Chain structure and immunomodulatory activity of a fructosylated chondroitin from an engineered Escherichia coli K4.

Polysaccharide K4 expressed from E. coli K4 has a similar structure with chondroitin, which can be used as a precursor to produce chondroitin sulfates. Here, we investigated the structure, conformation and biological activity of K4 from an engineered strain with high productivity. The NMR analysis suggested that K4 from wild-type strain with a low yield was a partially fructosylated chondroitin. While K4 from engineered strain was a fully fructosylated chondroitin. Light scattering analysis gave the Mw values of 6.15 × 104, 8.23 × 104 and 1.92 × 104 for K4-1, K4-2 and defructosylated K4 (DK4), respectively. The exponents of functions z1/2 = f(Mw) were in the range of 0.643-0.608, suggesting a random coil conformation for K4 and DK4. And the random coils K4 easily self-assembled into sphere-like aggregates in the dilute aqueous solution. Both K4 and DK4 exhibited significant immunomodulatory activities on RAW 264.7 cells at the dosage range of 5-500 µg/mL.

Biosynthesis of Chondroitin in Engineered Corynebacterium glutamicum.

Chondroitin, the precursor of chondroitin sulfate, which is an important polysaccharide, has drawn significant attention due to its applications in many fields. In the present study, a heterologous biosynthesis pathway of chondroitin was designed in a GRAS (generally recognized as safe) strain C. glutamicum. CgkfoC and CgkfoA genes with host codon preference were synthesized and driven by promoter Ptac, which was confirmed as a strong promoter via GFPuv reporter assessment. In a lactate dehydrogenase (ldh) deficient host, intracellular chondroitin titer increased from 0.25 to 0.88 g/l compared with that in a wild-type host. Moreover, precursor enhancement via overexpressing precursor synthesizing gene ugdA further improved chondroitin titers to 1.09 g/l. Chondroitin production reached 1.91 g/l with the engineered strain C. glutamicum ΔL-CgCAU in a 5-L fed-batch fermentation with a single distribution Mw of 186 kDa. This work provides an alternative, safe and novel means of producing chondroitin for industrial applications.

Enhancing fructosylated chondroitin production in Escherichia coli K4 by balancing the UDP-precursors.

Microbial production of chondroitin and chondroitin-like polysaccharides from renewable feedstock is a promising and sustainable alternative to extraction from animal tissues. In this study, we attempted to improve production of fructosylated chondroitin in Escherichia coli K4 by balancing intracellular levels of the precursors UDP-GalNAc and UDP-GlcA. To this end, we deleted pfkA to favor the production of Fru-6-P. Then, we identified rate-limiting enzymes in the synthesis of UDP-precursors. Third, UDP-GalNAc synthesis, UDP-GlcA synthesis, and chondroitin polymerization were combinatorially optimized by altering the expression of relevant enzymes. The ratio of intracellular UDP-GalNAc to UDP-GlcA increased from 0.17 in the wild-type strain to 1.05 in a 30-L fed-batch culture of the engineered strain. Titer and productivity of fructosylated chondroitin also increased to 8.43 g/L and 227.84 mg/L/h; the latter represented the highest productivity level achieved to date.

Prognostic impact of chondroitin-4-sulfotransferase CHST11 in ovarian cancer.

Ovarian cancer (OvCa) accounts for the highest tumor-related mortality among gynecological malignancies, but the underlying mechanisms are poorly understood. Glycosaminoglycans are abundantly present in ovarian tumors, and there is rising evidence that chondroitin sulfate (CS) as well as diverse carbohydrate sulfotransferases (CHSTs), the enzymes involved in the sulfation process of these structures, plays an important role in metastatic spread of tumor cells. mRNA expression levels of CHST3/7/11/12/13/15 were compared between malignant (86 OvCas) and non-malignant tumors (6 borderline tumors and 3 cystadenomas). CHST11 and CHST15 were further chosen for Western blot analysis in a cohort of 216 OvCas. Protein expression levels were correlated with clinicopathologic prognostic parameters and survival data. A significantly higher mRNA expression of CHST11, CHST12, and CHST15 was measured in ovarian cancer samples in comparison to non-malignant ones, and the same trend was observed for CHST13. For CHST3 and CHST7, no significant differences were found between the two groups. At protein level, high CHST11 expression was independently associated with unfavorable progression-free survival (PFS; p = 0.027). A similar trend was observed for CHST15, showing a nearly significant correlation between high expression levels and shorter recurrence-free survival in patients without macroscopic residual tumor after surgery (p = 0.053). We conclude that CHSTs involved in the synthesis of CS-A and CS-E might influence ovarian cancer progression, and we suggest CHST11 as independent unfavorable prognostic factor in this entity.

