Hyaluronates: relation between molecular conformations.

The discovery that both potassium and sodium salts of hyaluronic acid can exist in a double-strand helical conformation that will convert to the already known single-strand helical structures illustrates the remarkable conformational versatility of this biopolymer. X-ray diffraction was used to monitor variations in molecular conformation as a function of several independent, controllable variables, such as relative humidity, temperature, and applied tension. A scheme is presented for the interrelation of a range of hyaluronate conformations.

Hyaluronan: polysaccharide chaos to protein organisation.

Molecular dynamics simulations of hyaluronan have revealed the inherent flexibility of this glycosaminoglycan in solution. Crystal structures of hyaluronan-digesting enzymes have provided the first direct insights into the molecular basis of hyaluronan-protein interactions. Various studies on hyaluronan-binding proteins suggest there is considerable diversity in their mode of interaction with hyaluronan, which might result in many different bound conformations of the polysaccharide.

An enzyme-linked immunosorbent assay (ELISA) of denatured cartilage link protein.

Antiserum (MB007) was raised in rabbits to SDS-denatured cartilage link protein in order to develop an enzyme-linked immunosorbent assay (ELISA) to quantify link protein in cartilage extracts. The antibodies were characterized by using native and denatured link protein, either as the immobilised or the inhibiting antigen in the assay, and shown to bind more effectively to denatured link protein. At low concentrations, neither hyaluronate (0-30 micrograms/ml), proteoglycan (0-50 micrograms/ml) nor hyaluronate-binding region (0-3 micrograms/ml) competitively inhibited the link protein assay. However, at higher concentrations of proteoglycan (50 micrograms/ml-4 mg/ml) and hyaluronate-binding region (3-40 micrograms/ml) inhibition was observed. A more highly purified proteoglycan and a further purified hyaluronate-binding region preparation showed identical behaviour. The inhibition produced by proteoglycan and hyaluronate-binding region occurred at approximately equivalent molar concentrations (assuming Mr of 10(6) and 7 X 10(4), respectively). These results suggest that a significant proportion of these polyclonal antibodies recognize an epitope common to link protein and hyaluronate-binding region. However, the possibility that these effects are due to contamination with covalently bound link protein cannot be excluded. Trypsinated aggregates (0-10 micrograms/ml) produced no inhibition in the link protein ELISA, but as higher concentrations the inhibition was approximately 2000-fold lower than might have been expected from the link protein concentration present. Thus, the accessibility and/or binding of the antibodies to link protein was substantially decreased, illustrating masking of the link protein antigenic sites, as found by A. Ratcliffe and T.E. Hardingham (Biochem. J. 213 (1983) 371-378). These studies indicate that link protein in tissue extracts may be quantified in the concentration range 30-200 ng/ml and in the presence of hyaluronate, proteoglycan and hyaluronate-binding region, provided that both the immobilised and extracted link proteins are denatured.

Interaction between heparan sulphate chains. I. A gel chromatographic, light-scattering and structural study of aggregating and non-aggregating chains.

1. Heparan sulphate from bovine lung was fractionated with cetylpyridinium chloride. Solubilisation of complexes was accomplished by increasing concentrations of NaCl in a step-wise manner. Fractions I-IV, which were low-sulphated, contained more D-glucuronic acid than L-iduronic acid, fraction V contained equal proportions while fraction VI was L-iduronic acid-rich. 2. Gel chromatography of heparan sulphates II-IV in 0.5 M sodium acetate yielded extremely asymmetric profiles, while fractions V, VI and heparin did not. 3. Heparan sulphate IV was separated into aggregatable and non-aggregatable species by gel chromatography in 0.5 M sodium acetate. The particle/molecular weights of the two species were determined by light scattering. In 0.15 M NaCl or KCl the aggregatable chains yielded particle weights of 60 000-100 000 while the molecular weight was 20 000 (in 4.0 M guanidine HCl). Non-aggregatable chains afforded 'monomeric' values in 0.15 M NaCl or KCl. 4. Periodate oxidation of D-glucuronic acid residues in N-acetylated block regions followed by scission in alkali was used to fragment aggregating and non-aggregating heparan sulphate IV. The former chains yielded, on average, shorter oligosaccharides than did the latter. Reoxidation of the remaining D-glucuronic acid residues (adjacent to N-sulphated amino sugars) in the oligosaccharides followed by alkaline cleavage resulted in distinctly different fragmentation patterns in the two cases. The iduronate-containing oligosaccharides derived from aggregatable chains were markedly degraded into fragments ranging from glucosamine-L-iduronic acid-glucosamine-(C-3 fragment) to higher saccharides. Only higher saccharides were obtained from fragments of non-aggregatable chains. 5. It is concluded that self-associating heparan sulphates comprise both D-glucuronic acid- and L-iduronic acid-containing repeating units and that these units are arranged in an alternating or mixed fashion. These characteristics are analogous to those observed with self-associating dermatan sulphate species (Fransson, L.-A. and Cöster, L. (1979) Biochim. Biophys. Acta 582, 132-144).

