Quantitative continuous assay for hyaluronan synthase

A rapid, continuous, and convenient three-enzyme coupled UV absorption assay was developed to quantitate the glucuronic acid and N-acetylglucosamine transferase activities of hyaluronan synthase from Pasteurella multocida (PmHAS). Activity was measured by coupling the UDP produced from the PmHAS-catalyzed transfer of UDP-GlcNAc and UDP-GlcUA to a hyaluronic acid tetrasaccharide primer with the oxidation of NADH. Using a fluorescently labeled primer, the products were characterized by gel electrophoresis. Our results show that a truncated soluble form of recombinant PmHAS (residues 1-703) can catalyze the glycosyl transfers in a time- and concentration-dependent manner. The assay can be used to determine kinetic parameters, inhibition constants, and mechanistic aspects of this enzyme. In addition, it can be used to quantify PmHAS during purification of the enzyme from culture media.     

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.     

Fusion protein of the hyaluronan binding domain from human TSG-6 with luciferase for assay of hyaluronan

The gene expression plasmid, pET-Lmluc, for the fusion protein of the hyaluronan binding domain from human TSG-6 [product of tumor necrosis factor (TNF)-stimulated gene-6] and luciferase from Renilla reniformis was constructed. The fused gene was expressed in Escherichia coli and the resulted insoluble Lm-luc fusion protein was purified and refolded to recover both the hyaluronan binding capability and the luciferase activity. Hyaluronan as low as 1 ng ml^–1 was detected by using the indirect enzymatic immunological assay with the refolded Lm-luc fusion protein.     

Novel methods for the preparation and characterization of hyaluronan oligosaccharides of defined length.

Hyaluronan is a ubiquitous glycosaminoglycan of high molecular weight that acts as a structural component of extracellular matrices and mediates cell adhesion. There have been numerous recent reports that fragments of hyaluronan have different properties compared to the intact molecule. Though many of these results may be genuine, it is possible that some activities are due to minor components in the preparations used. Therefore, it is important that well-characterized and highly purified oligosaccharides are used in cell biological and structural studies so that erroneous results are avoided. We present methods for the purification of hyaluronan oligomers of defined size using size exclusion and anion-exchange chromatography following digestion of hyaluronan with testicular hyaluronidase. These preparations were characterized by a combination of electrospray ionization mass spectrometry, matrix-assisted laser desorption/ionization mass spectrometry with time-of-flight analysis, and fluorophore-assisted carbohydrate electrophoresis. Hyaluronan oligomers ranging from tetrasaccharides to 34-mers were separated. The 4- to 16-mers were shown to be homogeneous with regard to length but did contain varying amounts of chondroitin sulfate. This contaminant could have been minimized if digestion had been performed with medical-grade hyaluronan rather than the relatively impure starting material used here. The 18- to 34-mer preparations were mixtures of oligosaccharides of different lengths (e.g., the latter contained 87% 34-mer, 10% 32-mer, and 3% 30-mer) but were free of detectable chondroitin sulfate. In addition to oligomers with even numbers of sugar rings, novel 5- and 7-mers with terminal glucuronic acid residues were identified.     

An enzyme-linked immunosorbent-inhibition assay for quantitation of hyaluronan (hyaluronic acid) in biological fluids

An enzyme-linked immunosorbent-inhibition assay for quantitation of hyaluronic acid (HA) is described. The principle of the method depends on the specific binding of HA to the hyaluronic acid-binding region (HABR) of proteoglycan (PG) monomers. The remaining uncomplexed PG monomers were determined by incubation with specific monoclonal antibodies to HABR followed by addition of polyclonal antibodies against PG monomers and enzyme-conjugated antibodies. The HA in samples was quantified by comparing their inhibitory capacity in the assay against a standard inhibition curve obtained using highly purified HA. This method was used to quantitate HA at nanogram levels in normal sera and synovial fluids. The level in normal human sera was found to be 28 +/- 17 ng/ml which compared favorably with values obtained using a commercial radioassay kit on the same samples. The assay was used to measure HA in synovial fluid from patients with rheumatoid and osteoarthritis and the results obtained were comparable with data published by others.     

Importance of albumin binding in the assay for carnitine palmitoyltransferase.

