A sulfotransferase-sulfatase system in avian oviduct which catalyzes a conversion of UDP-N-acetylgalactosamine 4-sulfate to the 6-sulfate isomer

Magnum from quail oviduct was subfractionated to yield epithelium and tubular glands. The in vitro enzymatic activities involved in sulfated sugar nucleotide biosynthesis were assayed in these isolated tissues. The results demonstrated that the activities necessary for a series of reactions, UDP-N-acetylgalactosamine----UDP-N-acetylgalactosamine 4-sulfate----UDP-N-acetylgalactosamine 4,6- bisulfate ----UDP-N-acetylgalactosamine 6-sulfate, are located predominantly in the tubular gland. Both time course and pulse-chase studies with [35S]sulfate gave results that were consistent with this reaction scheme. A microsomal preparation from the magnum was shown to be capable of labeling all three sulfate sugar nucleotides with [35S]sulfate upon incubation with UDP-N-acetylgalactosamine and 3'- phosphoadenylyl [35S]sulfate. Again, their relative labeling rates were in the order necessary to allow for a synthesis of sulfated sugar nucleotides in the sequence described above. Furthermore, incubation of the microsomal preparation with UDP-N-[14C]acetylgalactosamine 4-sulfate and 3'- phosphoadenylyl sulfate resulted in the formation of UDP-N-[14C]acetylgalactosamine 6-sulfate. Also shown was the existence in the microsomal preparation of a sulfatase specific for the sulfate at position 4 of UDP-N-acetylgalactosamine 4,6- bisulfate . The results, together with those obtained in previous investigations, suggest that the tubular gland of quail oviduct contains a microsomal multienzyme system which catalyzes a series of sulfation and desulfation of N-acetylgalactosamine residues at the nonreducing terminal position of either sugar nucleotides or polysaccharide chains.     

Improved high-performance liquid chromatographic method for N-acetylgalactosamine-4-sulfate sulfatase (arylsulfatase B) activity determination using uridine diphosho-N-acetylgalactosamine-4-sulfate

UDP-N-acetylgalactosamine-4-sulfate (UDP-GalNAc-4-S) was isolated from hen oviduct (isthmus) with a yield of 31 μmol per 100 g of wet tissue and used for arylsulfatase B (ASB) activity determination. Two HPLC methods of separation and quantitation of the reaction product were described: (1) an original gradient elution method which makes it possible to determine the reaction product when only partially purified ASB was used and additional uridine derivatives were formed during incubation: (2) an improved, fast isocratic elution method which may be used in the case of purified ASB preparations, devoid of other nucleotide hydrolysing enzymes. For both methods the detection limit was 0.1 nmol of product with standard error of determination ?3%. Using the gradient elution method we have found that UDP-GalNAc-4-S was hydrolysed by bovine arylsulfatase B1 most efficiently at pH 5.0 and concentration 0.5 mM with K_m=85 μM.     

Heterogeneity of rat rib chondroitin sulfate and susceptibility to rat gastric chondrosulfatase

Cartilage chondroitin sulfate isolated directly from rat rib or from in vitro culture of rat rib constitutes a population of glycosaminoglycans which is heterogeneous with respect to size, degree of sulfation and content of N-acetylgalactosamine 4-sulfate. Fractions elute from Dowex-1 in order of increasing molecular size and degree of sulfation up to a certain limit. Unsulfated disaccharides and disulfated disaccharides are present in both the undersulfated chondroitin sulfate fractions and in the average or more representative chondroitin sulfate. A small content of disaccharide 6-sulfate is present in all fractions and appears to be an integral part of the chondroitin 4-sulfate molecules. Rat gastric chondrosulfatase hydrolyzes sulfate preferentially from the larger chondroitin 4-sulfate molecules, and the sulfate is removed primarily from the disaccharide 4-sulfate units.     