Hyaluronic acid: molecular conformation and interactions in the tetragonal form of the potassium salt containing extended chains.

X-ray diffraction data were used to determine the detailed crystal structure of a tetragonal form of potassium hyaluronate containing relatively extended 4-fold helical chains (rise per disaccharide h = 0.95 nm). The polysaccharide chains are left-handed (4(3] helices. Two antiparallel chains pack in a tetragonal unit cell (a = b = 0.996 nm, c = 3.788 nm) with P4(3)2(1)2 space group symmetry. The chain conformations are stabilized intramolecularly by O4-O5 hydrogen bonds across the beta(1 leads to 3) linkage and by a pair of intermolecular hydrogen bonds per disaccharide between adjacent antiparallel chains. Fourier difference synthesis revealed one potassium ion and two water molecules per disaccharide. Six polyanion oxygen atoms from three neighboring chains together with one of these water molecules form the co-ordination polyhedra. Further stability is brought about through inter- and intrachain water bridges involving both water molecules. The probable reason for the stability of this extended allomorph is discussed in terms of the preferred co-ordination geometry of the potassium ion.

Deducing polymeric structure from aqueous molecular dynamics simulations of oligosaccharides: predictions from simulations of hyaluronan tetrasaccharides compared with hydrodynamic and X-ray fibre diffraction data.

Molecular dynamics simulations of the two hyaluronan tetrasaccharides in water predict that over a period of 500 ps, their central linkages populate a single primary minima. Over the same period the peripheral linkages explore this minima, but also a secondary minima. Structures constructed using the primary minima were found to be extended left-handed helices of axial rise per disaccharide (h) 0.8 to 1.0 nm and 2.8 to 4.5 disaccharides per turn (n), in good agreement with n=3 and n=4 helices found by X-ray fibre diffraction studies. We have used the predicted average conformation from molecular dynamics to calculate the translational diffusion coefficients of the oligosaccharide series up to decasaccharide, and compared these with experimental measurements obtained using the method of capillary dispersion. Our calculated values are found to be in good agreement with experiment beyond the size of a tetrasaccharide. A partial digest of hyaluronan in the molecular mass range 10 to 100 kDa was fractionated by gel chromatography. Molecular weights were determined by in-line laser light-scattering measurements, and the translational diffusion coefficients of selected fractions were determined by dynamic laser light-scattering. A similar experiment was performed on hyaluronan with a molecular mass greater than 1MDa. The data suggest a change from rod-like to stiff coil behaviour beyond a molecular weight of 10 kDa. We have also examined the conformations available using the secondary minima, found at the peripheral linkages. In contrast to the extended structures previously described we have found left and right-handed helices with high values of n (5-10) and low values of h. Although there is no experimental evidence for these structures, they are of interest as, over short stretches, they would introduce folds, loops, and turns into the hyaluronan molecule. Such shapes may play an important role in the hydrodynamics of hyaluronan and its interaction with lipids and proteins.

Hyaluronic acid: molecular conformations and interactions in the orthorhombic and tetragonal forms containing sinuous chains.

