An enzyme capture assay for analysis of active hyaluronan synthases

We describe a sensitive assay for detection of active hyaluronan synthases (HASs) capable of synthesizing hyaluronan (HA) without use of radioactive uridine 5'-diphosphate sugar precursors. The HAS capture assay is based on the binding of a biotinylated HA binding protein (bHABP) to HA chains that are associated with HAS and the subsequent capture of bHABP-HA-HAS complexes with streptavidin-agarose. Specific HAS proteins (e.g., HAS1, not HAS2 or HAS3) captured in this pull-down approach are readily immunodetected by Western blot analysis using appropriate antibodies. The assay was used to detect active HAS proteins in cell membranes, purified recombinant Streptococcus equisimilis HAS (SeHAS), and in vitro translated human HAS1 or SeHAS. The HAS capture assay was also used to assess the fraction of HAS molecules that were active, which cannot be done using standard assays for synthase activity. Assay sensitivity for detection of purified SeHAS is <1 pmol.     

Measurement of high-molecular-weight hyaluronan in solid tissue using agarose gel electrophoresis

The size of hyaluronan in solid tissue was measured using a combination of agarose gel electrophoresis and a radiometric assay. Radiolabeled hyaluronan binding proteins, used in the radiometric assay, were also used to detect hyaluronan after transfer to a nylon membrane following gel electrophoresis. Lane intensity on the autoradiograph was linearly related to the amount applied to the gel between 10 and 100ng. The intensity was independent of the hyaluronan molecular weight for standards with molecular weights equal to or greater than 790,000. The radiometric assay was used to measure hyaluronan irrespective of size, while gel electrophoresis was used to measure hyaluronan with molecular weights greater than 0.79x10(6) or 4x10(6). Deferoxamine was used to inhibit depolymerization during the digestion of tissue samples with protease. The molecular weight pattern was similar for skin, skeletal muscle, heart, lung, small intestine, and large intestine despite large differences in hyaluronan content. For all tissues, 58% of the hyaluronan had a molecular weight greater than 4million. All tissues showed an absence of hyaluronan with a molecular weight below 790,000. The procedure can be used to study changes in hyaluronan size in tissue during inflammation and other pathological states.     

Preparation and application of biologically active fluorescent hyaluronan oligosaccharides

We report the production of biologically active hyaluronan (HA) oligosaccharides labeled with the fluorophore 2-aminobenzoic acid (2AA). Oligosaccharides from 4 to 40 residues in length were purified to homogeneity by ion exchange chromatography using a logarithmic gradient. Molecular weight and purity characterization of HA oligosaccharides is facilitated by 2AA derivatization because it enhances signals in MALDI-TOF MS and improves FACE (fluorophore-assisted carbohydrate electrophoresis) analysis by avoiding the inverted parabolic migration characteristic of 2-aminoacridone (AMAC)-labeled sugars. The small size and shape of the fluorophore maintains the biological activity of the derivatized oligosaccharides, as demonstrated by their ability to compete for polymeric HA binding to the G1-domain of human recombinant versican (VG1). An electrophoretic mobility shift assay was used to study VG1 binding to labeled HA 8-, 10-, 20-, 30-, and 40-mers, and although no stable VG1 binding was observed to labeled 8-mers, the equilibrium dissociation constant (100 nM) for VG1 with HA(10) was estimated from densitometry analysis of the free oligosaccharide. Interactions involving HA 20-, 30-, and 40-mers (proposed to be multivalent) could also be studied using this protocol. Oligosaccharides labeled with 2AA therefore show excellent potential as probes in fluorescence-based assays that investigate protein-carbohydrate interactions.     

Binding of hyaluronan to lysozyme at various pHs and salt concentrations.

Binding of hyaluronan (HA) to lysozyme immobilized on Sepharose-6B was investigated as a function of pH and NaCl concentration. High affinity binding (Kd = 1.0-2.0 x 10(-8) M) was observed at pH 7.5 and at 10-50 mM NaCl; the number of moles of HA bound to lysozyme was twice as high at 30 mM NaCl as at 10 mM. No specific binding was observed at and above 100 mM NaCl. Binding was suppressed in the presence of chaotropic agents such as guanidinium chloride and urea. These results suggest that binding between HA and lysozyme can occur in the extracellular matrix where an electrolyte concentration as low as 50 mM could be expected due to ionic exclusion by the highly negative charge concentration arising from the polyanions present.     

