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J D Esko

Publications and source records attributed to J D Esko.

At least 37 records · Page 2Linked to original sources
PubMed1999

Formation of HNK-1 determinants and the glycosaminoglycan tetrasaccharide linkage region by UDP-GlcUA:Galactose beta1, 3-glucuronosyltransferases.

While expression-cloning enzymes involved in heparan sulfate biosynthesis, we isolated a cDNA that encodes a protein 65% identical to the UDP-GlcUA:glycoprotein beta1, 3-glucuronosyltransferase (GlcUAT-P) involved in forming HNK-1 carbohydrate epitopes (3OSO3GlcUAbeta1,3Gal-) on glycoproteins. The cDNA contains an open reading frame coding for a protein of 335 amino acids with a predicted type II transmembrane protein orientation. Cotransfection of the cDNA with HNK-1 3-O-sulfotransferase produced HNK-1 carbohydrate epitopes in Chinese hamster ovary (CHO) cells and COS-7 cells. In vitro, a soluble recombinant form of the enzyme transferred GlcUA in beta-linkage to Galbeta1,3/4GlcNAcbeta-O-naphthalenemethanol, which resembles the core oligosaccharide on which the HNK-1 epitope is assembled. However, the enzyme greatly preferred Galbeta1, 3Galbeta-O-naphthalenemethanol, a disaccharide component found in the linkage region tetrasaccharide in chondroitin sulfate and heparan sulfate. During the course of this study, a human cDNA clone was described that was thought to encode UDP-GlcUA:Galbeta1,3Gal-R glucuronosyltransferase (GlcUAT-I), involved in the formation of the linkage region of glycosaminoglycans (Kitagawa, H., Tone, Y., Tamura, J., Neumann, K. W., Ogawa, T., Oka, S., Kawasaki, T., and Sugahara, K. (1998) J. Biol. Chem. 273, 6615-6618). The deduced amino acid sequences of the CHO and human cDNAs are 95% identical, suggesting that they are in fact homologues of the same gene. Transfection of a CHO cell mutant defective in GlcUAT-I with the hamster cDNA restored glycosaminoglycan assembly in vivo, confirming its identity. Interestingly, transfection of the mutant with GlcUAT-P also restored glycosaminoglycan synthesis. Thus, both GlcUAT-P and GlcUAT-I have overlapping substrate specificities. However, the expression of the two genes was entirely different, with GlcUAT-I expressed in all tissues tested and GlcUAT-P expressed only in brain. These findings suggest that, in neural tissues, GlcUAT-P may participate in both HNK-1 and glycosaminoglycan production.

Amino Acid Sequence↗
PubMed1999

Molecular cloning and expression of a third member of the heparan sulfate/heparin GlcNAc N-deacetylase/ N-sulfotransferase family.

N-Deacetylation and N-sulfation of N-acetylglucosamine residues in heparan sulfate and heparin initiate a series of chemical modifications that ultimately lead to oligosaccharide sequences with specific ligand binding properties. These reactions are catalyzed by GlcNAc N-deacetylase/N-sulfotransferase (NDST), a monomeric enzyme with two catalytic activities. Two genes encoding NDST isozymes have been described, one from rat liver (NDST1) and another from murine mastocytoma (NDST2). Both isozymes are expressed in tissues in varying amounts, but their relative contribution to heparan sulfate formation in any one tissue is unknown. We now report the identification of a third member of the NDST family, designated NDST3. A full-length cDNA clone (3.2 kilobase pairs) encoding a 873-amino acid protein was obtained from a human fetal/infant brain cDNA library. Human NDST3 (hNDST3) has a nucleotide sequence homologous but not identical to hNDST1 and NDST2. The deduced amino acid sequence shows 70% and 65% amino acid identity to that of hNDST1 and NDST2, respectively. A soluble chimera of hNDST3 and protein A exhibited both N-deacetylase and N-sulfotransferase activity, confirming its enzymatic identity. Northern blot analysis of human fetal brain poly(A)+ RNA showed a single transcript of 6.4 kilobase pairs. Reverse transcription polymerase chain reaction analysis revealed more restricted tissue expression of hNDST3 than hNDST1 and NDST2, and high levels in brain, liver, and kidney. Analysis of Chinese hamster ovary cells revealed expression of NDST1 and NDST2, but not NDST3. In a Chinese hamster ovary cell mutant exhibiting reduced N-sulfotransferase activity and reduced sulfation of heparan sulfate (Bame, K. J., and Esko, J. D. (1989) J. Biol. Chem. 264, 8059-8065), expression of NDST1 was greatly reduced, but NDST2 was expressed normally, suggesting that both enzymes are involved in heparan sulfate assembly. The discovery of multiple NDST isozymes suggests that the assembly of heparan sulfate is much complicated than previously appreciated.

