Purification, Characterization, and Molecular Cloning of a Novel Keratan Sulfate Hydrolase, Endo-β-N-acetylglucosaminidase, from Bacillus circulans

Abstract

Keratan sulfate (KS) is degraded by various enzymes including endo-β-galactosidase, keratanase, and keratanase II, which are used for the structural analysis of KS. We purified a novel KS hydrolase, endo-β-N-acetylglucosaminidase, from the cell pellet and conditioned medium of Bacillus circulans, by sequential chromatography using DE52 and phenyl-Sepharose columns with ∼63- and 180-fold purity and 58 and 12.5% recovery, respectively. Like keratanase II of Bacillus sp. Ks36, the enzyme, designated Bc keratanase II, hydrolyzed KS between the 4GlcNAcβ1–3Gal1 structure (endo-β-N-acetylglucosaminidase), but not hyaluronan, heparan sulfate, heparin, and chondroitin sulfate C, demonstrating a strict specificity to KS. The enzyme digested shark cartilage KS to disaccharides and tetrasaccharides and bovine cornea KS to hexasaccharide, indicating that it prefers highly sulfated KS. Distinct from keratanase II of strain Ks36, the enzyme digested shark cartilage KS at an optimal temperature of 55 °C. Based on partial peptide sequencing of the enzyme, we molecularly cloned the gene, which encodes a protein with a predicted molecular mass of ∼200 kDa. From the deduced protein sequence, Bc keratanase II contained a domain at the C terminus, homologous to the S-layer-like domain of pullulanase from Thermoanaerobacterium thermosulfurigenes and endoxylanase from Thermoanaerobacterium saccharolyticum, and a carbohydrate-binding domain, which may serve to specifically recognize KS chains. A full-length recombinant enzyme showed keratanase II activity. These results may prove useful for the structural analysis of KS toward achieving an understanding of its function. Keratan sulfate (KS) is degraded by various enzymes including endo-β-galactosidase, keratanase, and keratanase II, which are used for the structural analysis of KS. We purified a novel KS hydrolase, endo-β-N-acetylglucosaminidase, from the cell pellet and conditioned medium of Bacillus circulans, by sequential chromatography using DE52 and phenyl-Sepharose columns with ∼63- and 180-fold purity and 58 and 12.5% recovery, respectively. Like keratanase II of Bacillus sp. Ks36, the enzyme, designated Bc keratanase II, hydrolyzed KS between the 4GlcNAcβ1–3Gal1 structure (endo-β-N-acetylglucosaminidase), but not hyaluronan, heparan sulfate, heparin, and chondroitin sulfate C, demonstrating a strict specificity to KS. The enzyme digested shark cartilage KS to disaccharides and tetrasaccharides and bovine cornea KS to hexasaccharide, indicating that it prefers highly sulfated KS. Distinct from keratanase II of strain Ks36, the enzyme digested shark cartilage KS at an optimal temperature of 55 °C. Based on partial peptide sequencing of the enzyme, we molecularly cloned the gene, which encodes a protein with a predicted molecular mass of ∼200 kDa. From the deduced protein sequence, Bc keratanase II contained a domain at the C terminus, homologous to the S-layer-like domain of pullulanase from Thermoanaerobacterium thermosulfurigenes and endoxylanase from Thermoanaerobacterium saccharolyticum, and a carbohydrate-binding domain, which may serve to specifically recognize KS chains. A full-length recombinant enzyme showed keratanase II activity. These results may prove useful for the structural analysis of KS toward achieving an understanding of its function. Keratan sulfate (KS) 1The abbreviations used are: KS, keratan sulfate; CSC, chondroitin 6-sulfate; Gal(6S), 6-O-sulfogalactose; GlcNAc(6S), 6-O-sulfo-N-acetylglucosamine; HPLC, high performance liquid chromatography; GPC, gel permeation chromatography; CE, capillary electrophoresis; PBS, phosphate-buffered saline; GAG, glycosaminoglycan; SLH, surface layer homology. is a glycosaminoglycan (GAG) whose main structure comprises β1–3 repeats of the galactosyl β1–4GlcNAc disaccharide unit and is classified into three types based on linkage structure as follows: KS-I, KS-II, and KS-III (1Funderburgh J.L. Glycobiology. 