Enzymes Responsible for Synthesis of Corneal Keratan Sulfate Glycosaminoglycans
2007; Elsevier BV; Volume: 282; Issue: 41 Linguagem: Inglês
10.1074/jbc.m703695200
ISSN1083-351X
AutoresKazuko Kitayama, Yasutaka Hayashida, Kohji Nishida, Tomoya O. Akama,
Tópico(s)Carbohydrate Chemistry and Synthesis
ResumoKeratan sulfate glycosaminoglycans are among the most abundant carbohydrate components of the cornea and are suggested to play an important role in maintaining corneal extracellular matrix structure. Keratan sulfate carbohydrate chains consist of repeating N-acetyllactosamine disaccharides with sulfation on the 6-O positions of N-acetylglucosamine and galactose. Despite its importance for corneal function, the biosynthetic pathway of the carbohydrate chain and particularly the elongation steps are poorly understood. Here we analyzed enzymatic activity of two glycosyltransferases, β1,3-N-acetylglucosaminyltansferase-7 (β3GnT7) and β1,4-galactosyltransferase-4 (β4GalT4), in the production of keratan sulfate carbohydrate in vitro. These glycosyltransferases produced only short, elongated carbohydrates when they were reacted with substrate in the absence of a carbohydrate sulfotransferase; however, they produced extended GlcNAc-sulfated poly-N-acetyllactosamine structures with more than four repeats of the GlcNAc-sulfated N-acetyllactosamine unit in the presence of corneal N-acetylglucosamine 6-O sulfotransferase (CGn6ST). Moreover, we detected production of highly sulfated keratan sulfate by a two-step reaction in vitro with a mixture of β3GnT7/β4GalT4/CGn6ST followed by keratan sulfate galactose 6-O sulfotransferase treatment. We also observed that production of highly sulfated keratan sulfate in cultured human corneal epithelial cells was dramatically reduced when expression of β3GnT7 or β4GalT4 was suppressed by small interfering RNAs, indicating that these glycosyltransferases are responsible for elongation of the keratan sulfate carbohydrate backbone. Keratan sulfate glycosaminoglycans are among the most abundant carbohydrate components of the cornea and are suggested to play an important role in maintaining corneal extracellular matrix structure. Keratan sulfate carbohydrate chains consist of repeating N-acetyllactosamine disaccharides with sulfation on the 6-O positions of N-acetylglucosamine and galactose. Despite its importance for corneal function, the biosynthetic pathway of the carbohydrate chain and particularly the elongation steps are poorly understood. Here we analyzed enzymatic activity of two glycosyltransferases, β1,3-N-acetylglucosaminyltansferase-7 (β3GnT7) and β1,4-galactosyltransferase-4 (β4GalT4), in the production of keratan sulfate carbohydrate in vitro. These glycosyltransferases produced only short, elongated carbohydrates when they were reacted with substrate in the absence of a carbohydrate sulfotransferase; however, they produced extended GlcNAc-sulfated poly-N-acetyllactosamine structures with more than four repeats of the GlcNAc-sulfated N-acetyllactosamine unit in the presence of corneal N-acetylglucosamine 6-O sulfotransferase (CGn6ST). Moreover, we detected production of highly sulfated keratan sulfate by a two-step reaction in vitro with a mixture of β3GnT7/β4GalT4/CGn6ST followed by keratan sulfate galactose 6-O sulfotransferase treatment. We also observed that production of highly sulfated keratan sulfate in cultured human corneal epithelial cells was dramatically reduced when expression of β3GnT7 or β4GalT4 was suppressed by small interfering RNAs, indicating that these glycosyltransferases are responsible for elongation of the keratan sulfate carbohydrate backbone. In higher eukaryotes, high numbers of cells interact with each other and form complicated but well organized tissue structures. During the tissue formation in the developmental stage, cells recognize the surrounding environment and interact with neighboring cells and the extracellular matrix. Proteoglycans (PGs) 3The abbreviations used are: PG, proteoglycan; β3GnT7, β1,3-N-acetylglucosaminyltransferase-7; β4GalT4, β1,4-galactosyltransferase-4; CGn6ST, corneal N-acetylglucosamine 6-O sulfotransferase; KS, keratan sulfate; GAG, glycosaminoglycan; Gal, galactose; KSG6ST, keratan sulfate galactose 6-O sulfotransferase; Man, mannose; PAPS, 3′-phosphoadenosine 5′-phosphosulfate; β3GnT2, β1,3-N-acetylglucosaminyltransferease-2; β4GalT1, β1,4-galactosyltransferase-1; siRNA, small interfering RNA; HPLC, high pressure liquid chromatography; PBS, phosphate-buffered saline. 