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

Abstract

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 K_M and V_Max for BTH were determined by using hyaluronic acid as a substrate. K_M and V_Max 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.     

Development of a fluorescent substrate to measure hyaluronidase activity

A novel fluorescent substrate (termed FRET-HA) to quantitatively assess hyaluronidase activity was developed. Hyaluronan (HA), the major substrate for hyaluronidase, was dual labeled with fluorescein amine and rhodamine B amine. The fluorescein amine fluorescence signal was significantly quenched and the rhodamine B amine signal was significantly enhanced due to fluorescence resonance energy transfer (FRET). In the presence of bovine testes hyaluronidase, cleavage of HA disrupted FRET, resulting in a loss of the fluorescein amine quenching that was dependent on both enzyme concentration and time. Increase in the fluorescein amine signal could be conveniently monitored in both noncontinuous and continuous fashions. The K_m value for bovine testes hyaluronidase was determined using FRET-HA in a continuous fluorescent assay. Importantly, the estimated K_m value for bovine testes hyaluronidase using FRET-HA as the substrate was in excellent agreement with K_m values reported previously for this enzyme using native (i.e., unlabeled) HA. Therefore, FRET-HA is a reliable substrate for quantitatively assessing the HA/hyaluronidase molecular interaction. The simplicity, sensitivity, and versatility of the FRET-HA substrate suggest that it will have utility in a variety of assay platforms and should be a new tool for assessing hyaluronidase activity.     

Novel products in hyaluronan digested by bovine testicular hyaluronidase

When the products of hyaluronan (HA) digested by bovine testicular hyaluronidase (BTH) were analyzed by high-performance liquid chromatography (HPLC), minor peaks were detected just before the main even-numbered oligosaccharide peaks. The amount of each minor peak was dependent on the reaction conditions for transglycosylation, rather than hydrolysis, by the BTH. Mainly based on HPLC and MS analysis, each minor peak was found to correspond to its oligosaccharide with one N-acetyl group removed from the reducing terminal N-acetylglucosamine. Enzymatic studies showed that the N-deacetylation activity was closely related to reaction temperature, pH, and the concentration of NaCl contained in the buffer, and glycosaminoglycan types and chain lengths of substrates. These findings strongly suggest that the N-deacetylation reaction in minor peaks was due to a novel enzyme contaminant in the BTH, N-deacetylase, that carries out N-deacetylation at the reducing terminal N-acetylglucosamine of oligosaccharides and is dependent on HA hydrolysis by BTH.     

The cell surface hyaluronidase TMEM2 is essential for systemic hyaluronan catabolism and turnover

As a major component of the extracellular matrix, hyaluronan (HA) plays an important role in defining the biochemical and biophysical properties of tissues. In light of the extremely rapid turnover of HA and the impact of this turnover on HA biology, elucidating the molecular mechanisms underlying HA catabolism is key to understanding the in vivo functions of this unique polysaccharide. Here, we show that TMEM2, a recently identified cell surface hyaluronidase, plays an essential role in systemic HA turnover. Employing induced global Tmem2 knockout mice (Tmem2^iKO), we determined the effects of Tmem2 ablation not only on the accumulation of HA in bodily fluids and organs, but also on the process of HA degradation in vivo. Within 3 weeks of tamoxifen-induced Tmem2 ablation, Tmem2^iKO mice exhibit pronounced accumulation of HA in circulating blood and various organs, reaching levels as high as 40-fold above levels observed in control mice. Experiments using lymphatic and vascular injection of fluorescent HA tracers demonstrate that ongoing HA degradation in the lymphatic system and the liver is significantly impaired in Tmem2^iKO mice. We also show that Tmem2 is strongly expressed in endothelial cells in the subcapsular sinus of lymph nodes and in the liver sinusoid, two primary sites implicated in systemic HA turnover. Our results establish TMEM2 as a physiologically relevant hyaluronidase with an essential role in systemic HA catabolism in vivo, acting primarily on the surface of endothelial cells in the lymph nodes and liver.     

