.

Cloning and Expression of Heparinase Gene from a Novel Strain Raoultella NX-TZ-3-15.

Heparin is a class of highly sulfated, acidic, linear, and complex polysaccharide that belongs to the heparin/heparan sulfate (HS) glycosaminoglycans family. Enzymatic depolymerization of heparin by heparinases is a promising strategy for the production of ultra-low molecular weight heparins (ULMWHs) as anticoagulants. In the present study, a novel heparinase-producing strain Raoultella NX-TZ-3-15 was isolated and identified from soil samples. Herein, the heparinase gene MBP-H1 was cloned to the pBENT vector to enable expression in Escherichia coli. The optimized conditions made the activity of recombinant heparinase reach the highest level (2140 U/L). The overexpressed MBP-H1 was purified by affinity chromatography and a purity of more than 90% was obtained. The condition for biocatalysis was also optimized and three metal ions Ca2+, Co2+, and Mg2+ were utilized to activate the reaction. In addition, the kinetics regarding the new fusion heparinase was also determined with a Vm value of 11.29 µmol/min and a Km value of 31.2 µmol/L. In short, due to excellent Km and Vmax, the recombinant enzyme has great potential to be used in the clinic in medicine and industrial production of low or ultra-low molecule weight heparin.

Production, characteristics and applications of microbial heparinases.

Heparinases are enzymes that selectively cleave heparin and heparan sulfate chains, via cleavage of the glycosidic linkage between hexosamines and uronic acids, producing disaccharide and oligosaccharide products. While heparin is well known as an anti-coagulant drug, heparin and heparan sulfate are also involved in biological processes such as inflammation, cancer and angiogenesis and viral and bacterial infections and are of growing interest for their therapeutic potential. Recently, potential roles of heparin and heparan sulfate in relation to COVID-19 infection have been highlighted. The ability of heparinases to selectively cleave heparin chains has been exploited industrially to produce low molecular weight heparin, which has replaced heparin in several clinical applications. Other applications of heparinases include heparin and heparan sulfate structural analysis, neutralisation of heparin in blood and removal of the inhibitory effect of heparin on various enzymes. Heparinases are known to inhibit neovascularization and heparinase III is of interest for treating cancer and inhibiting tumour cell growth. Heparinase activity, first isolated from Pedobacter heparinus, has since been reported from several other microorganisms. Significant progress has been made in the production, characterisation and improvement of microbial heparinases in response to application demands in terms of heparinase yield and purity, which is likely to extend their usefulness in various applications. This review focuses on recent developments in the identification, characterisation and improvement of microbial heparinases and their established and emerging industrial, clinical and therapeutic applications.

Influence of saccharide modifications on heparin lyase III substrate specificities.

A library of 23 synthetic heparan sulfate (HS) oligosaccharides, varying in chain length, types, and positions of modifications, was used to analyze the substrate specificities of heparin lyase III enzymes from both Flavobacterium heparinum and Bacteroides eggerthii. The influence of specific modifications, including N-substitution, 2-O sulfation, 6-O sulfation, and 3-O sulfation on lyase III digestion was examined systematically. It was demonstrated that lyase III from both sources can completely digest oligosaccharides lacking O-sulfates. 2-O Sulfation completely blocked cleavage at the corresponding site; 6-O and 3-O sulfation on glucosamine residues inhibited enzyme activity. We also observed that there are differences in substrate specificities between the two lyase III enzymes for highly sulfated oligosaccharides. These findings will facilitate obtaining and analyzing the functional sulfated domains from large HS polymer, to better understand their structure/function relationships in biological processes.

The 3-O-sulfation of heparan sulfate modulates protein binding and lyase degradation.

Humans express seven heparan sulfate (HS) 3-O-sulfotransferases that differ in substrate specificity and tissue expression. Although genetic studies have indicated that 3-O-sulfated HS modulates many biological processes, ligand requirements for proteins engaging with HS modified by 3-O-sulfate (3-OS) have been difficult to determine. In particular, the context in which the 3-OS group needs to be presented for binding is largely unknown. We describe herein a modular synthetic approach that can provide structurally diverse HS oligosaccharides with and without 3-OS. The methodology was employed to prepare 27 hexasaccharides that were printed as a glycan microarray to examine ligand requirements of a wide range of HS-binding proteins. The binding selectivity of antithrombin-III (AT-III) compared well with anti-Factor Xa activity supporting robustness of the array technology. Many of the other examined HS-binding proteins required an IdoA2S-GlcNS3S6S sequon for binding but exhibited variable dependence for the 2-OS and 6-OS moieties, and a GlcA or IdoA2S residue neighboring the central GlcNS3S. The HS oligosaccharides were also examined as inhibitors of cell entry by herpes simplex virus type 1, which, surprisingly, showed a lack of dependence of 3-OS, indicating that, instead of glycoprotein D (gD), they competitively bind to gB and gC. The compounds were also used to examine substrate specificities of heparin lyases, which are enzymes used for depolymerization of HS/heparin for sequence determination and production of therapeutic heparins. It was found that cleavage by lyase II is influenced by 3-OS, while digestion by lyase I is only affected by 2-OS. Lyase III exhibited sensitivity to both 3-OS and 2-OS.