3The abbreviations used are: PG, proteoglycan; β3GnT7, β1,3-N-acetylglucosaminyltransferase-7; β4GalT4, β1,4-galactosyltransferase-4; CGn6ST, corneal N-acetylglucosamine 6-O sulfotransferase; KS, keratan sulfate; GAG, glycosaminoglycan; Gal, galactose; KSG6ST, keratan sulfate galactose 6-O sulfotransferase; Man, mannose; PAPS, 3′-phosphoadenosine 5′-phosphosulfate; β3GnT2, β1,3-N-acetylglucosaminyltransferease-2; β4GalT1, β1,4-galactosyltransferase-1; siRNA, small interfering RNA; HPLC, high pressure liquid chromatography; PBS, phosphate-buffered saline. are glycoproteins that carry linearly elongated polysaccharides called glycosaminoglycans (GAGs) on core proteins and that are largely found in the extracellular matrix and play important roles for development, maintenance, and function of the tissues. Production of the GAG chain takes place mostly in the Golgi apparatus. Several glycosyltransferases and carbohydrate-modifying enzymes such as sulfotransferases act on GAG elongation and modification. So far almost all of the enzymes involved in GAG production are cloned and analyzed for their enzymatic activities; however, the process of GAG production is still unknown in some GAGs because of the presence of multiple enzymes with redundant activities. Keratan sulfate (KS) PGs are major components of the cornea and also found in cartilage and brain. Because of the high concentration of these molecules in the cornea, their biological function has been extensively studied and found to include maintenance of corneal extracellular matrix structure (1Cornuet P.K. Blochberger T.C. Hassell J.R. Investig. Ophthalmol. Vis. Sci. 1994; 35: 870-877PubMed Google Scholar, 2Connon C.J. Meek K.M. Kinoshita S. Quantock A.J. Exp. 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Caterson B. Tanigami A. Nakayama J. Fukada M.N. Tano Y. Nishida K. Quantock A.J. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 13333-13338Crossref PubMed Scopus (61) Google Scholar). Corneal KS PGs consist of PG core proteins, such as lumican, keratocan, and mimecan, carrying KS GAGs in an N-linked manner (10Greiling H. Jolles P Proteoglycans. Birkhauser Verlag, Basel, Switzerland1994: 101-122Crossref Scopus (19) Google Scholar, 11Funderburgh J.L. IUBMB Life. 2002; 54: 187-194Crossref PubMed Scopus (111) Google Scholar, 12Funderburgh J.L. Glycobiology. 2000; 10: 951-958Crossref PubMed Scopus (333) Google Scholar). KS GAG is a linear carbohydrate chain made of sulfated disaccharide repeats of -3Galβ1–4GlcNAcβ1- with sulfate on the 6-O position of GlcNAc and galactose (10Greiling H. Jolles P Proteoglycans. Birkhauser Verlag, Basel, Switzerland1994: 101-122Crossref Scopus (19) Google Scholar, 11Funderburgh J.L. IUBMB Life. 2002; 54: 187-194Crossref PubMed Scopus (111) Google Scholar, 12Funderburgh J.L. Glycobiology. 2000; 10: 951-958Crossref PubMed Scopus (333) Google Scholar) and exhibiting modifications such as fucose and sialic acid (13Tai G.H. Huckerby T.N. Nieduszynski I.A. Biochem. J. 1993; 291: 889-894Crossref PubMed Scopus (29) Google Scholar, 14Tai G.H. Nieduszynski I.A. Fullwood N.J. Huckerby T.N. J. Biol. Chem. 1997; 272: 28227-28231Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Production of the KS GAG chain on PGs is processed by glycosyltransferases and sulfotransferases localized in the Golgi apparatus, and matured KS PGs are secreted into the extracellular matrix. Elongation of the carbohydrate backbone of the KS GAG chain is catalyzed by enzymes of two glycosyltransferase families, β1,3-N-acetylglucosaminyltransferase (β3GnT) and β1,4-galactosyltransferase (β4GalT), and sulfation of the chain is catalyzed by two carbohydrate sulfotransferases. Recent studies of carbohydrate sulfotransferases have identified