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1.
Chondroitin sulfates, dermatan sulfate, heparan sulfate, heparin, keratan sulfate, and oligosaccharides derived from these sulfated glycosaminoglycans have been used for the measurement of sulfatase activity of rat skin extracts. Chromatographic fractionation of the extracts followed by specificity studies demonstrated the existence of five different sulfatases, specific for 1) the nonreducing N-acetylglucosamine 6-sulfate end groups of heparin sulfate and keratan sulfate, 2) the nonreducing N-acetylgalactosamine (or galactose) 6-sulfate end groups of chondroitin sulfate (or keratan sulfate), 3) the nonreducing N-acetylgalactosamine 4-sulfate end groups of chondroitin sulfate and dermatan sulfate, 4) certain suitably located glucosamine N-sulfate groups of heparin and heparan sulfate, or 5) certain suitably located iduronate sulfate groups of heparan sulfate and dermatan sulfate. Two arylsulfatases, one of which was identical in its chromatographic behaviors with the third enzyme described above, were also demonstrated in the extracts. These results taken together with those previously obtained from studies on human fibroblast cultures suggest that normal skin fibroblasts contain at least five specific sulfatases and diminished activity of any one may result in a specific storage disease. 相似文献
2.
Four constitutive enzymes, capable of degrading keratan sulfate, were isolated from Pseudomonas sp.: a particulate endoglycosidase, a soluble endoglycosidase, a soluble exo-beta-D-galactosidase and a soluble exo-beta-D-N-acetylglucosaminidase. The endoglycosidases were shown to act only upon keratan sulfate forming beta-D-2-acetamido-2-deoxy-6-O-sulfoglucosyl-(1----3)-D-galactose, as the main product. This results indicates that the enzyme catalyses the hydrolysis of beta-D-galactose-(1----4)-N-acetylglucosamine linkages. It was also shown that this monosulfated disaccharide inhibits the particulate keratan sulfate endoglycosidase. The bovine nucleus pulposus keratan sulfate is depolymerized at a lower rate and extent when compared to the corneal keratan sulfate. The soluble endoglycosidase is very labile, in contrast to the particulate enzyme, which has been stored at -20 degrees C or at 4 degrees C for at least 12 months with no loss in activity. The particulate endoglycosidase and the soluble exo-beta-D-galactosidase and exo-beta-D-N-acetylglucosaminidase are induced when the bacteria is grown in adaptative media containing either 0.1% keratan sulfate or 0.1% chondroitin sulfate. Furthermore, particulate forms of the exoenzymes were detected. The soluble endoglycosidase specific activity, in contrast, is approximately the same in extracts of cells grown in glucose, keratan sulfate or chondroitin sulfate. A chondroitin sulfate lyase was also identified in the soluble extracts of Pseudomonas sp. cells. This enzyme depolymerizes chondroitin 4-sulfate, chondroitin 6-sulfate and hyaluronic acid forming unsaturated disaccharides as main products. It is also active upon the glucuronic-acid-containing regions of the dermatan sulfate molecules. The properties of the soluble enzymes, further purified by ion-exchange chromatography, and of the particulate keratan sulfate endoglycosidase are presented. 相似文献
3.
Partial purification and characterization of 3'-phosphoadenylylsulfate:keratan sulfate sulfotransferases 总被引:1,自引:0,他引:1
Two 3'-phosphoadenylylsulfate:keratan sulfate sulfotransferases were purified 600-fold and 340-fold, respectively, from isolated bovine cornea cells. Sulfotransferase I exhibited an apparent Mr = 220,000, whereas an Mr = 140,000 was calculated for sulfotransferase II. The final preparations were both devoid of chondroitin sulfate sulfotransferase activity. The position of sulfation was determined by proton nuclear magnetic resonance spectroscopy. Sixty per cent of the sulfate ester groups formed by sulfotransferase I were linked to the C-6 atom of galactosyl residues, the other ones to the C-6 atom of N-acetylglucosamine. Sulfotransferase II showed a different specificity: 23% of the newly formed sulfate ester groups were on galactosyl and 77% on N-acetylglucosaminyl residues. Both sulfotransferase preparations acted in a cooperative manner. In the presence of both sulfotransferases, the incorporation of [35S]sulfate into keratan sulfate was up to 75% higher than could be expected from the sum of individual activities. From the specific radioactivities of the oligosaccharides produced by digestion with endo-beta-galactosidase, it was also concluded that both enzyme species reacted best with keratan sulfate segments exhibiting a relatively high degree of sulfation. 相似文献
4.
