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

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.     

Purification and characterization of heparinase from Flavobacterium heparinum

Heparinase (EC 4.2.2.7) isolated from Flavobacterium heparinum was purified to homogeneity by a combination of hydroxylapatite chromatography, repeated gel filtration chromatography, and chromatofocusing. Homogeneity was established by the presence of a single band on both sodium dodecyl sulfate and acid-urea gel electrophoretic systems. Amino acid analysis shows that the enzyme contains relatively high amounts of lysine residues (9%) consistent with its cationic nature (pI 8.5) but contains only 4 cysteine residues/polypeptide. The molecular weight of heparinase was estimated to be 42,900 +/- 1,000 daltons by gel filtration and 42,700 +/- 1,200 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is very specific, acting only on heparin and heparan monosulfate out of 12 similar polysaccharide substrates tested. It has an activity maximum at pH 6.5 and 0.1 M NaCl and a stability maximum at pH 7.0 and 0.15 M NaCl. The Arrhenius activation energy was found to be 6.3 kcal/mol. However, the enzyme is very sensitive to thermal denaturation and loses activity very rapidly at temperatures over 40 degrees C. Kinetic studies of the heparinase reaction at 37 degrees C gave a Km of 8.04 X 10(-6) M and a Vm of 9.85 X 10(-5) M/min at a protein concentration of 0.5 microgram/ml. By adapting batch procedures of hydroxylapatite and QAE (quaternary aminoethyl)-Sephadex chromatography, gram quantities of heparinase that is nearly free of catalytic enzyme contaminants can be purified in 4-5 h.     

Sulfur regulation of heparinase and sulfatases in Flavobacterium heparinum.

Sulfur regulation of heparinase synthesis and sulfatase synthesis was studied in Flavobacterium heparinum. Heparinase synthesis was strongly repressed by sulfate and L-cysteine, while the activity of this enzyme showed little or no inhibition by these compounds. Heparinase was synthesized in the absence of heparin when L-methionine was used as the sole sulfur source. The sulfatases produced by F. heparinum, which include the sulfatases involved in heparin catabolism, were also studied. At least some of the sulfatase activity was regulated by sulfur compounds in a manner similar to heparinase regulation. L-Cysteic acid and taurine were not suitable sulfur sources to support the growth of F. heparinum.     

Enhanced soluble expression of recombinant Flavobacterium heparinum heparinase I in Escherichia coli by fusing it with various soluble partners

Heparinase I (HepA) was originally isolated from Flavobacterium heparinum (F. heparinum) and specifically cleaves heparin/heparan sulfate in a site-dependent manner, showing great promise for producing low molecular weight heparin (LMWH). However, expressing recombinant HepA is extremely difficult in Escherichia coli because it suffers from low yields, insufficient purity and insolubility. In this paper, we systematically cloned and fused the HepA gene to the C-terminus of five soluble partners, including translation initiation factor 2 domain I (IF2), glutathione S-transferase (GST), maltose-binding protein (MBP), small ubiquitin modifying protein (SUMO) and N-utilization substance A (NusA), to screen for their abilities to improve the solubility of recombinant HepA when expressed in E. coli. A convenient two-step immobilized metal affinity chromatography (IMAC) method was utilized to purify these fused HepA hybrids. We show that, except for NusA, the fusion partners dramatically improved the soluble expression of recombinant HepA, with IF2-HepA and SUMO-HepA creating almost completely soluble HepA (98% and 94% of expressed HepA fusions are soluble, respectively), which is the highest yield rate published to the best of our knowledge. Moreover, all of the fusion proteins show comparable biological activity to their unfused counterparts and could be used directly without removing the fusion tags. Together, our results provide a viable option to produce large amounts of soluble and active recombinant HepA for manufacturing.     

Sulfur regulation of heparinase and sulfatases in Flavobacterium heparinum

Sulfur regulation of heparinase synthesis and sulfatase synthesis was studied in Flavobacterium heparinum. Heparinase synthesis was strongly repressed by sulfate and L-cysteine, while the activity of this enzyme showed little or no inhibition by these compounds. Heparinase was synthesized in the absence of heparin when L-methionine was used as the sole sulfur source. The sulfatases produced by F. heparinum, which include the sulfatases involved in heparin catabolism, were also studied. At least some of the sulfatase activity was regulated by sulfur compounds in a manner similar to heparinase regulation. L-Cysteic acid and taurine were not suitable sulfur sources to support the growth of F. heparinum.     