2000; 10: 951-958Crossref PubMed Scopus (346) Google Scholar). KS-I is N-linked at the reducing end of GlcNAc to Asn residues in proteins. This type of KS is a major component of cornea and contributes to its transparency. KS-II is mainly localized in cartilage and is O-linked at the reducing end of GlcNAc to Ser/Thr in proteins, a link susceptible to alkaline treatment, compared with the N-link of KS-I. KS-III is localized in the brain and is O-linked to Ser/Thr via mannose. In addition to the linkage region, KS chains differ in sulfate content and the proportions of minor sugar components including sialic acid, fucose, and N-acetylgalactosamine. Thus, KS represents a variety of forms in tissues. Recent studies have revealed various in vivo functions of KS chains in embryogenesis, cancer invasion, immune responses, and cartilage metabolism. The KS chain is expressed in various tissues in the embryo and regulates organogenesis by affecting filtration and differentiation in the kidney (2Hemming F.J. Saxod R. Histochem. Cell Biol. 1998; 110: 189-200Crossref PubMed Scopus (6) Google Scholar), migration of primary germ cells in the gonads or of neural crest cells in the heart and gut (3Oakley R.A. Tosney K.W. Dev. Biol. 1991; 147: 187-206Crossref PubMed Scopus (240) Google Scholar), and axonal guidance in the gut and skin (2Hemming F.J. Saxod R. Histochem. Cell Biol. 1998; 110: 189-200Crossref PubMed Scopus (6) Google Scholar). A sialylated KS proteoglycan serves as a specific marker for human embryonal carcinoma, which may regulate the biological behavior of the tumor (4Badcock G. Pigott C. Goepel J. Andrews P.W. Cancer Res. 1999; 59: 4715-4719PubMed Google Scholar). 6-Sulfo-N-acetyllactosamine has been identified as an epitope of dendritic cell-specific monoclonal antibodies, M-DC8, DD1, and DD2, suggesting an important role for this saccharide structure in T cell stimulation by dendritic cells (5Schakel K. Kannagi R. Kniep B. Goto Y. Mitsuoka C. Zwirner J. Soruri A. von Kietzell M. Rieber E. Immunity. 2002; 17: 289Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). KS is known to be important for aggrecan degradation by aggrecanase-1, a key enzyme in cartilage metabolism (6Pratta M.A. Tortorella M.D. Arner E.C. J. Biol. Chem. 2000; 275: 39096-39102Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Many KS-containing proteoglycans, including lumican, keratocan, mimecan, fibromodulin, and phosphacan, have been identified and their in vivo roles studied by using knockout mice (7Jepsen K.J. Wu F. Peragallo J.H. Paul J. Roberts L. Ezura Y. Oldberg A. Birk D.E. Chakravarti S. J. Biol. Chem. 2002; 277: 35532-35540Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 8Ameye L. Aria D. Jepsen K. Oldberg A. Xu T. Young M.F. FASEB J. 2002; 16: 673-680Crossref PubMed Scopus (277) Google Scholar). As these proteoglycans function not only via their core protein but also via their GAG chains, detailed characterization of KS chains is necessary to define the structure for their specific functions. Any fingerprinting method for analyzing the structure of KS requires both fragmentation usually with KS-degrading enzymes and subsequent separation. At present, the various KS-degrading enzymes are classified into three groups based on their cleavage sites and substrate specificity. The first are the endo-β-galactosidases identified in Escherichia freundii (9Fukuda M.N. Matsumura G. J. Biol. Chem. 1976; 251: 6218-6225Abstract Full Text PDF PubMed Google Scholar), Coccobacterium sp. (10Hirano S. Meyer K. Biochem. Biophys. Res. Commun. 