A 6-sulfatase specific for sugasr of the galactose configuration was purified 81-fold from the crude extract of Actinobacillus sp. IFO-13310. This preparation contained activity towards both N-acetylgalactosamine 6-sulfate and galactose 6-sulfate (relative activity, 2.4 : 1). The enzyme also release inorganic sulfate from the non-reducing galactose 6-sulfate end group of a trisaccharide disulfate prepared from keratan sulfate by sequential degradation with endo-beta-galactosidase, N-acetylglucosamine-6-sulfatase and exo-beta-N-acetylglucosaminidase. In addition, a tetrasaccharide trisulfate bearing the non-reducing N-acetylglucosamine 6-sulfate end group, also enzymatically prepared from keratan sulfate, was degraded to give rise to inorganic sulfate, N-acetylglucosamine and galactose by the sequential action of this enzyme, N-acetylglucosamine-6-sulfatase, exo-beta-N-acetylglucosaminidase and exo-beta-galactosidase (Charonia lampas). 相似文献
5.
Akama TO Nakayama J Nishida K Hiraoka N Suzuki M McAuliffe J Hindsgaul O Fukuda M Fukuda MN 《The Journal of biological chemistry》2001,276(19):16271-16278
Human corneal N-acetylglucosamine 6-O-sulfotransferase (hCGn6ST) has been identified by the positional candidate approach as the gene responsible for macular corneal dystrophy (MCD). Because of its high homology to carbohydrate sulfotransferases and the presence of mutations of this gene in MCD patients who lack sulfated keratan sulfate in the cornea and serum, hCGn6ST protein is thought to be a sulfotransferase that catalyzes sulfation of GlcNAc in keratan sulfate. In this report, we analyzed the enzymatic activity of hCGn6ST by expressing it in cultured cells. A lysate prepared from HeLa cells transfected with an intact form of hCGn6ST cDNA or culture medium from cells transfected with a secreted form of hCGn6ST cDNA showed an activity of transferring sulfate to C-6 of GlcNAc of synthetic oligosaccharide substrates in vitro. When hCGn6ST was expressed together with human keratan sulfate Gal-6-sulfotransferase (hKSG6ST), HeLa cells produced highly sulfated carbohydrate detected by an anti-keratan sulfate antibody 5D4. These results indicate that hCGn6ST transfers sulfate to C-6 of GlcNAc in keratan sulfate. Amino acid substitutions in hCGn6ST identical to changes resulting from missense mutations found in MCD patients abolished enzymatic activity. Moreover, mouse intestinal GlcNAc 6-O-sulfotransferase had the same activity as hCGn6ST. This observation suggests that mouse intestinal GlcNAc 6-O-sulfotransferase is the orthologue of hCGn6ST and functions as a sulfotransferase to produce keratan sulfate in the cornea. 相似文献
6.
Enzymatic sulfation of galactose residue of keratan sulfate by chondroitin 6-sulfotransferase 总被引:4,自引:3,他引:1
We have previously found that the purified chondroitin 6-sulfotransferase(C6ST), which transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate(PAPS) to position 6 of N-acetylgalactosamine in chondroitin,catalyzed the sulfation of keratan sulfate, and that both theC6ST activity and the keratan sulfate sulfotransferase (KSST)activity were expressed in COS-7 cells when C6ST cDNA was transfected.In this report we describe some properties of the KSST activitycontained in the purified C6ST, and characterize the sulfatedproducts formed from keratan sulfate and partially desulfatedkeratan sulfate. Optimal pH, requirement for cationic activators,and Km value for PAPS of the KSST activity were very similarto those of the C6ST activity. 35S-Labeled glycosaminoglycansformed from keratan sulfate and partially desulfated keratansulfate were N-deacetylated by treatment with hydrazine/hydrazinesulfate and then cleaved with HNO2 at pH 4, and the resultingproducts were reduced with NaB3H4. Analysis of the degradationproducts with paper chromatography and high performance liquidchromatography provided evidence that C6ST transferred sulfateto position 6 of galactose residue which was glycosidicallylinked to N-acetylglucosamine 6-sulfate residue or to N-acetylglucosamineresidue. Northern blot analysis using poly (A)+ RNA from 12-d-oldchick embryos indicated that the message of C6ST was expressednot only in the cartilage but also in the cornea in which keratansulfate is actively synthesized. chondroitin sulfate keratan sulfate glycosaminoglycan sulfotransferase hydrazinolysis deaminative cleavage 相似文献
7.
