Cloning and Expression of Recombinant Chondroitinase ACII and Its Comparison to the Arthrobacter aurescens Enzyme.

Chondroitin sulfates are the glycosaminoglycan chains of proteoglycans critical in the normal development and pathophysiology of all animals. Chondroitinase ACII, a polysaccharide lyase originally isolated from Arthrobacter aurescens IAM 110 65, which is widely used in the analysis and study of chondroitin structure, is no longer commercially available. The aim of the current study is to prepare recombinant versions of this critical enzyme for the glycobiology research community. Two versions of recombinant chondroitinase ACII are prepared in Escherichia coli, and their activity, stability, specificity, and action pattern are examined, along with a non-recombinant version secreted by an Arthrobacter strain. The recombinant enzymes are similar to the enzyme obtained from Arthrobacter for all examined properties, except for some subtle specificity differences toward uncommon chondroitin sulfate substrates. These differences are believed to be due to either post-translational modification of the Arthrobacter-secreted enzyme or other subtle structural differences between the recombinant and natural enzymes. The secreted chondroitinase can serve as a suitable replacement for the original enzyme that is currently unavailable, while the recombinant ones can be applied generally in the structural determination of most standard chondroitin sulfates.

Isolation of Serratia marcescens as a chondroitinase-producing bacterium and purification of a novel chondroitinase AC.

A strain of Serratia marcescens that produced chondroitinase was isolated from soil. It produced a novel chondroitinase AC, which was purified to homogeneity. The enzyme was composed of two identical subunits of 35 kDa as revealed by SDS-PAGE and gel filtration. The isoelectric point for the chondroitinase AC was 7.19. Its optimal activity was at pH 7.5 and 40 degrees C. The purified enzyme was active on chondroitin sulfates A and C and hyaluronic acid, but was not with chondroitin sulfate B (dermatan sulfate), heparin or heparan sulfate. The apparent K(m) and V(max) of the chondroitinase AC for chondroitin sulfate A were 0.4 mg ml(-1) and 85 mmol min(-1) mg(-1), respectively, and for chondroitin sulfate C, 0.5 mg ml(-1) and 103 mmol min(-1) mg(-1), respectively.

Studies on hyaluronidase, chondroitin sulphatase, proteinase and phospholipase secreted by Candida species.

We studied the ability of different Candida species to produce, at the same time, hyaluronidase, chondroitin sulphatase, proteinase, and phospholipase to assess whether they could be related to Candida pathogenicity. Only C. albicans was able to produce the four enzymes tested (73%) and was highly virulent to mice. Strains, that lack the capacity to produce one or more of the enzymes assayed, seemed less virulent or avirulent, similarly to the spontaneous hyaluronidase, chondroitin sulphatase, phospholipase and proteinase-deficient C. albicans strain FCF 14, 1 which was non-pathogenic to mice. Among the other Candida species tested, none of them produced the four enzymes simultaneously, being less virulent in intravenously inoculated mice.

Isolation and characterization of an induced chondroitinase ABC from Flavobacterium heparinum.

During the investigation of alternative methods for the large scale preparation of chondroitinases AC, B and C from Flavobacterium heparinum, a new chondroitinase activity was observed. This new enzyme, like the other chondroitinases, acts as an eliminase, forming unsaturated sulfated disaccharides from dermatan and chondroitin sulfates. In contrast to the chondroitinases previously described, which are endoglycosidases, this chondroitinase ABC cleaves the glycosidic linkages in an exolytic fashion, beginning at the reducing end of the substrate molecules. The oligosaccharides formed as transient products by the action of either chondroitinases or testicular hyaluronidase upon dermatan and chondroitin sulfates are also rapidly degraded by the chondroitinase ABC, regardless of their size or the presence of delta-4,5 unsaturation in the terminal uronic acid residue. The maximum activity of the chondroitinase ABC occurs at 30 degrees C and at pH 6.0-7.5. Only 15% of the activity was observed at 37 degrees C, indicating that the enzyme is very sensitive to thermal denaturation. It is strongly inhibited by phosphate ions and is also inhibited by the unsaturated disaccharides formed.