enzymes responsible for sulfation of the corneal KS GAG chain as KS galactose 6-O sulfotransferase (KSG6ST) and corneal N-acetylglucosamine 6-O sulfotransferase (CGn6ST, also known as GlcNAc6ST-5 and GST4β) (15Fukuta M. Inazawa J. Torii T. Tsuzuki K. Shimada E. Habuchi O. J. Biol. Chem. 1997; 272: 32321-32328Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 16Akama T.O. Misra A.K. Hindsgaul O. Fukuda M.N. J. Biol. Chem. 2002; 277: 42505-42513Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Because CGn6ST only transfers sulfate on the nonreducing terminal GlcNAc but not onto internal GlcNAc, sulfation of GlcNAc residues of KS GAG is coupled with KS GAG elongation (16Akama T.O. Misra A.K. Hindsgaul O. Fukuda M.N. J. Biol. Chem. 2002; 277: 42505-42513Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). On the other hand, KSG6ST transfers sulfate on galactose located both internally and on the nonreducing terminal of the carbohydrate chain (15Fukuta M. Inazawa J. Torii T. Tsuzuki K. Shimada E. Habuchi O. J. Biol. Chem. 1997; 272: 32321-32328Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 17Torii T. Fukuta M. Habuchi O. Glycobiology. 2000; 10: 203-211Crossref PubMed Scopus (49) Google Scholar). KSG6ST also prefers a sulfated carbohydrate as substrate (15Fukuta M. Inazawa J. Torii T. Tsuzuki K. Shimada E. Habuchi O. J. Biol. Chem. 1997; 272: 32321-32328Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 17Torii T. Fukuta M. Habuchi O. Glycobiology. 2000; 10: 203-211Crossref PubMed Scopus (49) Google Scholar), suggesting that galactose sulfation occurs after production of the GlcNAc-sulfated poly-N-acetyllactosamine chain and that GlcNAc sulfation is necessary for sulfation of galactose residues by KSG6ST. Patients with macular corneal dystrophy type I, which is caused by deficiency of functional CGn6ST, have no detectable highly sulfated KS in the cornea (18Akama T.O. Nishida K. Nakayama J. Watanabe H. Ozaki K. Nakamura T. Dota A. Kawasaki S. Inoue Y. Maeda N. Yamamoto S. Fujiwara T. Thonar E.J. Shimomura Y. Kinoshita S. Tanigami A. Fukuda M.N. Nat. Genet. 2000; 26: 237-241Crossref PubMed Scopus (222) Google Scholar), serum, and cartilage (19Edward D.P. Thonar E.J. Srinivasan M. Yue B.J. Tso M.O. Ophthalmology. 1990; 97: 1194-1200Abstract Full Text PDF PubMed Scopus (49) Google Scholar, 20Yang C.J. SundarRaj N. Thonar E.J. Klintworth G.K. Am. J. Ophthalmol. 1988; 106: 65-71Abstract Full Text PDF PubMed Scopus (69) Google Scholar), suggesting that GlcNAc sulfation is required to produce highly sulfated KS GAG. Observations that macular corneal dystrophy patients with CGn6ST mutations develop corneal opacities (21Klintworth G.K. Front. Biosci. 2003; 8: 687-713Crossref PubMed Scopus (145) Google Scholar) and that mice lacking the orthologous sulfotransferase display corneal thinning with abnormalities in corneal extracellular matrix structure (9Hayashida Y. Akama T.O. Beecher N. Lewis P. Young R.D. Meek K.M. Kerr B. Hughes C.E. Caterson B. Tanigami A. Nakayama J. Fukada M.N. Tano Y. Nishida K. Quantock A.J. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 13333-13338Crossref PubMed Scopus (61) Google Scholar) indicate important roles for KS GAG sulfation in the function and maintenance of the cornea. However, unlike KS sulfation, mechanisms underlying KS GAG chain elongation are poorly understood. In humans, β3GnT and β4GalT enzymes are encoded by eight and seven genes, respectively. Among these, β3GnT7 and β4GalT4 are thought to be responsible for elongation of the KS carbohydrate chain because these enzymes have higher activity for sulfated than nonsulfated substrates (22Seko A. Dohmae N. Takio K. Yamashita K. J. Biol. Chem. 2003; 278: 9150-9158Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 23Seko A. Yamashita K. FEBS Lett. 2004; 556: 216-220Crossref PubMed Scopus (38) Google Scholar); however, direct evidence in support of this hypothesis has not been reported. Here, using soluble forms of recombinant enzymes and glycosidase-assisted column chromatography, we demonstrate that β3GnT7 and β4GalT4 can produce KS GAG carbohydrate in vitro. We also observed