N-acetylglucosamine 6-sulfate residues in keratan sulfate and heparan sulfate are desulfated by the same enzyme 总被引:3,自引:0,他引:3
We have prepared a series of oligosaccharides to assess the substrate specificity of exo sulfatase activity in cultured human skin fibroblasts toward N-acetylglucosamine-6-sulfate residues present in keratan sulfate (KS) and heparan sulfate (HS). Non-reducing end alpha-GlcNAc-6-SO4 residues (derived from HS) were desulfated by a specific sulfatase that when deficient leads to the accumulation of HS and the expression of mucopolysaccharidosis type IIID (Sanfilippo D). Under the in vitro conditions studied there are two pathways for the degradation of oligosaccharides containing non-reducing end beta-GlcNAc-6-SO4 residues (derived from KS). In one pathway beta-N-acetylglucosaminidase produces GlcNAc-6-SO4 which is then desulfated. In the other pathway the beta-GlcNAc-6-SO4 residue is desulfated and then cleaved by the action of an beta-N-acetylglucosaminidase activity. There was no detectable beta-N-acetylglucosaminidase activity in fibroblasts from a Tay-Sachs patient to produce GlcNAc-6-SO4 from beta-GlcNAc-6-SO4 residues in KS of oligosaccharides. There was approximately 10% of this normal beta-N-acetylglucosaminidase activity in fibroblasts from a Sandhoff patient, suggesting the A and S forms may be involved in this reaction. Desulfation of GlcNAc-6-SO4 residues in KS, HS and the monosaccharide GlcNAc-6-SO4 was considerably reduced or not detected in fibroblasts from a Sanfilippo D patient. As KS was not detected in the urine of a Sanfilippo D patient we propose that KS degradation in these patients proceeds by the action of a beta-N-acetylglucosaminidase activity to produce GlcNAc-6-SO4 which is not further degraded.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
8.
N-Acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST), which transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to position 6 of N-acetylgalactosamine 4-sulfate in chondroitin sulfate and dermatan sulfate, was purified 19,600-fold to apparent homogeneity from the squid cartilage. SDS-polyacrylamide gel electrophoresis of the purified enzyme showed a broad protein band with a molecular mass of 63 kDa. The protein band coeluted with GalNAc4S-6ST activity from Toyopearl HW-55 around the position of 66 kDa, indicating that the active form of GalNAc4S-6ST may be a monomer. The purified enzyme transferred sulfate from PAPS to chondroitin sulfate A, chondroitin sulfate C, and dermatan sulfate. The transfer of sulfate to chondroitin sulfate A and dermatan sulfate occurred mainly at position 6 of the internal N-acetylgalactosamine 4-sulfate residues. Chondroitin sulfate E, keratan sulfate, heparan sulfate, and completely desulfated N-resulfated heparin were not efficient acceptors of the sulfotransferase. When a trisaccharide or a pentasaccharide having sulfate groups at position 4 of N-acetylgalactosamine was used as acceptor, efficient sulfation of position 6 at the nonreducing terminal N-acetylgalactosamine 4-sulfate residue was observed. 相似文献
9.
An extract of bacterial cells Pseudomonas sp. IFO-13309 grown on medium containing 0.1% bovine cornea keratan sulfate of low sulfate content degraded exhaustively bovine cornea keratan sulfate to give 2-acetamido-2-deoxy-beta-D-gluco-pyranosyl 6-sulfate-(1 goes to 3)-D-galactose, isolated by gel filtration on Sephadex G-25 and purified by preparative paper chromatography. This was reduced with sodium borotritide to give 2-acetamido-2-deoxy-beta-D-glucopyranosyl 6-sulfate-(1 goes to 3)-D-[1-3H]galactitol, purified by gel filtration on Sephadex G-15, which was an excellent substrate for the measurement of 2-acetamido-2-deoxy-D-glucose 6-sulfate sulfatase. The reduced, radioactive monosulfated disaccharide was desulfated with methanolic 70mM hydrogen chloride and purified by gel filtration on Sephadex G-15 to give O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-(1 goes to 3)-D-[1-3H]galactitol, which allowed the measurement of (1 goes to 3)-N-acetyl-beta-D-glucosaminidase. This enzyme may participate in the normal degradation of keratan sulfate. 相似文献
10.