Alterations in the long-chain acyl-CoA binding to albumin in the carnitine palmitoyltransferase (CPT) assay appreciably affect the reaction at commonly used substrate concentrations. Since in the CPT assay the latter are typically well below saturation or Vmax. values, the measured enzyme activity depends on both the absolute quantity of albumin in the CPT assay and any biochemical modification of its binding. The present study verifies the striking dependence of the K0.5 for palmitoyl-CoA on albumin and the misleading 'activation' of the enzyme by compounds that also avidly bind to albumin. In assessing the intracellular physiological relevance of any modifier of CPT, the effects of protein binding in the assay assume particular importance. Indeed, any compound that alters CPT activity may do so, not directly, but as an assay artifact changing the free or unbound substrate concentrations.     

Importance of the pedicle length measurement in reduction mammaplasty

Various measurements are still considered to be important for making decisions regarding the approach in reduction mammaplasty, even though the vast majority of techniques used are based on an inferior pedicle. This study was performed to analyze the variability of the suprasternal notch-to-nipple and nipple-to-inframammary fold distance measurements when using the inferior pedicle technique. In 92 patients, 184 breasts having various volumes were measured preoperatively, and the variability of the measurements of the suprasternal notch-to-nipple and nipple-to-inframammary fold distance was analyzed. The values of the distance from the suprasternal notch to nipple were dispersed over a wide range with a trimodal distribution, with peaks at the intervals of 23 to 27 cm, 28 to 33 cm, and 34 to 45 cm. In contrast, the values of the nipple-to-inframammary fold distance showed a normal distribution pattern, with the peak at 14 cm. The distance from the suprasternal notch to nipple has a fair relationship to the inframammary fold-to-nipple distance, the latter being concentrated in a smaller range (r = 0.51). The former is often used to decide whether a free-nipple graft should be performed. However, the relative consistency of the latter measurement has led to the conclusion that the free-nipple graft technique need never be considered.     

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.     

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.     

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.     

Characterization of a motif for specific binding to hyaluronan in chicken SPACR

The chicken sialoprotein associated with cones and rods (SPACR) binds to hyaluronan (HA) in the interphotoreceptor matrix space, but the motif for HA binding is still unknown. This study was conducted to determine the critical site required for specific binding to HA. Western blotting study showed that SPACR binds biotinylated HA, and this interaction was specifically inhibited by unlabeled HA. A series of GST fusion proteins covering whole SPACR was prepared, and reactivity with HA was individually screened to narrow down the region for the binding. Further, putative HA-binding motif found near the carboxyl-terminus of SPACR was mutated by site-directed mutagenesis to identify the critical binding site. Finally, we showed that native SPACR derived from retina similarly binds to HA-affinity column under both reducing and non-reducing conditions. These results revealed that the specific putative HA-binding motif is located near the carboxyl-terminus of chicken SPACR, and suggested that a structural integrity such as folded structure is not largely involved in the HA binding.     

Step length measurement--theory and simulation for tethered bead constant-force single molecule assay

Linear molecular motors translocate along polymeric tracks using discrete steps. The step length is usually measured using constant-force single molecule experiments in which the polymer is tethered to a force-clamped microsphere. During the enzymatic cycle the motor shortens the tether contour length. Experimental conditions influence the achievable step length resolution, and ideally experiments should be conducted with high clamp-force using slow motors linked to small beads via stiff short tethers. We focus on the limitations that the polymer-track flexibility, the thermal motion of the microsphere, and the motor kinetics pose for step-length measurement in a typical optical tweezers experiment. An expression for the signal/noise ratio in a constant-force, worm-like chain tethered particle, single-molecule experiment is developed. The signal/noise ratio is related to the Fourier transform of the pairwise distance distribution, commonly used to determine step length from a time-series. Monte Carlo simulations verify the proposed theory for experimental parameter values typically encountered with molecular motors (polymerases and helicases) translocating along single- or double-stranded nucleic acids. The predictions are consistent with recent experimental results for double-stranded DNA tethers. Our results map favorable experimental conditions for observing single motor steps on various substrates but indicate that principal resolution limits are set by thermal fluctuations.     

Collagen/hyaluronan membrane as a scaffold for chondrocytes cultivation

Rabbit chondrocytes were cultivated in vitro using the collagen/hyaluronan membrane. The membrane did not show any adverse effects on chondrocyte viability during in vitro cultivation. The inoculated cells grew without any negative changes. According to the histochemical analyses: (i) hematoxylin and eosin; (ii) safranin O; and (iii) rabbit anti-human collagen type II staining, the rabbit chondrocytes maintained their morphology and phenotype during in vitro cultivation. The collagen/hyaluronan membrane became more stable and stiffer after long time cultivation. The proliferation of the chondrocytes stabilised the structure of the membrane. The collagen/hyaluronan membrane is suitable material for the chondrocyte growth and could provide functional tissue-engineered scaffold for cartilage repair.     