that suppressing expression of either β3GnT7 or β4GalT4 reduced highly sulfated KS GAG production in cultured human corneal cells, indicating that these glycosyltransferases are responsible for KS GAG elongation. Construction of Expression Vectors Encoding Soluble Forms of Enzymes—cDNAs encoding the catalytic domains of β4GalT4 and β3GnT7 were PCR-amplified using human placental Marathon-Ready cDNA (Clontech, Mountain View, CA) as template and the following primers: β4GalT4, 5′-ATTGATATCCCTAAAGCAAAGGAGTTCATGGC-3′ and 5′-TAACTCGAGTCATGCACCAAACCAGAAATCCACT-3′; and β3GnT7, 5′-TTTGATATCAGTCTCACCCCTGGTCAGTTTCTGCA-3′ and 5′-TTCCTCGAGAGCAAGTGCCCTGGCCTGTCCTAGTA-3′. Amplified DNAs were digested with EcoRV and XhoI and cloned into the corresponding sites of pcDNA3.1-HSH (24Angata K. Yen T.Y. El-Battari A. Macher B.A. Fukuda M. J. Biol. Chem. 2001; 276: 15369-15377Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). To construct an expression vector for soluble KSG6ST, we amplified cDNA encoding the catalytic domain of KSG6ST using the primer pair, 5′-ATTGGATCCATGCCCCGGGCTGGCAGAG and 5′-GGTGCTCGAGGTCACGAGAAGGGGCGGAAGTC-3′. The product was digested with BamHI and XhoI and cloned into the corresponding sites of pcDNA3.1-HSH. Construction of expression vectors for intact and soluble CGn6ST was described previously (25Akama T.O. Nakayama J. Nishida K. Hiraoka N. Suzuki M. McAuliffe J. Hindsgaul O. Fukuda M. Fukuda M.N. J. Biol. Chem. 2001; 276: 16271-16278Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Preparation of Soluble Enzymes—Expression vectors or an empty pcDNA3.1-HSH were transfected individually into Lec20 cells (26Lee J. Sundaram S. Shaper N.L. Raju T.S. Stanley P. J. Biol. Chem. 2001; 276: 13924-13934Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar), kindly provided by Dr. Pamela Stanley, using Lipofectamine Plus reagent (Invitrogen) according to the manufacturer's instructions. The cells were then cultured for 24 h with α-minimum essential medium (Irvine Scientific, Santa Ana, CA) containing 10% fetal bovine serum, and the medium was replaced with Opti-MEM medium (Invitrogen) supplemented with 40 μg/ml of l-proline and cultured cells for 24 h at 37 °C. We then concentrated the culture medium up to 200-fold using a Microcon YM-30 (Millipore Corp., Bedford, MA) and mixed that medium (1:1) with glycerol for storage at -20 °C. Protein production was confirmed by Western analysis using alkaline phosphatase-conjugated anti-T7 tag antibody (Novagen, Madison, WI) and the LumiPhos WB chemiluminescence solution (Pierce). KS Synthesis in Vitro—As a starting substrate, we prepared the monosulfated trisaccharide carbohydrate 35SO3−-6GlcNAcβ1–6Manα1–6Manα-octyl by treatment of the chemically synthesized trisaccharide GlcNAcβ1–6Manα1–6Manα-octyl with soluble CGn6ST enzyme as described (16Akama T.O. Misra A.K. Hindsgaul O. Fukuda M.N. J. Biol. Chem. 2002; 277: 42505-42513Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), except the amount of the trisaccharide (2.5 nmol) and 35S-PAPS (10 μCi; PerkinElmer Life Sciences) was changed to maximize labeling efficiency (5–10 × 106 cpm/2.5 nmol of substrate). For enzymatic KS synthesis in vitro, we incubated a 50-μl reaction mixture containing 50 mm HEPES-NaOH, pH 7.2, 5 mm MnCl2, 1 mm 5′-AMP, 0.25 mm UDP-galactose (Sigma), 0.3 mm UDP-GlcNAc (Sigma), 100 μg/ml protamine chloride, 1 mm PAPS (Sigma), 3 × 106 cpm of the starting substrate, and a mixture of concentrated medium of 0.5 μl of β4GalT4, 5 μl of β3GnT7, and 5 μl of CGn6ST with or without 4 μl of KSG6ST at 37 °C for 16 h. We then stopped the reaction by incubation in boiling water and removed insoluble debris by centrifugation before column chromatography. For additional KSG6ST treatment, the reaction mixture of β3GnT7/β4GalT4/CGn6ST was incubated in boiling water and incubated for 21 h at 37 °C after the addition of KSG6ST and PAPS. Column Chromatography—To determine the size of reaction products, we performed gel filtration chromatography with a column (1.0-cm diameter × 120-cm length) of Bio-Gel P-4 (Bio-Rad) equilibrated with 100 mm ammonium acetate buffer, pH 6.8. The