Kinetic investigation of recombinant human hyaluronidase PH20 on hyaluronic acid

The kinetic investigation of hyaluronidases using physiologically relevant hyaluronic acid (HA or hyaluronan) substrate will provide useful and important clues to their catalytic behavior and function in vivo. We present here a simple and sensitive method for kinetic measurement of recombinant human hyaluronidase PH20 (rHuPH20) on HA substrates with sizes ranging from 90 to 752 kDa. The method is based on 2-aminobenzamide labeling of hydrolyzed HA products combined with separation by size exclusion–ultra performance liquid chromatography coupled with fluorescence detection. rHuPH20 was found to follow Michaelis–Menten kinetics during the initial reaction time. Optimal reaction rates were observed in the pH range of 4.5–5.5. The HA substrate size did not have significant effects on the initial rate of the reaction. By studying HA substrates of 215, 357, and 752 kDa, the kinetic parameters K_m, V_max, and k_cat were determined to be 0.87–0.91 mg/ml, 1.66–1.74 nM s⁻¹, and 40.5–42.4 s⁻¹, respectively. This method allows for direct measurement of kinetics using physiologically relevant HA substrates and can be applied to other hyaluronidase kinetic measurements.     

Isoenzyme-specific differences in the degradation of hyaluronic acid by mammalian-type hyaluronidases

Bovine testicular hyaluronidase (BTH) has been used as a spreading factor for many years and was primarily characterized by its enzymatic activity. As recombinant human hyaluronidases are now available the bovine preparations can be replaced by the human enzymes. However, data on the pH-dependent activity of hyaluronidases reported in literature are inconsistent in part or even contradictory. Detection of the pH-dependent activity of PH-20 type hyaluronidases, i.e. recombinant human PH-20 (rhPH-20) and BTH, showed a shift of the pH optimum from acidic pH values in a colorimetric activity assay to higher pH values in a turbidimetric activity assay. Contrarily, recombinant human Hyal-1 (rhHyal-1) and bee venom hyaluronidase (BVH) exhibited nearly identical pH profiles in both commonly used types of activity assays. Analysis of the hyaluronic acid (HA) degradation products by capillary zone electrophoresis showed that hyaluronan was catabolized by rhHyal-1 continuously into HA oligosaccharides. BTH and, to a less extent, rhPH-20 exhibited a different mode of action: at acidic pH (pH 4.5) HA was degraded as described for rhHyal-1, while at elevated pH (pH 5.5) small oligosaccharides were produced in addition to HA fragments of medium molecular weight, thus explaining the pH-dependent discrepancies in the activity assays. Our results suggest a sub-classification of mammalian-type hyaluronidases into a PH-20/BTH and a Hyal-1/BVH subtype. As the biological effects of HA fragments are reported to depend on the size of the molecules it can be speculated that different pH values at the site of hyaluronan degradation may result in different biological responses.     

Inhibition of hyaluronan degradation by dextran sulphate facilitates characterisation of hyaluronan synthesis: An in vitro and in vivo study

