Synthesis of hyaluronate in differentiated teratocarcinoma cells. Characterization of the synthase.

Differentiation of teratocarcinoma cells led to induction of hyaluronate synthesis. The synthase was recovered in the membrane fraction of cell lysates. Hyaluronate was synthesized at the membranes and was then released as a soluble product. The synthase could be stimulated by a variety of phosphate esters which prevented the degradation of the substrates UDP-GlcNAc and UDP-GlcA and the release of the growing hyaluronic acid chain from the membrane. Hyaluronidases or oligosaccharides derived from hyaluronate did not affect the synthesis. The chains grew at a rate of 60 repeating units/min. Continuous new chain initiation occurred during prolonged synthesis. Digestion of pulse-chase-labelled hyaluronate with beta-N-acetylglucosaminidase and beta-glucuronidase showed that the chains grew at the reducing end.     

Binding of bivalent cations by hyaluronate in aqueous solution

The interaction between sodium hyaluronate and bivalent cations was investigated by conductometry, viscosimetry, circular dichroism and nuclear magnetic resonance spectroscopy. It is shown that the hyaluronate chains (Mn approximately 4.0 x 10(5)-1.7 x 10(6)g/mol) bind various bivalent cations (Ca2+, Mg2+, Mn2+, Fe2+, Cu2+, Zn2+, Cd2+ and Pb2+) at pH 6 in aqueous solutions. Hyaluronate deriving from Streptococcus equi was studied in comparison with dextran from Leuconostoc mesenteroides which was shown to develop no specific interactions with the bivalent cations. The molar relation between the bivalent cations and the disaccharide units of the resulting complex was determined with the result that one bivalent cation is bound by approximately five disaccharide units. Heavy metal ions (Cd2+, Pb2+) seem to bind stronger to the hyaluronate chain than their lighter counterparts (Ca2+, Mg2+). Circular dichroism spectra of the hyaluronate exhibit a cation-induced change in the n-pi* transition, indicating that the acetamide group of the aminoglucane unit is involved during the complexation. NMR spectra of hyaluronic acid in presence of paramagnetic manganese cations show strong interactions between the acetamide as well as the carboxylate groups and the cations. Based on these data, a structure of the binding complex is proposed which involves two disaccharide units.     

Polyacrylamide-gel electrophoresis and Alcian Blue staining of sulphated glycosaminoglycan oligosaccharides.

Oligosaccharide fragments of glycosaminoglycans may be separated for rapid analysis by electrophoresis through a 10% polyacrylamide matrix. An extensive ladder-like set of bands is observed for partial testicular hyaluronidase digests of chondroitin 4- or 6-sulphate, and for dermatan sulphate. Co-electrophoresis of purified oligosaccharides has established that the major bands of these patterns represent fragments differing in chain length by one disaccharide unit, with the smallest fragments having the greatest mobility. Additional minor bands, representing heterogeneity in the repeating unit structure, are also observed. There are slight differences in the mobilities of oligosaccharides derived from the three major types of sulphated glycosaminoglycans. Alcian Blue is employed for visualization of the digest fragments. Sample loads of 5-10 micrograms per band appear optimum. The smallest oligosaccharide which may be stained by this method is the hexasaccharide. After consideration of this effect, a good correlation is found to exist between densitometric scans of the gel-electrophoretic patterns and gel-filtration chromatographic profiles based on uronic acid concentration.     

Characterization of a high-Mr plasma-membrane-bound protein and assessment of its role as a constituent of hyaluronate synthase complex.

