Testicular Hyaluronidase Research Articles

Conformational Transitions in 3D Model of Bovine Testicular Hyaluronidase during Molecular Docking with Glycosaminoglycan Ligands

In silico molecular docking of the trimer repeating unit of chondroitin sulfate (sulfated hexasaccharide) and tetramer repeating unit of heparin (sulfated octasaccharide) to the 3D model of bovine testicular hyaluronidase by the methods of computational chemistry demonstrated the presence of eight significant binding sites for these ligands (cs1–cs8). The interaction of the active site of the enzyme with the heparin ligand, which inactivates the enzyme, and the protective effect of the chondroitin sulfate ligands bound to the surface sites of the biocatalyst molecule were theoretically studied using calculation approaches. We sequentially determined binding sites for the chondroitin sulfate ligands (in positions cs2, cs4, cs7, cs8 or cs1, cs2, cs4, cs7, cs8) critical for the protein structure stabilization, whose occupancy is theoretically sufficient to prevent irreversible deformations of the enzyme molecule when the heparin ligand is introduced into the cavity of its active site. Theoretical detection of these ‘sensibility points’ on the hyaluronidase globule indicates the possibility of regulating its functioning under the binding of the glycosaminoglycan ligands that initiate the fine formation of an effective type of the surface electrostatic potential. The interaction of the glycosaminoglycan ligands with hyaluronidase is mainly determined by electrostatic forces.

In vitro effects of 2, 4-Dichlorophenoxy acetic acid dimethylamine salt and enrofloxacin on BTH

In this study, the in vitro effects of enrofloxacin, a broad spectrum antibiotic, and 2, 4-Dichlorophenoxy acetic acid dimethylamine salt that, a herbicide, on the bovine testicular hyaluronidase (BTH) were analyzed. BTH was purified using the method of ammonium sulfate precipitation and using affinity gel (Sepharose-4B-L-tyrosine-m-anisidine). Then the inhibiting effects of enrofloxacin and 2, 4-Dichlorophenoxy acetic acid dimethylamine salt on the purified BTH enzyme were identified. IC50 values of these chemical substances were found to be 0.3101 and 0.4276 mM, respectively.

PH20 is not expressed in murine CNS and oligodendrocyte precursor cells.

ObjectiveExpression of Spam1/PH20 and its modulation of high/low molecular weight hyaluronan substrate have been proposed to play an important role in murine oligodendrocyte precursor cell (OPC) maturation in vitro and in normal and demyelinated central nervous system (CNS). We reexamined this using highly purified PH20.MethodsSteady‐state expression of mRNA in OPCs was evaluated by quantitative polymerase chain reaction; the role of PH20 in bovine testicular hyaluronidase (BTH) inhibition of OPC differentiation was explored by comparing BTH to a purified recombinant human PH20 (rHuPH20). Contaminants in commercial BTH were identified and their impact on OPC differentiation characterized. Spam1/PH20 expression in normal and demyelinated mouse CNS tissue was investigated using deep RNA sequencing and immunohistological methods with two antibodies directed against recombinant murine PH20.Results BTH, but not rHuPH20, inhibited OPC differentiation in vitro. Basic fibroblast growth factor (bFGF) was identified as a significant contaminant in BTH, and bFGF immunodepletion reversed the inhibitory effects of BTH on OPC differentiation. Spam1 mRNA was undetected in OPCs in vitro and in vivo; PH20 immunolabeling was undetected in normal and demyelinated CNS.InterpretationWe were unable to detect Spam1/PH20 expression in OPCs or in normal or demyelinated CNS using the most sensitive methods currently available. Further, “BTH” effects on OPC differentiation are not due to PH20, but may be attributable to contaminating bFGF. Our data suggest that caution be exercised when using some commercially available hyaluronidases, and reports of Spam1/PH20 morphogenic activity in the CNS may be due to contaminants in reagents.

Sulfated glycosaminoglycan-derived oligosaccharides produced from chicken connective tissue promote iron uptake in a human intestinal Caco-2 cell line.