The concentration and molecular weight of hyaluronan often dictates its physiological function. Consequently full characterisation of the anabolic products and turnover rates of HA could facilitate understanding of the role that HA metabolism plays in disease processes. In order to achieve this it is necessary to interrupt the dynamic balance between concurrent HA synthesis and degradation, achievable through the inhibition of the hyaluronidases, a group of enzymes which degrade HA. The sulphated polysaccharide, dextran sulphate has been demonstrated to competitively inhibit testicular hyaluronidase in a non-biological system, but its application to in vitro biological systems had yet to be developed and evaluated. This study determined the inhibitory concentrations of dextran sulphate against both testicular and Streptomyces hyaluronidase in a cell-free and breast cancer model followed by characterisation of the effect that hyaluronidase inhibition exerted on HA synthesis and degradation. The IC₁₀₀ of dextran sulphate for both hyaluronidases in a cell-free and biological system was determined to be ≥400 μg/ml. At concentrations up to 10 mg/ml the dextran sulphate did not effect breast cancer cell proliferation or morphology, while at 400 μg/ml HA degradation was totally inhibited, enabling an accurate quantitation of HA production as well as characterisation of the cell-associated and liberated HA. FACS quantitation of the HA receptor CD44, HA synthase and the hyaluronidases HYAL 1 and HYAL 2 demonstrated that dextran sulphate down-regulated CD44 and HA synthase while upregulating the hyaluronidases. These results suggest dynamic feedback signalling and complex mechanisms occur in the net deposition of HA in vivo. Published in 2004.     

bFGF induces changes in hyaluronan synthase and hyaluronidase isoform expression and modulates the migration capacity of fibrosarcoma cells

Background

Hyaluronan (HA) a glycosaminoglycan, is capable of transmitting extracellular matrix derived signals to regulate cellular functions. In this study, we investigated whether the changes in HT1080 and B6FS fibrosarcoma cell lines HA metabolism induced by basic fibroblast growth factor (bFGF) are correlated to their migration.

Methods

Real-time PCR, in vitro wound healing assay, siRNA transfection, enzyme digestions, western blotting and immunofluorescence were utilized.

Results

bFGF inhibited the degradation of HA by decreasing hyaluronidase-2 expression in HT1080 cells (p = 0.0028), increased HA-synthase-1 and -2 expression as we previously found and enhanced high molecular weight HA deposition in the pericellular matrix. Increased endogenous HA production (p = 0.0022) and treatment with exogenous high molecular weight HA (p = 0.0268) correlated with a significant decrease of HT1080 cell migration capacity. Transfection with siHAS2 and siHAS1 showed that mainly HAS1 synthesized high molecular weight HA regulates HT1080 cell motility. Induced degradation of the HA content by hyaluronidase treatment and addition of low molecular weight HA, resulted in a significant stimulation of HT1080 cells' motility (p < 0.01). In contrast, no effects on B6FS fibrosarcoma cell motility were observed.

Conclusions

bFGF regulates, in a cell-specific manner the migration capability of fibrosarcoma cells by modulating their HA metabolism.HA metabolism is suggested to be a potential therapeutic target in fibrosarcoma.     

CRISPR/Cas9 knockout of HAS2 in rat chondrosarcoma chondrocytes demonstrates the requirement of hyaluronan for aggrecan retention

Hyaluronan (HA) plays an essential role in cartilage where it functions to retain aggrecan. Previous studies have suggested that aggrecan is anchored indirectly to the plasma membrane of chondrocytes via its binding to cell-associated HA. However, reagents used to test these observations such as hyaluronidase and HA oligosaccharides are short term and may have side activities that complicate interpretation. Using the CRISPR/Cas9 gene editing approach, a model system was developed by generating HA-deficient chondrocyte cell lines. HA synthase-2 (Has2)-specific single guide RNA was introduced into two different variant lines of rat chondrosarcoma chondrocytes; knockout clones were isolated and characterized. Two other members of the HA synthase gene family were expressed at very low relative copy number but showed no compensatory response in the Has2 knockouts. Wild type chondrocytes of both variants exhibited large pericellular matrices or coats extending from the plasma membrane. Addition of purified aggrecan monomer expanded the size of these coats as the proteoglycan became retained within the pericellular matrix. Has2 knockout chondrocytes lost all capacity to assemble a particle-excluding pericellular matrix and more importantly, no matrices formed around the knockout cells following the addition of purified aggrecan. When grown as pellet cultures so as to generate a bioengineered neocartilage tissue, the Has2 knockout chondrocytes assumed a tightly-compacted morphology as compared to the wild type cells. When knockout chondrocytes were transduced with Adeno-ZsGreen1-mycHas2, the cell-associated pericellular matrices were restored including the capacity to bind and incorporate additional exogenous aggrecan into the matrix. These results suggest that HA is essential for aggrecan retention and maintaining cell separation during tissue formation.     