1971; 44: 1371-1375Crossref PubMed Scopus (26) Google Scholar), Flavobacterium keratolyticus (11Kitamikado M. Ito M. Li Y.T. J. Biol. Chem. 1981; 256: 3906-3909Abstract Full Text PDF PubMed Google Scholar), and Bacteroides fragilis (12Scudder P. Uemura K. Dolby J. Fukuda M.N. Feizi T. Biochem. J. 1983; 213: 485-494Crossref PubMed Scopus (58) Google Scholar), which hydrolyze the internal nonsulfated galactosidic bonds of KS. The second is the keratanase identified in Pseudomonas sp. IFO-13309 (13Nakazawa K. Suzuki S. J. Biol. Chem. 1975; 250: 912-917Abstract Full Text PDF PubMed Google Scholar). This enzyme also cleaves internal β1–4 galactosidic linkages in KS but differs in substrate specificity from the endo-β-galactosidase. It requires at least one N-acetylglucosamine 6-sulfate residue and does not hydrolyze the desulfated KS polymer. The third group is the keratanase II identified in Bacillus sp. Ks36, endo-β-N-acetylglucosaminidase (14Nakazawa K. Ito M. Yamagata T. Suzuki S. Greiling H. Scott J.E. Keratan Sulfate. The Biochemical Society, London1989: 99-110Google Scholar), which cleaves N-acetylglucosamine linkages of the KS chain, releasing Galβ1–4GlcNAc disaccharides with mono- or di-sulfates, or tetrasaccharides of Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAc, both of which contain an N-acetyllactosamine structure. At present, keratanase II is defined as a KS-degrading endo-β-N-acetylglucosaminidase. Fucose residues on adjacent N-acetylglucosamines interfere with digestion of KS by keratanase, resulting in a larger number of oligosaccharides. In contrast, keratanase II of Bacillus sp. efficiently digests KS chains even with fucose residues and therefore is usually used for structural analyses of KS. However, keratanase II of Bacillus sp. Ks36 is not thermostable and may contain β-galactosidase activity as suggested recently (15Plaas A.H. West L.A. Midura R.J. Glycobiology. 2001; 11: 779-790Crossref PubMed Scopus (50) Google Scholar). In the search for other KS-degrading enzymes useful for KS analysis, we screened numerous microorganisms isolated from soil samples and identified a highly thermostable keratanase II, endo-β-N-acetylglucosaminidase, in Bacillus circulans. The purified enzyme designated as Bc keratanase II digested KS to disaccharides or tetrasaccharides at an optimal temperature of 55 °C. Based on partial amino acid sequences of the enzyme, we further performed molecular cloning. The recombinant enzyme showed activity comparable with the native keratanase II. These results will provide us with another tool for the structural analysis of KS and preparation of sulfated N-acetyllactosamine. Enzymes, Substrates, and Chromatographic Materials—Bovine cornea keratan sulfate (KS), heparan sulfate, chondroitin 6-sulfate, hyaluronan, N-acetyllactosamine, GlcNAc, β-galactosidase from Streptococcus sp., β-N-acetylhexosaminidase from Penicillium oxalicum, chondroitinase ABC from Proteus vulgaris, keratanase from Pseudomonas sp., and keratanase II from Bacillus sp. were purchased from Seikagaku Corp. (Tokyo, Japan). Heparin was obtained from Sigma. Phenyl-Sepharose CL-4B was purchased from Amersham Biosciences. DEAE-cellulose DE52 was from Whatman. A protein marker kit and Bio-Gel P-2 were obtained from Bio-Rad. Muromac anion exchanger was obtained from Muromachi Technos (Tokyo, Japan). All other chemicals used in this study were of the highest grade commercially available. Preparation of KS and KS Oligosaccharides—Shark cartilage keratan sulfate (KS) and KS oligosaccharides were prepared as reported previously (16Brown G.M. Nieduszynski I.A. Morris H.G. Abram B.L. Huckerby T.N. Block J.A. Glycobiology. 1995; 5: 311-317Crossref PubMed Scopus (29) Google Scholar). Briefly, shark cartilage KS (1 g) was dissolved in 100 mm sodium acetate buffer, pH 6.0, and digested with Bacillus sp. keratanase II (5 units) at 37 °C for 24 h. After addition of 2 volumes of absolute ethanol, the sample was kept at 25 °C overnight and was centrifuged at 3,000 for The was and to The oligosaccharides dissolved in were to a Bio-Gel P-2 and in The as by at was to a Muromac anion exchanger and with a of of oligosaccharides were After these oligosaccharides were using and mass not the of the major KS oligosaccharides by keratanase II of Bacillus sp. Ks36 digestion of shark cartilage of of in a Preparation of KS and KS of shark cartilage KS and KS disaccharide was performed to the method of Y. M. Suzuki K. T. T. K. M. R. K. S. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). of shark cartilage KS or KS disaccharide was dissolved in of and of the preparation of desulfated KS, the was at °C for 2 h. After on an of was to the The sample was dissolved in and preparation of desulfated KS the was at °C for 2 and an of was to the and dissolved in The was to a to After the been with volumes of the desulfated KS disaccharide was with and of B. was on brain heart medium brain heart and pH with shark cartilage KS. After into a 100 of medium and shark cartilage KS, pH the were at 37 °C for in a at of the was into a of medium and shark cartilage KS, pH and further at 37 °C and with an for h. keratanase was to the method of and J. Biol. Chem. Full Text PDF PubMed Google Scholar). The was performed in of 100 mm acetate buffer, pH 6.0, 100 of KS. After for at 37 the was by of the mm and mm and of and for After to the sample was with of mm and and for at the of reducing sugar was from the in at A sample was used as a unit of the activity was defined as the of the enzyme that of reducing the and of Bc B. keratanase II keratanase was purified from or of cells in the of shark cartilage KS as All and were at °C. of obtained from of was in of phosphate-buffered and with both and for at 37 °C. After the was and the pellet was addition of sulfate, the KS-degrading activity was in the between and The sample at was dissolved in of mm pH the buffer, and to a DE52 with the The enzyme was using a of to After the addition of to a of the enzyme was to a phenyl-Sepharose CL-4B Amersham with mm pH and The enzyme was using a of in the from to using of conditioned medium kept at °C for 24 the protein was of The was in mm pH and the was to with The enzyme was with a The enzyme was to a DE52 24 with mm pH and was using a of to The protein was with of sulfate, in mm pH and to a The enzyme activity was to a DE52 and using a The enzyme was to a and the enzyme was to a phenyl-Sepharose and as of KS analysis of KS oligosaccharides was performed by the method of The were performed on a with a at The was in by the sample at the The was mm sodium pH The sample was and using a capillary was performed at a of The and were and were using from on a The migration of and are and of KS and molecular and of KS oligosaccharides were by gel permeation performance liquid chromatography with columns of and a of mm at °C. of KS was digested with of the enzyme in acetate buffer, pH 6.0, at 37 °C for 24 h. After at and °C for of the were used as the The was M. T. Suzuki S. K. Biophys. PubMed Scopus Google Scholar). was performed with a The of and are in The of amino was by with a a of at °C. was at The was from the at The of N-acetylglucosamine and N-acetyllactosamine were and of enzyme was to using a After with a protein in the gel was using mm pH mm and and digested with The was to a 2 with The resulting were with a of and at a of acid sequences of the resulting were with a protein of the Bc II was performed using the on the amino acid of peptide and on the amino acid of peptide and of the as the with a of at °C for by of °C for °C for and °C for 2 The which to a partial of the enzyme gene, was into a The and of the were by and were for using analysis with the as a was performed using the and and the of the as the with a of at °C for by of °C for °C for and °C for was performed using the and and of the as the with the was performed using the and and of the as the was performed using the and and of the as the The were into sequencing was performed by the chain using an domain search was performed using and domain search of Bc II and that encodes a full-length enzyme protein was by using as and and of the as and into the using and of the enzyme an domain, the and were Escherichia cells were with the recombinant and at 37 °C in of medium and the