Ten strains of Propionibacterium shermanii were tested for beta-galactosidase (beta-gal) activity. Of these ten strains, five yielded enhanced enzyme activity when cell suspensions were treated with toluene-acetone; on solvent treatment, the remaining five lost a considerable portion of the activity found in whole-cell suspensions. By using a strain yielding decreased activity upon solvent treatment, explanations for the loss in activity were sought through assays for possible alternative beta-galactoside utilization mechanisms. When this strain was assayed for beta-D-phosphogalactoside galactohydrolase by using orthonitrophenyl-beta-D-galactopyranoside-6-P04 as a substrate, the activity was wither lower or indiffernt as compared with beta-gal activity determined simultaneously. Cell suspensions of P. shermanii 7 and 22 (strains chosen for further work) grown separately on the individual substrates (lactose, glucose, galactose, and sodium lactate) did not show significant differences in beta-gal activity. Optimal temperature for beta-gal activity in untreated and toluene-acetone-treated cell suspensions of strain 7 was 52 C. With strain 22, of the temperatures tested, maximal activity in untreated cell suspensions was noted at 58 C and with solvent-treated cells at 32 C. In the cell-free extract (CFE) system, both strains exhibited maximal activity at 52 C. Optimal pH for untreated and solvent-treated cell suspensions of both strains was around 7.5. In the P. shermanii 22 CFE system, maximal activity occurred at pH 7.0; pH had very little effect on enzyme activity in P. shermanii 7 CFE. Sodium or potassium phosphate buffers in the assay system yielded the best activity. In the CFE system of these two strains, Mn2+ was definitely stimulatory, but in untreated and solvent-treated cell systems of these strains presence or absence of Mn2+ in the assay system had variable effects on enzyme activity. Maximal beta-gal activity was noted in P. shermanii 7 cells harvested after 28 h of growth at 32 C in sodium lactate broth. Sulfhydryl-group blocking agents inhibited enzyme activity in P. shermanii 22 CFE; the inhibition was partly reversed by dithiothreitol. 相似文献
11.
The residual liver acid beta-galactosidase (beta-gal) activity from a case of feline GM1 gangliosidosis was partially purified and characterized with respect to its pH optimum, kinetic properties, thermostability, isoelectric point, molecular weight, and antigenicity. In comparison to the normal enzyme, the mutant enzyme had the same pH optima for the three substrates tested, a reduced Km for 4-methylumbelliferyl-beta-gal, elevated Km's for GM1 and asialofetuin (ASF), and increased thermolability. In addition, the mutant beta-gal had a higher isoelectric point, a reduced molecular weight, and appeared to be antigenically different from normal. The results suggest that the mutation in the Birmingham GM1 cat is structural and that the residual enzyme activity is a structurally altered acid beta-gal. The apparent lack of antigenic identity between the mutant and normal enzymes, in contrast to the situation in many human GM1 patients, is most unusual. 相似文献
12.
Kitayama K Hayashida Y Nishida K Akama TO 《The Journal of biological chemistry》2007,282(41):30085-30096
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, beta1,3-N-acetylglucosaminyltansferase-7 (beta3GnT7) and beta1,4-galactosyltransferase-4 (beta4GalT4), 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 beta3GnT7/beta4GalT4/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 beta3GnT7 or beta4GalT4 was suppressed by small interfering RNAs, indicating that these glycosyltransferases are responsible for elongation of the keratan sulfate carbohydrate backbone. 相似文献
13.