Acetyltransferases↗
PubMed1999

Glycoside primers of Psittacanthus cucullaris.

Bioassay-directed chromatographic separation of the ethyl acetate extract of the whole plant of Psittacanthus cucullaris afforded a new phenolic xyloside, ellagic acid-4-O-beta-xyloside-3,3', 4'-trimethyl ether (1) together with four known compounds, ellagic acid-4-O-beta-xyloside-3,3'-dimethyl ether (2), gallic acid, beta-sitosterol, and beta-sitosterol beta-D-glucoside. The structure of the new compound was determined by spectroscopic methods. Like other beta-D-xylosides, compounds 1 and 2 stimulated the formation of glycosaminoglycan chains when fed to the cultured Chinese hamster ovary cells.

Animals↗
PubMed1998

Primers of glycosaminoglycan biosynthesis from Peruvian rain forest plants.

We have developed a rapid, high throughput screening assay for compounds that alter the assembly of glycosaminoglycan chains in Chinese hamster ovary cells. The assay uses autoradiography to measure the binding of newly synthesized [35S]proteoglycans and [35S]glycosaminoglycans to a positively charged membrane. Screening over 1000 extracts from a random plant collection obtained from the Amazon rain forest yielded five plants that stimulated glycosaminoglycan assembly in both wild-type cells and a mutant cell line defective in xylosyltransferase (the first committed enzyme involved in glycosaminoglycan biosynthesis). Fractionation of an extract of Maieta guianensis by silica gel and reverse-phase chromatography yielded two pure compounds with stimulatory activity. Spectroscopic analysis by NMR and mass spectrometry revealed that the active principles were xylosides of dimethylated ellagic acid. One of the compounds also contained a galloyl group at C-3 of the xylose moiety. These findings suggest that plants and other natural products may be a source of agents that can potentially alter glycosaminoglycan and proteoglycan formation in animal cells.

Animals↗
PubMed1997

Fucosylation of disaccharide precursors of sialyl LewisX inhibit selectin-mediated cell adhesion.

We showed previously that HL-60 and F9 mouse embryonal carcinoma cells will take up and deblock peracetylated Galbeta1-4GlcNAcbeta-O-naphthalenemethanol (Galbeta1-4GlcNAc-NM) and use the disaccharide as a primer of oligosaccharide chains (Sarkar, A. K., Fritz, T. A., Taylor, W. H., and Esko, J. D. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 3323-3327). We now report that another disaccharide, acetylated GlcNAcbeta1-3Gal-naphthalenemethanol (GlcNAcbeta1-3Gal-NM), has even greater potency and that both compounds will inhibit sialyl LewisX (sLex)-dependent cell adhesion. When fed to U937 cells, acetylated forms of Galbeta1-4GlcNAc-NM and GlcNAcbeta1-3Gal-NM primed oligosaccharides in a dose-dependent manner. Analysis of compounds assembled on Galbeta1-4GlcNAc-NM showed only one product, namely Galbeta1-4(Fucalpha1-3)GlcNAc-NM. In contrast, GlcNAcbeta1-3Gal-NM generated Galbeta1-4GlcNAcbeta1-3Gal-NM, Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Gal-NM, NeuAcalpha2-3Galbeta1-4GlcNAcbeta1-3Gal-NM, and NeuAcalpha2-3Galbeta1-4(Fucalpha1-3)GlcNAcbeta1- 3Gal-NM. Both compounds decreased the incorporation of [3H]fucose into cellular glycoconjugates, without affecting the incorporation of [3H]mannosamine, a precursor of sialic acid residues. Moreover, the overall extent of sialylation was not affected based on the reactivity of cells to fluorescein isothiocyanate-conjugated Maackia amurensis lectin. Priming inhibited expression of sLex on cell surface glycoconjugates, which reduced E-selectin-dependent cell adhesion to tumor necrosis factor-alpha-activated human umbilical vein endothelial cells. GlcNAcbeta1-3Gal-NM and Galbeta1-4GlcNAc-NM represent starting points for making enzyme-specific, site-directed inhibitors of glycosyltransferases that could act in living cells.