protein was by mm to the After the cells were by at for and in of the After the cell was on The the enzyme was by at for at °C and purified by chromatography on columns of acid and of Bc B. keratanase II was purified from a pellet or conditioned A obtained from the pellet is in II. From the the enzyme was purified by to with by a chromatography using DE52 and of DE52 and phenyl-Sepharose are in of the with in a or with a not From of with a specific activity of was from the conditioned the enzyme was purified by 180-fold with 12.5% by a chromatography using and From of conditioned of the enzyme with a specific activity of was analysis of the purified enzyme by method a to a of reducing and not suggesting that the protein has of Bc keratanase in a of Bc keratanase II. The purified protein was to a gel and the were by purified enzyme we the enzyme by using shark cartilage KS as a The enzyme was at to °C and activity at °C for in 100 mm acetate buffer, pH The optimal temperature for the enzyme was 55 °C. The enzyme and activity at and The pH of the keratanase II that the enzyme is at pH As in the enzyme activity was at pH in the of on the activity were the keratanase and on the enzyme as at as acid and as showed not we the substrate specificity of this enzyme by using various GAG chains including bovine cornea KS and shark cartilage KS. both KS chains were cartilage KS was hydrolyzed that of cornea KS. In contrast, the other GAG chains including acid, chondroitin sulfate, heparan sulfate, and were demonstrating a specificity to KS chains. activity was toward KS chains, indicating an absolute of the of a sulfated residue for specificity of Bc keratanase sulfate in a we the of the enzyme The keratanase was performed at various of cornea KS and cartilage KS at 37 pH The and were from that cartilage KS was to KS disaccharides and The for cornea KS and cartilage KS was and and the was and respectively. of KS digests of shark cartilage KS and bovine cornea KS by this enzyme, using a of the cartilage KS was digested to KS disaccharide and KS oligosaccharides in the cornea KS as and were not in both digestion were obtained these KS samples were digested with keratanase II of Bacillus sp. Ks36 not These results that this enzyme is an we the cleavage of the As the of the digests by the enzyme with by keratanase II of Bacillus sp. Ks36, we compared the structure of the major disaccharide of shark cartilage KS digests by the purified enzyme 2 in and its desulfated with various KS using and In the analysis, the migration of the KS disaccharide and desulfated disaccharide was and suggesting that these were and N-acetyllactosamine, and The of the desulfated disaccharide for analyzing the amino also with that of N-acetyllactosamine analysis of the desulfated disaccharide was performed using β-galactosidase or β-N-acetylhexosaminidase digestion to the sugar The of the desulfated disaccharide from to on with β-galactosidase β-N-acetylhexosaminidase The results were obtained in N-acetyllactosamine. These results that KS disaccharide from shark cartilage KS digested with the purified enzyme is to and that the enzyme was an keratanase II, to its we this enzyme as Bc keratanase II. and of the II enzyme from a gel was digested with and the digests were purified using a by peptide The sequences of three and were obtained and designated as respectively. Based on these peptide were as using of B. as a and a of and a of using enzyme was performed to the and The an of to a protein of amino of a of the was of the From the deduced amino acid sequence, the molecular and the of the enzyme were to be and A of amino a sequence, was between the and the of the purified enzyme, We for using search and domain and we with to A at the C of the enzyme contained three and of homologous to the domain of pullulanase from thermosulfurigenes and endoxylanase from A to the of carbohydrate-binding domain was in the of amino of the enzyme, which is in F. other homologous were identified in the