A simplified protocol for profiling heparin-contaminated circulating miRNAs: by microfluidic array.

Peripheral blood is a valuable, non-invasive source of biomarkers which include circulating miRNAs. Using microfluidic array-based techniques, miRNAs can be successfully measured in small amounts of blood plasma (< 0.5 mL) using cDNA pre-amplification. However, the use of heparin-based anticoagulants for blood collection hinders the detection of circulating miRNAs due to its inhibitory effect on PCR components. Although pre-treatment with heparinase have been shown to overcome heparin contamination in blood, its effect has not been described in array-based analyses or more sensitive applications with smaller sample volumes (i.e. 200 µL plasma) requiring pre-amplification. We show that the treatment of miRNA extracted from heparinised plasma with an optimised concentration of Bacteroides heparinase I prior to cDNA pre-amplification dramatically improves the number of detectable miRNA from 2 to 67 targets on the TaqMan® Array Human MicroRNA Cards. Furthermore, the titrated amount of heparinase (3 U) gave the best miRNA detection compared to those used in previous studies (6-24 U). This study provides novel data which demonstrates that heparinase treatment is compatible with protocols that involve pre-amplification of cDNA and microfluidic array-based techniques. This an improved methodology that permits miRNA-based biomarker analysis from small volume of heparinised plasma.

A highly active heparinase I from Bacteroides cellulosilyticus: Cloning, high level expression, and molecular characterization.

As one of the most extensively studied glycosaminoglycan lyases, heparinase I has been used in producing low or ultra-low molecular weight heparin. Its' important applications are to neutralize the heparin in human blood and analyze heparin structure in the clinic. However, the low productivity and activity of the enzyme have greatly hindered its applications. In this study, a novel Hep-I from Bacteroides cellulosilyticus (BcHep-I) was successfully cloned and heterologously expressed in E. coli BL21 (DE3) as a soluble protein. The molecular mass and isoelectric point (pI) of the enzyme are 44.42 kDa and 9.02, respectively. And the characterization of BcHep-I after purified with Ni-NTA affinity chromatography suggested that it is a mesophilic enzyme. BcHep-I can be activated by 1 mM Ca2+, Mg2+, and Mn2+, while severely inhibited by Zn2+, Co2+, and EDTA. The specific activity of the enzyme was 738.3 U·mg-1 which is the highest activity ever reported. The Km and Vmax were calculated as 0.17 mg·mL-1 and 740.58 U·mg-1, respectively. Besides, the half-life of 300 min at 30°C showed BcHep-I has practical applications. Homology modeling and substrate docking revealed that Gln15, Lys74, Arg76, Lys104, Arg149, Gln208, Tyr336, Tyr342, and Lys338 were mainly involved in the substrate binding of Hep-I, and 11 hydrogen bonds were formed between heparin and the enzyme. These results indicated that BcHep-I with high activity has great potential applications in the industrial production of heparin, especially in the clinic to neutralize heparin.

Filter-entrapment enrichment pull-down assay for glycosaminoglycan structural characterization and protein interaction.

Heparins are the most pharmaceutically important polysaccharides. These heparin-based anticoagulant/antithrombotic agents include unfractionated heparins, low molecular weight heparins (LMWHs) and ultralow molecular weight heparins (ULMWHs). Heparins exhibit their pharmacological and biological activities through interaction with heparin-binding proteins. The prototypical heparin-binding protein is antithrombin III (AT), responsible for heparin's anticoagulant/antithrombotic activity. This study describes a filter-trapping method to isolate the chains in enoxaparin, a LMWH, which bind to AT. We demonstrate this method using the ULMWH, fondaparinux, which consists of a single well defined AT binding site. The interacting chains of enoxaparin are then characterized by activity assays, top-down liquid chromatography-mass spectrometry, and capillary zone electrophoresis mass spectrometry. This filter-trapping assay is an improvement over affinity chromatography for isolating heparin chains interacting with heparin binding proteins.

Qualitative analysis of enzymatic and chemical depolymerized low molecular weight heparins by UHPLC coupled with electrospray ionization quadrupole time-of-flight-mass spectrometry.