Intralysosomal formation and metabolic fate of N-acetylglucosamine 6-sulfate from keratan sulfate

The physiological relevance of the ability of beta-N-acetylhexosaminidase A to liberate N-acetylglucosamine 6-sulfate from polymeric keratan sulfate was investigated. Upon intravenous injection into rats of [35S]sulfate-labeled proteokeratan sulfate up to 25% of the radioactivity excreted with the urine were identified as N-acetyl-glucosamine 6-sulfate. Within 24 h, however, excretion of inorganic sulfate rose at the expense of the sulfated monosaccharide. Upon incubation in vitro of liver lysosomes from rats treated with proteokeratan sulfate, inorganic sulfate and minor amounts of sulfated monosaccharide were found in the incubation fluid. Cultured rat peritoneal macrophages ingested proteokeratan sulfate with a clearance rate of 6-9 micrograms X h-1 X mg cell protein-1 and degraded it rapidly. Inorganic sulfate but not N-acetylglucosamine 6-sulfate was delivered to the culture medium. During a chase period the amount of intracellular N-acetylglucosamine 6-sulfate fell, and a corresponding amount of sulfate could be found extracellularly. Significant amount of N-acetylglucosamine 6-sulfate were only found in the culture medium when the cells were challenged with zymosan. These results suggest that N-acetylglucosamine 6-sulfate is a physiological intermediate during the degradation of keratan sulfate, but is usually hydrolyzed intralysosomally by N-acetylglucosamine-6-sulfate sulfatase. Genetic deficiency of the sulfatase in humans therefore results in excessive excretion of the sulfated amino sugar but not of keratan sulfate.     

Spondyloepiphyseal dysplasia, chondroitin sulfate type: a possible defect of PAPS--chondroitin sulfate sulfotransferase in humans

Four patients with an unusual form of spondyloepiphyseal dysplasia excreted in the urine undersulfated chondroitin 6-sulfate (Biochem. Med. $\text{7}$, 415–423, 1973). The sera of these patients show a low activity of PAPS — chondroitin sulfate sulfotransferase, while the undersulfated chondroitin sulfate present in their urine is a much better acceptor of ${}^{35}\text{SO}{}_4$ than standard chondroitin sulfate when they are incubated with [${}^{35}\text{S}$]PAPS and normal sulfotransferases. These results suggest that in these patients the skeletal lesions are secondary to a defect in the synthesis of chondroitin sulfate involving specifically the sulfotransferase activity.     

Cooperation of binding sites at the hydrophilic domain of cell-surface sulfatase Sulf1 allows for dynamic interaction of the enzyme with its substrate heparan sulfate

Background

Sulf1 is a cell-surface sulfatase removing internal 6-O-sulfate groups from heparan sulfate (HS) chains. Thereby it modulates the activity of HS-dependent growth factors. For HS interaction Sulf1 employs a unique hydrophilic domain (HD).

Methods

Affinity-chromatography, AFM-single-molecule force spectroscopy (SMFS) and immunofluorescence on living cells were used to analyze specificity, kinetics and structural basis of this interaction.

Results

Full-length Sulf1 interacts broadly with sulfated glycosaminoglycans (GAGs) showing, however, higher affinity toward HS and heparin than toward chondroitin sulfate or dermatan sulfate. Strong interaction depends on the presence of Sulf1-substrate groups, as Sulf1 bound significantly weaker to HS after enzymatic 6-O-desulfation by Sulf1 pretreatment, hence suggesting autoregulation of Sulf1/substrate association. In contrast, HD alone exhibited outstanding specificity toward HS and did not interact with chondroitin sulfate, dermatan sulfate or 6-O-desulfated HS. Dynamic SMFS revealed an off-rate of 0.04/s, i.e., ~ 500-fold higher than determined by surface plasmon resonance. SMFS allowed resolving the dynamics of single dissociation events in each force–distance curve. HD subdomain constructs revealed heparin interaction sites in the inner and C-terminal regions of HD.

Conclusions

Specific substrate binding of Sulf1 is mediated by HD and involves at least two separate HS-binding sites. Surface plasmon resonance K_D-values reflect a high avidity resulting from multivalent HD/heparin interaction. While this ensures stable cell–surface HS association, the dynamic cooperation of binding sites at HD and also the catalytic domain enables processive action of Sulf1 along or across HS chains.

General significance

HD confers a novel and highly dynamic mode of protein interaction with HS.     