reaction mixture samples were applied to the column, and the eluate was collected in 1-ml fractions. The elution pattern of carbohydrate products was monitored by counting 35S radioactivity using a liquid scintillation counter. For further analyses, we pooled radioactive fractions, desalted them by Sephadex G25 gel filtration (Sigma) equilibrated with 7% (v/v) 1-propanol/water medium, and lyophilized the samples again. To identify the degree of sulfation of products, we separated them by anion exchange HPLC using a Whatman Partisil SAX-10 column (4.6-mm diameter × 25-cm length). The column was first equilibrated with 60% acetonitrile/water, and then after loading a sample, the products were eluted under the following conditions: 60% acetonitrile/water for 5 min; a linear gradient from 60% acetonitrile/water to either 45 mm (up to tetrasulfated materials); or 70 mm (up to heptasulfated materials) KH2PO4 containing 60% acetonitrile/water for 75 min. The flow rate was 1 ml/min, 1-ml fractions were collected, and 35S radioactivity was monitored as described. To evaluate sulfation status, we prepared multi-sulfated carbohydrate standards by enzymatic reactions as described previously (16Akama T.O. Misra A.K. Hindsgaul O. Fukuda M.N. J. Biol. Chem. 2002; 277: 42505-42513Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Glycosidase-associated Carbohydrate Structure Determination—To determine nonreducing terminal structure of materials in P-4 gel filtration fractions, the purified materials were treated with glycosidases and analyzed as described above. For β-galactosidase and hexosaminidase A treatments, lyophilized samples were dissolved in water and incubated with either 25 milliunits of Jack bean β-galactosidase (Seikagaku Co., Tokyo, Japan) or 5 milliunits of human placental hexosaminidase A (Sigma) in a 30-μl mixture containing 50 mm sodium citrate buffer pH 3.5, at 37 °C for 5 min (β-galactosidase) or for 16 h (hexosaminidase A). The samples were then boiled for 5 min, and the digests were separated by column chromatography. For keratanase treatment, we incubated purified samples with 75 milliunits of keratanase from Pseudomonas sp. (Seikagaku Co.) in a 20-μl mixture containing 100 mm Tris-HCl, pH 7.5, at 37 °C for an hour, stopped the enzymatic reaction by boiling, and subjected samples to column chromatography. KS Synthesis in Cultured Human Corneal Epithelial Cells—SV40-immortalized human corneal epithelial cells were maintained in Dulbecco's modified Eagle's medium/Ham's F-12 50/50 mix medium (Mediatech, Inc., Herndon, VA) supplemented with 15% fetal bovine serum, 4.2 μg/ml of bovine insulin, 0.8 μg/ml of cholera toxin (Invitrogen), 8.3 μg/ml of mouse epidermal growth factor (Invitrogen) and 33 μg/ml gentamycin (Sigma). To produce sulfated KS, we co-transfected CGn6ST and KSG6ST expression vectors into 1 × 106 cells using Nucleofector electroporator (Amaxa Inc. Gaithersburg, MD) according to the manufacturer's instruction and cultured cells in the above medium at 37 °C for 48 h. To analyze effects of glycosyltransferase expression on KS production, we co-transfected CGn6ST and KSG6ST expression vectors together with specific glycosyltransferase siRNAs (sequence information is listed in supplemental Table S1), which were obtained from Ambion (Austin, TX). As a negative control, we used a commercially available negative control siRNA (NC#2; Ambion). Western Blot Analysis of Highly Sulfated KS—Transfected cells were washed with cold PBS five times, and lysates were prepared using 120 μl of lysis buffer containing 50 mm Tris-HCl, pH 7.4, 1% Nonidet P-40, 150 mm NaCl, a 1× proteinase inhibitor mixture (Sigma), and 1 mm phenylmethylsulfonyl fluoride. After SDS-PAGE, the proteins were electroblotted to an Immobilon-P transfer membrane (Millipore). The membranes were treated with blocking buffer (10% nonfat milk in PBS) at room temperature for 1 h and then probed with 5D4 anti-KS antibody (1:4000) (Seikagaku Co.) in blocking buffer for 1 h. The membranes were washed in 0.05% Tween 20 in PBS three times and reacted with horseradish peroxidase-conjugated goat anti-mouse IgG