Tackling Obstacles for Gene Therapy Targeting Neurons: Disrupting Perineural Nets with Hyaluronidase Improves Transduction

Gene therapy has been proposed for many diseases in the nervous system. In most cases for successful treatment, therapeutic vectors must be able to transduce mature neurons. However, both in vivo, and in vitro, where preliminary characterisation of viral particles takes place, transduction of neurons is typically inefficient. One possible explanation is that the extracellular matrix (ECM), forming dense perineural nets (PNNs) around neurons, physically blocks access to the cell surface. We asked whether co-administration of lentiviral vectors with an enzyme that disrupts the ECM could improve transduction efficiency. Using hyaluronidase, an enzyme which degrades hyaluronic acid, a high molecular weight molecule of the ECM with mainly a scaffolding function, we show that in vitro in mixed primary cortical cultures, and also in vivo in rat cortex, hyaluronidase co-administration increased the percentage of transduced mature, NeuN-positive neurons. Moreover, hyaluronidase was effective at doses that showed no toxicity in vitro based on propidium iodide staining of treated cultures. Our data suggest that limited efficacy of neuronal transduction is partly due to PNNs surrounding neurons, and further that co-applying hyaluronidase may benefit applications where efficient transduction of neurons in vitro or in vivo is required.     

Aggrecan is expressed by embryonic brain glia and regulates astrocyte development

Determination of the molecules that regulate astrocyte development has been hindered by the paucity of markers that identify astrocytic precursors in vivo. Here we report that the chondroitin sulfate proteoglycan aggrecan both regulates astrocyte development and is expressed by embryonic glial precursors. During chick brain development, the onset of aggrecan expression precedes that of the astrocytic marker GFAP and is concomitant with detection of the early glial markers GLAST and glutamine synthetase. In co-expression studies, we established that aggrecan-rich cells contain the radial glial markers nestin, BLBP and GLAST and later in embryogenesis, the astroglial marker GFAP. Parallel in vitro studies showed that ventricular zone cultures, enriched in aggrecan-expressing cells, could be directed to a GFAP-positive fate in G5-supplemented differentiation media. Analysis of the chick aggrecan mutant nanomelia revealed marked increases in the expression of the astrocyte differentiation genes GFAP, GLAST and GS in the absence of extracellular aggrecan. These increases in astrocytic marker gene expression could not be accounted for by changes in precursor proliferation or cell death, suggesting that aggrecan regulates the rate of astrocyte differentiation. Taken together, these results indicate a major role for aggrecan in the control of glial cell maturation during brain development.     

A convenient plate assay for the quantitation of hyaluronidase in hymenoptera venoms

A simple plate assay for hyaluronidase activity in biological samples is described. Hyaluronic acid is incorporated into agarose gels and the enzyme is allowed to diffuse from punched wells. The undigested hyaluronic acid is then precipitated with cetylpyridinium chloride and the diameters of the clear circles are proportional to the logarithm of the enzyme concentration applied to the well. The assay was utilized to examine commercially available hymenoptera venoms, manufactured for use in allergy diagnosis and treatment, for their content of hyaluronidase as a measure of lot-to-lot consistency. The assay permits the analyses of a large number of samples with good reproducibility, without the need for any special instrumentation. Based on the quantity of purified hyaluronidase reported in honey bee venom (T. P. King, A. K. Sobotka, L. Kochoumian, and L. M. Lichtenstein, 1976, Arch. Biochem. Biophys.172, 661–671) we estimate that the assay should detect 70 ng/ml of purified honey bee venom hyaluronidase.     