Spacrcan binding to hyaluronan and other glycosaminoglycans. Molecular and biochemical studies

Photoreceptors project from the outer retinal surface into a specialized glycocalyx, the interphotoreceptor matrix (IPM), which contains hyaluronan (HA) and two novel proteoglycans, Spacr and Spacrcan. This matrix must be stable enough to function in the attachment of the retina to the outer eye wall yet porous enough to allow movement of metabolites between these tissues. How this matrix is organized is not known. HA is a potential candidate in IPM organization since biochemical studies show that these proteoglycans bind HA. RHAMM (receptor for HA-mediated motility)-type HA binding motifs (HABMs) are present in their deduced amino acid sequence and may be the sites of this HA interaction. To test this hypothesis, we subcloned three fragments of mouse Spacrcan that contain the putative HABMs. We found that each recombinant fragment binds HA. Binding decreased when residues in the HABMs were mutated. This provides direct evidence that the RHAMM-type HABMs in Spacrcan are involved in hyaluronan binding. Since chondroitin sulfate and heparan sulfate proteoglycans are important for retinal development and function, we also evaluated the binding of these recombinant proteins to heparin and chondroitin sulfates, the glycosaminoglycan side chain of these proteoglycans. We found that each recombinant protein bound to both heparin and chondroitin sulfates. Binding to chondroitin sulfates involved these HABMs, because mutagenesis reduced binding. Binding to heparin was probably not mediated through these HABMs since heparin binding persisted following their mutagenesis. These studies provide the first evidence defining the sites of protein-carbohydrate interaction of molecules present in the IPM.     

Analysis of the interaction between hyaluronan and hyaluronan-binding proteins by capillary affinity electrophoresis: significance of hyaluronan molecular size on binding reaction

We developed a method for the analysis of the interaction between hyaluronan (HA) oligosaccharides and hyaluronan-binding proteins (HABPs) using capillary affinity electrophoresis (CAE). The method is based on high-resolution separation of fluorescent-labeled HA molecules in the presence of hyaluronan-binding proteins at different concentrations by capillary electrophoresis (CE) with laser-induced fluorescent detection. Hyaluronan-binding protein from bovine nasal cartilage interacts strongly with HA decasaccharide or larger oligosaccharides. Effect of the molecular size of HA oligomers clearly showed that longer carbohydrate chains than decasaccharide were required for recognition by HA binding protein. Interestingly, the interaction did not cause retardation of HA oligomers as observed in many binding reactions such as the interaction between pharmaceuticals and serum albumin, but showed disappearance of the oligomer peak. Although we cannot explain the accurate mechanism on the interaction, disappearance is probably due to low equilibrium rate between free and conjugate states. The present technique will be useful to compare the relative binding affinity, and to understand the mechanism on the interaction between hyaluronan and hyaluronan-binding proteins.     

Effect of hyaluronan to inhibit caspase activation in porcine granulosa cells

We studied the ability of hyaluronan (HA) to inhibit apoptosis in porcine granulosa cells. The granulosa layer with cumulus-oocyte complex is cultured in media supplemented with follicle stimulating hormone (FSH) and 4-MU an inhibitor of hyaluronan synthases. The concentration of HA significantly increased after supplemented with FSH, but significantly decreased with 4-MU. CD44, receptor of HA, expressed after cultured with FSH, decreased in addition low concentration of 4-MU, whereas not detected in high concentration of 4-MU, indicating parallel relation between the amount of HA and CD44 expression. The 4-MU treatment also decreased the expression of procaspase-3, -8, -9 suggesting that inhibition of HA synthesis leads to activation of these caspases. Moreover, addition of anti-CD44 antibody decreased the expression of procaspases suggesting that perturbation of HA-CD44 binding leads activation of caspases. Hence, HA has ability to inhibit apoptosis and HA-CD44 binding is important on apoptosis inhibitory mechanism in porcine granulosa cells.     