A high-Mr phosphoprotein (Mr 442,000) was purified from Nonidet-P-40-solubilized plasma membranes of cultured human skin fibroblasts. The protein comprised one 200,000-Mr subunit consisting of 116,000- and 84,000-Mr polypeptides and two identical 121,000-Mr subunits each consisting of 66,000- and 55,000-Mr polypeptides. The 200,000-Mr subunit and its polypeptides contained phosphotyrosine residues and were also [32P]phosphorylated at these residues from [gamma-32P]ATP in vitro by an intrinsic tyrosine kinase activity of the protein molecule in response to the presence of hyaluronate precursors, UDP-glucuronic acid and UDP-N-acetylglucosamine. The 121,000-Mr subunits and their polypeptides contained phosphoserine residues that could not be [32P]phosphorylated during autophosphorylation of the protein in vitro. The protein molecules separated from exponential- and stationary-growth-phase cells were identical in their quaternary structure, but appeared to exist in different proportions with respect to the state of phosphorylation of their 121,000-Mr subunits during different growth phases of the cell. Phosphorylation of polypeptides appeared to predispose in favour of their UDP-glucuronic acid- and UDP-N-acetylglucosamine-binding activities. The phosphorylated 116,000- and 84,000-Mr polypeptides of 200,000-Mr subunits possessed a single binding site for UDP-glucuronic acid and UDP-N-acetylglucosamine respectively. The phosphorylated 200,000-Mr subunit could also cleave the UDP moiety from UDP-glucuronic acid and UDP-N-acetylglucosamine precursors. The phosphorylated 121,000-Mr subunit possessed two binding sites with equal affinity towards UDP-glucuronic acid and UDP-N-acetylglucosamine but did not possess UDP-moiety-cleavage activity. The phosphorylation of 200,000-Mr subunit by an intrinsic kinase activity of the protein molecule appeared to elicit its oligosaccharide-synthesizing activity, whereas phosphorylation of 121,000-Mr subunits, presumably carried out in vivo, abolished this activity of the protein molecule. The oligosaccharides synthesized by the protein were about Mr 5000 and about 12 disaccharide units in length. Neither nucleotide sugars nor glycosyl residues nor newly synthesized oligosaccharides were bound covalently to the protein molecule. The UDP moiety of nucleotide sugar precursors did not constitute a link between protein molecule and oligosaccharide during its synthesis. Although isolated 442,000-Mr protein did not synthesize high-Mr hyaluronate in vitro, this protein molecule can be considered as a constituent of membrane-bound hyaluronate synthase complex because of its observed properties.     

Vacuum-ultraviolet circular dichroism of sodium hyaluronate oligosaccharides and polymer segments

The CD spectrum of an enzymatically derived sodium hyaluronate (NaHA) segment preparation with chain length 18 ± 3 disaccharide units [NaHAseg, ( NaGlcUA GlcNAc)_15–20°. NaGlcUA, sodium D -glucuronate; GlcNAc, 2-acetamido-2-deoxy-D -glucose] in H₂O was recorded to 180 nm using a computer-controlled vacuum-uv CD instrument. Near 190 nm the spectrum is of low intensity, similar to the sum of the free monosaccharide contributios, attributed to the π–π* transitions of the acetamido and carboxylate substituents. In contrast, much smaller oligosaccharides, also derived from high-molecular-weight NaHA by enzymatic digestions, show CD spectra in H₂O with prominent bands centered near 190 nm. The oligosaccharide spectra can be matched as linear combinations of interior sugar residue (? NaHAseg) and end sugar residue CD contributions. End residues from oligosaccharides of the type (NaGlcUA-GlcNAc)_n show a negative CD band near 190 nm. End residues from oligosaccharides of the reverse sequence (GlcNAc-NaGlcUA)_n show a positive CD band near 190 nm. Averaging of the two end-residue spectral contributions yields an approximate match for the spectrum of NAHAseg below 200 nm. It is proposed that the low intensity CD of NaHA in the π–π* region is the result of large-magnitude, oppositely signed contributions, which can be visulized by studying oligosaccharides.     

Binding of hyaluronate and chondroitin sulphate to liver endothelial cells.

Hyaluronate is taken up and metabolized in liver endothelial cells by means of a receptor. To characterize the interaction with the receptor, two preparations of 3H-labelled hyaluronate, of Mr 4 X 10(5) and 6.4 X 10(6), and a series of hyaluronate oligosaccharides were bound to cultured liver endothelial cells at 7 degrees C. The dissociation constant varied between 4.6 X 10(-6) M for an octasaccharide and 9 X 10(-12) M for the largest polymer. The Mr-dependence for the series of oligosaccharides was explained by the increased probability of binding due to the repetitive sequence along the chain. The high affinity of high-Mr hyaluronate for the receptor could also be mainly ascribed to this effect, which rules out any major contribution of co-operative multiple-site attachment to the cell surface. Each liver endothelial cell can bind 10(5) oligosaccharides, about 10(4) molecules with Mr 4 X 10(5) and about 10(3) molecules with Mr 6.4 X 10(6). This is explained by mutual exclusion of large molecules from the cell surface. Chondroitin sulphate is also bound to liver endothelial cells. Inhibition studies showed that it binds to the same receptor as hyaluronate and with an affinity that is about 3-fold higher than that of hyaluronate of the same degree of polymerization.     