The conformation, packing and probable mode of cation binding have been determined for compact potassium hyaluronate chains (average rise per disaccharide h = 0.89 nm) organized in an orthorhombic unit cell (a = 1.173 nm, b = 0.925 nm, c = 3.542 nm). The space group symmetry is P2(1)2(1)2(1) and the unit cell contains two antiparallel polysaccharide chains that are disturbed 4(3) helices. Each chain is stabilized intramolecularly by four hydrogen bonds and between adjacent antiparallel chains there are two intermolecular hydrogen bonds per crystallographic tetrasaccharide repeat. Fourier difference synthesis revealed two potassium ions and two water molecules in each asymmetric unit. Both potassium ions show octahedral co-ordination geometries and link adjacent antiparallel polysaccharide chains. The water molecules provide further intermolecular association through water bridges. A comparison of this potassium hyaluronate structure with the orthorhombic and tetragonal sodium hyaluronate structures containing similar compact hyaluronate chains revealed that the apparently isomorphous orthorhombic sodium and potassium salts did not have the same packing arrangements. The relative orientations of the sinuous hyaluronate chains in the sodium and potassium salts are about 90 degrees apart. The locations of the cations and hence the coordination schemes of K+ and Na+ are therefore very different. These analyses have delineated, for the first time, how hyaluronate chains can respond differentially to two different monovalent cations.

The large chondroitin sulphate proteoglycan versican in mammalian vitreous.

Hyaluronan is a major component of the vitreous gel. Hyaluronan-binding macromolecules, including the aggregating proteoglycans, have been shown to perform an important role in maintaining the structural integrity of a number of tissues. However, there have not previously been any biochemical data to establish the presence of these types of macromolecules in vitreous. Bovine vitreous gel was solubilized (apart from a residual collagenous pellet) in 4 M guanidine hydrochloride and after dialysis into phosphate buffered saline analyzed by gel filtration chromatography with in-line measurement of refractive index and multi-angle laser light scattering. The concentration of hyaluronan in whole vitreous was found to be 0.57 mg/ml. The average molecular weight of the hyaluronan was found to be 170,000 (after isolation of the vitreous hyaluronan by isopycnic centrifugation in 0.5 M guanidine hydrochloride and papain digestion). Following Superose 12 gel filtration chromatography of the Streptomyces hyaluronan lyase digested vitreous extract, a pool of material was identified at or near the void volume of the column, and this material was shown to contain sulphated proteoglycans. Analysis of fractions following Superose 12 gel filtration chromatography by Western blotting showed that this pool of material contained the chondroitin sulphate proteoglycans versican and type IX collagen. Link protein was also identified in vitreous extracts by Western blotting. In whole vitreous, the concentration of versican was found to be 21.4+/-2.8 microg/ml and of link protein 0.62+/-0.07 microg/ml. Versican and link protein were thus present in approximately 1:1 molar ratios, but hyaluronan was present in a molar excess of 150 times. Therefore, aggregating proteoglycans are present in vitreous but, assuming that they bind to hyaluronan in-vivo, their overall density along the hyaluronan is much lower than that found in other tissues.

Monoclonal antibody recognizing a core epitope on mucin.

Monoclonal antibody TH1 (IgM) was prepared by immunizing mice with deglycosylated (TFMSA-treated) cystic fibrosis mucin. TH1 reacted strongly with TFMSA treated cystic fibrosis mucin but not with the fully glycosylated mucin, indicating reactivity with a core mucin epitope. TH1 showed no reactivity with ovine mucin (98% of glycans as sialyl-Tn) but reacted strongly with desialylated ovine mucin, indicating the epitope for this mab was the Tn-antigen (O-linked GalNAc). However, TH1 showed no reactivity with Tn-positive red blood cells, and the binding of TH1 was not inhibited by GalNAc at 2.5 mg/ml, illustrating the importance of the peptide sequence to which the GalNAc is attached. TH1 stained the majority of cancers of the colon, lung, stomach, ovary, breast, and cervix, and the cellular distribution of this antigen in normal tissue suggested reactivity with immature mucin. This antibody appears to be a useful reagent for the detection of immature mucin.

Identification of glycoproteins on nitrocellulose membranes and gels.

In this article we describe a procedure for the detection of glycoproteins on gels employing the periodic acid-Schiff's reagent. In addition, a number of staining protocols and direct binding ELISA, employing antibodies and lectins, are described for the identification and quantitation of glycoproteins after their immobilization by dot, slot, or Western blotting onto nitrocellulose membranes. We document, in detail, the conditions (i.e., the effect of solvent and detergents) for the immobilization of one specific family of O-linked glycoproteins, namely mucins. However, taking into account our suggestions, these procedures should be applicable to other types of glycoprotein.

