A novel human Gal-3-O-sulfotransferase: molecular cloning, characterization, and its implications in biosynthesis of (SO(4)-3)Galbeta1-4(Fucalpha1-3)GlcNAc

Based on sequence homology with the previously cloned human cerebroside sulfotransferase (CST) cDNA, a novel sulfotransferase was cloned by screening a human fetal brain cDNA library. The novel sulfotransferase gene was present on human chromosome 11q13; the location was different from human CST and from that of the recently cloned human beta-Gal 3'-sulfotransferase (GP3ST). The isolated cDNA contained an open reading frame that encoded a predicted protein of 431 amino acid residues with type II transmembrane topology. The amino acid sequence showed 33% identity with that of human CST and 38% with that of human GP3ST. The recombinant enzyme expressed in Chinese hamster ovary cells catalyzed transfer of sulfate to position 3 of non-reducing beta-galactosyl residues in Galbeta1-4GlcNAc. Type 2 chains served as good acceptors, whereas type 1 chains served as poor acceptors, and intermediate activity was found toward Galbeta1-3GalNAc. Therefore, the substrate specificity was different from that of GP3ST. CST activity was not detected in the newly cloned enzyme. Northern blotting analysis showed that the sulfotransferase mRNA was strongly expressed in the thyroid and moderately expressed in the brain, heart, kidney, and spinal cord. Co-transfection of the enzyme cDNA and fucosyltransferase III into COS-7 cells resulted in expression of (SO(4)-3)Galbeta1-4(Fucalpha1-3)GlcNAc and a small amount of (SO(4)-3)Galbeta1-3(Fucalpha1-4)GlcNAc. These results indicated that the newly cloned enzyme is a novel Gal-3-O-sulfotransferase and is involved in biosynthesis of the (SO(4)-3)Galbeta1-4(Fucalpha1-3)GlcNAc structure.     

Effects of cycloheximide, brefeldin A, suramin, heparin and primaquine on proteoglycan and glycosaminoglycan biosynthesis in human embryonic skin fibroblasts.

(1) We have isolated radiolabelled proteoglycans and glycosaminoglycans produced by human embryonic skin fibroblasts in the presence of (a) cycloheximide to inhibit protein synthesis or (b) brefeldin A to impede transport between the endoplasmic reticulum and the Golgi complex or (c) suramin, heparin or primaquine to interfere with internalization, recycling and degradation. Effects on glycosaminoglycan synthesis were assayed separately by using exogenous p-nitrophenyl beta-D-xylopyranoside (and [3H]galactose) or 125I-labelled p-hydroxyphenyl beta-D-xylopyranoside as initiators. (2) Inhibition of protein synthesis or blocking of transport to the Golgi complex prevented production of most of the proteoglycans with one exception: Cell-associated heparan sulphate-proteoglycan was still produced at 20% of the control level. (3) Treatment with suramin or heparin resulted in decreased deposition of proteoglycan in the pericellular matrix but increased accumulation of cell-associated proteoglycan. Primaquine blocked all proteoglycan synthesis. (4) In the presence of cycloheximide, exogenous beta-D-xyloside initiated galactosaminoglycan production. In contrast, in brefeldin A-treated cells, synthesis was completely abolished. Not even formation of the linkage-region trisaccharide could be detected. (5) These results suggest that exogenous xyloside enters the endoplasmic reticulum and is subsequently transported to the trans-Golgi complex where all further steps involved in glycosaminoglycan assembly takes place. (6) Heparan sulphate proteoglycan produced by brefeldin A-treated cells could be derived from (a) an intracellular pool of preformed core protein located to the trans-Golgi complex, or (b) resident proteoglycan that was either deglycanated/reglycanated or chain-extended. As combined treatment with suramin and brefeldin A markedly reduced cell-associated proteoglycan production, the latter possibility is favoured.     

Analysis of glycosaminoglycan chains from different proteoglycan populations in human embryonic skin fibroblasts.

