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Lidholt, Kerstin
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Publications (10 of 21) Show all publications
Roman, E., Roberts, I., Lidholt, K. & Kusche-Gullberg, M. (2003). Overexpression of UDP-glucose dehydrogenase in Escherichia coli results in decreased biosynthesis of K5 polysaccharide. Biochemical Journal, 374(3), 767-772
Open this publication in new window or tab >>Overexpression of UDP-glucose dehydrogenase in Escherichia coli results in decreased biosynthesis of K5 polysaccharide
2003 (English)In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 374, no 3, p. 767-772Article in journal (Refereed) Published
Abstract [en]

The Escherichia coli K5 capsular polysaccharide (glycosaminoglycan) chains are composed of the repeated disaccharide structure: -GlcAbeta1,4-GlcNAcalpha1,4-(where GlcA is glucuronic acid and GlcNAc is N-acetyl-D-glucosamine). The GlcA, present in most glycosaminoglycans, is donated from UDP-GlcA, which, in turn, is generated from UDP-glucose by the enzyme UDP-glucose dehydrogenase (UDPGDH). The formation of UDP-GlcA is critical for the biosynthesis of glycosaminoglycans. To investigate the role of UDPGDH in glycosaminoglycan biosynthesis, we used K5 polysaccharide biosynthesis as a model. E. coli was transformed with the complete gene cluster for K5 polysaccharide production. Additional transformation with an extra copy of UDPGDH resulted in an approx. 15-fold increase in the in vitro UDPGDH enzyme activity compared with the strain lacking extra UDPGDH. UDP-GlcA levels were increased 3-fold in overexpressing strains. However, metabolic labelling with [14C]glucose showed, unexpectedly, that overexpression of UDPGDH lead to decreased formation of K5 polysaccharide. No significant difference in the K5 polysaccharide chain length was observed between control and overexpressing strains, indicating that the decrease in K5-polysaccharide production most probably was due to synthesis of fewer chains. Our results suggest that K5-polysaccharide biosynthesis is strictly regulated such that increasing the amount of available UDP-GlcA results in diminished K5-polysaccharide production.

Keywords
bacterial capsule, Escherichia coli, glycosaminoglycan, K5 polysaccharide, UDP-glucose dehydrogenase, UDP-glucuronic acid
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-92360 (URN)10.1042/BJ20030365 (DOI)000185546800021 ()12775214 (PubMedID)
Available from: 2004-11-17 Created: 2004-11-17 Last updated: 2017-12-14Bibliographically approved
Hodson, N., Griffiths, G., Cook, N., Pourhossein, M., Gottfridson, E., Lind, T., . . . Roberts, I. (2000). Identification that KfiA, a protein essential for the biosynthesis of the Escherichia coli K5 capsular polysaccharide, is an alpha -UDP-GlcNAcglycosyltransferase: The formation of a membrane-associated K5 biosynthetic complex requires KfiA, KfiB and KfiC.. Journal of Biological Chemistry, 275(35), 27311-27315
Open this publication in new window or tab >>Identification that KfiA, a protein essential for the biosynthesis of the Escherichia coli K5 capsular polysaccharide, is an alpha -UDP-GlcNAcglycosyltransferase: The formation of a membrane-associated K5 biosynthetic complex requires KfiA, KfiB and KfiC.
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2000 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 275, no 35, p. 27311-27315Article in journal (Refereed) Published
Abstract [en]

The Escherichia coli K5 capsular polysaccharide consists of the repeat structure -4)GlcA-beta(1,4)-GlcNAc-alpha(1-and requires the KfiA, KfiB, KfiC, and KfiD proteins for its synthesis, Previously, the KfiC protein was shown to be a beta-UDP-GlcA glycosyltransferase, and KfiD was shown to be a UDP-Glc dehydrogenase. Here, we demonstrate that KfiA is an alpha-UDP-GlcNAc glycosyltransferase and that biosynthesis of the K5 polysaccharide involves the concerted action of the KfiA and KfiC proteins. By site-directed mutagenesis, we determined that the acidic motif of DDD, which is conserved between the C family of glycosyltransferases, is essential for the enzymatic activity of KfiA III addition, by Western blot analysis, we determined that association of KfiA with the cytoplasmic membrane requires KfiC but not KfiB, whereas the interaction of KfiC with the cytoplasmic membrane was dependent on both KfiA and KfiB. Likewise, KfiB was only detectable in cytoplasmic membrane fractions when both KfiA and KfiC were present. These data suggest that the interaction between the KfiA, KfiB, and KfiC proteins is essential for the stable association of these proteins with the cytoplasmic membrane and the biosynthesis of the K5 polysaccharide.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-52413 (URN)10.1074/jbc.M004426200 (DOI)000089144800086 ()10859322 (PubMedID)
Available from: 2008-10-17 Created: 2008-10-17 Last updated: 2018-05-24Bibliographically approved
Senay, C., Lind, T., Muguruma, K., Tone, Y., Kitagawa, H., Sugahara, K., . . . Kusche-Gullberg, M. (2000). The EXT1/EXT2 tumor suppressors: catalytic activities and role in heparan sulfate biosynthesis. EMBO Reports, 1(3), 282-286
Open this publication in new window or tab >>The EXT1/EXT2 tumor suppressors: catalytic activities and role in heparan sulfate biosynthesis
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2000 (English)In: EMBO Reports, ISSN 1469-221X, E-ISSN 1469-3178, Vol. 1, no 3, p. 282-286Article in journal (Refereed) Published
Abstract [en]