Mutations affecting the structural genes and the genes coding for modifying enzymes for ribosomal proteins in Escherichia coli.

Summary Temperature-sensitive mutants of an Escherichia coli K-12 strain PA3092 have been isolated following mutagenesis with nitrosoguanidine, and their ribosomal proteins analyzed by two-dimensional gel electrophoresis This method was found to be very efficient in obtaining mutants with various structural alterations in ribosomal proteins. Thus a total of some 160 mutants with alterations in 41 different ribosomal proteins have so far been isolated. By characterizing these mutants, we could isolate, not only those mutants with alterations in the structural genes for various ribosomal proteins, but also those with impairments in the modification of proteins S5, S18 and L12. Furthermore, a mutant has been obtained which apparently lacks the protein S20 (L26) with a concomitant reduction to a great extent of the polypeptide synthetic activity of the small subunit. The usefulness of these mutants in establishing the genetic architecture of the genes coding for the ribosomal proteins and their modifiers is discussed.     

Structure of heparin. Characterization of the products formed from heparin by the action of a heparinase and a heparitinase from Flavobacterium heparinum.

The total degradation of heparin by the joint action of a purified heparinase and a heparitinase from Flavobacterium heparinum is reported. The heparinase acts directly upon heparin, yielding 52% of a trisulfated disaccharide (O-(alpha-L-ido-4-enepyranosyluronic acid 2-sulfate)-(1leads to 4)-2sulfoamino-2-deoxy-D-glucose 6-sulfate) and 40% of a tetrasaccharide besides small amounts of hexa- and disaccharides. The tetrasaccharide is in turn completely degraded by the heparitinase, forming trisulfated disaccharide and disulfated disaccharide (O-(alpha-D-glyco-4-enepyranosyluronic acid)-(1leads to 4)-2-sulfoamino-2-deoxy-D-glucose 6-sulfate) in equal amounts. These and other results indicate that the tri- and disulfated disaccharides are linked alternately, in a proportion of 3:1, respectively. The primary structure of heparin and the mode of action of the heparinase and the heparitinase are proposed based on the analysis of the different products formed by the action of the enzymes.     

Isolation of the lux genes from Photobacterium leiognathi and expression in Escherichia coli

Genes necessary for luminescence (lux genes) in the marine bacterium Photobacterium leiognathi, strain PL721, were isolated and expressed in Escherichia coli. A 15-kb fragment obtained from a partial digestion of PL721 DNA with HindIII was cloned into the plasmid pACYC184, resulting in the hybrid plasmid pSD721. When pSD721 was transformed into E. coli ED8654, the resulting transformants were luminous with no additions to the cells, indicating that it contained the structural genes coding for the alpha and beta subunits of luciferase (luxA and luxB), and for components involved in aldehyde biosynthesis. Hybridization analysis with luxA and luxB 32P probes confirmed the location of these two genes on the 15-kb insert. When pSD721 was transformed into four different strains of E. coli, luminescence expression varied widely in amount and in pattern. In some strains, luminescence developed like an autoinducible system, and at maximum induction was very bright, even with no addition of aldehyde, while in others, luminescence was 100-fold less, and no induction was seen. In no case was luminescence affected by shifts in temperature, osmolarity, or iron concentration. These results indicate that, while the complete lux regulon is apparently contained on the 15-kb cloned fragment, the regulation of the lux regulon in pSD721 is subject to host controls by E. coli, controls which vary widely among different E. coli strains.     

Cloning and expression in Escherichia coli of mercuric ion resistance coding genes from Zymomonas mobilis.

From a genomic library of Zymomonas mobilis prepared in Escherichia coli, two clones (carrying pZH4 and pZH5) resistant to the mercuric ion were isolated. On partial restriction analysis these two clones appeared to have the same 2.9 kb insert. Mercuric reductase activity was assayed from the Escherichia coli clone carrying pZH5 and it was Hg(2+)-inducible, NADH dependent and also required 2-mercaptoethanol for its activity. The plasmid pZH5 encoded three polypeptides, mercuric reductase (merA; 65 kDa), a transport protein (merT 18-17 kDa) and merC (15 kDa) as analysed by SDS-PAGE. Southern blot analysis showed the positive signal for the total DNA prepared from Hgr Z. mobilis but not with the Hgs strain which was cured for a plasmid (30 kb). These results were also confirmed by isolating this plasmid from Hgr Z. mobilis and transforming into E. coli. Moreover the plasmid pZH5 also hybridized with the mer probes derived from Tn21.     