of Bc keratanase II. we expressed a full-length enzyme using a and its activity. A of the cell digested KS with an activity of the activity of the native enzyme expressed by B. circulans. the KS digests were using were obtained as digests with the native enzyme indicating that the recombinant protein has the keratanase II activity. a recombinant enzyme the domain was of the activity was in the not demonstrating that the domain is for the of the In this we the and molecular of a novel keratan sulfate hydrolase, endo-β-N-acetylglucosaminidase, from B. circulans. Like the keratanase II previously identified in Bacillus sp. Ks36, this enzyme cleaves KS chains releasing sulfated N-acetyllactosamine and therefore is defined as a keratanase II. The deduced amino acid of the enzyme, designated as Bc keratanase II, that the the of the carbohydrate-binding domain in the and the surface layer domain at the C The full-length protein expressed by a has enzyme activity comparable with the native enzyme, and analysis of the the of the domain to Bc keratanase II has at least three compared with the enzyme of Bacillus sp. B. a larger of the enzyme, which a and of pellet of compared with 24 from Bacillus sp. we have purified the native enzyme by using a of protein from of with keratanase II of Bacillus sp. Ks36 has been purified by a chromatography by using and sulfate with a of Bc keratanase II is thermostable with an optimal temperature of 55 °C. the enzyme is by and the activity of Bacillus sp. keratanase II is by and and by these as keratanase II of B. be useful for analysis, which is a highly and analysis of KS chains (15Plaas A.H. West L.A. Midura R.J. Glycobiology. 2001; 11: 779-790Crossref PubMed Scopus (50) Google Scholar). Recent studies revealed that keratanase II of Bacillus sp. Ks36 contained as a β-galactosidase activity that cleaves the disaccharide to and (15Plaas A.H. West L.A. Midura R.J. Glycobiology. 2001; 11: 779-790Crossref PubMed Scopus (50) Google Scholar). we have not activity in the purified Bc keratanase II, and even present, using this enzyme at a temperature 37 °C may of keratanase II is of sulfated N-acetyllactosamine. The that is specific to of dendritic cells an important role for this saccharide structure in T cell stimulation by dendritic cells (5Schakel K. Kannagi R. Kniep B. Goto Y. Mitsuoka C. Zwirner J. Soruri A. von Kietzell M. Rieber E. Immunity. 2002; 17: 289Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). The N-acetyllactosamine structure has been in a for and E. M. S. 250: PubMed Scopus Google Scholar, P.W. Full Text PDF PubMed Scopus Google Scholar, S. A. J. PubMed Scopus Google Scholar, J. R.A. E.C. Biochem. Biophys. Res. Commun. PubMed Scopus Google Scholar), which of As is a for KS oligosaccharides may have a function or may serve as a for their It is that keratanase II cleaves highly sulfated KS but not the desulfated of on activity have been in other enzymes as ABC and ABC digests chondroitin sulfate chondroitinase digests chondroitin However, the of KS-degrading enzymes strict compared with that of of at least one of the GlcNAc residues adjacent to a residue is for the of keratanase of Pseudomonas sp. but not for (14Nakazawa K. Ito M. Yamagata T. Suzuki S. Greiling H. Scott J.E. Keratan Sulfate. The Biochemical Society, London1989: 99-110Google Scholar). analysis, shark cartilage KS not to contain GlcNAc not The of on the substrate specificity to be We have performed molecular of keratanase II for the first and we have further expressed an enzyme with comparable activity to the native suggesting that this E. may be to of for and As the carbohydrate-binding domain in the is also in the of F. this domain may be important for specific of the sulfated KS chains. analysis using various forms will the and for At present, the of KS chains has not been and KS-degrading enzyme has been identified in may of a of the enzyme, which may to of of KS metabolism. We S. Suzuki and K. for We also Nieduszynski for of the