Interactions of cartilage proteoglycans with hyaluronate. Inhibition of the interaction by modified oligomers of hyaluronate.

Oligomers of hyaluronic acid were prepared by digestion of hyaluronic acid from rooster combs with testicular hyaluronidase (hyaluronate 4-glycanohydrolase, EC 3.2.1.35), leech head hyaluronidase (hyaluronate 3-glycanohydrolase, EC 3.2.1.36), and with fungal hyaluronidase (hyaluronate lyase from Streptomyces hyalurolyticus). The oligomers were fractionated by gel permeation, using Sephadex G-50. Oligomers isolated after incubation of the hyaluronic acid with the testicular hyaluronidase were further modified. To prepare oligomers with N-acetylglucosamine at both ends, terminal nonreducing glucuronic acid residues were removed with beta-glucuronidase. Reducing terminal N-acetylglucosamine residues were removed by reaction under mildly alkaline conditions. The reducing terminal N-acetylglucosamine residues were also reduced with sodium borohydride to form N-acetylglucosaminitol. The potentials of the various oligosaccharides to bind to the proteoglycan from bovine nasal septum cartilage were estimated by determining their effectiveness as inhibitors of the proteoglycan-hyaluronate interaction. The present study shows that, to bind maximally to the proteoglycan, the hyaluronate oligosaccharide must be at least 10 sugar residues in length and be terminated at the nonreducing and reducing ends with a glucuronate residue and an N-acetylglucosamine residue, respectively. Sugar residues extended beyond this basic decasaccharide, do not interact with the hyaluronate binding site on the proteoglycan.     

Competitive inhibition evidence for specific intermolecular interactions in hyaluronate solutions

Intermolecular associations in hyaluronate systems have been investigated by a competitive inhibition approach, monitored by low deformation oscillatory measurements of dynamic viscosity and rigidity. Solutions of sodium hyaluronate isolated by a mild procedure from rooster combs showed, under physiological conditions of pH and ionic strength, coupling behaviour typical of a transient polymer network. On addition of an equal concentration of hyaluronate segments (~60 disaccharide units), all evidence of coupling is lost and the solution rheology approximates closely to that typical of isolated chains. Such behaviour is clearly incompatible with entanglement coupling, such as occurs between synthetic polymers in solution, but parallels the behaviour of gelling polysaccharides, where co-operative interchain association is known to occur. Similar inhibition is observed in hyaluronate viscoelastic “putties” and “gels”, where further intermolecular association is promoted by suppression of interchain electrostatic repulsion and reduction of water activity. The effects are particularly pronounced for hyaluronate of lower molecular weight, where crosslinking in putties and gels may approach the minimum requirements for a cohesive network. No inhibition is observed with very short chain segments (~4 disaccharide residues) nor with longer segments (~400 disaccharides). On the basis of this evidence we suggest that hyaluronate chains interact by formation of specific co-operative junctions analogous to those characterised for plant structural polysaccharides, but having substantially shorter lifetimes. The magnitude of rheological changes on suppression of these fleeting associations by competitive inhibition suggests that they are likely to dominate the physical properties of hyaluronate in vivo.     

Kinetic properties of Streptococcus pneumoniae hyaluronate lyase

Streptococcus pneumoniae hyaluronate lyase is a surface antigen of this bacterial pathogen, which causes significant mortality and morbidity in human populations worldwide. The primary function of this enzyme is the degradation of hyaluronan, a major component of the extracellular matrix of the tissues of practically all vertebrates. The enzyme uses a processive mode of action to degrade hyaluronan to a final product, an unsaturated disaccharide hyaluronan unit. This catalysis proceeds via a five-step proton acceptance and donation mechanism that includes substrate binding, catalysis, release of the disaccharide product, translocation of the remaining hyaluronan substrate, and proton exchange with microenvironment. Based on the analysis of the three-dimensional structure of the native enzyme and its complexes with hexasaccharide substrate and disaccharide product, several residues have been chosen for mutation studies. These mutated residues included the catalytic residues Asn349, His399, Tyr408, and residues responsible for substrate binding and translocation, Arg243 and Asn580. The comparison of the kinetic properties of the wild-type with the mutant enzymes allowed for the characterization of every mutant and the correlation of the kinetic properties of the enzyme with its structure. The comparison of the wild-type hyaluronate lyase with other polysaccharide-degrading enzymes, the hydrolases endonuclease and glucoamylase, shows striking similarity of K(m)s for all of these different enzymes.     