Acetylation↗
PubMed1997

Turnover of heparan sulfate depends on 2-O-sulfation of uronic acids.

To study how the pattern of sulfation along a heparan sulfate chain affects its turnover, we examined heparan sulfate catabolism in wild-type Chinese hamster ovary cells and mutant pgsF-17, defective in 2-O-sulfation of uronic acid residues (Bai, X., and Esko, J. D. (1996) J. Biol. Chem. 271, 17711-17717). Heparan sulfate from the mutant contains normal amounts of 6-O-sulfated glucosamine residues and iduronic acid and somewhat higher levels of N-sulfated glucosamine residues but lacks any 2-O-sulfated iduronic or glucuronic acid residues. Pulse-chase experiments showed that both mutant and wild-type cells transport newly synthesized heparan sulfate proteoglycans to the plasma membrane, where they shed into the medium or move into the cell through endocytosis. Internalization of the cell-associated molecules leads to sequential endoglycosidase (heparanase) fragmentation of the chains and eventual lysosomal degradation. In wild-type cells, the chains begin to degrade within 1 h, leading to the accumulation of intermediate (10-20-kDa) and small (4-7-kDa) oligosaccharides. Mutant cells did not generate these intermediates, although internalization and intracellular trafficking of the heparan sulfate chains appeared normal, and the chains degraded with normal kinetics. This difference was not due to defective heparanase activities in the mutant, since cytoplasmic extracts from mutant cells cleaved wild-type heparan sulfate chains in vitro. Instead, the heparan sulfate chains from the mutant were relatively resistant to degradation by cellular heparanases. These findings suggest that 2-O-sulfated iduronic acid residues in heparan sulfate are important for cleavage by endogenous heparanases but not for the overall catabolism of the chains.

Animals↗
PubMed1997

Cell adhesion to a motif shared by the malaria circumsporozoite protein and thrombospondin is mediated by its glycosaminoglycan-binding region and not by CSVTCG.

The malaria circumsporozoite protein (CS), thrombospondin (TSP), and several other proteins including the terminal complement proteins and the neural adhesion molecules F-spondin and Unc-5, share a cell adhesive sequence. In CS this sequence is designated as region II-plus (EWSPCSVTCGNGIQVRIK) and in TSP it is found in the type I repeats. Previous studies aimed at fine mapping the amino acid residues required for cell adhesion have yielded discrepant results. Here we show in three different cell lines that the downstream basic residues are required for cell adhesion whereas the CSVTCG sequence is not. Using mutant Chinese hamster ovary cells selected for deficiencies in proteoglycan synthesis, we show that in wild type cells, heparan sulfate proteoglycans are the binding sites for this motif. This finding is supported by additional experiments with two other cell lines demonstrating that treatment with heparitinase but not chondroitinase abolishes cell adhesion to peptides representing this motif. Using Chinese hamster ovary cell mutants deficient in heparan sulfate proteoglycans but possessing chondroitin sulfate proteoglycans, we show that cell surface chondroitin sulfate proteoglycans can also mediate binding to this motif although higher concentrations of peptides are required for adhesion. Chondroitinase, but not heparitinase, treatment of these cells destroys cell surface-binding sites. Taken together, these results indicate that cell adhesion to this motif involves an interaction between the downstream positively-charged residues and the negatively charged glycosaminoglycan chains of heparan sulfate, or in some cases chondroitin sulfate, proteoglycans on the cell surface.