Food grade sulfated glycosaminoglycan (GAG) polysaccharides were successfully extracted from chicken cartilage and skin. Their respective oligosaccharides were obtained by bovine testicular hyaluronidase digestion. The antioxidant capacity was analyzed to determine the possible mechanism explaining how GAGs promote iron uptake by the Caco-2 cells. The sulfated GAG oligosaccharides derived from cartilage possessed the greatest DPPH scavenging and ferric reducing activities (p<0.05), but limited ferrous chelating activities. Both the cartilage and skin sulfated GAG polysaccharides showed greater ferritin formation compared to the negative control (p<0.05). Depolymerisation of GAG polysaccharides to oligosaccharides further improved ferritin formation by twofold. This research establishes that chicken sulfated GAG polysaccharides can enhance iron uptake by Caco-2 cells. The enhanced iron uptake through enzymatic GAG depolymerisation could be due to the combined effects of reduced molecular weight, increased amount of hydroxyl terminal groups and ferric reducing activities.

FT-IR Microspectroscopy of Rat Ear Cartilage.

Rat ear cartilage was studied using Fourier transform-infrared (FT-IR) microspectroscopy to expand the current knowledge which has been established for relatively more complex cartilage types. Comparison of the FT-IR spectra of the ear cartilage extracellular matrix (ECM) with published data on articular cartilage, collagen II and 4-chondroitin-sulfate standards, as well as of collagen type I-containing dermal collagen bundles (CBs) with collagen type II, was performed. Ear cartilage ECM glycosaminoglycans (GAGs) were revealed histochemically and as a reduction in ECM FT-IR spectral band heights (1140–820 cm-1) after testicular hyaluronidase digestion. Although ear cartilage is less complex than articular cartilage, it contains ECM components with a macromolecular orientation as revealed using polarization microscopy. Collagen type II and GAGs, which play a structural role in the stereo-arrangement of the ear cartilage, contribute to its FT-IR spectrum. Similar to articular cartilage, ear cartilage showed that proteoglycans add a contribution to the collagen amide I spectral region, a finding that does not recommend this region for collagen type II quantification purposes. In contrast to articular cartilage, the symmetric stretching vibration of –SO3- groups at 1064 cm-1 appeared under-represented in the FT-IR spectral profile of ear cartilage. Because the band corresponding to the asymmetric stretching vibration of –SO3- groups (1236–1225 cm-1) overlapped with that of amide III bands, it is not recommended for evaluation of the –SO3- contribution to the FT-IR spectrum of the ear cartilage ECM. Instead, a peak (or shoulder) at 1027–1016 cm-1 could be better considered for this intent. Amide I/amide II ratios as calculated here and data from the literature suggest that protein complexes of the ear cartilage ECM are arranged with a lower helical conformation compared to pure collagen II. The present results could motivate further studies on this tissue under pathological or experimental states involving ear cartilage.

Characterisation of separated end hyaluronan oligosaccharides from leech hyaluronidase and evaluation of angiogenesis

Hyaluronan oligosaccharides (o-HAs), especially saturated o-HAs, have attracted intensive attention due to their potential applications in medical treatments. In this study, the hydrolysis process of leech hyaluronidase (LHase) towards the hyaluronan was investigated by HPLC and HPLC/ESI–MS. The proportions of hyaluronan tetrasaccharide (HA4) with hexasaccharide (HA6), end products, were illustrated to have a relationship with the amount of LHase. Higher yield of HA4 was achieved with higher activity of LHase. After optimisation of the packing resin and operation parameters (balanced pH, elution concentration, elution volume and elution flow rate), the highly pure HA4 and HA6 were efficiently separated and prepared by combining ion exchange Q-Sepharose Fast Flow and size exclusion column chromatography. Compared with o-HAs (average Mr of 4000Da), HA4 and HA6 were demonstrated to show higher activity for promoting angiogenesis, which was similar with the corresponding HA4 and HA6 produced by bovine testicular hyaluronidase. The pure HA4 and HA6 that prepared from LHase will attract intensive studies and be used in potential applications in near future.