Preliminary study pointing out a significant alteration in the biochemical composition of MUC2 in colorectal mucinous carcinoma.

OBJECTIVES: In this study, we characterized colonic MUC2 mucin from a mucinous carcinoma cell line and tried to find out carcinoma-associated alterations by comparing the results with those obtained from its benign phenotype previously. DESIGN AND METHODS: The molecular size distribution of the extracted molecules and their reactivity with two different MUC2 polypeptide antibodies indicated the presence of precursor and mature forms of the mucin in both cell lines. Isopycnic density gradient centrifugation gave good resolution of mature and precursor forms of MUC2 as assessed by agarose gel electrophoresis. Using this approach, we compared the different forms of MUC2 between benign and malign colonic cells. RESULTS: In the comparison, we detected some aberrant glycosylated MUC2 molecules in mucinous carcinoma cell line. Agarose gel electrophoretic analysis of the low-density fractions indicated that these molecules are more charged than precursors, however, they are smaller and/or less glycosylated than mature MUC2 molecules. CONCLUSION: The identification of unusual partially glycosylated forms of the major colonic mucin MUC2 is novel and unexpected. Implication of defective processes in the post translational modification/ processing of MUC2 opens a new field in the cancer mucin biology.

Immunochemical analysis of high molecular-weight human salivary mucins (MG1) using monoclonal antibodies.

Using four Mabs with different specificities for salivary mucins, an ELISA has been developed in which human whole saliva, glandular salivas, salivary protein fractions and purified, high molecular-weight, mucin fractions (MG1) isolated from human submandibular and sublingual glandular tissues have been immunochemically analysed. All four Mabs reacted with MG1s. Three of them reacted with the purified, low molecular-weight salivary mucins (MG2). None was reactive with parotid saliva. MG1 preparations isolated from submandibular and sublingual glandular tissues of one and the same individual displayed different patterns of reactivity with these Mabs, indicating that they differ immunochemically. Analysis of the MG1s in salivas derived from individual salivary glands showed differences in immunochemical composition. These results indicate that the MG1 fraction in human whole saliva consists of several immunochemically different species.

Macromolecular architecture and hydrodynamic properties of human cervical mucins.

Cervical mucus glycoproteins (mucins) were extracted by using slow stirring in 6M-guanidinium chloride supplemented with proteinase inhibitors. Subsequent purification was achieved by isopycnic density-gradient centrifugation in CsCl/guanidinium chloride. The whole mucins (Mr 10 X 10(6) - 15 X 10(6)) were degraded into subunits (Mr 2 X 10(6) - 3 X 10(6)) by reduction. Trypsin digestion of subunits afforded glycopeptides (T-domains) with Mr 0.4 X 10(6). The relationship between the intrinsic viscosity and Mr for the whole mucins and the fragments suggests that cervical mucins are linear flexible macromolecules. This view is supported by hydrodynamic data.

Molecular organization of the mucins and glycocalyx underlying mucus transport over mucosal surfaces of the airways.

Mucus, with its burden of inspired particulates and pathogens, is cleared from mucosal surfaces of the airways by cilia beating within the periciliary layer (PCL). The PCL is held to be "watery" and free of mucus by thixotropic-like forces arising from beating cilia. With radii of gyration ~250 nm, however, polymeric mucins should reptate readily into the PCL, so we assessed the glycocalyx for barrier functions. The PCL stained negative for MUC5AC and MUC5B, but it was positive for keratan sulfate (KS), a glycosaminoglycan commonly associated with glycoconjugates. Shotgun proteomics showed KS-rich fractions from mucus containing abundant tethered mucins, MUC1, MUC4, and MUC16, but no proteoglycans. Immuno-histology by light and electron microscopy localized MUC1 to microvilli, MUC4 and MUC20 to cilia, and MUC16 to goblet cells. Electron and atomic force microscopy revealed molecular lengths of 190-1,500 nm for tethered mucins, and a finely textured glycocalyx matrix filling interciliary spaces. Adenoviral particles were excluded from glycocalyx of the microvilli, whereas the smaller adenoassociated virus penetrated, but were trapped within. Hence, tethered mucins organized as a space-filling glycocalyx function as a selective barrier for the PCL, broadening their role in innate lung defense and offering new molecular targets for conventional and gene therapies.