Determination of hyaluronan molecular mass distribution in human breast milk

Hyaluronan (HA) in human milk mediates host responses to microbial infection via TLR4- and CD44-dependent signaling. Signaling by HA is generally size specific. Because pure HA with average molecular mass (M) of 35 kDa can elicit a protective response in intestinal epithelial cells, it has been proposed that human milk HA may have a bioactive low-M component. Here we report the size distribution of HA in human milk samples from 20 unique donors. A new method for HA analysis, employing ion exchange (IEX) chromatography to fractionate HA by size and specific quantification of each size fraction by competitive enzyme-linked sorbent assay (ELSA), was developed. When separated into four fractions, milk HA with M ? 20 kDa, M ∼ 20 to 60 kDa, and M ∼ 60 to 110 kDa comprised averages of 1.5, 1.4, and 2.0% of the total HA, respectively. The remaining 95% was HA with M ? 110 kDa. Electrophoretic analysis of the higher M HA from 13 samples showed nearly identical M distributions, with an average M of approximately 440 kDa. This higher M HA component in human milk is proposed to bind to CD44 and to enhance human beta defensin 2 (HBD2) induction by the low-M HA components.     

Pigment epithelium-derived factor binds to hyaluronan. Mapping of a hyaluronan binding site

Pigment epithelium-derived factor (PEDF) is a multifunctional serpin with antitumorigenic, antimetastatic, and differentiating activities. PEDF is found within tissues rich in the glycosaminoglycan hyaluronan (HA), and its amino acid sequence contains putative HA-binding motifs. We show that PEDF coprecipitation with glycosaminoglycans in media conditioned by human retinoblastoma Y-79 cells decreased after pretreatments with hyaluronidase, implying an association between HA and PEDF. Direct binding of human recombinant PEDF to highly purified HA was demonstrated by coprecipitation in the presence of cetylpyridinium chloride. Binding of PEDF to HA was concentration-dependent and saturable. The PEDF-HA interactions were sensitive to increasing NaCl concentrations, indicating an ionic nature of these interactions and having affinity higher than PEDF-heparin. Competition assays showed that PEDF can bind heparin and HA simultaneously. PEDF chemically modified with fluorescein retained the capacity for interacting with HA but lacked heparin affinity, suggesting one or more distinct HA-binding regions on PEDF. The HA-binding region was examined by site-directed mutagenesis. Single-point and cumulative alterations at basic residues within the putative HA-binding motif K189A/K191A/R194A/K197A drastically reduced the HA-binding activity without affecting heparin- or collagen I binding of PEDF. Cumulative alterations at sites critical for heparin binding (K146A/K147A/R149A) decreased HA affinity but not collagen I binding. Thus these clusters of basic residues (BXBXXBXXB and BX3AB2XB motifs) in PEDF are functional regions for binding HA. In the spatial PEDF structure they are located in distinct areas away from the collagen-binding site. The HA-binding activity of PEDF may contribute to deposition in the extracellular matrix and to its reported antitumor/antimetastatic effects.     

An improved technique for hyaluronan histochemistry using microwave irradiation

Summary A hyaluronan binding protein (HABP), extracted from cartilage, was biotin-labelled and used for histochemical localization of hyaluronan (HA) in tissue sections. Various tissues were fixed for a mixture of formaldehyde and glutaraldehyde during microwave irradiation. The microwave oven when set at 700 W and 45°C yielded an intense and specific staining of HA. Under these conditions the relative proportion of the two aldehydes did not influence the staining intensity. Aldehyde fixation during microwave irradiation for HA histochemistry, (1) saves time, (2) eliminates the use of cetylpyridinium chloride (CPC), and (3) improves the reproducibility.     