Studies on glycosaminoglycans of regenerating rabbit ear cartilage

Cartilage regeneration in the adult rabbit ear was examined with respect to glycosaminoglycan (GAG) synthesis at various stages of the regeneration process. Increased hyaluronic acid and chondroitin sulfate synthesis was first seen 31 days after wounding, when a metachromatic cartilage matrix could be distinguished from blastemal cells. Analysis of cartilage and the overlying skin separately showed that 90% of the labeled chondroitin sulfate was found in the cartilage being regenerated. DEAE-cellulose chromatography of GAG preparations from 35-day regenerating cartilages showed hyaluronic acid and chondroitin sulfate peaks eluting in the same position as those isolated from normal cartilages. The identity of the hyaluronic acid and chondroitin sulfate peaks was confirmed by their susceptibility to Streptomyces hyaluronidase and chondroitinase ABC, respectively. Although the degree of sulfation in normal and regenerated cartilages was similar, the ratio of chondroitin 6-sulfate to chondroitin 4-sulfate was increased in regenerated cartilages. GAG preparations from unlabeled cartilages were digested with chondroitinase ABC and the disaccharide digestive products were identified and quantitiated. Normal cartilage had a $\text{Δ}\text{Di-6S}\text{Δ}\text{Di-4S}$ ratio of 0.27; the same ratio for the regenerated cartilage was 1.58.     

Dermatan sulfate and chondroitin 6-sulfate conformations

X-ray diffraction patterns show that dermatan sulfate in oriented, crystalline films occurs as two or three or eight-fold helices. The two-fold helix has a greater axial rise per disaccharide residue $\text{[}\text{h}\text{= 9.6}\text{A}\text{?}\text{]}$ than the corresponding chondroitin 6-sulfate helix $\text{[}\text{h}\text{= 9.3}\text{A}\text{?}\text{]}$. Three-fold dermatan sulfate and chondroitin 6-sulfate helices both have $\text{h}\text{= 9.5}\text{A}\text{?}$. Consequently the α-L-iduronate residues in dermatan sulfate helices have the C1 chair conformation like β-D-glucuronate in chondroitin 6-sulfate. Since the eight-fold dermatan sulfate helix has $\text{h}\text{= 9.3}\text{A}\text{?}$ rather less than the eight-fold chondroitin 6-sulfate helix $\text{[}\text{h}\text{= 9.8}\text{A}\text{?}\text{]}$ the possibility of α-L-iduronate 1C chairs cannot be ruled out for it. Computer methods have been used to produce molecular models. In these the polysaccharide chains are almost linear. Each backbone conformation can accommodate a variety of arrangements of charged side groups.     

Molecular cloning of squid N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase and synthesis of a unique chondroitin sulfate containing E-D hybrid tetrasaccharide structure by the recombinant enzyme

N-Acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) transfers sulfate to position 6 of GalNAc(4SO(4)) residues in chondroitin sulfate (CS). We previously purified squid GalNAc4S-6ST and cloned a cDNA encoding the partial sequence of squid GalNAc4S-6ST. In this paper, we cloned squid GalNAc4S-6ST cDNA containing a full open reading frame and characterized the recombinant squid GalNAc4S-6ST. The cDNA predicts a Type II transmembrane protein composed of 425 amino acid residues. The recombinant squid GalNAc4S-6ST transferred sulfate preferentially to the internal GalNAc(4SO(4)) residues of chondroitin sulfate A (CS-A); nevertheless, the nonreducing terminal GalNAc(4SO(4)) could be sulfated efficiently when the GalNAc(4SO(4)) residue was included in the unique nonreducing terminal structure, GalNAc(4SO(4))-GlcA(2SO(4))-GalNAc(6SO(4)), which was previously found in CS-A. Shark cartilage chondroitin sulfate C (CS-C) and chondroitin sulfate D (CS-D), poor acceptors for human GalNAc4S-6ST, served as the good acceptors for the recombinant squid GalNAc4S-6ST. Analysis of the sulfated products formed from CS-C and CS-D revealed that GalNAc(4SO(4)) residues included in a tetrasaccharide sequence, GlcA-GalNAc(4SO(4))-GlcA(2SO(4))-GalNAc(6SO(4)), were sulfated efficiently by squid GalNAc4S-6ST, and the E-D hybrid tetrasaccharide sequence, GlcA-GalNAc(4,6-SO(4))-GlcA(2SO(4))-GalNAc(6SO(4)) was generated in the resulting sulfated glycosaminoglycans. These observations indicate that the recombinant squid GalNAc4S-6ST is a useful enzyme for preparing a unique chondroitin sulfate containing the E-D hybrid tetrasaccharide structure.     