antibody (1:4000) in blocking buffer for 1 h. After washing membranes in 0.05% Tween 20 in PBS three times, signals were detected using the ECL Plus reagent (GE Healthcare, Piscataway, NJ). Signal intensity was calculated using NIH Image 1.62 software. Quantitative Reverse Transcription-PCR Analysis—Total RNA was isolated from transfected cells by RNeasy Mini kit (Qiagen) according to the manufacturer's instruction. After DNase I treatment, we subjected samples to reverse transcription using Superscript II reverse transcriptase (Invitrogen). Quantitative PCR analysis was carried out using Power SYBR Green PCR master mix (Applied Biosystems, Foster City, CA) with a specific primer set for each glycosyltransferase gene (see supplemental Table S2). Amplification of DNA products was monitored by the Mx 3000 QPCR system (Stratagene, La Jolla, CA) using the following reaction conditions: initial denaturation at 95 °C for 10 min followed by 40 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 1 min, and extension at 72 °C for 30 s. Cyclophilin A mRNA served as the internal control. GlcNAc-sulfated Poly-N-acetyllactosamine Production by β3GnT7, β4GalT4, and CGn6ST in Vitro—To analyze enzymatic activity of β3GnT7 and β4GalT4 for KS carbohydrate synthesis in vitro, we constructed expression vectors encoding soluble glycosyltransferases and the sulfotransferases, KSG6ST and CGn6ST, and transfected Lec20 cells, a cell line derived from Chinese hamster ovary cells, with vectors individually to secrete enzymes into the medium (Fig. 1). Because Lec20 cells do not produce endogenous β4GalT1 (26Lee J. Sundaram S. Shaper N.L. Raju T.S. Stanley P. J. Biol. Chem. 2001; 276: 13924-13934Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar), most glycosyltransferase activity in the medium originates from the transfected expression vector (Fig. 1B). Enzymes prepared from the culture medium were mixed in several combinations and incubated with a monosulfated trisaccharide substrate, 35SO3−-6GlcNAcβ1–6Manα1–6Manα-octyl, and carbohydrate donor substrates, UDP-GlcNAc, UDP-Gal, and PAPS. After 16 h of incubation at 37 °C, the reaction products were analyzed by P-4 gel filtration column chromatography (Fig. 2). When the substrate was reacted with a mixture of β3GnT7 and β4GalT4 glycosyltransferases, two major products were detected as molecules larger than the substrate (Fig. 2A), indicating that the two enzymes can add carbohydrate onto the carbohydrate substrate. When we incubated the substrate with a mixture of β3GnT7, β4GalT4, and CGn6ST, we detected several products of much larger size (Fig. 2B) than products from a mixture lacking CGn6ST (Fig. 2A).FIGURE 2P-4 gel filtration patterns of enzymatic KS synthesis products in vitro. Radiolabeled monosulfated trisaccharide substrate was treated with a mixture of two enzymes, β3GnT7 and β4GalT4 (A) or a mixture of three enzymes, β3GnT7, β4GalT4, and CGn6ST (B), and analyzed on a P-4 column. Detected fractions were collected separately (fractions I-IV in B), and further analyzed by P-4 column chromatography without glycosidase treatment (closed diamonds), after hexosaminidase A treatment (open circles), after β-galactosidase treatment (closed circles), or after sequential treatments with β-galactosidase followed with hexosaminidase A (closed triangles). Analyzed chromatograms of fractions I, II, III, and IV are shown in C–F, respectively. Arrows at S, I, II, III, IV, and II-1 indicate retention positions of starting substrate, fractions I, II, III, IV, and II-1, respectively. Fractions I–IV and hexosaminidase A-treated fractions (II-1, II-2, III-1, III-2, IV-1, and IV-2) are collected separately and analyzed by SAX-10 HPLC.