Quantification and characterization of enzymatically produced hyaluronan with fluorophore-assisted carbohydrate electrophoresis

Hyaluronan (HA) is a polysaccharide with high-potential medical applications, depending on the chain length and the chain length distribution. Special interest goes to homogeneous HA oligosaccharides, which can be enzymatically produced using Pasteurella multocida hyaluronan synthase (PmHAS). We have developed a sensitive, simple, and fast method, based on fluorophore-assisted carbohydrate electrophoresis (FACE), for characterization and quantification of polymerization products. A chromatographic pure fluorescent template was synthesized from HA tetrasaccharide (HA4) and 2-aminobenzoic acid. HA4-fluor and HA4 were used as template for PmHAS-mediated polymerization of nucleotide sugars. All products, fluorescent and nonfluorescent, were analyzed with gel electrophoresis and quantified using lane densitometry. Comparison of HA4- and HA4-fluor-derived polymers showed that the fluorophore did not negatively influence the PmHAS-mediated polymerization. Only even-numbered oligosaccharide products were observed using HA4-fluor or HA4 as template. The fluorophore intensity was linearly related to its concentration, and the limit of detection was determined to be 7.4 pmol per product band. With this assay, we can now differentiate oligosaccharides of size range DP2 (degree of polymerization 2) to approximately DP400, monitor the progress of polymerization reactions, and measure subtle differences in polymerization rate. Quantifying polymerization products enables us to study the influence of experimental conditions on HA synthesis.     

Mammalian hyaluronan synthases

Three mammalian hyaluronan (HA) synthase genes, HAS1, HAS2, and HAS3, have been cloned and expressed, allowing the mechanisms for regulation of HA biosynthesis and function to be studied. The hyaluronan synthase (HAS) isoforms differ in kinetic characteristics and product size. The expression of each HAS isoform is controlled in a different fashion when mammalian cells are stimulated by various cytokines and the expression patterns are both spatially and temporally regulated during embryonic development. The existence of three different HAS isoforms with different characteristics implies that the broad range of biological and physiological roles performed by HA are regulated by controlling the activities and expression of the HAS isoforms. This review focuses on recent findings on the regulatory mechanisms for controlling HA biosynthesis and provides new insights into the enzymic basis for the functional regulation of HA.     

The molecular basis of the solution properties of hyaluronan investigated by confocal fluorescence recovery after photobleaching.

Hyaluronan (HA) is a highly hydrated polyanion, which is a network-forming and space-filling component in the extracellular matrix of animal tissues. Confocal fluorescence recovery after photobleaching (confocal-FRAP) was used to investigate intramolecular hydrogen bonding and electrostatic interactions in hyaluronan solutions. Self and tracer lateral diffusion coefficients within hyaluronan solutions were measured over a wide range of concentrations (c), with varying electrolyte and at neutral and alkaline pH. The free diffusion coefficient of fluoresceinamine-labeled HA of 500 kDa in PBS was 7.9 x 10(-8) cm(2) s(-1) and of 830 kDa HA was 5.6 x 10(-8) cm(2) s(-1). Reductions in self- and tracer-diffusion with c followed a stretched exponential model. Electrolyte-induced polyanion coil contraction and destiffening resulted in a 2.8-fold increase in self-diffusion between 0 and 100 mM NaCl. Disruption of hydrogen bonds by strong alkali (0.5 M NaOH) resulted in further larger increases in self- and tracer-diffusion coefficients, consistent with a more dynamic and permeable network. Concentrated hyaluronan solution properties were attributed to hydrodynamic and entanglement interactions between domains. There was no evidence of chain-chain associations. At physiological electrolyte concentration and pH, the greatest contribution to the intrinsic stiffness of hyaluronan appeared to be due to hydrogen bonds between adjacent saccharides.     

Practical determination of hyaluronan by a new noncompetitive fluorescence-based assay on serum of normal and cirrhotic patients