Hypotheses on the evolution of hyaluronan: a highly ironic acid.

Hyaluronan is a high-molecular-weight glycosaminoglycan (GAG) prominent in the extracellular matrix. Emerging relatively late in evolution, it may have evolved to evade immune recognition. Chondroitin is a more ancient GAG and a possible hyaluronan precursor. Epimerization of a 4-hydroxyl in N-acetylgalactosamine in chondroitin to N-acetylglucosamine of hyaluronan is the only structural difference other than chain length between these two polymers. The axial 4-hydroxyl group extends out perpendicular from the equatorial plane of N-acetylgalactosamine in chondroitin. We suspect that this hydroxyl is a prime target for immune recognition. Conversion of a thumbs-up hydroxyl group into a thumbs-down position in the plane of the sugar endows hyaluronan with the ability to avoid immune recognition. Chitin is another potential precursor to hyaluronan. But regardless whether of chondroitin or of chitin origin, an ancient chondroitinase enzyme sequence seems to have been commandeered to catalyze the cleavage of the new hyaluronan substrate. The evolution of six hyaluronidase-like sequences in the human genome from a single chondroitinase as found in Caenorhabditis elegans can now be traced. Confirming our previous predictions, two duplication events occurred, with three hyaluronidase-like sequences occurring in the genome of Ciona intestinalis (sea squirt), the earliest known chordate. This was probably followed by en masse duplication, with six such genes present in the genome of zebra fish onwards. These events occurred, however, much earlier than predicted. It is also apparent on an evolutionary time scale that in several species, this gene family is continuing to evolve.

The structural basis for a coordinated reaction catalyzed by a bifunctional glycosyltransferase in chondroitin biosynthesis.

Bifunctional chondroitin synthase K4CP catalyzes glucuronic acid and N-acetylgalactosamine transfer activities and polymerizes a chondroitin chain. Here we have determined that an N-terminal region (residues 58-134) coordinates two transfer reactions and enables K4CP to catalyze polymerization. When residues 58-107 are deleted, K4CP loses polymerase activity while retaining both transfer activities. Peptide (113)DWPSDL(118) within this N-terminal region interacts with C-terminal peptide (677)YTWEKI(682). The deletion of either sequence abolishes glucuronic acid but not N-acetylgalactosamine transfer activity in K4CP. Both donor bindings and transfer activities are lost by mutating (677)YTWEKI(682) to (677)DAWEDI(682). On the other hand, acceptor substrates retain their binding to K4CP mutants. The characteristics of these K4CP mutants highlight different states of the enzyme reaction, providing an underlying structural basis for how these peptides play essential roles in coordinating the two glycosyltransferase activities for K4CP to elongate the chondroitin chain.

High cell density cultivation of Escherichia coli K4 in a microfiltration bioreactor: a step towards improvement of chondroitin precursor production.

BACKGROUND: The bacteria Escherichia coli K4 produces a capsular polysaccharide (K4 CPS) whose backbone is similar to the non sulphated chondroitin chain. The chondroitin sulphate is one of the major components of the extra-cellular matrix of the vertebrate connective tissues and a high value molecule, widely employed as active principle in the treatment of osteoarthritis. It is usually obtained by extraction from animal tissues, but the risk of virus contaminations, as well as the scarceness of raw material, makes this productive process unsafe and unable to satisfy the growing market demand. In previous studies a new biotechnological process to produce chondroitin from Escherichia coli K4 capsular polysaccharide was investigated and a 1.4 g·L(-1) K4 CPS concentration was reached using fed-batch fermentation techniques. In this work, on the trail of these results, we exploited new fermentation strategies to further improve the capsular polysaccharide production. RESULTS: The inhibitory effect of acetate on the bacterial cells growth and K4 CPS production was studied in shake flask conditions, while a new approach, that combined the optimization of the feeding profiles, the improvement of aeration conditions and the use of a microfiltration bioreactor, was investigated in three different types of fermentation processes. High polysaccharide concentrations (4.73 ± 0.2 g·L(-1)), with corresponding average yields (0.13 ± 0.006 gK4 CPS·gcdw(-1)), were obtained; the increase of K4 CPS titre, compared to batch and fed-batch results, was of 16-fold and 3.3-fold respectively, while average yield was almost 3.5 and 1.4 fold higher. CONCLUSION: The increase of capsular polysaccharide titre confirmed the validity of the proposed fermentation strategy and opened the way to the use of the microfiltration bioreactor for the biotechnological production of chondroitin.