Sequence determination of synthesized chondroitin sulfate dodecasaccharides.

Chondroitin sulfate (CS) is a linear acidic polysaccharide composed of repeating disaccharide units of glucuronic acid and N-acetyl-d-galactosamine. The polysaccharide is modified with sulfate groups at different positions by a variety of sulfotransferases. CS chains exhibit various biological and pathological functions by interacting with cytokines and growth factors and regulating their signal transduction. The fine structure of the CS chain defines its specific biological roles. However, structural analysis of CS has been restricted to disaccharide analysis, hampering the understanding of the structure-function relationship of CS chains. Here, we chemo-enzymatically synthesized CS dodecasaccharides having various sulfate modifications using a bioreactor system of bacterial chondroitin polymerase mutants and various CS sulfotransferases. We developed a sequencing method for CS chains using the CS dodecasaccharides. The method consists of (i) labeling a reducing end with 2-aminopyridine (PA), (ii) partial digestion of CS with testicular hyaluronidase, followed by separation of PA-conjugated oligosaccharides with different chain lengths, (iii) limited digestion of these oligosaccharides with chondroitin lyase AC II into disaccharides, followed by labeling with 2-aminobenzamide, (iv) CS disaccharide analysis using a dual-fluorescence HPLC system (reversed-phase ion-pair and ion-exchange chromatography), and (v) estimation of the composition by calculating individual disaccharide ratios. This CS chain sequencing allows characterization of CS-modifying enzymes and provides a useful tool toward understanding the structure-function relationship of CS chains.

An Improved Immunostaining and Imaging Methodology to Determine Cell and Protein Distributions within the Bone Environment.

Bone is a dynamic tissue that undergoes multiple changes throughout its lifetime. Its maintenance requires a tight regulation between the cells embedded within the bone matrix, and an imbalance among these cells may lead to bone diseases such as osteoporosis. Identifying cell populations and their proteins within bone is necessary for understanding bone biology. Immunolabeling is one approach used to visualize proteins in tissues. Efficient immunolabeling of bone samples often requires decalcification, which may lead to changes in the structural morphology of the bone. Recently, methyl-methacrylate embedding of non-decalcified tissue followed by heat-induced antigen retrieval has been used to process bone sections for immunolabeling. However, this technique is applicable for bone slices below 50-µm thickness while fixed on slides. Additionally, enhancing epitope exposure for immunolabeling is still a challenge. Moreover, imaging bone cells within the bone environment using standard confocal microscopy is difficult. Here we demonstrate for the first time an improved methodology for immunolabeling non-decalcified bone using a testicular hyaluronidase enzyme-based antigen retrieval technique followed by two-photon fluorescence laser microscopy (TPLM) imaging. This procedure allowed us to image key intracellular proteins in bone cells while preserving the structural morphology of the cells and the bone.

Hyaluronic acid alkyl derivative: A novel inhibitor of metalloproteases and hyaluronidases

Extracellular matrix (ECM) degradation, one of the main features of osteoarthritis, is driven by at least two major classes of enzymes: matrix metalloproteases (MMPs) and hyaluronidases. Among certain glycosaminoglycans, including natural and chemically cross-linked HAs, which are currently used as viscosupplements, the hyaluronic acid (HA) alkyl-amides (Hyadd) were here selected as the strongest MMP and hyaluronidase inhibitors. We used C. histolyticum collagenase (ChC) and bovine testicular hyaluronidase (BTH) as representative models of human MMPs and hyaluronidases, respectively. The role of the alkyl moiety was investigated using HA derivatives with varying alkyl lengths and degrees of derivatization. The selected compound was then screened against 10 different human MMPs in vitro, and the results were validated ex vivo in human synovial fluid. Hyadd-C16, identified as a lead compound, showed the highest inhibition potency against MMP13 and MMP8. The in vitro results were confirmed by the inhibition of human MMP13 (Ki=106.1μM) and hyaluronidase-2 in the synovial fluid of patients with osteoarthritis. This study demonstrates the unique properties of Hyadd-C16, including its remarkable enzymatic inhibitory activity, which is conferred by the hydrophobic chain, and its high biocompatibility and water solubility of the HA backbone.