1. The structure of chondroitin/dermatan and heparan-sulphate chains from various proteoglycan populations derived from cultured human skin fibroblasts have been examined. Confluent cell cultures were biosynthetically labelled with [3H]-glucosamine and 35SO4(2-), and proteoglycans were purified according to buoyant density, size and charge density [Schmidtchen, A., Carlstedt, I., Malmstr?m, A. & Fransson, L.-A. (1990) Biochem. J. 265, 289-300]. Some proteoglycan fractions were further fractionated according to hydrophobicity on octyl-Sepharose in Triton X-100 gradients. The glycosaminoglycan chains, intact or degraded by chemical or enzymic methods were then analysed by gel chromatography on Sepharose CL-6B, Bio-Gel P-6, ion exchange HPLC and gel electrophoresis. 2. Three types of dermatan-sulphate chains were identified on the basis of disaccharide composition and chain length. They were derived from the large proteoglycan, two small proteoglycans and a cell-associated proteoglycan with core proteins of 90 kDa and 45 kDa. Intracellular, free dermatan-sulphate chains were very similar to those of the small proteoglycans. 3. Heparan-sulphate chains from different proteoglycans had, in spite of small but distinct differences in size, strikingly similar compositional features. They contained similar amounts of D-glucuronate, L-iduronate (with or without sulphate) and N-sulphate groups. They all displayed heparin-lyase-resistant domains with average molecular mass of 10-15 kDa. The heparan-sulphate chains from proteoglycans with 250-kDa and 350-kDa cores were the largest greater than 50 kDa), containing an average of four or five domains, in contrast to heparan-sulphate chains from the small heparan-sulphate proteoglycans which had average molecular mass of 45 kDa and consisted of three or four such domains. Free, cell-associated heparan-sulphate chains were heterogeneous in size (5-45 kDa). 4. These results suggest that the core protein may have important regulatory functions with regard to dermatan-sulphate synthesis. On the other hand, synthesis of heparan sulphate may be largely controlled by the cell that expresses a particular proteoglycan core protein.     

Modulation of the morphology and glycosaminoglycan biosynthesis of human monocytes, induced by culture substrates

Monocytes were isolated from human blood and cultured in vitro on plastic culture dishes or on fibronectin-coated dishes. After 5 days in vitro, the cells on plastic dishes displayed marked morphological changes compared with day 1, with an epithelioid appearance resembling that of foreign-body cells. This transition was inhibited in cells cultured on fibronectin-coated dishes. 35S-labelled polysaccharides were isolated from the culture media after 24h incubation periods with inorganic [35S]sulphate. The cells cultured for 5 days on a plastic substrate synthesized, and secreted into the medium, an oversulphated galactosaminoglycan previously shown to contain 4,6-di-O-sulphated N-acetylgalactosamine units [Kolset, Kjellén, Seljelid & Lindahl (1983) Biochem. J. 210, 661-667]. In contrast, 35S-labelled polysaccharide produced by cells cultured on plastic for 1 day only, or on fibronectin for either 1 or 5 days, contained only minor amounts of such disulphated sugar units. These findings indicate that the formation of oversulphated chondroitin sulphate is coupled to the conversion of monocytes into epithelioid cells. Furthermore, they suggest that the overall process is induced by contact with artificial substrates, and that it may be regarded as the equivalent of a foreign-body reaction in vivo.     

Comparison of collagen and glycosaminoglycan synthesis in attaching control and diabetic human skin fibroblasts