Two Novel Functions of Hyaluronidase-2 (Hyal2) Are Formation of the Glycocalyx and Control of CD44-ERM Interactions

It has long been predicted that the members of the hyaluronidase enzyme family have important non-enzymatic functions. However, their nature remains a mystery. The metabolism of hyaluronan (HA), their major enzymatic substrate, is also enigmatic. To examine the function of Hyal2, a glycosylphosphatidylinositol-anchored hyaluronidase with intrinsically weak enzymatic activity, we have compared stably transfected rat fibroblastic BB16 cell lines with various levels of expression of Hyal2. These cell lines continue to express exclusively the standard form (CD44s) of the main HA receptor, CD44. Hyal2, CD44, and one of its main intracellular partners, ezrin-radixin-moesin (ERM), were found to co-immunoprecipitate. Functionally, Hyal2 overexpression was linked to loss of the glycocalyx, the HA-rich pericellular coat. This effect could be mimicked by exposure of BB16 cells either to Streptomyces hyaluronidase, to HA synthesis inhibitors, or to HA oligosaccharides. This led to shedding of CD44, separation of CD44 from ERM, reduction in baseline level of ERM activation, and markedly decreased cell motility (50% reduction in a wound healing assay). The effects of Hyal2 on the pericellular coat and on CD44-ERM interactions were inhibited by treatment with the Na⁺/H⁺ exchanger-1 inhibitor ethyl-N-isopropylamiloride. We surmise that Hyal2, through direct interactions with CD44 and possibly some pericellular hyaluronidase activity requiring acidic foci, suppresses the formation or the stability of the glycocalyx, modulates ERM-related cytoskeletal interactions, and diminishes cell motility. These effects may be relevant to the purported in vivo tumor-suppressive activity of Hyal2.     

A complete hyaluronan hydrodynamic characterization using a size exclusion chromatography-triple detector array system during in vitro enzymatic degradation

Size exclusion chromatography coupled with triple detection (online laser light scattering, refractometry, and viscosimetry) (SEC-TDA) was applied for the study of hyaluronan (HA) fragments produced during hydrolysis catalyzed by bovine testicular hyaluronidase (BTH). The main advantage this approach provides is the complete hydrodynamic characterization without requiring further experiments. HA was hydrolyzed using several BTH amounts and for increasing incubation times. Fragments were characterized in terms of weight and number average molecular weights (M_w and M_n, respectively), polydispersity index (M_w/M_n), hydrodynamic radius (R_h), and intrinsic viscosity ([η]). The Mark-Houwink-Sakurada (MHS) curves (log [η] versus log M_w) were then derived directly. Fragments covering a whole range of M_w (10-900 kDa) and size (R_h = 4-81 nm) and presenting a rather narrow distribution of molar masses (M_w/M_n = 1.6-1.7) were produced. From the MHS curves, HA conformation resulted in a change from a random coil toward a rigid rod structure while decreasing the M_w. HA enzymatic hydrolysis in the presence of a BTH inhibitor was also monitored, revealing that inhibition profiles are affected by ionic strength. Finally, a comparison of the kinetic data derived from SEC-TDA with the data from rheological measurements suggested different strengths of the two methods in the determination of the depolymerization rate depending on the hydrolysis conditions.     

The cell surface hyaluronidase TMEM2 plays an essential role in mouse neural crest cell development and survival