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To identify the carbohydrate structure of the products, we collected products separately and analyzed them by glycosidase treatment followed by column chromatography. Fraction I, containing the most abundant product, was slightly larger than the starting substrate by P-4 gel filtration column chromatography, and its size was not altered by hexosaminidase A treatment (Fig. 2C). However, a component of fraction I was converted to a digested fraction with the same retention position as the starting substrate by β-galactosidase treatment (Fig. 2C). This result indicates that the product in fraction I was a tetrasaccharide carbohydrate resulting from addition of one galactose on the nonreducing terminal of the starting trisaccharide substrate by β4GalT4. This tetrasaccharide product was next analyzed by SAX-10 anion exchange chromatography, and the product was found to be a monosulfated carbohydrate (Fig. 3A). From these results, we conclude that the product was monosulfated tetrasaccharide Galβ1-4( 35SO3−-6)GlcNAcβ1–6Manα1-6Manα1-octyl. We next analyzed the carbohydrate structure found in fraction II (Fig. 2B) using the same strategy. A chromatogram of fraction II on a P-4 column showed a broader peak (Fig. 2D), but the pattern was converted to a sharper peak by β-galactosidase treatment (fraction II′ in Fig. 2D). Meanwhile, hexosaminidase A treatment, which hydrolyzes both nonsulfated GlcNAc and 6-O-sulfated GlcNAc located on nonreducing terminal of a carbohydrate substrate (27Kresse H. Fuchs W. Glossl J. Holtfrerich D. Gilberg W. J. Biol. Chem. 1981; 256: 12926-12932Abstract Full Text PDF PubMed Google Scholar), converted constituents of fraction II into two components (II-1 and II-2 in Fig. 2D). Because the retention positions of fractions II′ and II-1 were very close but not identical and because both peaks overlapped with the original broad peak of fraction II (Fig. 2D), we conclude that fraction II contains a mixture of fractions II′ and II-1 and that the II-1 component is hydrolyzed by β-galactosidase and converted to component II′ (Fig. 2D). We also performed sequential digestion of fraction II components with β-galactosidase followed by hexosaminidase A and found that all products were converted to a single fraction having the same elution position as fraction II-2 and also fraction I (Fig. 2D). These results indicate that the carbohydrate backbone structure of fraction II is a mixture of a pentasaccharide, GlcNAcβ1–3Galβ1–4GlcNAcβ1–6Manα1–6Manα1-octyl (fraction II′), and a hexasaccharide, Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAcβ1–6Manα1–6Manα1-octyl (fraction II-1), and that hexosaminidase A treatment converted the pentasaccharide product (fraction II′) into a tetrasaccharide carbohydrate, Galβ1–4GlcNAcβ1–6Manα1–6Manα1-octyl (fraction II-2). The sulfation status of components of II, II-1, and II-2 was analyzed by a SAX-10 column (Fig. 3, B–D). Fraction II was separated into two components, monosulfated material and disulfated material (Fig. 3B). Fraction II-1 was also separated into two components, both of which eluted at the same positions as fraction II, but the proportion of mono- to disulfated materials in fraction II-1 was nearly 1:1 (Fig. 3C). Because increased sulfation was apparently due to the presence of CGn6ST, disulfated components found in fractions II and II-1 were sulfated on GlcNAc in the carbohydrate products. Fraction II-2, which was originally a pentasaccharide component in fraction II and was converted to a tetrasaccharide by hexosaminidase A, was identified as a single component of monosulfated carbohydrate (Fig. 3D). However, because hexosaminidase A hydrolyzes the nonreducing terminal GlcNAc both with or without sulfate modification, the degree of sulfation of fraction II-2 does not represent the sulfation status of pentasaccharide components originally found in fraction II. Thus, we estimated the presence of disulfated pentasaccharide components and calculated the amount of mono- and disulfated pentasaccharide components in fraction II (see supplemental Tables S3–S5). From these results, we conclude that fraction II consists of four molecules: mono- and disulfated hexasaccharide, Galβ1–4(±SO3−-6)GlcNAcβ1–3Galβ1–4( 35SO3−-6)GlcNAc-β1–6Manα1–6Manα1-octyl, and mono- and disulfated pentasaccharide, ±SO3−-6GlcNAcβ1–3Galβ1–4( 35SO3−-6) GlcNAcβ1–6Manα1–6Manα1-octyl. Similar to fraction II, components of fractions III and IV were separated into two fractions by he
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