Enzymatic synthesis of hyaluronan hybrid urinary trypsin inhibitor

Human urinary trypsin inhibitor is a proteoglycan that has a single low-sulfated chondroitin 4-sulfate chain at the seryl residue in position 10 of the core protein as a glycosaminoglycan moiety, and is used as an anti-inflammatory medicine based on the protease inhibitory activity of the core protein. However, the functions of the glycosaminoglycan moiety have not yet been elucidated in detail. In the present study, the glycosaminoglycan chains of a native urinary trypsin inhibitor were remodeled to hyaluronan chains, with no changes to the core protein, using transglycosylation as a reverse reaction of the hydrolysis of bovine testicular hyaluronidase, and the properties of the hybrid urinary trypsin inhibitor were then analyzed. The trypsin inhibitory activitiy of the hyaluronan hybrid urinary trypsin inhibitor was similar to that of the native type; however, its inhibitory effect on the hydrolysis of hyaluronidase were not as strong as that of the native type. This result demonstrated that the native urinary trypsin inhibitor possessed hyaluronidase inhibitory activity on its chondroitin sulfate chain. The hyaluronan hybrid urinary trypsin inhibitors obtained affinity to a hyaluronan-binding protein not exhibited by the native type. The interactions between the hyaluronan hybrid urinary trypsin inhibitors and phosphatidylcholine (abundant in the outer layer of plasma membrane) were stronger than that of the native type. Hyaluronan hybrid urinary trypsin inhibitors may be useful for investigating the functions of the glycosaminoglycan chains of urinary trypsin inhibitors and hyaluronan, and our hybrid synthesizing method may be used widely in research for future medical applications.

Modulation of bleomycin-induced lung fibrosis by pegylated hyaluronidase and dopamine receptor antagonist in mice.

Hyaluronidases are groups of enzymes that degrade hyaluronic acid (HA). To stop enzymatic hydrolysis we modified testicular hyaluronidase (HYAL) by activated polyethylene oxide with the help of electron-beam synthesis. As a result we received pegylated hyaluronidase (pegHYAL). Spiperone is a selective D2 dopamine receptor antagonist. It was demonstrated on the model of a single bleomycin damage of alveolar epithelium that during the inflammatory phase monotherapy by pegHYAL or spiperone reduced the populations of hematopoietic stem /progenitor cells in the lung parenchyma. PegHYAL also reduced the levels of transforming growth factor (TGF)-β, interleukin (IL)-1β, tumor necrosis factor (TNF)-α in the serum and lungs, while spiperone reduced the level of the serum IL-1β. Polytherapy by spiperone and pegHYAL caused the increase of the quantity of hematopoietic stem/ progenitor cells in the lungs. Such an influx of blood cell precursors was observed on the background of considerable fall level of TGF-β and the increase level of TNF-α in the serum and lungs. These results show pegHYAL reduced the bleomycin-induced fibrosis reaction (production and accumulation of collagen) in the lung parenchyma. This effect was observed at a single and repetitive bleomycin damage of alveolar epithelium, the antifibrotic activity of pegHYAL surpassing the activity of testicular HYAL. The antifibrotic effect of pegHYAL is enhanced by an additional instillation of spiperone. Therapy by pegHYAL causes the flow of CD31‒CD34‒CD45‒CD44+CD73+CD90+CD106+-cells into the fibrous lungs. These cells are incapable of differentiating into fibroblast cells. Spiperone instillation separately or together with pegHYAL reduced the MSC-like cells considerably. These data enable us to assume, that pegHYAL is a new and promising instrument both for preventive and therapy of toxic pneumofibrosis. The blockage of D2 dopamine receptors with the following change of hyaluronan matrix can be considered as a new strategy in treatment of pneumofibrosis.