H Inoue K Otsu M Yoneda K Kimata S Suzuki Y Nakanishi 《The Journal of biological chemistry》1986,261(10):4460-4469
Heparan sulfate, keratan sulfate, chondroitin, chondroitin 4/6-sulfate (80% 4-sulfate and 20% 6-sulfate), and UDP-N-acetylgalactosamine 4-sulfate were used as acceptors for the measurement of 3'-phosphoadenylyl sulfate: glycosaminoglycan sulfotransferase activities in human serum. Chromatographic fractionation of the serum followed by determination of the sulfotransferase activities demonstrated the existence of at least four different sulfotransferases capable of introducing sulfate to 1) position 6 of the internal N-acetylgalactosamine units of chondroitin, 2) position 6 of the nonreducing terminal N-acetylgalactosamine 4-sulfate unit of chondroitin 4/6-sulfate, 3) position 2 (amino group) of the glucosamine units in heparan sulfate, and 4) the sugar units in keratan sulfate, respectively. The fourth activity was separated into two subfractions with different specificities for the structure of neighboring sugars of the sulfate-accepting sugar units. No major variations in the sulfotransferase activities on added receptors were found to occur in sera from individuals 22-41 years old. In contrast, the activities in sera of various mammalian and avian species showed a species-specific variation. With mouse skin fibroblasts cultured in serum-free medium, preferential secretion of several sulfotransferases could be demonstrated. The results, taken together, suggest that the appearance of the sulfotransferases in serum is not a fortuitous event due to nonspecific cell death, but the result of an elaborate mechanism for enzyme secretion by a cell or tissue system. 相似文献
14.
A glycosidase which attacks corneal keratan sulfate was purified from extracts of Pseudomonas sp. IFO-13309. When corneal keratan sulfate was degraded by the purified enzyme, Sephadex G-50 chromatography indicated the presence of a number of oligosaccharides differing in size and sulfate content. The characterization of two major fractions of the oligosaccharides indicated that the point of enzyme attack is limited to the endo-beta-D-galactoside bonds in which nonsulfated D-galactose residues participate. The enzyme, unlike ordinary exo-beta-D-galactosidases, did not catalyze the hydrolysis of phenyl beta-D-galactoside. Moreover, beta-D-galactosyl-(1 leads to 3)-2-acetamido-2-deoxy-beta-D-glucosyl-(1 leads to 3)-beta-D-galactosyl-(1 leads to 4)-D-glucose ("lacto-N-tetraose") was completely refractory to the action of this enzyme, suggesting that a structure of the type, X-(1 leads to 3)-beta-D-galactosyl-(1 leads to 4)-Y, is not the only specificity-determining factor, i.e. neighboring sugars, X and Y, or even larger portions of substrate molecule must have an important effect. Compared with corneal keratan sulfate, keratan sulfates from human nucleus pulposus and shark cartilage were attacked at lower rates with a resultant production of oligosaccharides of relatively large size. The result is in agreement with the view that considerable variations exist in the structure of keratan sulfates of different origin, and further suggests that the enzyme may serve as a useful reagent in studying these variations. 相似文献
15.
Enzymic cleavage of beta-N-acetylglucosamine residues of keratan sulphate was studied in vitro by using substrate a [3H]glucosamine-labelled desulphated keratan sulphate with N-acetylglucosamine residues at the non-reducing end. Both lysosomal beta-N-acetylhexosaminidases A and B are proposed to participate in the degradation of keratan sulphate on the basis of the following observations. Homogenates of fibroblasts from patients with Sandhoff disease, but not those from patients with Tay--Sachs disease, were unable to release significant amounts of N-acetyl[3H]glucosamine. On isoelectric focusing of beta-N-acetylhexosaminidase from human liver the peaks of keratan sulphate-degrading activity coincided with the activity towards p-nitrophenyl beta-N-acetylglucosaminide. A monospecific antibody against the human enzyme reacted with both enzyme forms and precipitated the keratan sulphate-degrading activity. Both isoenzymes had the same apparent Km of 4mM, but the B form was approximately twice as active as the A form when compared with the activity towards a chromogenic substrate. Differences were noted in the pH--activity profiles of both isoenzymes. Thermal inactivation of isoenzyme B was less pronounced towards the polymeric substrate than towards the p-nitrophenyl derivative. 相似文献
16.