Summary Cultured fibroblasts derived from normal subjects and juvenile diabetics attach in the absence of serum to plastic culture dishes and secrete macromolecules, including collagenous components, hyaluronic acid, and proteoglycans into the medium and onto the plastic surface where they form a microexudate carpet. Most diabetic fibroblasts examined did not spread as well as normal cells during a 4-hr interval after the initial attachment. There were no significant differences between normal and diabetic cells with respect to proline and lysine incorporation and lysine hydroxylation. The percentage glycosylation of hydroxylysine was marginally higher in the media proteins of diabetic cells, but glycosylation in both normal and diabetic cells was elevated over that typically observed in human skin collagen. Collagenous components were estimated to constitute approximately 15–20% of the microexudate carpet fraction in both normal and diabetic cell strains. Diabetic fibroblasts exhibited a marginally lower ratio of heparan sulfate to chondroitin sulfate in the cell surface to matrix microexudate carpet fraction (trypsinate) than did normal fibroblasts. The hyaluronate and chondroitin sulfate contents of this fraction of diabetic cells were not significantly different from those of normal cells. This work was supported by Grants AM 11821 and AM 19606 from the National Institutes of Health, United States Public Health Service, by a grant from the American Diabetes Association (Leon W. Cunningham, Principal Investigator) and by the Vanderbilt Diabetes Endocrinology Center grant, AM 17026. R. E. Branson was the recipient of a research fellowship of the Juvenile Diabetes Foundation and of a National Institutes of Health Postdoctoral Fellowship AM 05636.     

L-663,536 (MK-886) (3-[1-(4-chlorobenzyl)-3-t-butyl-thio-5-isopropylindol-2-yl]-2,2 - dimethylpropanoic acid), a novel, orally active leukotriene biosynthesis inhibitor

L-663,536 (3-[1-(4-chlorobenzyl)-3-t-butyl-thio-5-isopropylindol-2-yl]-2, 2-dimethylpropanoic acid) is a potent inhibitor of leukotriene (LT) biosynthesis in intact human polymorphonuclear leukocytes (PMN) (IC50, 2.5 nM). Similarly, L-663,536 inhibited A23187-induced LTB4 formation by rat peripheral blood and elicited PMN. At concentrations where inhibition of leukotriene biosynthesis occurred in human whole blood (1.1 microM), no effect was seen on cyclooxygenase or 12-lipoxygenase, an effect also observed in washed human platelets. The compound had no effect on rat or porcine 5-lipoxygenase indicating that L-663,536 is not a direct 5-lipoxygenase inhibitor. When administered in vivo L-663,536 was a potent inhibitor of antigen-induced dyspnea in inbred rats pretreated with methysergide (ED50, 0.036 mg/kg p.o.) and of Ascaris-induced bronchoconstriction in squirrel monkeys (1 mg/kg p.o.). The compound inhibited leukotriene biosynthesis in vivo in a rat pleurisy model (ED50, 0.2 mg/kg p.o.), an inflamed rat paw model (ED50, 0.8 mg/kg), a model of leukotriene excretion in rat bile following antigen provocation, and a model in the guinea-pig ear where leukotriene synthesis was induced by topical challenge with ionophore A23187 (ED50, 2.5 mg/kg p.o. and 0.6 micrograms topically). The results indicate that L-663,536 is a potent inhibitor of leukotriene biosynthesis both in vitro and in vivo indicating that the compound is suitable for studying the role of leukotrienes in a variety of pathological situations.     

Tissue form of type VII collagen from human skin and dermal fibroblasts in culture

The triple-helical domain of type VII collagen was isolated from human placental membranes by mild digestion with pepsin, and polyclonal antibodies were raised in rabbits against this protein. After affinity purification the antibodies specifically recognized type VII collagen in both the triple-helical and the unfolded state. They also reacted with the fragments P1 and P2, derived from the triple-helical domain by further proteolysis with pepsin, but did not crossreact with other biochemical components of the dermal connective tissue. In skin the presence of a fragment of type VII collagen, similar to that isolated from placenta, was demonstrated by SDS-PAGE and immunoblotting. Type VII collagen represented less than 0.001% of the total collagen extracted by pepsin digestion from newborn or adult skin. The tissue form of type VII collagen was obtained from dermis after artificial epidermolysis with strongly denaturing buffers under conditions reducing disulfide bonds. The protein was identified by immunoblotting with the antibodies. The molecule was composed of three polypeptides with an apparent molecular mass of about 250 kDa, each. Similar large-molecular-mass chains could be identified by immunoblotting in extracts of human fibroblasts in culture.     