Amino Acid Sequence↗
PubMed1997

Dengue virus infectivity depends on envelope protein binding to target cell heparan sulfate.

Dengue virus is a human pathogen that has reemerged as an increasingly important public health threat. We found that the cellular receptor utilized by dengue envelope protein to bind to target cells is a highly sulfated type of heparan sulfate. Heparin, highly sulfated heparan sulfate, and the polysulfonate pharmaceutical Suramin effectively prevented dengue virus infection of target cells, indicating that the envelope protein-target cell receptor interaction is a critical determinant of infectivity. The dengue envelope protein sequence includes two putative glycosaminoglycan-binding motifs at the carboxy terminus; the first could be structurally modeled and formed an unusual extended binding surface of basic amino acids. Similar motifs were also identified in the envelope proteins of other flaviviridae. Developing pharmaceuticals that inhibit target cell binding may be an effective strategy for treating flavivirus infections.

Amino Acid Sequence↗
PubMed1997

Partial purification and substrate specificity of heparan sulfate alpha-N-acetylglucosaminyltransferase I: synthesis, NMR spectroscopic characterization and in vitro assays of two aryl tetrasaccharides.

Studies of heparan sulfate biosynthesis on beta-D-xylosides have led to the hypothesis that heparan sulfate alpha-N-acetylglucosaminyltransferase I (alpha-GlcNAc-TI) recognizes structures at the reducing end of the proteoglycan linkage tetrasaccharide. We report here the in vivo and in vitro testing of this hypothesis using four synthetic substrates, benzyl- and 2-naphthalenemethanyl-beta-D-xylosides, and two proteoglycan linkage tetrasaccharides containing benzyl alcohol or naphthalmethanol aglycones, viz., GlcAbeta(1 --> 3)Gal beta(1 --> 3)Gal beta(1 --> 4)Xyl beta-O-Bn (BNT) and GlcAbeta(1 --> 3)Gal beta(1 --> 3)Gal beta(1 --> 4)Xyl beta-O-NM (NMT). The aryl tetrasaccharides were chemically synthesized and the 1H and 13C resonances were assigned by two-dimensional NMR spectroscopy. The inter-residue spatial constraints, determined by the 2D NOESY data, revealed essentially identical conformations for the interglycosidic linkages and Xyl-O-CH2Ar linkages in both compounds. Interestingly, the aromatic rings in both tetrasaccharides undergo rapid internal rotation across the CH2-Ar bond. These tetrasaccharides were used to assay heparan sulfate alpha-GlcNAc-TI from homogenates of wild-type CHO cells. alpha-GlcNAc-TI was also purified approximately 900-fold from rat liver and assayed with BNT and NMT. At nearly all concentrations tested, alpha-GlcNAc-TI activity from both CHO cell homogenates and rat liver was greater with the NMT. When fed to CHO cells, benzyl-beta-D-xyloside primed heparan sulfate poorly relative to 2-naphthalenemethanyl-beta-D-xyloside. Thus, the in vitro enzyme activity is consistent with the in vivo priming data that suggests that alpha-GlcNAc-TI can directly recognize structure at the reducing end of the linkage tetrasaccharide. These studies provide an in vivo basis for the possible role of core protein sequences in the biosynthesis of specific glycosaminoglycans.

Animals↗
PubMed1996

Unusual beta-D-xylosides that prime glycosaminoglycans in animal cells.