Importance of internal regions and the overall length of tropomyosin for actin binding and regulatory function

Tropomyosin (Tm) binds along actin filaments, one molecule spanning four to seven actin monomers, depending on the isoform. Periodic repeats in the sequence have been proposed to correspond to actin binding sites. To learn the functional importance of length and the internal periods we made a series of progressively shorter Tms, deleting from two up to six of the internal periods from rat striated alpha-TM (dAc2--3, dAc2--4, dAc3--5, dAc2--5, dAc2--6, dAc1.5--6.5). Recombinant Tms (unacetylated) were expressed in Escherichia coli. Tropomyosins that are four or more periods long (dAc2--3, dAc2--4, and dAc3--5) bound well to F-actin with troponin (Tn). dAc2--5 bound weakly (with EGTA) and binding of shorter mutants was undetectable in any condition. Myosin S1-induced binding of Tm to actin in the tight Tm-binding "open" state did not correlate with actin binding. dAc3--5 and dAc2--5 did not bind to actin even when the filament was saturated with S1. In contrast, dAc2--3 and dAc2--4 did, like wild-type-Tm, requiring about 3 mol of S1/mol of Tm for half-maximal binding. The results show the critical importance of period 5 (residues 166--207) for myosin S1-induced binding. The Tms that bound to actin (dAc2--3, dAc2--4, and dAc3--5) all fully inhibited the actomyosin ATPase (+Tn) in EGTA. In the presence of Ca(2+), relief of inhibition by these Tms was incomplete. We conclude (1) four or more actin periods are required for Tm to bind to actin with reasonable affinity and (2) that the structural requirements of Tm for the transition of the regulated filament from the blocked-to-closed/open (relief of inhibition by Ca(2+)) and the closed-to-open states (strong Tm binding to actin-S1) are different.     

Immobilisation of sulphated hyaluronan for improved biocompatibility

Hyaluronan (Hyal) was modified by the insertion of sulphate to hydroxyl groups. A series of heparin-like compounds with controllable properties was obtained. The physicochemical and biological behaviours of all these sulphated hyaluronan acids (HyalSx) and their complexes with heavy metal ions (Cu2+ and Zn2+) were investigated. HyalS, derivatives showed a good anticoagulant activity and low platelet aggregation which increased with increasing degree of sulphation. Moreover HyalSx and their Cu2+ complexes were demonstrated to favour the growth of human endothelial cells. However, the utilisation of HyalSx as a material is hindered by its high solubility in physiological solution. Our approach to improve its stability was directed to the synthesis of new HyalSx-based hydrogels and on the preparation of new biocompatible polymeric surfaces obtained through covalent photoimmobilisation of HyalSx. The reaction of primary ovine chondrocytes and B10D2 endothelial cells was studied on both matrices in terms of cell number, F-actin and CD44 receptor immunostaining. Analysis of cell movement showed that the cells respond to HyalSx showing good adhesion and spreading. These results suggest that HyalSx containing materials could be used as biomaterials to aid cartilage repair and vessel endothelisation.     

Domain motions of hyaluronan lyase underlying processive hyaluronan translocation

Hyaluronan lyase (Hyal) is a surface enzyme occurring in many bacterial organisms including members of Streptococcus species. Streptococcal Hyal primarily degrades hyaluronan‐substrate (HA) of the extracellular matrix. This degradation appears to facilitate the spread of this bacterium throughout host tissues. Unlike purely endolytic degradation of its other substrates, unsulfated chondroitin or some chondroitin sulfates, the degradation of HA by Hyal proceeds by processive exolytic cleavage of one disaccharide at a time following an initial endolytic cut. Molecular dynamics (MD) studies of Hyal from Streptococcus pneumoniae are presented that address the enzyme's molecular mechanism of action and the role of domain motions for processive functionality. The analysis of extensive sub‐microsecond MD simulations of this enzyme action on HA‐substrates of different lengths and the connection between the domain dynamics of Hyal and the translocation of the HA‐substrate reveals that opening/closing and twisting domain motions of the Hyal are intimately linked to processive HA degradation. Enforced simulations confirmed this finding as the domain motions in SpnHyal were found to be induced by enforced substrate translocation. These results establish the dynamic interplay between Hyal flexibility and substrate translocation and provide insight into the processive mechanism of Hyal. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Pep-1 as a novel probe for the in situ detection of hyaluronan.

