Depolymerization of hyaluronan by sonication

High molecular weight hyaluronan (M _r 400 000) obtained from human umbilical cord was depolymerized by sonication for 10 h into small molecules and finally into molecules of constant size (M _r 11 000). The molecular size of the depolymerized hyaluronan was unaltered even under different conditions of sonication. After sonication, the main sugar residues at the reducing and non-reducing termini of depolymerized hyaluronan wereN-acetylglucosamine (86%) and glucuronic acid (98%), respectively. Hyaluronans derived from rooster comb (M _r 1×10⁶) andStreptococcus zooepidemicus (M _r 1.2×10⁶) were deploymerized into molecules of different but characteristic sizes by sonication. On the other hand, neither chondroitin sulfate nor glycogen was depolymerized by sonication. These results suggest that high molecular weight hyaluronan may have some weak linkages related toN-acetylglucosamine in the chain, which are extremely sensitive to sonication. At present, however, the nature of these linkages is still unclear.Abbreviations HA hyaluronan - PA 2-aminopyridine     

Labelling of high molecular weight hyaluronan with125I-tyrosine: studiesin vitro andin vivo in the rat

Studies on the metabolism of the polysaccharide hyaluronan has previously been hampered by the lack of radioactive hyaluronan of high molecular weight (MW) and high specific activity. In the present study¹²⁵I-tyrosine (T)-labelled hyaluronan was produced after CNBr-activation of the polysaccharide. A specific activity of approximately 0.1 MBq µg^–1 was achieved using 100 µg of 0.5×10⁶ Da hyaluronan labelled for 2 h with 18 MBq¹²⁵I. The¹²⁵I-T-hyaluronan kept a high MW-profile upon gel filtration chromatography and was found to be cleared from the circulation with the kinetics and organ distribution reported for biosynthetically labelled hyaluronan of high MW. The¹²⁵I-labelled polysaccharide is also taken up by liver endothelial cells bothin vivo andin vitro, indicating that the labelling does not interfere with the binding to specific cell-surface receptors found on these cells. The intracellular degradation is slower than that earlier reported for biosynthetically labelled hyaluronan and seems to be halted at the level of low MW oligo- or mono-saccharides that eventually leave the organism via the urine. Scintigraphic images of rats after intravenous injection of¹²⁵I-T-hyaluronan showed rapid uptake in the liver and a redistribution of radioactivity from liver to urine with time. Our results indicate that the¹²⁵I-T-hyaluronan is suitable for studies of hyaluronan-metabolism in a number of ways. The gamma emitters¹²⁵I and¹³¹I are easy to monitor and can be used also forin vivo 3D-imaging using single photon emission computer tomography.Abbreviations CNBr cyanogen bromide - T-HA tyrosine-labelled hyaluronan     

Cartilage link protein interacts with neurocan, which shows hyaluronan binding characteristics different from CD44 and TSG-6

The interaction of neurocan with hyaluronan was qualitatively characterized with alkaline phosphatase fusion proteins secreted by mammalian cells. The wild type neurocan hyaluronan binding domain fused to alkaline phosphatase bound to immobilized hyaluronan under physiological as well as moderately hypertonic conditions, whereas its ability to bind to immobilized chondroitin sulfate dropped rapidly with increasing salt concentration. Strong hyaluronan binding ability was still evident when in both link modules within the hyaluronan binding domain a basic amino acid was mutated, which is well conserved among link modules of hyaluronan binding proteins. A strong enhancement of the binding of neurocan to immobilized hyaluronan was observed after preincubation of the immobilized hyaluronan with cartilage link protein. Moreover, this preincubation mediated also the binding of a fusion protein representing only the immunoglobulin module of neurocan linked to alkaline phosphatase, which showed no binding to immobilized hyaluronan alone. The interaction of the neurocan immunoglobulin module with link protein could also be shown by overlay blot analysis. These observations suggest that the hyaluronan binding characteristics of paired link modules are different from those of single link modules, and that the reported temporal co-expression of cartilage link protein and of neurocan in developing brain implicates the possibility of a cooperative function of these molecules.     