Cloning and expression in Escherichia coli of a gene coding for a chondroitin lyase from Bacteroides thetaiotaomicron.

We cloned the gene for one of the two chondroitin lyases of Bacteroides thetaiotaomicron into the cosmid vector pHC79 and subcloned it into pBR328. No proteins the size of B. thetaiotaomicron chondroitin lyase I or II (104 to 108 kilodaltons) were detectable in maxicell or in vitro transcription-translation preparations. However, partial purification of the chondroitin lyase activity from the Escherichia coli subclone showed that its properties were similar to those of the B. thetaiotaomicron chondroitin lyases. Antibodies to the chondroitin lyase that was produced in E. coli cross-reacted with the B. thetaiotaomicron chondroitin lyase II but not with chondroitin lyase I. The molecular weight of the enzyme produced in E. coli, as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel filtration, was slightly lower than those of the two chondroitin lyases from B. thetaiotaomicron; the enzyme had a higher affinity for bacterial membranes and for heparin-agarose, and cyanogen bromide digestion products of the chondroitin lyase produced in E. coli differed slightly from those of B. thetaiotaomicron chondroitin lyase II. gamma delta mutagenesis was used to locate the chondroitin lyase gene on the subcloned 7.8-kilobase EcoRI fragment. The size of the gene was approximately 3.3 kilobases, as expected for a protein with a molecular weight of 104,000.

Galactosamine inhibition of protein synthesis in Bacteroides thetaiotaomicron.

Galactosamine does not support growth of Bacteroides thetaiotaomicron. Despite this, galactosamine was more effective than utilizable carbohydrates such as glucose in preventing synthesis of the inducible enzymes alpha-glucosidase and chondroitin lyase. Galactosamine also stopped overall protein synthesis. By contrast glucose and other utilizable carbohydrates increased the rate of protein synthesis. Addition of glucose to bacteria which had been treated with galactosamine restored the ability of the bacteria to synthesize protein and to produce inducible enzymes. Moreover, when B. thetaiotaomicron was incubated with [1-14C]galactosamine for 30 min at 37 degrees C, about one-third of the label which was taken up by the cells comigrated with glucosamine-6-phosphate on a thin-layer chromatogram. Thus galactosamine appears to be phosphorylated by the bacteria. After 2 h incubation of the bacteria with [1-14C]galactosamine, there was a significant increase in the amount of label which could be extracted from acidified extracellular fluid with diethyl ether. This indicates that galactosamine can be metabolized to the level of volatile fatty acids. The rate of uptake of galactosamine and the amount of labeled fatty acids produced from galactosamine were both much lower than the values obtained when glucosamine was the substrate. Thus, although some metabolism of galactosamine occurs, the rate is apparently too slow to enable galactosamine to support growth of B. thetaiotaomicron.

Induction of chondroitin sulfate lyase activity in Bacteroides thetaiotaomicron.

Chondroitin sulfate lyase (EC 4.2.2.4) was present constitutively at low levels (0.06 to 0.08 U/mg of protein) in cells of Bacteroides thetaiotaomicron which were growing on glucose or other monosaccharides. When these uninduced bacteria were incubated with chondroitin sulfate A (5 mg/ml), chondroitin sulfate lyase specific activity increased more than 10-fold within 90 min. Synthesis of ribonucleic acid and of protein was required for induction, and induction was sensitive to oxygen. The disaccharides which resulted from chondroitinase action did not act as inducers, nor did tetrasaccharides or hexasaccharides obtained by digestion of chondroitin sulfate with bovine testicular hyaluronidase. None of these substances was taken up by uninduced cells; they may not have been able to penetrate the outer membrane. The smallest oligomer capable of acting as an inducer was the outer membrane. The smallest oligomer capable of acting as an inducer was the octassacharide. Oligomers larger than the octassacharide induced chondroitin lyase activity nearly as well as intact chondroitin sulfate.