Complete heparin digestion with heparin lyase I and II results in a mixture of hexasaccharides and tetrasaccharides with 3-O-sulfo group-containing glucosamine residues at their reducing ends. Because these tetrasaccharides are derived from antithrombin III-binding sites of heparin, we examined whether this method could be applied to estimate the anticoagulant activity of heparin. Therefore, this paper presents a new low molecular weight heparin sample preparation method-chemical depolymerization. Qualitative analysis of the studied compounds and a comparison of their composition are an important contribution to the structural analysis of low molecular weight heparins, which has not been fully conducted so far. Qualitative on-line liquid chromatography-mass spectrometric analysis of these resistant oligosaccharides is also described in this paper.

Cloning, expression, and characterization of a novel heparinase I from Bacteroides eggerthii.

Heparinase I (Hep I) specifically degrades heparin to oligosaccharide or unsaturated disaccharide and has been widely used in preparation of low molecular weight heparin (LMWH). In this work, a novel Hep I from Bacteroides eggerthii VPI T5-42B-1 was cloned and overexpressed in Escherichia coli BL21 (DE3). The enzyme has specific activity of 480 IU·mg-1 at the optimal temperature and pH of 30 °C and pH 7.5, and the Km and Vmax were 3.6 mg·mL-1 and 647.93 U·mg-1, respectively. The Hep I has good stability with t1/2 values of 350 and 60 min at 30 and 37 °C, respectively. And it showed a residual relative activity of 70.8% after 21 days incubation at 4 °C. Substrate docking study revealed that Lys99, Arg101, Gln241, Lys270, Asn275, and Lys292 were mainly involved in the substrate binding of Hep I. The shorter hydrogen bonds formed between heparin and these residues suggested the higher specific activity of BeHep I. And the minimum conformational entropy value of 756 J·K-1 provides an evidence for the improved stability of this enzyme. This Hep I could be of interest in the industrial preparation of LMWH for its high specific activity and good stability.

Oligosaccharides mapping of nitrous acid degraded heparin through UHPLC-HILIC/WAX-MS.

Building blocks characterization is a significant approach for understanding the molecular structure of heparin and its derivatives. Nitrous acid (HONO) depolymerization of heparin generates oligosaccharides that maintain the epimerization conformation on C5 of the uronic acids, reflecting the authentic structure of the parental chain. HONO treatment at pH 1.5 selectively cleaves the bond between N-sulfated glucosamine and hexuronic acid, resulting mainly disaccharides, as well as tetra-, tri-, and mono-saccharides. The tetrasaccharides are derived from the structure of N-acetylated domains while tri-, and mono-saccharides are derived from the reducing or the non-reducing end of the heparin chain. The resulted oligosaccharides were separated and analyzed using a UHPLC-HILIC/WAX-MS method. We succeeded in the identification of 19 tetrasaccharides, 19 trisaccharides and 4 monosaccharides species, majority of which is structurally characterized. By comparing the theoretical possibilities and actual occurrence of the well-characterized tetrasaccharides, we demonstrated that the biosynthesis of heparin is a systematic process.

Identification of a heparosan heptasaccharide as an effective anti-inflammatory agent by partial desulfation of low molecular weight heparin.

Low molecular weight heparin (LMWH) possesses a dual function of anticoagulation and anti-inflammation. While the structures and mechanisms on its anticoagulation have been widely studied, the structural features responsible for the anti-inflammatory activity of LMWH remain to be explored. In the present study, guided by an anti-inflammation assay, a non-anticoagulant species was generated from partial desulfation of LMWH to fully retain the anti-inflammatory activity, from which five fractions were further separated and three of them were characterized by enzymatic degradation, hydrophobic labeling, C18-based HPLC and LC-MS/MS analyses. The structure-activity relationship revealed that the sulfate groups in LMWH are critical to distinguish and separate the activities of anticoagulation and anti-inflammation, leading to the identification of a synthetic heparosan-type heptasaccharide as a potent anti-inflammatory agent. The present strategy enables the simplification of complex polysaccharides to bioactive synthetic oligosaccharides for therapeutic utility.

Structure-based engineering of heparinase I with improved specific activity for degrading heparin.

BACKGROUND: Heparinase I from Pedobacter heparinus (Ph-HepI), which specifically cleaves heparin and heparan sulfate, is one of the most extensively studied glycosaminoglycan lyases. Enzymatic degradation of heparin by heparin lyases not only largely facilitates heparin structural analysis but also showed great potential to produce low-molecular-weight heparin (LMWH) in an environmentally friendly way. However, industrial applications of Ph-HepI have been limited by their poor yield and enzyme activity. In this work, we improve the specific enzyme activity of Ph-HepI based on homology modeling, multiple sequence alignment, molecular docking and site-directed mutagenesis. RESULTS: Three mutations (S169D, A259D, S169D/A259D) exhibited a 50.18, 40.43, and 122.05% increase in the specific enzyme activity and a 91.67, 108.33, and 75% increase in the yield, respectively. The catalytic efficiencies (kcat/Km) of the mutanted enzymes S169D, A259D, and S169D/A259D were higher than those of the wild-type enzyme by 275, 164, and 406%, respectively. Mass spectrometry and activity detection showed the enzyme degradation products were in line with the standards of the European Pharmacopoeia. Protein structure analysis showed that hydrogen bonds and ionic bonds were important factors for improving specific enzyme activity and yield. CONCLUSIONS: We found that the mutant S169D/A259D had more industrial application value than the wild-type enzyme due to molecular modifications. Our results provide a new strategy to increase the catalytic efficiency of other heparinases.