Binding and degradation of hyaluronan by human breast cancer cell lines expressing different forms of CD44: Correlation with invasive potential

In the present study, we examined a panel of human breast cancer cell lines with regard to their expression of CD44 and ability to bind and degrade hyaluronan. The cell lines expressed varying amounts of different molecular weight forms of CD44 (85–200 kDa) and, in general, those that expressed the greatest amounts of CD44 were the most invasive as judged by in vitro assays. In addition, the ability to bind and degrade hyaluronan was restricted to the cell lines expressing high levels of CD44, and both these functions were blocked by an antibody to CD44 (Hermes-1). Moreover, the rate of [³H]hyaluronan degradation was highly correlated with the amount of CD44 (r = 0.951, P < 0.0001), as well as with the invasive potential of the cells. Scatchard analysis of the [³H]hyaluronan binding of these cells revealed the existence of significant differences in both their binding capacity and their dissociation constant. To determine the source of this deviation, the different molecular weight forms of CD44 were partially separated by gel filtration chromatography. In all cell lines, the 85 kDa form was able to bind hyaluronan, although with different affinities. In contrast, not all of the high molecular weight forms of CD44 had this ability. These results illustrate the diversity of CD44 molecules in invasive tumor cells, and suggest that one of their major functions is to degrade hyaluronan. © 1994 Wiley-Liss, Inc.     

Expression of scFv‐Mel‐Gal4 triple fusion protein as a targeted DNA‐carrier in Escherichia Coli

Liver‐directed gene therapy has become a promising treatment for many liver diseases. In this study, we constructed a multi‐functional targeting molecule, which maintains targeting, endosome‐escaping, and DNA‐binding abilities for gene delivery. Two single oligonucleotide chains of Melittin (M) were synthesized. The full‐length cDNA encoding anti‐hepatic asialoglycoprotein receptor scFv C1 (C1) was purified from C1/pIT2. The GAL4 (G) gene was amplified from pSW50‐Gal4 by polymerase chain reaction. M, C1 and G were inserted into plasmid pGC4C26H to product the recombinant plasmid pGC‐C1MG. The fused gene C1MG was subsequently subcloned into plasmid pET32c to product the recombinant plasmid C1MG/pET32c and expressed in Escherichia coli BL21. The scFv‐Mel‐Gal4 triple fusion protein (C1MG) was purified with a Ni²⁺ chelating HiTrap HP column. The fusion protein C1MG of roughly 64 kD was expressed in inclusion bodies; 4.5 mg/ml C1MG was prepared with Ni²⁺ column purification. Western blot and immunohistochemistry showed the antigen‐binding ability of C1MG to the cell surface of the liver‐derived cell line and liver tissue slices. Hemolysis testing showed that C1MG maintained membrane‐disrupting activity. DNA‐binding capacity was substantiated by luciferase assay, suggesting that C1MG could deliver the DNA into cells efficiently on the basis of C1MG. Successful expression of C1MG was achieved in E. coli, and C1MG recombinant protein confers targeting, endosome‐escaping and DNA‐binding capacity, which makes it probable to further study its liver‐specific DNA delivery efficacy in vivo. Copyright © 2013 John Wiley & Sons, Ltd.     

Accessible hyaluronan receptors identical to ICAM-1 in mouse mast-cell tumours

Immunohistochemical studies of the hyaluronan (HA)-receptor (R), originally found on liver endothelial cells (LEC) and related to the intercellular adhesion molecule 1 (ICAM-1), showed that polyclonal antibodies against HARLEC (HA receptor on LEC) also stain structures in mouse mastocytomas, mainly vessels. To test if intravenously administered HA might target the tumour receptorsin vivo, mice carrying an inoculated mastocytoma in one hind leg muscle were injected in the tail vein with¹²⁵I-tyrosine (T)-labelled HA and killed 75 min after injection when organs and tissues were checked for radioactivity. When doses exceeding the binding capacity of the liver were injected, a significant increase in radioactivity (up to five-fold) within the tumour tissue was found. The weight adjusted difference between control and tumour tissue was greater for smaller tumours, probably due to necrosis in the larger. HA-staining of tumours from animals receiving¹²⁵I-T-HA, showed HA in areas that also stained weakly for ICAM-1 using monoclonal antibodies. ICAM-1 staining was dramatically increased after hyaluronidase treatment of the sections, indicating that the HA is bound to these receptors and thereby blocks antibody recognition.Abbreviations ICAM-1 intercellular adhesion molecule 1 - HA hyaluronan - HARLEC hyaluronan receptor on liver endothelial cells - MW molecular weight     