On the structure of heparitin sulfates. Analyses of the products formed from heparitin sulfates by two heparitinases and a heparinase from Flavobacterium heparinum.

The analyses of the products formed from heparitin sulfates by the action of two heparitinases and a heparinase from Flavorbacterium heparinum is reported. Heparitin sulfates A and B are degraded by heparitinase I yielding two disaccharides, one of them composed of N-acetylucosamine and an unsaturated uronic, joined by alpha(1 lead to 4) linkage, and the other, with the same composition but with an O-sulfate at the hexosamine moiety. A third disaccharide is also formed from heparitin sulfate B, by the action of the same enzyme, composed of glucosamine N-sulfate and an unsaturated uronic acid joined probably by alpha(1 lead to 4) linkage. Besides these three disaccharides, heparitin sulfate B yields, by the action of heparitinase I, an oligosaccharide (with an average molecular weight of 6000) which is completely degraded by the heparitinase II yielding a disaccharide composed of glucosamine 2,6-disulfate and unsaturated uronic acid. All the disaccharides are further degraded by alpha-glycuronidase from Flavobacterium heparinum yielding the respective monosaccharides. Based on these and other analyses the possible structures of the heparitin sulfates are proposed.     

Purification and characterization of heparin lyases from Flavobacterium heparinum.

Heparin lyase I has been purified from Flavobacterium heparinum and has been partially characterized (Yang, V. C., Linhardt, R. J., Berstein, H., Cooney, C. L., and Langer, R. (1985) J. Biol. Chem. 260, 1849-1857). There has been no report of the purification of the other polysaccharide lyases from this organism. Although all three of these heparin/heparan sulfate lyases are widely used, with the exception of heparin lyase I, there is no information on their purity or their physical and kinetic characteristics. The absence of pure heparin lyases and a lack of understanding of the optimal catalytic conditions and substrate specificity has stood in the way of the use of these enzymes as reagents for the specific depolymerization of heparin and heparan sulfate into oligosaccharides for structure and activity studies. This paper describes a single, reproducible scheme to simultaneously purify all three of the heparin lyases from F. heparinum to apparent homogeneity. Heparin lyase I (heparinase, EC 4.2.2.7), heparin lyase II (no EC number), and heparin lyase III (heparitinase, EC 4.2.2.8) have molecular weights (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and isoelectric points (by isoelectric focusing) of M(r) 42,800, pI 9.1-9.2, M(r) 84,100, pI 8.9-9.1, M(r) 70,800, pI 9.9-10.1, respectively. Their amino acid analyses and peptide maps demonstrate that while these proteins are different gene products they are closely related. The kinetic properties of the heparin lyases have been determined as well as the conditions to optimize their activity and stability. These data should improve the application of these important enzymes in the study of heparin and heparan sulfate.     

Heparinase II from Flavobacterium heparinum. HPLC analysis of the saccharides generated from chemically modified heparins.