Hyaluronan (HA) is expressed by most tissues, including skin. Localization of HA in the skin is assessed by histology with HA-binding protein (HABP) serving as the probe. Reports have suggested that HA expression in skin is altered in a number of diseases. However, interlaboratory variations in HABP staining profiles, even in normal skin, suggest a need to standardize methods and/or identify new probes. We report the staining patterns of a HA-binding peptide (termed "Pep-1") in human and mouse skin. After acetone fixation, Pep-1 stained HA in the intercellular spaces of the epidermis, whereas staining in the dermis was weak and diffuse in both human and mouse skin. HABP staining of the epidermis and dermis were comparable in human skin but failed to stain the vital epidermis of mouse skin. In human skin, Pep-1 stained the basal, spinous, and granular layers, whereas HABP failed to stain the basal layer. Precipitation of HA in situ resulted in dermal staining but weak staining in the epidermis for HABP and Pep-1. Our results may suggest that Pep-1 is sensitive to HA conformation. Furthermore, Pep-1 may represent a new probe to study HA expression in the skin.     

An engineered hyaluronan synthase: characterization for recombinant human hyaluronan synthase 2 Escherichia coli

The Class I hyaluronan synthase (HAS) is a unique glycosyltransferase synthesizing hyaluronan (HA), a polysaccharide composed of GlcUA and GlcNAc, by using one catalytic domain that elongates two different monosaccharides. As for the synthetic mechanism, there are two alternative manners for the sugar elongation process. Some bacterial HASs add new sugars to the non-reducing end of the acceptor to grow polymers. On the other hand, some vertebrate enzymes seem to transfer sugars to the reducing end. Expression of vertebrate HASs as active and soluble proteins will accelerate further precise insight into mechanisms of sugar elongation reactions by natural HASs. Since large scale production of HA polymers and oligomers would become powerful tools both for basic studies and new biotechnology to create functional carbohydrates in medicinal purposes, advent of an efficient method for the expression of HASs in Escherichia coli is strongly expected. Here we communicate the first success of the production of recombinant human HAS2 proteins composed of only the catalytic region in E. coli as the active form. It was demonstrated that an engineered HAS2 expressed in E. coli exhibited significant activity to synthesize a mixture of HAS oligomers from 8-mer (HA8) to 16-mer (HA16). Engineered HAS2 prepared herein elongated sugars from exogenous tetrasaccharide to form polymers with a direction to the non-reducing end. According to the present results, large scale production of engineered recombinant HASs is to be performed using E. coli that will provide practical and economic advantages in manufacturing enzymes for use in the synthesis of various oligomeric HA molecules and their industrial applications.     

Identification of a common hyaluronan binding motif in the hyaluronan binding proteins RHAMM, CD44 and link protein.

We have previously identified two hyaluronan (HA) binding domains in the HA receptor, RHAMM, that occur near the carboxyl-terminus of this protein. We show here that these two HA binding domains are the only HA binding regions in RHAMM, and that they contribute approximately equally to the HA binding ability of this receptor. Mutation of domain II using recombinant polypeptides of RHAMM demonstrates that K423 and R431, spaced seven amino acids apart, are critical for HA binding activity. Domain I contains two sets of two basic amino acids, each spaced seven residues apart, and mutation of these basic amino acids reduced their binding to HA--Sepharose. These results predict that two basic amino acids flanking a seven amino acid stretch [hereafter called B(X7)B] are minimally required for HA binding activity. To assess whether this motif predicts HA binding in the intact RHAMM protein, we mutated all basic amino acids in domains I and II that form part of these motifs using site-directed mutagenesis and prepared fusion protein from the mutated cDNA. The altered RHAMM protein did not bind HA, confirming that the basic amino acids and their spacing are critical for binding. A specific requirement for arginine or lysine residues was identified since mutation of K430, R431 and K432 to histidine residues abolished binding. Clustering of basic amino acids either within or at either end of the motif enhanced HA binding activity while the occurrence of acidic residues between the basic amino acids reduced binding. The B(X7)B motif, in which B is either R or K and X7 contains no acidic residues and at least one basic amino acid, was found in all HA binding proteins molecularly characterized to date. Recombinant techniques were used to generate chimeric proteins containing either the B(X7)B motifs present in CD44 or link protein, with the amino-terminus of RHAMM (amino acids 1-238) that does not bind HA. All chimeric proteins containing the motif bound HA in transblot analyses. Site-directed mutations of these motifs in CD44 sequences abolished HA binding. Collectively, these results predict that the motif of B(X7)B as a minimal binding requirement for HA in RHAMM, CD44 and link protein, and occurs in all HA binding proteins described to date.