Intracellular Refolding Assay

This protocol describes a method to measure the enzymatic activity of molecular chaperones in a cell-based system and the possible effects of compounds with inhibitory/stimulating activity. Molecular chaperones are proteins involved in regulation of protein folding¹ and have a crucial role in promoting cell survival upon stress insults like heat shock², nutrient starvation and exposure to chemicals/poisons³. For this reason chaperones are found to be involved in events like tumor development, chemioresistance of cancer cells⁴ as well as neurodegeneration⁵. Design of small molecules able to inhibit or stimulate the activity of these enzymes is therefore one of the most studied strategies for cancer therapy⁷ and neurodegenerative disorders⁹. The assay here described offers the possibility to measure the refolding activity of a particular molecular chaperone and to study the effect of compounds on its activity. In this method the gene of the molecular chaperone investigated is transfected together with an expression vector encoding for the firefly luciferase gene. It has been already described that denaturated firefly luciferase can be refolded by molecular chaperones^10,11. As normalizing transfection control, a vector encoding for the renilla luciferase gene is transfected. All transfections described in this protocol are performed with X-treme Gene ¹¹ (Roche) in HEK-293 cells. In the first step, protein synthesis is inhibited by treating the cells with cycloheximide. Thereafter protein unfolding is induced by heat shock at 45°C for 30 minutes. Upon recovery at 37°C, proteins are re-folded into their active conformation and the activity of the firefly luciferase is used as read-out: the more light will be produced, the more protein will have re-gained the original conformation. Non-heat shocked cells are set as reference (100% of refolded luciferase).     

Synthesis of hyaluronan of distinctly different chain length is regulated by differential expression of Xhas1 and 2 during early development of Xenopus laevis

The localization of hyaluronan has been determined in tailbud stage embryos of Xenopus laevis using a neurocan-alkaline phosphatase fusion protein. This polysaccharide was located between the germ layers and enriched in mesenchyme, the lumen of the neural tube, the embryonic gut, the hepatic cavity and the heart. A full-length cDNA for a hyaluronan synthase, Xhas2 has been cloned. The expression pattern of Xhas1 and 2 is closely similar to the distribution of hyaluronan in the embryo. Xhas1 produces hyaluronan with a molecular mass of around 40–200 kDa, while the product formed by Xhas2 has a molecular mass above 1 million Da.     

Hydrocortisone regulation of hyaluronan metabolism in human skin organ culture

We studied the influence of hydrocortisone (HC) on hyaluronan (HA) metabolism in explants of human skin, a model retaining normal three-dimensional architecture of dermal connective tissue and dynamic growth and stratification of epidermal keratinocytes. The synthesis of hyaluronan and proteoglycans (PGs), and DNA, were determined with ³H-glucosamine and ³H-thymidine labelings, respectively. The total content and histological distribution of hyaluronan was studied utilizing a biotinylated aggrecan-link protein complex. A low concentration of HC (10^?9 M) stimulated the incorporation of ³H-glucosamine into hyaluronan in epidermis by 23% and reduced the disappearance rate of hyaluronan by 25% in chase experiments, resulting in a 74% increase in total hyaluronan (per epidermal dry weight) after a 5-day culture in 10^?9 M HC. On the other hand, a high concentration of HC (10^?5 M) reduced both synthesis (-42%) and degradation (-46%) of epidermal hyaluronan during 24 h labeling and chase periods. The cumulative effect of a 5-day treatment was a 24% decrease of total epidermal hyaluronan. The high dose (10^?5 M) also reduced keratinocyte DNA synthesis and epidermal thickness. In dermis, only the high (10^?5 M) concentration of HC was effective, inhibiting the incorporation of ³H-glucosamine into hyaluronan by 28%. No significant influences on total hyaluronan content or the disappearance rate of hyaluronan in dermal tissue was found. All HC concentrations lacked significant effects on newly synthesized PGs in epidermal and dermal tissues, but reduced the labeled PGs diffusing into culture medium. A low physiological concentration of HC thus maintains active synthesis and high concentration of hyaluronan in epidermal tissue, while high pharmacological doses of HC slows hyaluronan turnover and reduces its content in epidermis, an effect correlated with enhanced terminal differentiation, reduced proliferation rate and reduced number of vital keratinocyte layers. © 1995 Wiley-Liss, Inc.     