A terminal 6-sulfotransferase catalyzing a synthesis of N-acetylgalactosamine 4,6-bissulfate residue at the nonreducing terminal position of chondroitin sulfate

A soluble enzyme from quail oviduct which incorporates sulfate into position 6 of the nonreducing N-acetylgalactosamine 4-sulfate end group of chondroitin sulfate has been purified. This enzyme (termed "terminal 6-sulfotransferase") was partially separated from a 6-sulfotransferase present in the same tissue which catalyzes the incorporation of sulfate into interior portion of unsulfated chondroitin. The basic requirements for the terminal 6-sulfotransferase reaction were shown to be 3'-phosphoadenylyl sulfate (donor) and chondroitin 4-sulfate (acceptor). The substitution of unsulfated chondroitin (prepared from squid skin) for chondroitin 4-sulfate resulted in a total loss of activity. These results suggest that the organization of the proteoglycan-synthesizing apparatus may well involve hitherto unrecognized mechanisms for the sulfation of chondroitin chains.     

Tissue specific distribution of sulfated mucopolysaccharides in mammals

A comparative study on the distribution of sulfated mucopolysaccharides in several tissues of five mammalian species is reported. It is shown that each tissue has a characteristic composition differing from each other regarding the relative amount, type and molecular size of chondroitin sulfate A/C, chondroitin sulfate B and heparan sulfate. It is also shown that the same tissue from different mammals has the same types and proportions of sulfated mucopolysaccharides, but with different molecular weights. Exception to this rule was observed for the distribution of heparin which was present only in a few tissues of the five mammals studied.The possible involvement of the sulfated mucopolysaccharides in cell recognition and/or adhesiveness is discussed in view of this characteristic distribution.     

6-Hydroxy- and 6-methoxyflavonoids from Neurolaena lobata and N. macrocephala

Twelve flavonoids including one new sulfate were isolated from Neurolaena lobata, and six known flavonoids were obtained from N. macrocephala. The new compound isolated from N. lobata is 6-hydroxykaempferol 3-methyl ether 7-sulfate, and the known flavonoids are 6-hydroxykaempferol 3,7-di-dimethyl ether, 6-hydroxykaempferol, 3-methyl ether 7-glucoside, 6-hydroxykaempferol 7-glucoside, quercetagetin and its 7-glucoside, quercetagetin 3,6- and 3,7-dimethyl ethers, quercetagetin 3-methyl ether 7-glucoside and 7-sulfate, 6-hydroxyluteolin 3′-methyl ether and 6-hydroxyluteolin 7-glucoside. The known flavonoids identified from N. macrocephala are quercetagetin 3,6- and 3, 7-dimethyl ethers, quercetagetin 6-methyl ether 7-glucoside, quercetagetin 3,6-dimethyl ether 7-glucoside, quercetagetin 7-glucoside and quercetagetin 3-methyl ether 7-sulfate.     

Synthesis of [75Se]trimethylselenonium iodide from [75Se]selenocystine

N-Acetylglucosamine-6-sulfate sulfatase activity was assayed by incubation of the radiolabeled monosaccharide N-acetylglucosamine [1-14C]6-sulfate (GlcNAc6S) with homogenates of leukocytes and cultured skin fibroblasts and concentrates of urine derived from normal individuals, patients affected with N-acetylglucosamine-6-sulfate sulfatase deficiency (Sanfilippo D syndrome, mucopolysaccharidosis type IIID), and patients affected with other mucopolysaccharidoses. The assay clearly distinguished affected homozygotes from normal controls and other mucopolysaccharidosis types. The level of enzymatic activity toward GlcNAc6S was compared with that toward a sulfated disaccharide and a sulfated trisaccharide prepared from heparin. The disaccharide was desulfated at the same rate as the monosaccharide and the trisaccharide at 30 times that of the monosaccharide. Sulfatase activity toward glucose 6-sulfate and N-acetylmannosamine 6-sulfate was not detected. Sulfatase activity in fibroblast homogenates with GlcNAc6S exhibited a pH optimum at pH 6.5, an apparent Km of 330 mumol/liter, and inhibition by both sulfate and phosphate ions. The use of radiolabeled GlcNAc6S substrate for the assay of N-acetylglucosamine-6-sulfate sulfatase in leukocytes and skin fibroblasts for the routine enzymatic detection of the Sanfilippo D syndrome is recommended.     