A practical fluorescence-based assay method for determination of hyaluronan (HA) was developed. Plates were coated with hyaluronan-binding proteins (HABP) obtained from bovine cartilage and successively incubated with samples containing standard solutions of hyaluronan or serum from normal and cyrrhotic patients, biotin-conjugated HABP, and europium-labeled streptavidin. After release of europium from streptavidin with enhancement solution the final fluorescence is measured in a fluorometer. The method is specific for HA even in the presence of substantial amounts of other glycosaminoglycans (chondroitin, dermatan sulfate, and heparan sulfate, and heparin) or proteins. It is possible to quantify HA between 0.2 and 500.0 microg/L. Analyses of HA concentration in 545 normal subjects and 40 cirrhotic patients gave average values of 14.5 and 542.0 microg/L, respectively. It was also shown that older subjects (> or =51 years old) have more HA (28.0 microg/L) than younger subjects (12.0 to 14.0 microg/L). This new sandwich technique has shown high precision and sensitivity similar to those of a recently described fluorescence-based assay method, being able to measure HA in amounts as small as 0.2 microg/L. In addition, this noncompetitive assay avoids preincubation, consumes less time (<5 h) than the previous competitive fluorescence-based assay (>72 h), and avoids the use of radioactive materials.     

Serum hyaluronan concentration determined by radiometric assay in patients with pretibial myxedema and Graves' ophthalmopathy

The serum concentration of hyaluronan (HA) was measured by radiometric assay in patients with pretibial myxedema (PTM) and Graves' ophthalmopathy (GO). The mean HA concentration in the patients (n = 8) was 21.2 +/- 15.3 (mean +/- SD) microgram/l, while that of Graves' disease without skin or eye involvement (n = 7) was 23.5 +/- 11.0 (mean +/- SD) microgram/l and that of the control (n = 8) was 25.5 +/- 16.4 (mean +/- SD) microgram/l. We conclude that local accumulation of glycosaminoglycan in PTM or GO is not associated with an increase in the serum HA concentration.     

Size determination of hyaluronan using a gas-phase electrophoretic mobility molecular analysis

Hyaluronan (HA) is a linear non-sulfated polysaccharide mainly found in the extracellular matrix. The size of HA can vary from a few disaccharides up to at least 25,000 units, reaching molecular weights of 10?10(3)?kDa. HA has many biological functions, and both its size and tissue concentration play an important role in many physiological and pathological processes. It is relatively easy to determine the HA concentration using enzyme-linked binding protein assays, but the molecular weight of HA has so far been shown to be a more challenging task to measure. Here, we present a method for size determination of HA using gas-phase electrophoretic mobility molecular analysis (GEMMA), which utilizes the electrophoretic mobility of molecules in air to estimate the molecular weight of the analyte. We show that this method gives reliable molecular weight estimations of HA in the range of 30-2400?kDa, which covers almost its whole biological range. The average measuring time for one GEMMA spectrum is between 5 and 10?min using only 6?pg of HA. In addition, the peak area in a GEMMA spectrum can be used to estimate the HA concentration in the sample. The high sensitivity and small sample volumes make GEMMA an excellent tool for both size determinations and estimation of concentration of samples with low HA concentration, as is the case for HA extracted from small tissue samples.     

Defined megadalton hyaluronan polymer standards

The utility of polymer standards for the calibration of average molecular mass estimates often is limited by the polydispersity--the breadth of the size distribution--of the standard. Here monodisperse synthetic hyaluronan (or hyaluronic acid [HA]) complexes in the approximately 1- to 8-megadalton (MDa) range were prepared in two steps. First, synchronized stoichiometrically controlled in vitro reactions yielded linear narrow size distribution biotinylated HA chains. Second, streptavidin protein was added at substoichiometric levels to prepare a series of complexes with one, two, three, or four HA chains per streptavidin molecule. The dendritic-like molecules approximate the mobility of natural linear HA chains on agarose gels, making the complexes useful as defined size standards for high-molecular weight HA preparations.     

Hyaluronan and a cell-associated hyaluronan binding protein regulate the locomotion of ras-transformed cells