Stratification of chondroitin sulfate binding sites in 3D-model of bovine testicular hyaluronidase and effective size of glycosaminoglycan coat of the modified protein.

A 3D-model of bovine testicular hyaluronidase (BTH) was constructed based on established tertiary structure of human hyaluronidase Hyal1 using a molecular homological modeling method in silico. The analysis of the BTH 3D-model demonstrated lysine residue stratification during enzyme modification. The 3D-model of chondroitin sulfate (CHS)-modified hyaluronidase (BTH-CHS) was obtained by modeling covalent binding of lysine residues with benzoquinone-activated CHS. The degree of enzyme modification and the length of CHS chains were varied during 3D modeling. The importance of deep BTH modification degree for the formation of active and stable enzyme derivatives was shown, as determined earlier experimentally. The effective size of the CHS coat for productive BTH modification was confirmed. It is theoretically achieved at the increase in molecular mass of BTH-CHS derivative to approximately 140-180kDa and can be practically obtained, according to experimental data, using CHS of different molecular mass (30-50 as well as 120-140kDa).

High performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) for the sensitive determination of hyaluronan oligosaccharides.

High performance anion exchange chromatography (HPAEC) with pulsed amperometric detection (PAD) was optimized for the analysis of oligosaccharides derived from the extracellular matrix component hyaluronan. Using this sensitive approach, the separation of oligosaccharides consisting of two (molecular weight ca. 0.8 kDa) up to 25-30 (molecular weight: ca. 9.5-11.4 kDa) disaccharide moieties was possible. Standard oligosaccharides (comprising 2-4 repetitive disaccharides) were detectable at very low amounts of 0.2-0.3 pmol (20-30 nM). Including 10 min of column equilibration, a complex mixture of low molecular weight hyaluronan can be analyzed within 40 min. The HPAEC method was successfully applied to the study of the size-dependency of both the action of bovine testicular hyaluronidase (BTH) and the precipitation of hyaluronan by cetyltrimethylammonium bromide (CTAB), a physicochemical reaction often used for the determination of hyaluronan and hyaluronidase activity.

Lack of in vitro antihyaluronidase activity of methanolic leaf extract of Indigofera tinctoria L and methanolic stem bark extract of Stereospermum suaveolens DC

Objective: To assess the antihyaluronidase activity of methanolic leaf extract of Indigofera tinctoria L (I. tinctoria) (family: Fabaceae/Leguminosae) and stem bark extract of Stereospermum suaveolens DC (S. suaveolens) (family: Bignoniaceae) in vitro with a view to develop an antiaging skin formulation. Materials and Methods: The antihyaluronidase activity of different concentrations (0.19 mg/mL, 0.38 mg/mL, 0.75 mg/mL, 1.5 mg/mL, and 3.0 mg/mL) of methanolic leaf extract of I. tinctoria, methanolic stem bark extract of S. suaveolens, and reference drug epigallocatechin gallate (EGCG) of different concentrations (12.5 μg/mL, 25 μg/mL, 50 μg/mL, 100 μg/mL, and 200 μg/mL) were determined spectrophotometrically using hyaluronic acid (from rooster combs) and bovine testicular hyaluronidase. Results: There is no in vitro antihyaluronidase activity in the methanolic extracts of I. tinctoria leaves and S. suaveolens stem bark even at high concentrations. On the contrary, EGCG, the reference agent, showed marked concentration-dependent (r2 = 0.92) antihyluronidase activity [in terms of percentage inhibition: half maximal inhibitory concentration (IC 50 ) 92.64 ± 0.64 μg/mL]. Conclusion: It is unlikely that skin antiaging effects of I. tinctoria leaves and S. suaveolens stem bark, as claimed in traditional and folk medicines in Sri Lanka, are mediated via antihyaluronidase activity.

Inhibitory effect of chondroitin sulfate oligosaccharides on bovine testicular hyaluronidase.