Proteolytic cleavage of the rat heparan sulfate 6-O-endosulfatase SulfFP2 by furin-type proprotein convertases

Heparan sulfate 6-O-endosufatases Sulf1 and Sulf2 hydrolyze the 6-O-sulfate of the glucosamine residues in heparin and heparan sulfate, thereby regulating multiple signaling pathways. A previous study reported that human Sulf1 and Sulf2 were proteolytically processed in a manner sensitive to a furin inhibitor. However, the exact sites of cleavage, the sequence motifs for proteolysis, and the effect of the cleavage on enzyme activity remain unknown. Here we show that the cleavage of rat Sulf2 (also called SulfFP2) occurs at two arginine residues, 543 and 570, in the hydrophilic domain. Both sites reside in the consensus sequence for the cleavage by furin-type proprotein convertases, and the consensus motifs are essential for cleavages. The cleavage at arginine 570 is sensitive to a furin inhibitor. Furthermore, the uncleavable form of SulfFP2 shows sulfatase activity comparable to the cleavable SulfFP2, indicating that the cleavage is not indispensable for activation of SulfFP2.     

Purification of heparinase and heparitinase by affinity chromatography on glycosaminoglycan-bound ah-sepha-rose 4b

Heparinase and heparitinase were separated from an extract of Flavobacterium heparinum, induced with heparin by using column chromatography on hydroxylapatite. As the heparinase preparation contained chondroitinases B and C, chondroitinase B was removed by rechromatography on a hydroxylapatite column. Chondroitinase C was then eliminated by column chromatography on O-phosphono(“phospho”)-cellulose. The heparinase preparation thus obtained was free from sulfoamidase for 2-deoxy-2-sulfoamino-D-glucose (GlcN-2S), sulfatase for 2-amino-2-deoxy-6-O-sulfo D-glucose (GlcN-6S), as well as (δ_4,5glycosiduronase for the unsaturated disaccharides obtained from heparin. The remaining sulfatase for 4-deoxy-α-L-thero-hex-4-enopyranosyluronic acid 2-sulfate (δUA-2S) in the heparinase preparation was removed by affinity chromatography with dermatan sulfate-bound AH-Sepharose 4B coated with dermatan sulfate. The heparitinase preparation separated by column chromatography on hydroxyla patite was purified by affinity chromatography with heparin-bound AH-Sepharose 4B coated with heparin. Sulfatase for 2-amino-2-deoxy-6-O-sulfo-D-glucose (GlcN-6S) and δ_4,5glycosiduronase for the unsaturated disaccharides obtained from heparin were removed by this chromatography. Sulfatase for 4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid 2-sulfate (δUA-2S) remaining in the heparitinase preparation was finally removed by column chromatography on hydroxylapatite. The recoveries of the purified preparations of heparinase and heparitinase were estimated to be 39 and 50%, respectively, from the crude enzyme fractions obtained by the first column chromatography on hydroxyl- patite. The purified heparinase and heparitinase were free from all enzymes that could degrade the sulfated unsaturated disaccharides produced from heparin with heparinase.     

Analysis of cartilage differentiation from skeletal muscle grown on bone matrix: II. Chondroitin sulfate synthesis and reaction to exogenous glycosaminoglycans