4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, a nicotine derivative, induces apoptosis of endothelial cells

Smoking causes endothelial cell (EC) injury; however, neither the components of cigarette smoke nor the mechanisms responsible for this injury are understood. The nitrosated derivative of nicotine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), has been implicated in the carcinogenic effects of tobacco; however, the effects of NNK on the cardiovascular system are largely unknown. NNK binds to beta1- and beta2-adrenergic receptors. Because beta-adrenergic receptor activation causes arachidonic acid (AA) release and cellular injury, we postulated that NNK causes EC injury by a mechanism that involves beta-adrenergic-mediated release of AA. NNK stimulated [3H]AA release from ECs, and this effect was mediated by both beta1- and beta2-adrenergic receptors because pretreatment with atenolol or ICI 118,551 inhibited the response. NNK also induced EC apoptosis, as measured by terminal deoxyribonucleotide transferase-mediated dUTP nick-end labeling and annexin V staining. NNK-mediated apoptosis was attenuated by pretreatment with atenolol or ICI 118,551. Furthermore, depletion of cellular AA by incubation with eicosapentaenoic acid abolished the apoptotic effect of NNK. These data suggest that NNK causes EC apoptosis by a mechanism that involves beta1- and beta2-adrenergic receptor-mediated release of AA.     

Biosynthesis and release of glycoproteins by human skin fibroblasts in culture

1. Confluent human skin fibroblasts maintained in a chemically defined medium incorporate l-[1-³H]fucose in a linear manner with time into non-diffusible macromolecules for up to 48h. Chromatographic analysis demonstrated that virtually all the macromolecule-associated ³H was present as [³H]fucose. 2. Equilibrium CsCl-density-gradient centrifugation established that [³H]fucose-labelled macromolecules released into the medium were predominantly glycoproteins. Confirmation of this finding was provided by molecular-size analyses of the [³H]fucose-labelled material before and after trypsin digestion. 3. The [³H]fucose-labelled glycoproteins released into fibroblast culture medium were analysed by gel-filtration chromatography and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. These techniques demonstrated that the major fucosylated glycoprotein had an apparent mol.wt. of 230000–250000; several minor labelled species were also detected. 4. Dual-labelling experiments with [³H]fucose and ¹⁴C-labelled amino acids indicated that the major fucosylated glycoprotein was synthesized de novo by cultured fibroblasts. The non-collagenous nature of this glycoprotein was established by three independent methods. 5. Gel-filtration analysis before and after reduction with dithiothreitol showed that the major glycoprotein occurs as a disulphide-bonded dimer when analysed under denaturing conditions. Further experiments demonstrated that this glycoprotein was the predominant labelled species released into the medium when fibroblasts were incubated with [³⁵S]cysteine. 6. The relationship between the major fucosylated glycoprotein and a glycoprotein, or group of glycoproteins, variously known as fibronectin, LETS protein, cell-surface protein etc., is discussed.     

Glycosylation of human LDL and its metabolism in human skin fibroblasts

Extrachromosomal DNA of heterogeneous size has been isolated from bursal lymphocytes and splenocytes of five week old chickens, and from splenocytes of mice. This DNA contains covalently closed circles, open circles, and open circles with tails. Open circular molecules with and without tails are more frequent than covalently closed species, and the total number of small circular DNA molecules per cell is in the range of 100–200 copies.     

Inhibitory effect of Aucubin isolated from Eucommia ulmoides against UVB-induced matrix metalloproteinase-1 production in human skin fibroblasts

Of 30 herbal plants tested, the methanol extracts of Eucommia ulmoides (52%), Evodia officinalis (45%), and Pleuropterus multiflorus (41%) each showed a potent inhibitory effect on the matrix metalloproteinase-1 (MMP-1) production in ultraviolet B (UVB)-irradiated human fibroblasts. Aucubin was isolated as the MMP-1 inhibitor from E. ulmoides, and significantly suppressed the production of MMP-1 by nearly 57% compared to the control. It also reduced MMP-1 mRNA expression. These results suggest that aucubin is a photoprotective phytochemical, and could be used as a potential agent in preventing photoaging.     