Saccharides produced by the action of heparinase II on native pig mucosal heparin (heparin IS), de-N-sulphated heparin (heparin IH), N-acetylheparin (heparin IA), de-N/O-sulphated heparin (heparin IVH), de-O-sulphated heparin (heparin IVS) and de-O-sulphated N-acetylheparin (heparin IVA) were analysed by reversed-phase HPLC using Spherisorb ODS2. Fractions obtained by gel filtration with Bio-Gel P-4 were similarly examined. Heparin IS gave delta UA-2S----GlcNS-6S (IS) as the major unsaturated disaccharide and lesser amounts of delta UA----GlcNS-6S (IIS), delta UA-2S----GlcNS (IIIS), delta UA----GlcNS (IVS), delta UA-2S----GlcNAc-6S (IA), delta UA----GlcNAc-6S (IIA), delta UA-2S----GlcNAc (IIIA) and delta UA----GlcNAc (IVA). Heparins IA, IVA and IVS gave as the predominant unsaturated disaccharide that corresponding to the major repeat structure of the polymer. These were respectively delta UA-2S----GlcNAc-6S (IA), delta UA-GlcNAc (IVA) and delta UA----GlcNS (IVS). Minor disaccharides from the heterogeneous structure in native pig heparin and from residual O-sulphates after the de-O-sulphating process were detected. Heparin IH was degraded more slowly than any of the N-substituted heparins. The predominant unsaturated disaccharide was IH, which was derived from the major repeating unit. In addition, disaccharides IIH, IIIH, IA, IIA and IVA were detected. Heparin IVH showed little degradation, the unsaturated disaccharide IVH not being detected after 24 h. Disaccharide IVA was obtained from the heterogeneous sequence in heparin IVH. Several higher oligosaccharides were identified in the gel-filtration fractions including saccharides from the linkage region (for heparin IS and IVA) and the anti-thrombin binding site (for heparin IS only). A tetrasaccharide and hexasaccharide, with the structures delta UA----GlcNAc----UA----GlcNAc and delta UA----GlcNAc----UA----GlcNAc----UA----GlcNAc, were present in the HPLC profiles of heparins IA and IVA.     

Isolation of DNA encoding sucrase genes from Streptococcus salivarius and partial characterization of the enzymes expressed in Escherichia coli.

Restriction enzyme fragments containing two sucrase genes have been isolated from a cosmid library of Streptococcus salivarius DNA. The genes were expressed in Escherichia coli cells, and the properties of both enzymes were studied in partially purified protein extracts from E. coli. One gene encoding an invertase-type sucrase was subcloned on a 2.4-kilobase-pair fragment. The sucrase enzyme had a Km for sucrose of 48 mM and a pH optimum of 6.5. The S. salivarius sucrase clone showed no detectable hybridization to a yeast invertase clone. Two overlapping subclones which had 1 kilobase pair of DNA in common were used to localize a fructosyltransferase gene. The fructosyltransferase had a Km of 93 mM and a pH optimum of 7.0. The product of the fructosyltransferase was a levan. A fructosyltransferase clone from Bacillus subtilis did not hybridize to S. salivarius DNA. The properties of the enzymes were compared with those of previously characterized sucrases.     

Recombinant expression, purification, and kinetic characterization of chondroitinase AC and chondroitinase B from Flavobacterium heparinum

Glycosaminoglycans (GAGs) are a family of complex polysaccharides involved in a diversity of biological processes, ranging from cell signaling to blood coagulation. Chondroitin sulfate (CS) and dermatan sulfate (DS) comprise a biologically important subset of GAGs. Two of the important lyases that degrade CS/DS, chondroitinase AC (EC 4.2.2.5) and chondroitinase B (no EC number), have been isolated and cloned from Flavobacterium heparinum. In this study, we outline an improved methodology for the recombinant expression and purification of these chondroitinases, thus enabling the functional characterization of the recombinant form of the enzymes for the first time. Utilizing an N-terminal 6x histidine tag, the recombinant chondroitinases were produced by two unique expression systems, each of which can be purified to homogeneity in a single chromatographic step. The products of exhaustive digestion of chondroitin-4SO(4) and chondroitin-6SO(4) with chondroitinase AC and dermatan sulfate with chondroitinase B were analyzed by strong-anion exchange chromatography and a novel reverse-polarity capillary electrophoretic technique. In addition, the Michaelis-Menten parameters were determined for these enzymes. With chondroitin-4SO(4) as the substrate, the recombinantly expressed chondroitinase AC has a K(m) of 0.8 microM and a k(cat) of 234 s(-1). This is the first report of kinetic parameters for chondroitinase AC with this substrate. With chondroitin-6SO(4) as the substrate, the enzyme has a K(m) of 0.6 microM and a k(cat) of 480 s(-1). Recombinantly expressed chondroitinase B has a K(m) of 4.6 microM and a k(cat) of 190 s(-1) for dermatan sulfate as its substrate. Efficient recombinant expression of the chondroitinases will facilitate the structure-function characterization of these enzymes and allow for the development of the chondroitinases as enzymatic tools for the fine characterization and sequencing of CS/DS.