Self-association of hyaluronate segments in aqueous NaCl solution

The potential for self-association by hyaluronate (HA) chains in 0.15 M NaCl was investigated, using low molecular weight HA segments as a model system. HA segments were derived from the polymer by controlled enzymatic digestion, and purified by gel filtration chromatography. Seven samples of narrow molecular weight distribution were analyzed by sensitivity-enhanced polyacrylamide gel electrophoresis, and found to have the following weight-average numbers of repeating disaccharide units: A, 90; B, 51; C, 38; D, 31; E, 23; F, 18; G, 13. The segment preparations were studied in 0.15 M NaCl by capillary viscometry, low angle laser light scattering, and circular dichroism spectroscopy. The data indicate concentration-dependent intermolecular association of short segments, and a capability for intramolecular association (hairpin formation) by larger HA segments.     

Degradation of even-numbered reduced and non-reduced hyaluronate oligosaccharides with D-glucuronic acid or N-acetyl-D-glucosamine as non-reducing terminal by chondroitin ABC and AC lyases.

Chondroitin ABC and AC lyases split hexosaminidic linkages in galactosaminoglycans and hyaluronic acid. Even-numbered oligosaccharides from hyaluronic acid with either D-glucuronic acid or N-acetylglucosamine in non-reducing position were used, prior to and after reduction with sodium borohydride, as substrates for chondroitin ABC and AC lyases. These substrates allowed elucidation of the effects of the nearest neighborhood of the bond to be split on the action of the enzymes. The results indicate that chondroitin ABC lyase acts strictly as an endolyase towards hyaluronate and requires the presence of a disaccharide in both reducing and non-reducing positions of the endohexosaminidic bond to be split. None of the hexosaminidic bonds of the tetrasaccharide GlcNAc-GlcUA-GlcNAc-GlcUA is split by chondroitin ABC lyase. In contrast chondroitin AC lyase acts also as an exoglycosidase towards hyaluronate and recognizes only the amino sugar and the uronic acid residue that are linked via the hexosaminidic bond which is split. Thus, the N-acetylglucosamine and glucuronic acid residues at both ends of a tetrasaccharide with the structure GlcNAc-GlcUA-GlcNAc-GlcUA are liberated.     

The distribution of repeating [Gal beta 1,4GlcNAc beta 1,3] sequences in asparagine-linked oligosaccharides of the mouse lymphoma cell lines BW5147 and PHAR 2.1

The occurrence and distribution of the repeating disaccharide [Gal beta 1,4GlcNAc beta 1,3] in the different types of Asn-linked oligosaccharides in mouse lymphoma BW5147 cells have been studied. Glycopeptides were prepared from cells grown in medium containing [6-3H]galactose, and the bi-, tri-, and tetraantennary Asn-linked oligosaccharides were fractionated by serial lectin affinity chromatography on concanavalin A-Sepharose, pea lectin -Sepharose, leukoagglutinating phytohemagglutinin-agarose, and Datura stramonium agglutinin-agarose. As described in this report, the latter lectin binds glycopeptides that contain either the repeating N-acetyllactosamine sequence or an outer mannose residue substituted at C-2 and C-6 by N-acetyllactosamine. The isolated glycopeptides were subjected to methylation analysis, specific exoglycosidase treatments, and digestion with Escherichia freundii endo-beta-galactosidase. Our data indicate that approximately two-thirds of the tetraantennary and one-half of the triantennary Asn-linked oligosaccharides contain repeating N-acetyllactosamine sequences in at least one branch. Many of the repeating sequences contain an additional galactose residue linked alpha 1,3 to a penultimate galactose residue. By contrast, less than 10% of the biantennary oligosaccharides contain the repeating disaccharide. The distribution of the repeating N-acetyllactosamine unit was also examined in a cell line ( PHAR 2.1) that is deficient in UDP-GlcNAc:alpha-mannoside beta 1,6-N-acetylglucosaminyltransferase. These cells are unable to synthesize tetraantennary and certain triantennary species and instead accumulate biantennary oligosaccharides. The total content of repeating N-acetyllactosamine units is greatly decreased in this line, and those that are present are found predominantly in triantennary Asn-linked oligosaccharides. These results demonstrate that the repeating N-acetyllactosamine sequence occurs commonly in complex-type Asn-linked oligosaccharides in BW5147 cells but is confined primarily to tri- and teraantennary species.     