Novel tandem biotransformation process for the biosynthesis of a novel compound, 4-(2,3,5,6-tetramethylpyrazine-1)-4'-demethylepipodophyllotoxin

According to the structure of podophyllotoxin and its structure-function relationship, a novel tandem biotransformation process was developed for the directional modification of the podophyllotoxin structure to directionally synthesize a novel compound, 4-(2,3,5,6-tetramethylpyrazine-1)-4'-demethylepipodophyllotoxin (4-TMP-DMEP). In this novel tandem biotransformation process, the starting substrate of podophyllotoxin was biotransformed into 4'-demethylepipodophyllotoxin (product 1) with the demethylation of the methoxyl group at the 4' position by Gibberella fujikuroi SH-f13, which was screened out from Shennongjia prime forest humus soil (Hubei, China). 4'-Demethylepipodophyllotoxin (product 1) was then biotransformed into 4'-demethylpodophyllotoxone (product 2) with the oxidation of the hydroxyl group at the 4 position by Alternaria alternata S-f6, which was screened out from the gathered Dysosma versipellis plants in the Wuhan Botanical Garden, Chinese Academy of Sciences. Finally, 4'-demethylpodophyllotoxone (product 2) and ligustrazine were linked with a transamination reaction to synthesize the target product 4-TMP-DMEP (product 3) by Alternaria alternata S-f6. Compared with podophyllotoxin (i.e., a 50% effective concentration [EC(50)] of 529 μM), the EC(50) of 4-TMP-DMEP against the tumor cell line BGC-823 (i.e., 0.11 μM) was significantly reduced by 5,199 times. Simultaneously, the EC(50) of 4-TMP-DMEP against the normal human proximal tubular epithelial cell line HK-2 (i.e., 0.40 μM) was 66 times higher than that of podophyllotoxin (i.e., 0.006 μM). Furthermore, compared with podophyllotoxin (i.e., log P = 0.34), the water solubility of 4-TMP-DMEP (i.e., log P = 0.66) was significantly enhanced by 94%. For the first time, the novel compound 4-TMP-DMEP with superior antitumor activity was directionally synthesized from podophyllotoxin by the novel tandem biotransformation process developed in this work.     

Interspecies somatic hybrid cultures from human epidermal cells and 3T3-4E mouse fibroblasts

Interspecific somatic hybrids were obtained by fusion of adult human epidermal cells with Mouse fibroblasts 3T3-4E, deficient in thymidine kinase. These hybrids were identified by their morphology and by the presence of markers from the parental cells. Some characters of keratinocytes such as keratin subunits 50 and 51 K were present in primary cultures and disappeared after serial passages, whereas bullous pemphigoid basement membrane zone antigens persisted for at least 20 passages. At the 7th passage, a metacentric chromosome, and more often a submetacentric chromosome, presumably of human origin, were observed in some cells.     

Biflavonoids isolated from Selaginella tamariscina regulate the expression of matrix metalloproteinase in human skin fibroblasts

The methanol extract from Selaginella tamariscina significantly inhibited UV irradiation induced activity of matrix metalloproteinase-1 (MMP-1) in primary fibroblasts from human skin. Using the technique of bioassay-directed chromatographic separation, five biflavonoids were isolated from the ethyl acetate soluble fraction of S. tamariscina. Here, we investigated the effect of these five biflavonoids on the regulation of MMP-1 and -2 in UV irradiated cultured dermal fibroblasts from human neonatal foreskins. Among these biflavonoids, sumaflavone and amentoflavone showed significant MMP-1 inhibitory activity in primary human dermal fibroblasts after UV irradiation. The IC(50) values of sumaflavone, amentoflavone and retinoic acid, which was used as a positive control, were 0.78, 1.8, and 10microM, respectively.     