A simple procedure using human basic fibroblast growth factor (FGF) was utilized for the selection of COS cell mutants with defects in the biosynthesis or expression of heparan sulfate proteoglycan (HSPG). Our approach was based on the strong binding affinity exhibited by COS cells to human basic FGF that had been adsorbed to plastic dishes. Cell binding to basic FGF could be inhibited by heparin and heparan sulfate (HS), but not by chondroitin sulfate, dermatan sulfate, keratan sulfate, or hyaluronic acid, suggesting that the cell binding involved an interaction between basic FGF and cell surface heparin-like molecules. COS cells were treated with ethyl methanesulfonate and four stable mutants were subsequently isolated that did not bind strongly to basic FGF adsorbed to plastic. These mutants cell lines (CM-2, CM-8, CM-9, and CM-15) exhibited significantly reduced 35SO4 incorporation into HS (40-70% depending on the cellular pool analyzed). In one of these cell lines, CM-15, the incorporation of [6-3H]glucosamine into HS was unaltered, suggesting that the extent of oligosaccharide polymerization was equivalent to that observed for the wild-type cells. Structural analysis revealed that N-sulfated glucosamine residues were present much less frequently in HS derived from these cells as compared with that derived from wild-type cells. Furthermore, CM-15 was found to be three-fold deficient in HS N-sulfotransferase activity, but contained wild-type levels of HS O-sulfotransferase activities.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
17.
Akama TO Misra AK Hindsgaul O Fukuda MN 《The Journal of biological chemistry》2002,277(45):42505-42513
Among the enzymes of the carbohydrate sulfotransferase family, human corneal GlcNAc 6-O-sulfotransferase (hCGn6ST, also known as human GlcNAc6ST-5/GST4beta) and human intestinal GlcNAc 6-O-sulfotransferase (hIGn6ST or human GlcNAc6ST-3/GST4alpha) are highly homologous. In the mouse, intestinal GlcNAc 6-O-sulfotransferase (mIGn6ST or mouse GlcNAc6ST-3/GST4) is the only orthologue of hCGn6ST and hIGn6ST. In the previous study, we found that hCGn6ST and mIGn6ST, but not hIGn6ST, have sulfotransferase activity to produce keratan sulfate (Akama, T. O., Nakayama, J., Nishida, K., Hiraoka, N., Suzuki, M., McAuliffe, J., Hindsgaul, O., Fukuda, M., and Fukuda, M. N. (2001) J. Biol. Chem. 276, 16271-16278). In this study, we analyzed the substrate specificities of these sulfotransferases in vitro using synthetic carbohydrate substrates. We found that all three sulfotransferases can transfer sulfate to the nonreducing terminal GlcNAc of short carbohydrate substrates. Both hCGn6ST and mIGn6ST, but not hIGn6ST, transfer sulfate to longer carbohydrate substrates that have poly-N-acetyllactosamine structures, suggesting the involvement of hCGn6ST and mIGn6ST in production of keratan sulfate. To clarify further the involvement of hCGn6ST in biosynthesis of keratan sulfate, we reconstituted the biosynthetic pathway in vitro by sequential enzymatic treatment of a synthetic carbohydrate substrate. Using four enzymes, beta1,4-galactosyltransferase-I, beta1,3-N-acetylglucosaminyltransferase-2, hCGn6ST, and keratan sulfate Gal 6-O-sulfotransferase, we were able to synthesize in vitro a product that conformed to the basic structural unit of keratan sulfate. Based on these results, we propose a biosynthetic pathway for N-linked keratan sulfate on corneal proteoglycans. 相似文献
18.