Hyaluronan (HA) and one of its cell binding sites, fibroblast hyaluronan binding protein (HABP), is shown to contribute to the regulation of 10T1/2 cell locomotion that contain an EJ-ras-metallothionein (MT-1) hybrid gene. Promotion of the ras-hybrid gene with zinc sulfate acutely stimulates, by 6-10-fold, cell locomotion. After 10 h, locomotion drops to two- to threefold above that of uninduced cells. Several observations indicate increased locomotion is partly regulated by HA. These include the ability of a peptide that specifically binds HA (HABR) to reduce locomotion, the ability of HA (0.001-0.1 micrograms/ml), added at 10-30 h after induction to stimulate locomotion back to the original, acute rate, and the ability of an mAb specific to a 56-kD fibroblast HABP to block locomotion. Further, both HA and HABP products are regulated by induction of the ras gene. The effect of exogenous HA is blocked by HABR, is dose-dependent and specific in that chondroitin sulfate or heparan have no significant effect. Stimulatory activity is retained by purified HA and lost upon digestion with Streptomyces hyaluronidase indicating that the activity of HA resides in its glycosaminoglycan chain. Uninduced cells are not affected by HA, HABR, or mAb and production of HA or HABP is not altered during the experimental period. These results suggest that ras-transformation activates an HA/HABP locomotory mechanism that forms part of an autocrine motility mechanism. Reliance of induced cells on HA/HABP for locomotion is transient and specific to the induced state.     

Mouse LYVE-1 is an endocytic receptor for hyaluronan in lymphatic endothelium

The glycosaminoglycan hyaluronan is a key substrate for cell migration in tissues during inflammation, wound healing, and neoplasia. Unlike other matrix components, hyaluronan (HA) is turned over rapidly, yet most degradation occurs not locally but within distant lymph nodes, through mechanisms that are not yet understood. While it is not clear which receptors are involved in binding and uptake of hyaluronan within the lymphatics, one likely candidate is the lymphatic endothelial hyaluronan receptor LYVE-1 recently described in our laboratory (Banerji, S., Ni, J., Wang, S., Clasper, S., Su, J., Tammi, R., Jones, M., and Jackson, D.G. (1999) J. Cell Biol. 144, 789-801). Here we present evidence that LYVE-1 is involved in the uptake of hyaluronan by lymphatic endothelial cells using a new murine LYVE-1 orthologue identified from the EST data base. We show that mouse LYVE-1 both binds and internalizes hyaluronan in transfected 293T fibroblasts in vitro and demonstrate using immunoelectron microscopy that it is distributed equally among the luminal and abluminal surfaces of lymphatic vessels in vivo. In addition, we show by means of specific antisera that expression of mouse LYVE-1 remains restricted to the lymphatics in homozygous knockout mice lacking a functional gene for CD44, the closest homologue of LYVE-1 and the only other Link superfamily HA receptor known to date. Together these results suggest a role for LYVE-1 in the transport of HA from tissue to lymph and imply that further novel hyaluronan receptors must exist that can compensate for the loss of CD44 function.     

Immunoenzymoassay of the hyaluronic acid-hyaluronectin interaction: application to the detection of hyaluronic acid in serum of normal subjects and cancer patients

The binding of a hyaluronic acid-binding glycoprotein, hyaluronectin (HN), isolated from human brain, to hyaluronic acid (HA) was investigated with the enzyme-linked immunosorbent assay technique using plastic microtest plates coated with a 50 mg/liter solution of HA in 0.1 M bicarbonate. Optimum conditions for HN binding to HA were in 0.2 M NaCl buffered with 0.1 M sodium phosphate at pH 7. An assay for HA in solution was set up exploiting the fact that HN binding could be inhibited by soluble HA. HA was preincubated for 1 h in a test tube with a 30-ng/ml HN solution (v/v) in the buffer containing 0.1% bovine serum albumin. Incubation on HA-coated microtest plate lasted 4 h and maximum sensitivity was achieved when incubation was carried out at 4 degrees C. HN bound to the plate was revealed by means of alkaline phosphatase-conjugated anti-HN antibodies. The test was used to measure HA inhibitory activity after depolymerization by ferrous ions. No difference was found between inhibitory activity or smaller fragments and that of high-molecular-weight HA. The assay was applied to determination of HA in sera. Specificity was demonstrated by Streptomyces hyaluronidase digestion of reactive material in sera. Other glycosaminoglycans did not interfere with the assay. Recovery of HA was good and intra- and interassay variation coefficients were 6 +/- 2.2 and 12%. In 103 blood donor sera, HA was found at 22.4 +/- 16.7 micrograms/liter. HA was elevated in most of the cancer patient sera tested.