Hyaluronan and chondroitin sulfates are prominent components of the extracellular matrices of animal tissues; however, their functions in relation to their oligosaccharide structures have not yet been fully elucidated. The oligosaccharides of hyaluronan and chondroitin sulfate were prepared and used to investigate their effects on the hydrolysis and transglycosylation reactions of bovine testicular hyaluronidase when hyaluronan was used as a substrate. Hydrolysis and transglycosylation activities were assessed in independent reaction systems by analyzing the products by HPLC. The hydrolysis and transglycosylation reactions of bovine testicular hyaluronidase were dose-dependently inhibited by chondroitin sulfate oligosaccharides, but not by hyaluronan or chondroitin oligosaccharides. A kinetic analysis of the hydrolysis reaction using hyaluronan octasaccharide revealed that the inhibition mode by chondroitin sulfate oligosaccharides was competitive.

In vitro anti-hyaluronidase activity of Sri Lankan low grown orthodox orange pekoe grade black tea (Camellia sinensis L.)

ABSTRACT Objective To access the anti-hyaluronidase activity of Sri Lankan low grown orthodox orange pekoe (OP) grade black tea with a view to develop an anti-aging skin formulation. Methods Five concentrations (0.125, 0.250, 0.500, 1.000 and 2.000 mg/mL) of black tea brew (BTB) were made using a freeze dried sample of Sri Lankan low grown orthodox OP grade black tea which was prepared according to international organization for standardization specification. Epigallocatechin gallate (EGCG) was used as the reference agent (concentrations tested: 0.012, 0.025, 0.050, 0.100 and 0.200 mg/mL). Anti-hyaluronidase activity of BTB and EGCG in vitro were ascertained spectrometrically using hyaluronic acid (from rooster comb) and bovine testicular hyaluronidase. Results The results revealed that BTB had moderate [IC 50 =(1.09±0.12) mg/mL] and dose dependent ( r 2 =0.94) anti-hyaluronidase activity. EGCG also exhibited dose dependent ( r 2 =0.93, P 50 =(0.09±0.00) mg/mL] to BTB. Conclusions Sri Lankan low grown orthodox OP grade black tea has promising anti-hyaluronidase activity in vitro and has the potential to be used as an anti-aging cosmaceutical. In addition, it may prove useful as a beverage in the management of allergy, some joint diseases and envenomation.

A new affinity method for purification of bovine testicular hyaluronidase enzyme and an investigation of the effects of some compounds on this enzyme

In this study, a new affinity gel for the purification of bovine testicular hyaluronidase (BTH) was synthesized. l-Tyrosine was added as the extension arm to the Sepharose-4B activated with cyanogen bromide. m-Anisidine is a specific inhibitor of BTH enzyme. m-Anisidine was clamped to the newly formed Sepharose-4B-l-tyrosine as a ligand. As a result, an affinity gel having the chemical structure of Sepharose-4B-l-tyrosine-m-anisidine was obtained. BTH purified by ammonium sulfate precipitation and affinity chromatography was obtained with a 16.95% yield and 881.78 degree of purity. The kinetic constants KM and VMax for BTH were determined by using hyaluronic acid as a substrate. KM and VMax values obtained from the Lineweaver–Burk graph were found to be 2.23 mM and 19.85 U/mL, respectively. In vitro effects of some chemicals were determined on purified BTH enzyme. Some chemically active ingredients were 1,1-dimethyl piperidinium chloride, β-naphthoxyacetic acid and gibberellic acid. Gibberellic acid showed the best inhibition effect on BTH.