Effect of Enzyme Preparation from the Marine Mollusk Littorina kurila on Fucoidan from the Brown Alga Fucus distichus

A fucoidanase preparation from the marine mollusk Littorina kurila cleaved some glycosidic bonds in fucoidan from the brown alga Fucus distichus, but neither fucose nor lower oligosaccharides were produced. The main product isolated from the incubation mixture was a polysaccharide built up of disaccharide repeating units -->3)-alpha-L-Fucp-(2,4-di-SO3(-))-(1-->4)-alpha-L-Fucp-(2SO3(-))-(1-->, the structure coinciding with the idealized formula proposed for the initial substance. A polymer fraction with the same carbohydrate chain but sulfated only at positions 2 and nonstoichiometrically acetylated at positions 3 and 4 of fucose residues was isolated as a minor component. It is suggested that the native polysaccharide should contain small amounts of non-sulfated and non-acetylated fucose residues, and only their glycosidic bonds are cleaved by the enzyme. The enzymatic hydrolysis showed that irregular regions of the native polysaccharide containing acetylated and partially sulfated repeating units were assembled in blocks.     

X-ray fibre diffraction study of conformational changes in hyaluronate induced in the presence of sodium,potassium and calcium cations

Hyaluronate purified from all cations by ion exchange chromatography was introduced to the cations sodium, potassium and calcium in a controlled way. The conformations formed in the presence of these ions were studied as a function of ionic strength, hydrogen ion activity, humidity and temperature using X-ray fibre diffraction. In sodium hyaluronate above pH 4.0 a contracted helix is found which approximates to a four-fold helix with an axial rise per disaccharide of 0.84 nm. There is no requirement for water molecules in the unit cell as the Na⁺ can be coordinate by the hyaluronate chains alone. On crystallizing hyaluronate below pH 4.0 an extended 2-fold helix with an axial rise per disaccharide of 0.98 nm is formed. In the presence of potassium above pH 4.0 a conformation similar, but not identical, to that of sodium was found where the helix backbone is again four-fold with an axial rise per disaccharide h=0.90 nm. To maintain the coordination of the potassium ion, four water molecule/disaccharide are required and on removal of these the conformation is destabilized going to a new helix where n = 4 and h = 0.97 nm. Below pH 4.0 the conformation is a contracted 4-fold helix with h = 0.82 nm. In this structure two antiparallel chains intertwine to form a double helix. The packing of the double helical units is stabilized by water molecules, the unit cell requiring 8 water molecules/disaccharide. Formation of the calcium hyaluronate complex above pH 3.5 yields a three-fold helix with h = 0.95 nm. The requirement for water in the unit cell to maintain full crystallinity is high, at 9 water molecules/disaccharide; however, on removal of this water, though the crystallinity is disrupted, the conformation remains constant. The acid form of calcium-hyaluronate yields an equivalent conformation to that of sodium under the same condition, i.e. a helix with n = 2, h = 0.98 nm. The presence of small quantities of calcium in what are otherwise potassium or sodium solutions of hyaluronate yield the 3-fold conformation for hyaluronate. Thus calcium has an important role to play in deciding the dominating conformation present in hyaluronate. The variety of conformations yielded by the different cations indicates a subtle interaction between hyaluronate and its environment, in which the balance between the cations will control to some degree the interactions between hyaluronate chains and thus affect the mechanical properties of the matrix which they form. The conformations of individual chains are all stabilized in varying degrees by intra-chain hydrogen bonds.     