Substrate recognition by bacterial solute-binding protein is responsible for import of extracellular hyaluronan and chondroitin sulfate from the animal host

ABSTRACT

Glycosaminoglycans (GAGs) such as hyaluronan and chondroitin in animal extracellular matrices contain disaccharide-repeating units. In a Gram-negative pathogenic Streptobacillus moniliformis, which belongs to Fusobacteria phylum and resides in rodent oral cavities, the solute-binding protein (Smon0123)-dependent ATP-binding cassette transporter imports unsaturated hyaluronan/chondroitin disaccharides into the cytoplasm after GAG lyase-dependent depolymerization. Here we show substrate recognition of unsaturated hyaluronan disaccharide by Smon0123. Moreover, Smon0123 exhibited no affinity for unsaturated chondroitin disaccharides containing three sulfate groups, distinct from non-sulfated, mono-sulfated, and di-sulfated chondroitin disaccharides previously identified as substrates. Crystal structure of Smon0123 with unsaturated hyaluronan disaccharide demonstrates that several residues, including Trp284 and Glu410, are crucial for binding to unsaturated hyaluronan/chondroitin disaccharides, whereas arrangements of water molecules at binding sites are found to be substrate dependent through comparison with substrate-bound structures determined previously. These residues are well conserved in Smon0123-like proteins of fusobacteria, and probably facilitate the fusobacterial residence in hyaluronan-rich oral cavities.     

A competitive enzyme-linked immunosorbent assay-like method for the measurement of urinary hyaluronan.

A highly sensitive method for measurement of urinary hyaluronan with a minimum molecular mass of 2,000 Da was developed without using HPLC or radioisotopes. This competitive enzyme-linked immunosorbent assay-like method, used competitive binding of free hyaluronan in the sample and biotin-labeled standard hyaluronan to hyaluronan binding protein in solid phase. A total of 150 healthy individuals from both sexes at ages from 0 to 100 years was examined by the established method. Hyaluronan of 384 +/- 80 ng/mg creatinine (mean +/- SD) was constantly excreted into urine of 24-40-year-old healthy adults. The urinary hyaluronan levels were significantly higher before age 1 (p < 0.001) and rather high after 90 years compared to the other groups. The average molecular weight of urinary hyaluronan (5,500 Da) was constant through all generations. Sex difference of urinary hyaluronan was not observed both quantitatively or qualitatively.     

A rapid increase in macrophage-derived versican and hyaluronan in infectious lung disease

The goals of this study were to characterize the changes in chondroitin sulfate proteoglycans and hyaluronan in lungs in acute response to gram-negative bacterial infection and to identify cellular components responsible for these changes. Mice were treated with intratracheal (IT) live Escherichia coli, E. coli lipopolysaccharide (LPS), or PBS. Both E. coli and LPS caused rapid selective increases in mRNA expression of versican and hyaluronan synthase (Has) isoforms 1 and 2 associated with increased immunohistochemical and histochemical staining for versican and hyaluronan in the lungs. Versican was associated with a subset of alveolar macrophages. To examine whether macrophages contribute to versican and hyaluronan accumulation, in vitro studies with primary cultures of bone marrow-derived and alveolar macrophages were performed. Unstimulated macrophages expressed very low levels of versican and hyaluronan synthase mRNA, with no detectible versican protein or hyaluronan product. Stimulation with LPS caused rapid increases in versican mRNA and protein, a rapid increase in Has1 mRNA, and concomitant inhibition of hyaluronidases 1 and 2, the major hyaluronan degrading enzymes. Hyaluronan could be detected following chloroquine pre-treatment, indicating rapid turnover and degradation of hyaluronan by macrophages. In addition, the effects of LPS, the M1 macrophage classical activation agonist, were compared to those of IL-4/IL-13 or IL-10, the M2a and M2c alternative activation agonists, respectively. Versican and Has1 increased only in response to M1 activation. Finally, the up-regulation of versican and Has1 in the whole lungs of wild-type mice following IT LPS was completely abrogated in TLR-4^−/− mice. These findings suggest that versican and hyaluronan synthesis may play an important role in the innate immune response to gram-negative lung infection.     