Characterization of hyaluronan, hyaluronidase PH20, and HA synthase HAS2 in inflammation and cancer

Hyaluronan (HA, an extracellular matrix glycosaminoglycan) has been reported to have a variety of biological activities, including regulation of inflammation. While high molecular weight HA (HMW HA) is recognized to be anti-inflammatory, the activity of low molecular weight HA (LMW HA, the degradation catabolite of hyaluronidase) in inflammation is less clear. Because of reagent contamination of both hyaluronidase and HA used in many studies, many reported aspects of the associated biology need reinvestigation. There are many reports which have shown that LMW HA is pro-inflammatory; however, some recent publications raised serious doubts that LMW HA and hyaluronidase PH20 are not pro-inflammatory. Endotoxin and other contaminants in the reagents used in previous reports (i.e., bovine testicular hyaluronidase [BTH] Hyal type I-S and IV-S from Sigma) may be responsible for the observed inflammation. Further investigation has shown that the most purified BTH (type VI-S from Sigma) contains no endotoxin, but has substantial level of peptidoglycan, which is also pro-inflammatory. Caution should be taken when conducting studies of inflammation using HA and hyaluronidase that may contain endotoxin and peptidoglycan, as well as other pro-inflammatory contaminants. We have characterized HA affinity to its receptor CD44. While HMW HA binds to CD44 strongly, LMW HA degraded by PH20 hyaluronidase does not bind to CD44 on the cell surface. This may explain the observation that recombinant human hyaluronidase PH20 (rHuPH20) inhibits leukocyte migration upon inflammatory stimulation by interrupting CD44 interaction with HA, mediated by HMW HA. PEGPH20, a pegylated rHuPH20, has the same inhibitory activity on leukocyte transmigration as rHuPH20. Further investigation of leukocyte-inhibiting activity of rHuPH20 may reveal further clinical applications, such as wound healing and arthritis. Another report which is difficult to understand is that the ultra-high molecular weight HA produced by naked mole rat hyaluronan synthase-2 (nmrHAS2) contributes to cancer resistance, probably due to its specific anti-inflammatory activity. In our study, nmrHAS2 is cancer-promoting in human cancer cells, to a similar extent as human HAS2. The proposed cancer resistant activity of nmrHAS2 may apply selectively to its host animal species, the naked mole rat.

Chemoenzymatic Synthesis of Oligohyaluronan–Lipid Conjugates

Herein, we describe an efficient and high-yielding method to synthesize hyaluronan oligosaccharide–lipid conjugates. This strategy is based on first covalently attaching diphytanoyl glycerophosphatidylethanolamine (DiPhPE) to commercially available high molecular weight hyaluronic acid (HA), via the carboxylate group of the glucuronic acid using carbodiimide chemistry. The HA-lipid conjugate mixture is then digested with bovine testicular hyaluronidase to yield HA-DiPhPE conjugates that have a narrow distribution of moderately sized HA oligosaccharides. These HA-lipid conjugates can be incorporated into liposomes or micelles to selectively target CD44 that is overexpressed on many cancer or cancer initiating cells.

Improvement of the digestibility of sulfated hyaluronans by bovine testicular hyaluronidase: a UV spectroscopic and mass spectrometric study.

Glycosaminoglycans (GAGs) such as hyaluronan (HA) and chondroitin sulfate (CS) are important, natural polysaccharides which occur in biological (connective) tissues and have various biotechnological and medical applications. Additionally, there is increasing evidence that chemically (over)sulfated GAGs possess promising properties and are useful as implant coatings. Unfortunately, a detailed characterization of these GAGs is challenging: although mass spectrometry (MS) is one of the most powerful tools to elucidate the structures of (poly)saccharides, MS is not applicable to high mass polysaccharides, but characteristic oligosaccharides are needed. These oligosaccharides are normally generated by enzymatic digestion. However, chemically modified (particularly sulfated) GAGs are extremely refractive to enzymatic digestion. This study focuses on the investigation of the digestibility of GAGs with different degrees of sulfation by bovine testicular hyaluronidase (BTH). It will be shown by using an adapted spectrophotometric assay that all investigated GAGs can be basically digested if the reaction conditions are carefully adjusted. However, the oligosaccharide yield correlates reciprocally with the number of sulfate residues per polymer repeating unit. Finally, matrix-laser desorption and ionization (MALDI) MS will be used to study the released oligosaccharides and their sulfation patterns.