Crude Heparin Preparations Unveil the Presence of Structurally Diverse Oversulfated Contaminants.

Nowadays, pharmaceutical heparin is purified from porcine and bovine intestinal mucosa. In the past decade there has been an ongoing concern about the safety of heparin, since in 2008, adverse effects associated with the presence of an oversulfated chondroitin sulfate (OSCS) were observed in preparations of pharmaceutical porcine heparin, which led to the death of patients, causing a global public health crisis. However, it has not been clarified whether OSCS has been added to the purified heparin preparation, or whether it has already been introduced during the production of the raw heparin. Using a combination of different analytical methods, we investigate both crude and final heparin products and we are able to demonstrate that the sulfated contaminants are intentionally introduced in the initial steps of heparin preparation. Furthermore, the results show that the oversulfated compounds are not structurally homogeneous. In addition, we show that these contaminants are able to bind to cells in using well known heparin binding sites. Together, the data highlights the importance of heparin quality control even at the initial stages of its production.

Single conical track-etched nanopore for a free-label detection of OSCS contaminants in heparin.

The heparin contamination by oversulfated chondroitin (OSCS) was at the origin of one major sanitary problem of last decade. Here we propose a novel strategy to detect OSCS from heparin solution based on conical nanopore functionalized with poly-L-lysine deposition to ensure its re-usability. This sensor is an excellent to detect low heparin concentration (from 25 ng/ml to 3 µg/ml) using the modification of ionic current rectification. It also allows following the kinetic of heparin degradation by heparinase with a good correlation with results obtained by classical methods. The sensor is sensitive to the inhibition of heparinase by OSCS until a concentration of 200 pg/ml representing 0.01% in weight in a heparin. This resolution is one order of magnitude lower than the one obtained by chromatography. For the first time, it was reached without fluorescence labeling.

Negative-Ion Mode Capillary Isoelectric Focusing Mass Spectrometry for Charge-Based Separation of Acidic Oligosaccharides.

Glycosaminoglycans (GAGs) are biologically and pharmacologically important linear, anionic polysaccharides containing various repeating disaccharides sequences. The analysis of these polysaccharides generally relies on their chemical or enzymatic breakdown to disaccharide units that are separated, by chromatography or electrophoresis, and detected, by UV, fluorescence, or mass spectrometry (MS). Isoelectric focusing (IEF) is an important analytical technique with high resolving power for the separation of analytes exhibiting differences in isoelectric points. One format of IEF, the capillary isoelectric focusing (cIEF), is an attractive approach in that it can be coupled with mass spectrometry (cIEF-MS) to provide online focusing and detection of complex mixtures. In the past three decades, numerous studies have applied cIEF-MS methods to the analysis of protein and peptide mixtures by positive-ion mode mass spectrometry. However, polysaccharide chemists largely rely on negative-ion mode mass spectrometry for the analysis of highly sulfated GAGs. The current study reports a negative-ion mode cIEF-MS method using an electrokinetically pumped sheath liquid nanospray capillary electrophoresis-mass spectrometry (CE-MS) coupling technology. The feasibility of this negative-ion cIEF-MS method and its potential applications are demonstrated using chondroitin sulfate and heparan sulfate oligosaccharides mixtures.

Cell Membrane Derived Platform To Study Virus Binding Kinetics and Diffusion with Single Particle Sensitivity.

Discovery and development of new antiviral therapies essentially rely on two key factors: an in-depth understanding of the mechanisms involved in viral infection and the development of fast and versatile drug screening platforms. To meet those demands, we present a biosensing platform to probe virus-cell membrane interactions on a single particle level. Our method is based on the formation of supported lipid bilayers from cell membrane material. Using total internal reflection fluorescence microscopy, we report the contribution of viral and cellular components to the interaction kinetics of herpes simplex virus type 1 with the cell membrane. Deletion of glycoprotein C (gC), the main viral attachment glycoprotein, or deletion of heparan sulfate, an attachment factor on the cell membrane, leads to an overall decrease in association of virions to the membrane and faster dissociation from the membrane. In addition to this, we perform binding inhibition studies using the antiviral compound heparin to estimate its IC50 value. Finally, single particle tracking is used to characterize the diffusive behavior of the virus particles on the supported lipid bilayers. Altogether, our results promote this platform as a complement to existing bioanalytical assays, being at the interface between simplified artificial membrane models and live cell experiments.