EVOLUTION OF BIOLUMINESCENCE IN MARINE DINOFLAGELLATES

Bioluminescence is broadly distributed in marine dinoflagellates and has been intensively studied in Lingulodinium (Gonyaulax) polyedra. In this species, bioluminescence is regulated in a circadian fashion; the enzyme (luciferase) and the luciferin (substrate)‐binding protein are synthesized and degraded on a daily basis. Synthesis of both proteins is regulated at the level of translation. The L. polyedra luciferase gene is composed of three contiguous domains that are greater than 75% identical at the nucleic acid level. Possible explanations for the high degree of sequence conservation include: (1) the domains evolved through a recent duplication event; (2) the sequence similarity is maintained by a molecular process such as gene conversion; or (3) there is a functional role associated with the primary nucleic acid sequence, such as in the translational regulation of luciferase expression. The phylogenetic relationship of dinoflagellates predicted from 18S rDNA genes provides a framework for examining the molecular evolution of the regulation of luciferase expression and of genes encoding luciferase and the luciferin‐binding protein. In particular, we are examining the evolution of the circadian rhythm of bioluminescence and of luciferase abundance, the presence/absence of the luciferin‐binding protein, and the molecular structure of the luciferase gene. We anticipate that this approach will distinguish between regions of the luciferase molecule that are conserved for enzyme function versus those concerned with the regulation of protein expression. In addition, it will provide insight into the evolution of the regulatory processes and pathways.     

Thermostabilization of protein A-luciferase fusion protein by single amino acid mutation

A gene expression plasmid, pMALU7, for coding a fusion protein between protein A (SpA) and mutated firefly luciferase (Luc), was constructed. The fused gene was expressed in Escherichia coli and the resulting protein (SpA-LucTS) was purified with affinity chromatography. By changing a single amino acid, from Glu to Lys at the position 354 in the luciferase moiety, the thermostability of luciferase was improved.     

Functional expression of rat adenosine A1 receptor in the dimorphic zygomycete<Emphasis Type="Italic"> Mucor circinelloides</Emphasis>

We have produced the rat adenosine A1 receptor in Mucor circinelloides using a translational fusion to the endogenous glucoamylase (GlaM) gene. The fusion protein produced from an episomal plasmid was correctly processed as judged by western blotting, since only a 33 kDa band was detected in membrane preparations from M. circinelloides expressing the receptor. This corresponds to the mass of the full-length receptor released from the fused GlaM protein. The presence of a high affinity binding site with a K_d value of 0.5 nM for the receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) in membrane preparations suggests that the receptor was correctly folded and inserted into the membranes. A receptor expression level of 100–300 fmol/mg total membrane protein was achieved as judged by binding of the antagonist [³H]-DPCPX.     

Direct quantification of the rupture force of single hyaluronan/hyaluronan binding protein bonds

The non-covalent bond between aggrecan and hyaluronan is critical for maintaining the normal structure and function of the extracellular matrix in articular cartilage. The failure of this bond can cause the loss of aggrecan and destruction of the extracellular matrix of articular cartilage. In this study, the rupture force of the single bond between hyaluronan and hyaluronan binding protein - the complex of the hyaluronan binding region of aggrecan and link protein - was directly measured with a nanomechanical testing system as 40+/-11 pN. The results were compared to a theoretical prediction based on a smart version of the Monte Carlo simulation.     