The biosynthesis of glycosaminoglycans (GAG) takes place while the polysaccharide chains are usually attached to a proteoglycan core protein. Cells also will assemble GAG chains on beta-D-xylosides containing hydrophobic aglycones. In order to evaluate the relationship of the structure of the sugar to priming activity of the glycoside, we synthesized beta-D-xyloside analogs in which the hydroxyls were substituted with hydrogen, fluorine, -O-methyl, amino, -O-isopropyl, and -O-benzyl groups. Epimers at the 2-, 3-, and 4-position of xylose also were made. Their ability to prime GAGs was tested in Chinese hamster ovary cells by measuring 35SO4 incorporation into polysaccharide chains and by assaying the transfer of galactose to the xylosides by galactosyltransferase I (UDP-D-galactose:xylose beta1-4-galactosyltransferase) in vitro. All of the analogs failed to act as primers of GAGs in vivo and as substrates in vitro with the following exceptions. Substitution of 2-OH and 3-OH with -OCH3 were active at high concentration (1 mM), but the deoxygenated derivatives were inactive. Efficient priming also occurred on a derivative with fluorine instead of the 3-OH group, suggesting that the oxygen atoms at C-2 and C-3 were involved as hydrogen bond acceptors. Methylated and deoxy analogs at C-4 were inactive, due to the loss of the acceptor hydroxyl group. Interestingly, benzyl-beta-D-threo-pentopyranos-4-uloside (4-keto derivative) and benzyl-4-methyl-beta-D-xyloside, with a methyl group in place of an axial hydrogen at C-4, primed GAG chains. Priming by these unusual xylosides suggests the possibility of designing inhibitors of GAG synthesis based on xyloside analogs with reactive groups in key positions.

Animals↗
PubMed1996

An animal cell mutant defective in heparan sulfate hexuronic acid 2-O-sulfation.

The interaction of heparan sulfate with protein ligands depends on unique oligosaccharide sequences containing iduronic acid (IdUA), N-sulfated glucosamine residues, and O-sulfated sugars. To study the role of O-sulfation in greater detail, we isolated a Chinese hamster ovary cell mutant defective in 2-O-sulfation of iduronic acid. The mutant, pgsF-17, was identified by a colony blotting assay in which colonies of mutagen-treated cells were replica plated to two disks of polyester cloth. One disk was blotted with 125I-labeled basic fibroblast growth factor (bFGF) to measure binding to cell surface proteoglycans. The other disk was incubated with 35SO4 to measure proteoglycan biosynthesis. Autoradiography revealed a colony that did not bind 125I-bFGF, but incorporated 35SO4 normally (mutant pgsF-17). Complete deaminative cleavage of heparan sulfate revealed that material from pgsF-17 lacked IdUA(2OSO3)-GlcNSO3 and IdUA(2OSO3)-GlcNSO3(6OSO3), but contained a higher proportion of glucuronic acid GlcUA-GlcNSO3(6OSO3) and IdUA-GlcNSO3(6OSO3). Assay of the 2-O-sulfotransferase that acts on IdUA residues showed that mutant 17 lacked enzyme activity. Interestingly, the alteration resulted in accumulation of GlcNSO3 groups, suggesting that under normal conditions 2-O-sulfation decreases GlcNAc N-deacetylation/N-sulfation, and that the reactions occur simultaneously. The formation of IdUA and 6-O-sulfated glucosaminyl residues appears to be independent of 2-O-sulfation. pgsF-17 also lacks 2-O-sulfated GlcUA residues, suggesting that the same enzyme is responsible for 2-O-sulfation of IdUA and GlcUA residues. Mutant 17 provides a useful tool for studying the regulation of heparan sulfate biosynthesis and the relationship of heparan sulfate fine structure to its biological function.

Animals↗
PubMed1996

Influence of core protein sequence on glycosaminoglycan assembly.

Recent studies have revealed a correlation between amino acid sequences around glycosylation sites in proteoglycans and the ability of cells to initiate and process glycosaminoglycan chains. Initiation depends on Ser-Gly/Ala dipeptides that have one or more acidic amino acids in close proximity. The formation of heparan sulfate chains depends on a nearby cluster of acidic residues, hydrophobic amino acids, and the close spacing of glycosylation sites.