The C. elegans hyaluronidase: a developmentally significant enzyme with chondroitin-degrading activity at both acidic and neutral pH.

Mammalian hyaluronidases degrade hyaluronan and some structurally related glycosaminoglycans. We generated a deletion mutant in the Caenorhabditis elegans orthologue of mammalian hyaluronidase, hya-1. Mutant animals are viable and grossly normal, but exhibit defects in vulval morphogenesis and egg-laying and showed increased staining with alcian blue, consistent with an accumulation of glycosaminoglycan. A hya-1::GFP reporter was expressed in a restricted pattern in somatic tissues of the animal with strongest expression in the intestine, the PLM sensory neurons and the vulva. Total protein extracts from wild-type animals exhibited chondroitin-degrading but not hyaluronan-degrading activity. Chondroitinase activities were observed at both neutral and acidic pH conditions while both neutral and acidic activities were absent in extracts from hya-1 mutant strains. We also evaluated the function of oga-1, which encodes the C. elegans orthologue of MGEA-5, a protein with hyaluronan-degrading activity in vitro. oga-1 is expressed in muscles, vulval cells and the scavenger-like coelomocytes. An oga-1 mutant strain exhibited egg-laying and vulval defects similar to those of hya-1; chondroitinase activity was unaffected in this mutant.

Analysis of the biosynthesis genes and chemical components of the capsule of Avibacterium paragallinarum.

The aim of this study was to investigate biosynthesis genes and chemical components of the capsule of Avibacterium paragallinarum. The sequence of a 10-kb region containing the capsule biosynthetic locus of Av. paragallinarum was determined. Two reference strains, i.e., 221 (serovar A) and H18 (serovar C), together with four Taiwanese field strains (all serovar C) were sequenced. The results showed that there are two genotypes (I and II) of the capsule biosynthetic locus in Av. paragallinarum, and the capsule genotype is independent of the serovar. The capsule biosynthetic loci of genotypes I and II consisted of six and five genes, respectively. The genotype I genes encoded proteins that are most similar to proteins from Pasteurella multocida capsule types A and F while the genotype II genes encoded proteins most similar to proteins from P. multocida capsule type D and Escherichia coli K5. The results suggested that genotype I strains contain hyaluronan or chondroitin in the capsule wall while genotype II contain heparosan. Enzymatic digestion of the capsule materials extracted from Av. paragallinarum showed that genotype I strains contained chondroitin while genotype II strains contained heparosan in the capsule. This is the first report on the existence of different genotypes of capsule biosynthesis genes in Av. paragallinarum and the presence of chondroitin and heparosan as chemical components of the capsule of Av. paragallinarum.

Biosynthesis of chondroitin and heparan sulfate in chinese hamster ovary cells depends on xylosyltransferase II.

Xylosyltransferase (XylT) catalyzes the initial enzymatic reaction in the glycosaminoglycan assembly pathway for proteoglycan biosynthesis. Its activity is thought to be rate-limiting. Two xylosyltransferases have been found using genomic analyses, and one of these, XylT1, has been shown to have xylosyltransferase activity. On the other hand, the less studied XylT2 in recombinant form lacks xylosyltransferase activity and has no known function. Wild-type Chinese hamster ovary cells express abundant Xylt2 mRNA levels and lack detectable Xylt1 mRNA levels. Analysis of a previously described Chinese hamster ovary cell xylosyltransferase mutant (psgA-745) shows that it harbors an Xylt2 nonsense mutation and fails to assemble glycosaminoglycans onto recombinant biglycan. Transfection of this cell line with a murine Xylt2 minigene results in the production of recombinant chondroitin sulfate-modified biglycan core protein and restoration of fibroblast growth factor binding to cell surface-associated heparan sulfate. Expression analyses on 10 different human transformed cell lines detect exclusive XYLT2 expression in two and co-expression of XYLT1 and XYLT2 in the others but at disparate ratios where XYLT2 expression is greater than XYLT1 in most cell lines. These results indicate that XylT2 has a significant role in proteoglycan biosynthesis and that cell type may control which family member is utilized.