Hyaluronan (HA) is a major extracellular matrix component whose tissue levels are dynamically regulated during embryonic development. Although the synthesis of HA has been shown to exert a substantial influence on embryonic morphogenesis, the functional importance of the catabolic aspect of HA turnover is poorly understood. Here, we demonstrate that the transmembrane hyaluronidase TMEM2 plays an essential role in neural crest development and the morphogenesis of neural crest derivatives, as evidenced by the presence of severe craniofacial abnormalities in Wnt1-Cre–mediated Tmem2 knockout (Tmem2^CKO) mice. Neural crest cells (NCCs) are a migratory population of cells that gives rise to diverse cell lineages, including the craniofacial complex, the peripheral nervous system, and part of the heart. Analysis of Tmem2 expression during NCC formation and migration reveals that Tmem2 is expressed at the site of NCC delamination and in emigrating Sox9-positive NCCs. In Tmem2^CKO embryos, the number of NCCs emigrating from the neural tube is greatly reduced. Furthermore, linage tracing reveals that the number of NCCs traversing the ventral migration pathway and the number of post-migratory neural crest derivatives are both significantly reduced in a Tmem2^CKO background. In vitro studies using Tmem2-depleted mouse O9-1 neural crest cells demonstrate that Tmem2 expression is essential for the ability of these cells to form focal adhesions on and to migrate into HA-containing substrates. Additionally, we show that Tmem2-deficient NCCs exhibit increased apoptotic cell death in NCC-derived tissues, an observation that is corroborated by in vitro experiments using O9-1 cells. Collectively, our data demonstrate that TMEM2-mediated HA degradation plays an essential role in normal neural crest development. This study reveals the hitherto unrecognized functional importance of HA degradation in embryonic development and highlights the pivotal role of Tmem2 in the developmental process.     

A recommended procedure for the estimation of bovine testicular hyaluronidase in the presence of human serum

A procedure is described for the assay of bovine testicular hyaluronidase in human blood following intravenous administration of the enzyme. Inhibition of hyaluronidase by the reported nonspecific serum inhibitor is minimal. However, the presence of human serum does alter the pH profile of hyaluronidase and enhances the activity of the enzyme at low pH values. Preliminary data indicates that the effects caused by serum on the pH optimum and activity of the enzyme are largely associated with the albumin fraction and are not due to the presence of endogenous serum hyaluronidase. The activation effect is not specific for any particular blood type and is independent of whether serum or citrated plasma is used. A similar effect to that of serum on hyaluronidase activity is produced by different buffer mixtures or increased NaCl concentration. It is recommended that bovine testicular hyaluronidase be measured at pH 4.0 in 0.1 m sodium citrate buffer containing 0.15 m NaCl as under these conditions the addition of human serum or citrated plasma does not alter the pH optimum of the enzyme. These recommendations necessitate certain modifications of the reducing N-acetylhexosamine assay method of Reissig et al. (J. L. Reissig, J. L. Strominger, and L. F. Leloir, 1955, J. Biol. Chem.217, 959–966).     

Kinetics of Hyal-1 and PH-20 hyaluronidases: comparison of minimal substrates and analysis of the transglycosylation reaction

The availability of recombinant expression systems for the production of purified human hyaluronidases PH-20 and Hyal-1 facilitated the first detailed analysis of the enzymatic reaction products. The human recombinant enzymes, both expressed by Drosophila Schneider-2 (DS-2) cells, were compared to bovine testicular hyaluronidase (BTH), a commercially available hyaluronidase preparation, which has long been considered a prototype of mammalian hyaluronidases. The conversion of low molecular weight hyaluronic acid (HA) fragments was detected by a capillary zone electrophoresis (CZE) method. Surprisingly, the HA hexasaccharide, which is generally accepted to be the minimum substrate of BTH, was not a substrate of recombinant human PH-20 and Hyal-1. However, HA octasaccharide was converted efficiently by both enzymes, thus representing the minimum substrate for human PH-20 and Hyal-1. Additionally, BTH was shown to catabolize the HA hexasaccharide at pH 4.0 mainly by hydrolysis, while at pH 6.0 transglycosylation prevailed. Human PH-20 was found to catalyze both hydrolysis and transglycosylation of the HA octasaccharide. On the contrary, human Hyal-1 converted the HA octasaccharide mainly by hydrolysis with transglycosylation products occurring only at high substrate concentrations (> or = 500 microM). The differences between the hyaluronidase subtypes and isoenzymes were much more prominent than expected. Obviously, the different hyaluronidase subtypes have evolved into very specialized enzymes with respect to their catalytic mechanism of action.     

