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Isolation and expression in Escherichia coli of cslA and cslB, genes coding for the chondroitin sulfate-degrading enzymes chondroitinase AC and chondroitinase B, respectively, from Flavobacterium heparinum.

ABSTRACT: In medium supplemented with chondroitin sulfate, Flavobacterium heparinum synthesizes and exports two chondroitinases, chondroitinase AC (chondroitin AC lyase; EC 4.2.2.5) and chondroitinase B (chondroitin B lyase; no EC number), into its periplasmic space. Chondroitinase AC preferentially depolymerizes chondroitin sulfates A and C, whereas chondroitinase B degrades only dermatan sulfate (chondroitin sulfate B). The genes coding for both enzymes were isolated from F. heparinum and designated cslA (chondroitinase AC) and cslB (chondroitinase B). They were found to be separated by 5.5 kb on the chromosome of F. heparinum, transcribed in the same orientation, but not linked to any of the heparinase genes. In addition, the synthesis of both enzymes appeared to be coregulated. The cslA and cslB DNA sequences revealed open reading frames of 2,103 and 1,521 bp coding for peptides of 700 and 506 amino acid residues, respectively. Chondroitinase AC has a signal sequence of 22 residues, while chondroitinase B is composed of 25 residues. The mature forms of chondroitinases AC and B are comprised of 678 and 481 amino acid residues and have calculated molecular masses of 77,169 and 53,563 Da, respectively. Truncated cslA and cslB genes have been used to produce active, mature chondroitinases in the cytoplasm of Escherichia coli. Partially purified recombinant chondroitinases AC and B exhibit specific activities similar to those of chondroitinases AC and B from F. heparinum.

SUBMITTER: Tkalec AL

PROVIDER: S-EPMC91781 | biostudies-literature | 2000 Jan

REPOSITORIES: biostudies-literature

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Isolation and expression in Escherichia coli of cslA and cslB, genes coding for the chondroitin sulfate-degrading enzymes chondroitinase AC and chondroitinase B, respectively, from Flavobacterium heparinum.

Tkalec A L AL Fink D D Blain F F Zhang-Sun G G Laliberte M M Bennett D C DC Gu K K Zimmermann J J JJ Su H H

Applied and environmental microbiology 20000101 1

In medium supplemented with chondroitin sulfate, Flavobacterium heparinum synthesizes and exports two chondroitinases, chondroitinase AC (chondroitin AC lyase; EC 4.2.2.5) and chondroitinase B (chondroitin B lyase; no EC number), into its periplasmic space. Chondroitinase AC preferentially depolymerizes chondroitin sulfates A and C, whereas chondroitinase B degrades only dermatan sulfate (chondroitin sulfate B). The genes coding for both enzymes were isolated from F. heparinum and designated csl ...[more]

PMID: 10618199

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Project description:The success of peripheral nerve regeneration is highly dependent on the regrowth of axons within the endoneurial basal lamina tubes that promote target-oriented pathfinding and appropriate reinnervation. Restoration of nerve continuity at this structural level after nerve transection injury by direct repair and nerve grafting remains a major surgical challenge. Recently, biological approaches that alter the balance of growth inhibitors and promoters in nerve have shown promise to improve appropriate axonal regeneration and recovery of peripheral nerve function. Chondroitin sulfate proteoglycans (CSPGs) are known inhibitors of axonal growth. This growth inhibition is mainly associated with a CSPG's glycosaminoglycan chains. Enzymatic degradation of these chains with chondroitinase eliminates this inhibitory activity and, when applied in vivo, can improve the outcome of nerve repair. To date, these encouraging findings were obtained with chondroitinase ABC (a pan-specific chondroitinase). The aim of this study was to examine the distribution of CSPG subtypes in rodent, rabbit, and human peripheral nerve and to test more selective biological enzymatic approaches to improve appropriate axonal growth within the endoneurium and minimize aberrant growth. Here we provide evidence that the endoneurium, but not the surrounding epineurium, is rich in CSPGs that have glycosaminoglycan chains readily degraded by chondroitinase C. Biochemical studies indicate that chondroitinase C has degradation specificity for 6-sulfated glycosaminoglycans found in peripheral nerve. We found that chondroitinase C degrades and inactivates inhibitory CSPGs within the endoneurium but not so much in the surrounding nerve compartments. Cryoculture bioassays (neurons grown on tissue sections) show that chondroitinase C selectively and significantly enhanced neuritic growth associated with the endoneurial basal laminae without changing growth-inhibiting properties of the surrounding epineurium. Interestingly, chondroitinase ABC treatment increased greatly the growth-promoting properties of the epineurial tissue whereas chondroitinase C had little effect. Our evidence indicates that chondroitinase C effectively degrades and inactivates inhibitory CSPGs present in the endoneurial Schwann cell basal lamina and does so more specifically than chondroitinase ABC. These findings are discussed in the context of improving nerve repair and regeneration and the growth-promoting properties of processed nerve allografts.

Dataset Information

Isolation and expression in Escherichia coli of cslA and cslB, genes coding for the chondroitin sulfate-degrading enzymes chondroitinase AC and chondroitinase B, respectively, from Flavobacterium heparinum.

Publications

Isolation and expression in Escherichia coli of cslA and cslB, genes coding for the chondroitin sulfate-degrading enzymes chondroitinase AC and chondroitinase B, respectively, from Flavobacterium heparinum.

Similar Datasets

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