In the first paper in this series (Nathanson, M. A., and Hay, E. D. (1980). Develop. Biol. 78, 301–331), we described the ultrastructural alterations that take place when embryonic skeletal muscle is induced to form hyaline cartilage by demineralized bone matrix in vitro. In this paper, we analyze the pattern of appearance of chondroitin sulfates and dermatan sulfate in injured muscle in situ and in explants of muscle cultured either on bone matrix or on collagen gel. We also investigate the effects of exogenous glycosaminoglycans on the cultures to determine whether chondroitin sulfate (Ch-S) and hyaluronic acid (HA) can enhance or inhibit the biochemical differentiation of cartilage under these conditions. Our results indicate that during the first morphological phase, 1–3 days in vitro, there is an increased sulfate uptake, a shift in the relative abundance of Ch-S, and an increase in the ratio of chondroitin-4-sulfate (Ch-4-S) to chondroitin-6-sulfate (Ch-6-S); this change is correlated with the transformation of myoblasts to fibroblast-like cells in both types of cultures. A similar increase in the $\text{Ch-4-S}\text{Ch-6-S}$ ratio occurs in injured muscle in situ, suggesting that phase I is a regenerative response. Explants on bone matrix sustain Ch-4-S levels between 4 and 5 days (phase II) and show a large increase in Ch-4-S and sulfate incorporation when they form cartilage at 6–10 days (phase III). Explants on collagen gels regenerate muscle at 4–10 days with decreasing $\text{Ch-4-S}\text{Ch-6-S}$ ratios and decreasing sulfate incorporation. The data demonstrate that an environmental influence, such as trauma, is sufficient to alter the biosynthetic expression of skeletal muscle and that under appropriate conditions (such as the presence of bone matrix) this response may be augmented, leading to the synthesis of extracellular matrix components at ratios characteristic of cartilage. Exogenous Ch-S and HA did not significantly effect this overall pattern. These results are discussed in relation to the morphological observations presented in the preceding paper.     

Cell recognition and adhesiveness: a possible biological role for the sulfated mucopolysaccharides.

The sulfated mucopolysaccharide composition of different neonate, adult and tumoral tissues is reported. It is shown that each tissue has a characteristic composition with respect to the relative amount, type and molecular size of chondroitin sulfate $\text{A}\text{C}$, chondroitin sulfate B and heparitin sulfate. Neonate and tumor tissues contain large amounts of chondrotin sulfate $\text{A}\text{C}$ which is nearly absent in most adult and normal tissues respectively. Based on these and other results a possible role for the sulfated mucopolysaccharides in cell recognition and adhesiveness is proposed.     

Uridine diphospho-N-acetylgalactosamine-4-sulfate sulfohydrolase activity of human arylsulfatase B and its deficiency in the Maroteaux-Lamy syndrome.

The hydrolysis of UDP-N-acetylgalactosamine-4-sulfate by human arylsulfatase B has been demonstrated with an enzyme preparation purified 200-fold from placenta. No hydrolysis was observed with arylsulfatase A. UDP-N-acetylgalactosamine-4-sulfate is the first fully characterized physiological compound shown to be a substrate for arylsulfatase B, confirming that arylsulfatase B is an N-acetylgalactosamine-4-sulfate sulfohydrolase. Cultured fibroblasts derived from patients with Maroteaux-Lamy syndrome were deficient in UDP-N-acetylgalactosamine-4-sulfate sulfohydrolase to the same extent that they were deficient in arylsulfatase B.     

<Emphasis Type="Italic">Aspergillus niger</Emphasis> metabolism of citrus furanocoumarin inhibitors of human cytochrome P450 3A4

Fungi metabolize polycyclic aromatic hydrocarbons by a number of detoxification processes, including the formation of sulfated and glycosidated conjugates. A class of aromatic compounds in grapefruit is the furanocoumarins (FCs), and their metabolism in humans is centrally involved in the “grapefruit/drug interactions.” Thus far, the metabolism by fungi of the major FCs in grapefruit, including 6′, 7′-epoxybergamottin (EB), 6′, 7′-dihydroxybergamottin (DHB), and bergamottin (BM), has received little attention. In this study, Aspergillus niger was observed to convert EB into DHB and a novel water-soluble metabolite (WSM). Bergaptol (BT) and BM were also metabolized by A. niger to the WSM, which was identified as BT-5-sulfate using mass spectrometry, UV spectroscopy, chemical hydrolysis, and ¹H and ¹³C nuclear magnetic resonance spectroscopy. Similarly, the fungus had a capability of metabolizing xanthotoxol (XT), a structural isomer of BT, to a sulfated analog of BT-5-sulfate, presumably XT-8-sulfate. A possible enzyme-catalyzed pathway for the grapefruit FC metabolism involving the cleavage of the geranyl group and the addition of a sulfate group is proposed.