A CONSIDERATION OF THE PERMEABILITY OF CARTILAGE TO INORGANIC SULFATE

On the basis of an examination of autoradiograms of knee-joints fixed so as to remove chondroitin sulfate or inorganic sulfate, or to minimize the loss of both, it is suggested that the cartilage is permeable to inorganic sulfate in vivo and in vitro. In vivo and in vitro, almost as rapidly as it enters the cartilage, inorganic sulfate is utilized by the cells in the synthesis of chondroitin sulfate. The net result is a continuing low concentration of inorganic sulfate in the cartilage.     

Hyaluronan Anchored to Activated CD44 on Central Nervous System Vascular Endothelial Cells Promotes Lymphocyte Extravasation in Experimental Autoimmune Encephalomyelitis

The extravasation of lymphocytes across central nervous system (CNS) vascular endothelium is a key step in inflammatory demyelinating diseases including multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). The glycosaminoglycan hyaluronan (HA) and its receptor, CD44, have been implicated in this process but their precise roles are unclear. We find that CD44^−/− mice have a delayed onset of EAE compared with wild type animals. Using an in vitro lymphocyte rolling assay, we find that fewer slow rolling (<1 μm/s) wild type (WT) activated lymphocytes interact with CD44^−/− brain vascular endothelial cells (ECs) than with WT ECs. We also find that CD44^−/− ECs fail to anchor HA to their surfaces, and that slow rolling lymphocyte interactions with WT ECs are inhibited when the ECs are treated with a pegylated form of the PH20 hyaluronidase (PEG-PH20). Subcutaneous injection of PEG-PH20 delays the onset of EAE symptoms by ∼1 day and transiently ameliorates symptoms for 2 days following disease onset. These improved symptoms correspond histologically to degradation of HA in the lumen of CNS blood vessels, decreased demyelination, and impaired CD4⁺ T-cell extravasation. Collectively these data suggest that HA tethered to CD44 on CNS ECs is critical for the extravasation of activated T cells into the CNS providing new insight into the mechanisms promoting inflammatory demyelinating disease.     

Hyaluronidase and esterase activities of the venom of the poisonous brown recluse spider

Venom of Loxosceles reclusa free from impurities was expressed from venom glands collected by microdissection. Polyacrylamide gel electrophoresis of the venom at pH 8.3 demonstrated 7 or 8 major plus 3 or 4 minor components. Upon electrophoresis at pH 4.9 two major components plus 3 or 4 minor components were noted. Monophoretic hyaluronidase prepared by Sephadex gel filtration and electrophoresis at pH 8.3 exhibited optimum activity from pH 5.0 to 6.6. Sodium dodecyl sulfate gel electrophoresis of purified hyaluronidase revealed two components with estimated molecular weights of 33,000 and 63,000. The purified hyaluronidase exhibited activity against chondroitin sulfate, types A, B, and C at approximately 20–30% of that upon hyaluronic acid. The enzyme was inhibited 10–20% by the heavy metal ions, Fe⁺³ and Cu⁺². Rabbit antivenom inhibited the spreading effect of whole venom in vivo and completely inhibited hyaluronidase in vitro.Incorporation of [¹⁴C]leucine into the spider venom led to the separation of hyaluronidase from the dermonecrotic activity of the venom.The venom demonstrated activity against carbobenzoxy-l-tyrosine-p-nitrophenyl ester and β-naphthylacetate which was inhibited approximately 65% by 2.5 × 10^?3m levels of EDTA and EGTA but not by 2.5 × 10^?4mo-phenanthroline. The esterase activity resisted concentrations of p-chloromercuribenzoate which totally inactivated papain. The venom appeared devoid of collagenase, dipeptidase, acetylcholinesterase, phosphodiesterase, ribonuclease A, and deoxyribonuclease.