Amino Acid Sequence↗
PubMed1996

Synthesis and secretion of lipoprotein lipase in heparan sulfate-deficient Chinese hamster ovary cells.

Synthesis and secretion of lipoprotein lipase was studied in two mutants of Chinese hamster ovary (CHO) cells which, due to a lack of xylosyl transferase (pgsA-745) or galactosyl transferase (pgsB-761), respectively, were deficient in heparan sulfate and chondroitin sulfate. One of the mutants (pgsB-761) was two- to threefold more active in synthesis and secretion of catalytically active lipoprotein lipase than the other mutant, which was about as active as the wild-type (K1) cells. A similar relation was found when lipoprotein lipase was metabolically labelled with 35S-methionine and then immunoprecipitated. Heparin stimulated secretion from all three cell types to a similar extent (about twofold). Heparin-releasable binding of 125I-labelled lipoprotein lipase was lower to either of the mutant cells than to the wild-type cells. Binding to the wild-type cells was reduced by heparitinase, while the low binding to the mutants was not affected. By immunogold labelling of cryosections, lipoprotein lipase was detected on the plasma membranes and on the inside of secretory vesicles of both wild-type and mutant cells, suggesting that some carrier could be involved. Inhibition of vesicular transport by monensin caused accumulation of lipoprotein lipase in the cells. In wild-type cells the lipase was mainly on the inside of vesicular structures, while in the mutants the main part was associated with membranous bodies that formed within the vesicles during a chase period. These results suggest that if lipoprotein lipase needs a carrier during intracellular assembly and transport, this function can be fulfilled by some structure other than heparan sulfate.

Animals↗
PubMed1995

Synthesis and glycosaminoglycan priming activity of three disaccharides related to the linkage region tetrasaccharide of proteoglycans.

To test if disaccharides might serve as primers of oligosaccharide synthesis in animal cells, we synthesized 2-naphthyl O-(beta-D-galactopyranosyl)-(1 --> 4)-beta-D-xylopyranoside, 2-naphthyl O-(beta-D-galactopyranosyl)-(1 --> 3)-beta-D-galactopyranoside, and 2-naphthyl O-(beta-D-glucopyranosyluronic acid)-(1 --> 3)-beta-D-galactopyranoside. These three disaccharides are related to subunits of the linkage tetrasaccharide of heparan sulfate and chondroitin sulfate chains in animal cell proteoglycans. The disaccharides were synthesized with coupling efficiencies of 40-70% using thioglycosides or by activating the monosaccharides with trichloroacetimidate. The structures of these compounds were confirmed by 1H NMR, 13C NMR and elemental analysis. The ability of these disaccharides to prime glycosaminoglycan chains was examined in a Chinese hamster ovary cell mutant, p gsA 745, which lacks xylosyltransferase. The missing enzyme renders the cells dependent on exogenous primers for making glycosaminoglycan chains. 2-Naphthyl O-(beta-D-galactopyranosyl)-(1 --> 3)-beta-D-galactopyranoside and 2-naphthyl O-(beta-D-glucopyranosyluronic acid)-(1 --> 3)-beta-D-galactopyranoside did not stimulate glycosaminoglycan synthesis, but 2-naphthyl O-(beta-D-galactopyranosyl)-(1 --> 4)-beta-D-xylopyranoside at high concentration primed chains. The peracetylated derivative (2-naphthyl O-(2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-(1 --> 4)- 2,3-di-O-acetyl-beta-D-xylopyranoside) primed chains at lower concentration (100 microM), suggesting that cells took up the compound and removed the acetyl groups apparently in the compartment where glycosaminoglycan synthesis occurs.

Acetylation↗
PubMed1995

Repetitive Ser-Gly sequences enhance heparan sulfate assembly in proteoglycans.