Exploration of the action pattern of Streptomyces hyaluronate lyase using high-resolution capillary electrophoresis

Hyaluronic acid was treated exhaustively with a hyaluronate lyase (hyaluronidase, EC 4.2.2.1) from Streptomyces hyalurolyticus to obtain a tetrasaccharide and a hexasaccharide product in a molar ratio of 1 to 1.2. The tetrasaccharide product was fluorescently labeled at the reducing end by reductive amination with 7-amino 1,3-naphthalene disulfonic acid (AGA) and the structure of the conjugate was determined spectroscopically. Partial treatments of hyaluronic acid with hyaluronate lyase afforded complex mixtures of oligosaccharides that were similarly fluorescently labeled. These labeled oligosaccharide mixtures were analyzed using high-resolution capillary electrophoresis. The resulting electropherograms showed the content of each hyaluronic acid derived oligosaccharide, having a degree of polymerization (dp) from 4 to 50, throughout the enzymatic reaction. Computer simulation studies gave comparable kinetic profiles suggesting that hyaluronate lyase exhibits a random endolytic action pattern. Interestingly, oligosaccharides of certain size (dp) were under-represented in these oligosaccharide mixtures suggesting that linkages at spacings of 10 to 12 saccharide units are somewhat resistant to this enzyme. The cause of this resistance might be the result of secondary or higher order structural features present in the hyaluronic acid polymer.     

Characterization of Staphylococcus aureus cell wall glycan strands, evidence for a new beta-N-acetylglucosaminidase activity

Using sequential digestion with the glycyl-glycine endopeptidase lysostaphin followed by the pneumococcal N-acetylmuramyl-L-alanine amidase (amidase), the glycan strands of the peptidoglycan of Staphylococcus aureus were purified and analyzed by a combination of reverse-phase-high pressure liquid chromatography (HPLC) and mass spectrometry. Reverse-phase-HPLC resolved the glycan strands to a family of major peaks, which represented oligosaccharides composed of repeating disaccharide units (N-acetylglucosamine-[beta-1, 4]-N-acetylmuramic acid) with different degrees of polymerization and terminating with N-acetylmuramic acid residues at the reducing ends. The method allowed separation of strands up to 23-26 disaccharide units with a predominant length between 3 and 10 and an average degree of polymerization of approximately 6. Glycan strands with a higher degree of polymerization (>26 disaccharide units) represented 10-15% of the total UV absorbing glycan material. A unique feature of the staphylococcal glycan strands was the presence of minor satellite peaks that were present throughout the HPLC elution profile eluting either just prior or shortly after the major oligosaccharide peaks. A number of observations including mass spectrometric analysis suggest that the satellites are the products of an N-acetylglucosaminidase activity that differs from the atl gene product and that appears to be involved with modification of the glycan strand structure.     

A model of the platelet factor 4 complex with heparin.

A model of heparin bound to bovine platelet factor 4 (BPF4) was completed using a graphically designed heparin molecule and the crystallographic coordinates of the native bovine platelet factor 4 tetramer. The oligosaccharides had a chain length of at least eight disaccharide units with the major repeating disaccharide unit consisting of (1----4)-O-(alpha-L-idopyranosyluronic acid 2-sulfate)-(1----4)-(2-deoxy-2-sulfamino-2-D-glucopyranosyl 6-sulfate). Each disaccharide unit carried a -4.0 charge. The structure of BPF4 was solved to 2.6 A resolution with R = 0.237. Each monomer of BPF4 contains an alpha-helix lying across 3 strands of antiparallel beta-sheet. Each helix has four lysines, which have been implicated in heparin binding. These lysine residues are predominantly on one side of the helix and are solvent accessible. Electrostatic calculations performed on the BPF4 tetramer show a ring of strong, positive charge which runs perpendicularly across the helices. Included in this ring of density is His-38, which has been shown by NMR to have a large pKa shift when heparin binds to BPF4. Our model of heparin bound to PF4 has the anionic polysaccharide perpendicular to the alpha-helices, wrapped about the tetramer along the ring of positive charge, and salt linked to all four lysines on the helix of each monomer.     

Constant and variable domains of different disaccharide structure in corneal keratan sulphate chains.