Melanin counter act puromycin-induced inhibition of collagen and DNA biosynthesis in human skin fibroblasts

Puromycin is an experimental anti-tumor antibiotic acting through inhibition of protein synthesis. Because of its untoward side effects (as inner ear and renal lesions) the antibiotic was not approved for clinical trials. The mechanism underlying the organ specificity of the side effect is not understood. In view of the fact that a number of drugs form with melanin complexes that affect their pharmacological activity, we determined whether puromycin interacts with melanin and how this process affects biosynthesis of collagen in cultured human skin fibroblasts. Our results indicate that puromycin forms complexes with melanin. The amount of puromycin bound to melanin increases with increase of initial drug concentration. The Scatchard plot analysis of the drug binding to melanin has shown that at least two classes of independent binding sites are implicated in the puromycin-melanin complex formation: one class of strong binding sites with the association constant K1 = 1.84 x 10(6) M(-1), and the second class of weak binding sites with the association constant K2 = 5.26 x 10(3) M(-1). The number of total binding sites were n1 = 0.1260 and n2 = 0.2861 mumol puromycin per 1 mg melanin. We found that puromycin induced inhibition of collagen and DNA biosynthesis (IC50 approximately 2 microM). Melanin at 100 microg/ml produced about 20% inhibition of DNA synthesis, but it had no effect on collagen biosynthesis in cultured fibroblasts. However, the addition of melanin (100 microg/ml) to puromycin - treated cells (2 microM) abolished the inhibitory action of puromycin on collagen and DNA biosynthesis. We have suggested that IGF-I receptor expression, involved in collagen metabolism, may be one of the targets for puromycin - induced inhibition of collagen biosynthesis. It was found that melanin abolished puromycin induced decrease in the expression of IGF-I receptor as well MAP kinases expression: ERK1 and ERK2 as shown by Western immunoblot analysis. These data suggest that tissue specific pharmacological activity of puromycin may depend on the melanin abundance in tissues.     

Heterogeneity of tyrosinases isolated from rat skin, normal human skin, and human melanoma tumors

An electrophoretically homogeneous preparation of tyrosinase (Mr = 61 kDa) was isolated from rat skin. The purification procedure which consisted in chromatographic separation of Triton X-100-solubilized proteins included four main steps, namely: gel filtration, anion-exchange chromatography and two consecutive affinity chromatography steps. Isoelectrofocusing revealed the presence of 9 isoforms possessing an L-DOPA oxidase activity, of which proteins with pI of 4.26 and 4.33 were the major ones. The specific activity of the preparation was 43 nmol/min/mg of protein. Human skin epidermis was practically devoid of the L-DOPA-oxidase activity which was due not only to the absence of tyrosinase but also to the presence of a large amount of a 66 kDa protein able to inhibit the oxidation of L-DOPA to DOPA-chrom. The tyrosinase preparation from human melanoma consisted, predominantly, of two isoforms (48 and 69 kDa) which upon isoelectrofocusing displayed a high heterogeneity at pH around 3-5. The specific activity of the melanoma preparation markedly exceeded that of normal skin tyrosinase and was equal to 290 nmol/min/mg of protein.     

Syntheses of beta-D-GalpNAc4SO3-(1-->4)-L-IdopA2SO3, a disaccharide fragment of dermatan sulfate, and of its methyl alpha-L-glycoside derivative

The syntheses of sodium (sodium 2-acetamido-2-deoxy-4-O-sulfonato-beta-D-galactopyranosyl)-(1-->4)-(sodium 2-O-sulfonato-L-idopyran)uronate, a disaccharide fragment of dermatan sulfate, and of its methyl alpha-L-glycoside derivative are reported for the first time. The use of 4-O-acetyl-3,6-di-O-benzyl-2-deoxy-2-trichloroacetamido-1-O-trichloroacetimidoyl-alpha-D-galactopyranose, readily prepared from a D-gluco precursor, allowed the stereocontrolled and high yielding coupling with the low reactive 4-hydroxyl group of L-iduronic acid ester derivatives. Classical transformation of the disaccharide products into the target molecules was achieved in high yield.