We showed previously that the synthesis of heparan sulfate on betaglycan occurs at a Ser-Gly dipeptide flanked by a cluster of acidic residues and an adjacent tryptophan (Zhang, L., and Esko, J.D. (1994) J. Biol. Chem. 269, 19295-19299). A survey of the protein data base revealed that most heparan sulfate proteoglycans contain repetitive (Ser-Gly)n segments (n = 2) and a nearby cluster of acidic residues. To study the role of these amino acid sequences in controlling heparan sulfate synthesis, we have examined the assembly of glycosaminoglycans on Chinese hamster ovary (CHO) cell syndecan-1. The glycosylation sites were mapped by making chimeric proteoglycans containing segments of CHO syndecan-1 cDNA fused to Protein A. Two sites near the transmembrane domain (-EGS205GEQ- and -ETS215GEN-) were used solely for chondroitin sulfate synthesis, whereas three sites near the N terminus (-DGS35GDDSDNFS45GS47GTG-) supported both heparan sulfate and chondroitin sulfate synthesis. The strongest sites for heparan sulfate synthesis consisted of the repeat unit, -S45GS47G-. An unusual coupling phenomenon occurred across the adjacent SG dipeptides, leading to a greater proportion of heparan sulfate than predicted by the behavior of each site acting independently. The clusters of acidic residues adjacent to the heparan sulfate sites play important roles as well. These sequence motifs suggest a set of rules for predicting whether heparan sulfate assembles at glycosylation sites in proteoglycan core proteins.

Amino Acid Sequence↗
PubMed1995

Accumulation of a pentasaccharide terminating in alpha-N-acetylglucosamine in an animal cell mutant defective in heparan sulfate biosynthesis.

Heparan sulfate biosynthesis initiates by the transfer of alpha-D-GlcNAc from UDP-GlcNAc to the D-GlcA moiety of the linkage tetrasaccharide, GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-core protein. The enzyme catalyzing this reaction differs from the alpha-GlcNAc transferase involved in chain polymerization based on genetic and enzymatic studies of an animal cell mutant defective in chain polymerization (Fritz, T. A., Gabb, M. M., Wei, G., and Esko, J. D. (1994) J. Biol. Chem. 269, 28809-28814). In this report we show that this mutant also accumulates a pentasaccharide intermediate containing alpha-GlcNAc. A fusion protein was made from the IgG-binding domain of protein A and a segment of the proteoglycan, betaglycan. This segment contained one glycosaminoglycan attachment site that primes only chondroitin sulfate and another that primes both heparan sulfate and chondroitin sulfate (Zhang, L., and Esko, J. D. (1994) J. Biol. Chem. 264, 19295-19299). Expression of the chimera in the mutant resulted in the accumulation of an oligosaccharide that labeled with [6-3H]GlcN. The oligosaccharide comigrated with a pentasaccharide standard derived from chondroitin sulfate, but acid hydrolysis gave 98% [3H]GlcN. Heparin lyase III digestion yielded [3H]GlcNAc, suggesting that the GlcNAc residue was alpha-linked to the nonreducing terminus. Enzymatic treatment of [6-3H]Gal-labeled material yielded the tetrasaccharide, delta GlcA-[3H]Gal-[3H]Gal-xylitol. These findings suggest that pentasaccharide had the structure, GlcNAc alpha 1-4GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl. Its accumulation in a Chinese hamster ovary cell mutant defective in the polymerizing alpha-GlcNAc transferase provides in vivo evidence that two alpha-GlcNAc transferases catalyze the formation of heparan sulfate.

Acetylglucosamine↗
PubMed1995

Killing of trypanosomes by the human haptoglobin-related protein.

African trypanosomes cause disease in humans and animals. Trypanosoma brucei brucei affects cattle but not humans because of its sensitivity to a subclass of human high density lipoproteins (HDLs) called trypanosome lytic factor (TLF). TLF contains two apolipoproteins that are sufficient to cause lysis of T. b. brucei in vitro. These proteins were identified as the human haptoglobin-related protein and paraoxonase-arylesterase. An antibody to haptoglobin inhibited TLF activity. TLF was shown to exhibit peroxidase activity and to be inhibited by catalase. These results suggest that TLF kills trypanosomes by oxidative damage initiated by its peroxidase activity.

Amino Acid Sequence↗