Four peptidokeratan sulphate fractions of different Mr and degree of sulphation were cut from the pig corneal keratan sulphate distribution spectrum. After exhaustive digestion with keratanase, the fragments were separated on DEAE-Sephacel and Bio-Gel P-10 and analysed for their Mr, degree of sulphation and amino sugar and neutral sugar content. It was found that every glycosaminoglycan chain is constructed of a constant domain of non-sulphated and monosulphated disaccharide units and a variable domain of disulphated disaccharide units. Total neuraminic acid of the four peptidokeratan sulphates was recovered from their isolated linkage-region oligosaccharides. In kinetic studies, the four peptidokeratan sulphates were investigated for Mr distribution after various incubation times with keratanase. There was a continuous shift towards lower Mr and no appearance of a distinct intermediate-sized product at any degradation time. The linkage-region oligosaccharide was already being liberated after a very short incubation period. From the results of these kinetic investigations in connection with the results of neuraminic acid analyses it is suggested that there exists only one disaccharide chain per peptidokeratan sulphate molecule. A model of corneal keratan sulphate is postulated. One of the alpha-mannose residues in the linkage region is bound to an oligosaccharide consisting of a lactosamine and a terminal sialic acid. The other alpha-mannose residue is attached to the disaccharide chain. This chain contains one or two non-sulphated disaccharide units at the reducing end, followed by 10-12 monosulphated disaccharide units. The disulphated disaccharide moiety of variable length is positioned at the non-reducing end of the chain.     

Structure and flexibility of Streptococcus agalactiae hyaluronate lyase complex with its substrate. Insights into the mechanism of processive degradation of hyaluronan

Streptococcus agalactiae hyaluronate lyase degrades primarily hyaluronan, the main polysaccharide component of the host connective tissues, into unsaturated disaccharide units as the end product. Such function of the enzyme destroys the normal connective tissue structure of the host and exposes the tissue cells to various bacterial toxins. The crystal structure of hexasaccharide hyaluronan complex with the S. agalactiae hyaluronate lyase was determined at 2.2 A resolution; the mechanism of the catalytic process, including the identification of specific residues involved in the degradation of hyaluronan, was clearly identified. The enzyme is composed structurally and functionally from two distinct domains, an alpha-helical alpha-domain and a beta-sheet beta-domain. The flexibility of the protein was investigated by comparing the crystal structures of the S. agalactiae and the Streptococcus pneumoniae enzymes, and by using essential dynamics analyses of CONCOORD computer simulations. These revealed important modes of flexibility, which could be related to the protein function. First, a rotation/twist of the alpha-domain relative to the beta-domain is potentially related to the mechanism of processivity of the enzyme; this twist motion likely facilitates shifting of the ligand along the catalytic site cleft in order to reposition it to be ready for further cleavage. Second, a movement of the alpha- and beta-domains with respect to each other was found to contribute to a change in electrostatic characteristics of the enzyme and appears to facilitate binding of the negatively charged hyaluronan ligand. Third, an opening/closing of the substrate binding cleft brings a catalytic histidine closer to the cleavable substrate beta1,4-glycosidic bond. This opening/closing mode also reflects the main conformational difference between the crystal structures of the S. agalactiae and the S. pneumoniae hyaluronate lyases.     

Mechanism of hyaluronan binding and degradation: structure of Streptococcus pneumoniae hyaluronate lyase in complex with hyaluronic acid disaccharide at 1.7 A resolution

Hyaluronic acid (HA) is an important constituent of the extracellular matrix; its bacterial degradation has been postulated to contribute to the spread of certain streptococci through tissue. Pneumococci and other streptococci produce hyaluronate lyase, an enzyme which depolymerizes HA, thus hyaluronate lyase might contribute directly to bacterial invasion. Although two different mechanisms for lyase action have been proposed, there was no crystallographic evidence to support those mechanisms. Here, we report the high-resolution crystal structure of Streptococcus pneumoniae hyaluronate lyase in the presence of HA disaccharide product, which ultimately provides the first crystallographic evidence for the binding of HA to hyaluronate lyase. This structural complex revealed a key interaction between the Streptococcus peneumoniae hyaluronate lyase protein and the product, and supports our previously proposed novel catalytic mechanism for HA degradation based on the native Streptococcus peneumoniae hyaluronate lyase structure. The information provided by this complex structure will likely be useful in the development of antimicrobial pharmaceutical agents.