Melita Dvorak-Ewell Dan Wendt Chuck Hague Terri Christianson Vish Koppaka Danielle Crippen Emil Kakkis Michel Vellard 《PloS one》2010,5(8)
Mucopolysaccharidosis IVA (MPS IVA; Morquio A syndrome) is a lysosomal storage disorder caused by deficiency of N-acetylgalactosamine-6-sulfatase (GALNS), an enzyme that degrades keratan sulfate (KS). Currently no therapy for MPS IVA is available. We produced recombinant human (rh)GALNS as a potential enzyme replacement therapy for MPS IVA. Chinese hamster ovary cells stably overexpressing GALNS and sulfatase modifying factor-1 were used to produce active (∼2 U/mg) and pure (≥97%) rhGALNS. The recombinant enzyme was phosphorylated and was dose-dependently taken up by mannose-6-phosphate receptor (Kuptake = 2.5 nM), thereby restoring enzyme activity in MPS IVA fibroblasts. In the absence of an animal model with a skeletal phenotype, we established chondrocytes isolated from two MPS IVA patients as a disease model in vitro. MPS IVA chondrocyte GALNS activity was not detectable and the cells exhibited KS storage up to 11-fold higher than unaffected chondrocytes. MPS IVA chondrocytes internalized rhGALNS into lysosomes, resulting in normalization of enzyme activity and decrease in KS storage. rhGALNS treatment also modulated gene expression, increasing expression of chondrogenic genes Collagen II, Collagen X, Aggrecan and Sox9 and decreasing abnormal expression of Collagen I. Intravenous administration of rhGALNS resulted in biodistribution throughout all layers of the heart valve and the entire thickness of the growth plate in wild-type mice. We show that enzyme replacement therapy with recombinant human GALNS results in clearance of keratan sulfate accumulation, and that such treatment ameliorates aberrant gene expression in human chondrocytes in vitro. Penetration of the therapeutic enzyme throughout poorly vascularized, but clinically relevant tissues, including growth plate cartilage and heart valve, as well as macrophages and hepatocytes in wild-type mouse, further supports development of rhGALNS as enzyme replacement therapy for MPS IVA. 相似文献
19.
Poon CJ Plaas AH Keene DR McQuillan DJ Last K Fosang AJ 《The Journal of biological chemistry》2005,280(25):23615-23621
Keratan sulfate is thought to influence the cleavage of aggrecan by metalloenzymes. We have therefore produced a recombinant substrate, substituted with keratan sulfate, suitable for the study of aggrecanolysis in vitro. Recombinant human G1-G2 was produced in primary bovine keratocytes using a vaccinia virus expression system. Following purification and digestion with specific hydrolases, fluorophore-assisted carbohydrate electrophoresis was used to confirm the presence of the monosulfated Gal-GlcNAc6S and GlcNAc6s-Gal disaccharides and the disulfated Gal6S-GlcNAc6S disaccharides of keratan sulfate. Negligible amounts of fucose or sialic acid were detected, and the level of unsulfated disaccharides was minimal. Treatment with keratanases reduced the size of the recombinant G1-G2 by approximately 5 kDa on SDS-PAGE. Treatment with N-glycosidase F also reduced the size of G1-G2 by approximately 5 kDa and substantially reduced G1-G2 immunoreactivity with monoclonal antibody 5-D-4, indicating that keratan sulfate on the recombinant protein is N-linked. Cleavage of G1-G2 by aggrecanase was markedly reduced when keratan sulfate chains were removed by treatment with keratanase, keratanase II, endo-beta-galactosidase, or N-glycosidase F. These results indicate that modification of oligosaccharides in the aggrecan interglobular domain with keratan sulfate, most likely at asparagine residue 368, potentiates aggrecanase activity in this part of the core protein. 相似文献
20.
Adult human articular chondrocytes were used to investigate why keratan sulfate/polylactosamine chains are deficient on the lumican residing in the matrix of adult articular cartilage, whereas they are present on the lumican residing in the matrix of juvenile cartilage. Under serum-free conditions with either monolayer cultures, agarose cultures, or micromass cultures, the adult chondrocytes synthesized a form of lumican possessing keratan sulfate/polylactosamine chains. Thus, the adult chondrocytes are capable of producing a proteoglycan form of lumican and this appears to be the default synthesis preference. The micromass culture system proved useful for demonstrating that growth factors/cytokines present in the extracellular milieu are capable of influencing the structure of the keratan sulfate/polylactosamine chains on the secreted lumican. Of particular note was the ability of IL-1beta to promote the secretion of a form of lumican deficient in keratan sulfate/polylactosamine chains, whereas with bFGF, IGF-1 and TGFbeta keratan sulfate/polylactosamine chains were present, though their size or degree of substitution varied. Thus, growth factors/cytokines are able to modulate the molecular form of lumican. Furthermore, additional studies showed that this modulation was not due to the degradation of keratan sulfate/polylactosamine chains following proteoglycan secretion, but represented a direct effect on synthesis. 相似文献