Reprint of: A rapid increase in macrophage-derived versican and hyaluronan in infectious lung disease

The goals of this study were to characterize the changes in chondroitin sulfate proteoglycans and hyaluronan in lungs in acute response to gram-negative bacterial infection and to identify cellular components responsible for these changes. Mice were treated with intratracheal (IT) live Escherichia coli, E. coli lipopolysaccharide (LPS), or PBS. Both E. coli and LPS caused rapid selective increases in mRNA expression of versican and hyaluronan synthase (Has) isoforms 1 and 2 associated with increased immunohistochemical and histochemical staining for versican and hyaluronan in the lungs. Versican was associated with a subset of alveolar macrophages. To examine whether macrophages contribute to versican and hyaluronan accumulation, in vitro studies with primary cultures of bone marrow-derived and alveolar macrophages were performed. Unstimulated macrophages expressed very low levels of versican and hyaluronan synthase mRNA, with no detectible versican protein or hyaluronan product. Stimulation with LPS caused rapid increases in versican mRNA and protein, a rapid increase in Has1 mRNA, and concomitant inhibition of hyaluronidases 1 and 2, the major hyaluronan degrading enzymes. Hyaluronan could be detected following chloroquine pre-treatment, indicating rapid turnover and degradation of hyaluronan by macrophages. In addition, the effects of LPS, the M1 macrophage classical activation agonist, were compared to those of IL-4/IL-13 or IL-10, the M2a and M2c alternative activation agonists, respectively. Versican and Has1 increased only in response to M1 activation. Finally, the up-regulation of versican and Has1 in the whole lungs of wild-type mice following IT LPS was completely abrogated in TLR-4^−/− mice. These findings suggest that versican and hyaluronan synthesis may play an important role in the innate immune response to gram-negative lung infection.     

Expression of Coat Protein of an Ordinary Strain of Potato virus Y in Escherichia coli and Production of Polyclonal Antibodies for Diagnosis

The coat protein gene (CP) of an ordinary strain of Potato virus Y (PVY^O) was cloned into the expression vector, pET‐28a(+). The insert was sequenced and analysis showed that the CP gene was in frame with intact N‐terminal 6X histidine tags. An approximately 35 kDa recombinant fusion protein was observed in inclusion bodies of induced Escherichia coli BL21 cells. This fusion protein was purified and used as antigen to raise polyclonal antibodies in rabbits. In Western blot and dot blot immuno‐binding assay (DIBA), both PVY^O‐CP IgG and PVY^O IgG strongly reacted with the recombinant CP. The PVY^O‐CP IgG could detect PVY^O in infected samples up to 1 : 3200 dilutions. A PVY^O‐CP ELISA kit was prepared and compared with conventional ELISA kit based on purified virus particles (PVY^O ELISA kit). The PVY^O‐CP ELISA kit consistently detected the PVY^O in DAS‐ELISA of field samples and was as effective as PVY^O ELISA kit.     

TSG-6 modulates the interaction between hyaluronan and cell surface CD44

Interactions between CD44 and hyaluronan are implicated in the primary adhesion of lymphocytes to endothelium at inflammatory locations. Here we show that preincubation of hyaluronan with full-length recombinant TSG-6 or its Link module domain (Link_TSG6) enhances or induces the binding of hyaluronan to cell surface CD44 on constitutive and inducible cell backgrounds, respectively. These effects are blocked by CD44-specific antibodies and are absent in CD44-negative cells. Enhancement of CD44-mediated interactions of lymphoid cells with hyaluronan by TSG-6 proteins was seen under conditions of flow at shear forces that occur in post-capillary venules. Increases in the number of rolling cells were observed on substrates comprising TSG-6-hyaluronan complexes as compared with a substrate containing hyaluronan alone. In ligand competition experiments, cell surface-bound TSG-6-hyaluronan complexes were more potent than hyaluronan alone in inhibiting cell adhesion to immobilized hyaluronan. Link_TSG6 mutants with impaired hyaluronan binding function had a reduced ability to modulate ligand binding by cell surface CD44. However, some mutants that exhibited close to wild-type hyaluronan binding were found to have either reduced or increased activity, suggesting that some amino acid residues outside of the hyaluronan binding site might be involved in protein self-association, potentially leading to the formation of cross-linked hyaluronan fibers. In turn, cross-linked hyaluronan could increase the binding avidity of CD44 by inducing receptor clustering. The ability of TSG-6 to modulate the interaction of hyaluronan with CD44 has important implications for CD44-mediated cell activity at sites of inflammation, where TSG-6 is expressed.