The D-glucuronyltransferase and N-acetyl-D-glucosaminyltransferase reactions in heparan sulfate biosynthesis have been associated with two genes, EXT1 and EXT2, which are also implicated in the inherited bone disorder, multiple exostoses. Since the cell systems used to express recombinant EXT proteins synthesize endogenous heparan sulfate, and the EXT proteins tend to associate, it has not been possible to define the functional roles of the individual protein species. We therefore expressed EXT1 and EXT2 in yeast, which does not synthesize heparan sulfate. The recombinant EXT1 and EXT2 were both found to catalyze both glycosyltransferase reactions in vitro. Coexpression of the two proteins, but not mixing of separately expressed recombinant EXT1 and EXT2, yields hetero-oligomeric complexes in yeast and mammalian cells, with augmented glycosyltransferase activities. This stimulation does not depend on the membrane-bound state of the proteins.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-133863 (URN)10.1093/embo-reports/kvd045 (DOI)11256613 (PubMedID)
Available from: 2010-11-16 Created: 2010-11-16 Last updated: 2018-05-24
Lind, T., Falk, E., Hjertson, E., Kusche-Gullberg, M. & Lidholt, K. (1999). cDNA cloning and expression of UDP-glucose dehydrogenase from bovine kidney. Glycobiology, 9(6), 595-600
Open this publication in new window or tab >>cDNA cloning and expression of UDP-glucose dehydrogenase from bovine kidney
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1999 (English)In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 9, no 6, p. 595-600Article in journal (Refereed) Published
Abstract [en]

We have isolated a cDNA encoding UDP-glucose dehydrogenase from a bovine kidney cDNA-library, the first mammalian cDNA clone published. [After submission of the manuscript, a study appeared describing the molecular cloning and characterization of  the human and mouse UDP-glucose dehydrogenase genes(Spicer et al., 1998).] The enzyme catalyzes the conversion of UDP-glucose to UDP-glucuronicacid, an essential precursor in glycosaminoglycan biosynthesis. The cDNA has an open reading frame of 1482 nucleotides coding for a 55 kDa protein. Expression of the enzyme in COS-7 cells showed a 3-fold increase inUDP-glucose dehydrogenase activity; also, the C-terminal 23 amino acidswas shown not to be necessary for enzyme activity. Northernblots from human and mouse tissues reveal high expression in liver and low in skeletal muscle. Human tissues have a majortranscript size of 3.2 kilobases and a minor of 2.6 whereas mousetissues have a single 2.6 kilobase transcript. We have also developed a sensitive and direct assay using UDP-[14C]Glc as a substrate for detection of small amounts of UDPGDH activity. 

Keywords
UDP-Glc dehydrogenase, UDP-GlcA, glycosaminoglycan, proteoglycan, biosynthesis
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-92359 (URN)10336992 (PubMedID)
Available from: 2004-11-17 Created: 2004-11-17 Last updated: 2018-11-13
Tsuchida, K., Lind, T., Kitagawa, H., Lindahl, U., Sugahara, K. & Lidholt, K. (1999). Purification and characterization of fetal bovine serum beta-N-acetyl-D-galactosaminyltransferase and beta-D-glucuronyltransferase involved in chondroitin sulfate biosynthesis. European Journal of Biochemistry, 264(2), 461-467
Open this publication in new window or tab >>Purification and characterization of fetal bovine serum beta-N-acetyl-D-galactosaminyltransferase and beta-D-glucuronyltransferase involved in chondroitin sulfate biosynthesis
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1999 (English)In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 264, no 2, p. 461-467Article in journal (Refereed) Published
Abstract [en]

beta-N-Acetylgalactosaminyltransferase II and beta-glucuronyltransferase II, involved in chondroitin sulfate biosynthesis, transfer an N-acetylgalactosamine (GalNAc) and glucuronic acid (GlcA) residue, respectively, through beta-linkages to an acceptor chondroitin oligosaccharide derived from the repeating disaccharide region of chondroitin sulfate. They were copurified from fetal bovine serum approximately 2500-fold and 850-fold, respectively, by sequential chromatographies on Red A-agarose, phenyl-Sepharose, S-Sepharose and wheat germ agglutinin-agarose. Identical and inseparable chromatographic profiles of both glycosyltransferase activities obtained through the above chromatographic steps and gel filtration suggest that the purified enzyme activities are tightly coupled, which could imply a single enzyme with dual transferase activities; beta-N-acetylgalactosaminyltransferase and beta-glucuronyltransferase, reminiscent of the heparan sulfate polymerase reaction. However, when a polymerization reaction was performed in vitro with the purified serum enzyme preparation under the polymerization conditions recently developed for the chondroitin-synthesizing system, derived from human melanoma cells, each monosaccharide transfer took place, but no polymerization occurred. These results may suggest that the purified serum enzyme preparation contains both beta-N-acetylgalactosaminyltransferase II and beta-glucuronyltransferase II activities on a single polypeptide or on the respective polypeptides forming an enzyme complex, but is different from that obtained from melanoma cells in that it transfers a single GalNAc or GlcA residue but does not polymerize chondroitin.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-61027 (URN)10.1046/j.1432-1327.1999.00635.x (DOI)000082367500023 ()10491092 (PubMedID)
Available from: 2008-10-17 Created: 2008-10-17 Last updated: 2018-05-24
Griffiths, G., Cook, N. J., Gottfridson, E., Lind, T., Lidholt, K. & Roberts, I. S. (1998). Characterization of the glycosyltransferase enzyme from the Escherichia coli K5 capsule gene cluster and identification and characterization of the glucuronyl active site.. Journal of Biological Chemistry, 273(19), 11752-7
Open this publication in new window or tab >>Characterization of the glycosyltransferase enzyme from the Escherichia coli K5 capsule gene cluster and identification and characterization of the glucuronyl active site.
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1998 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 273, no 19, p. 11752-7Article in journal (Refereed) Published
Abstract [en]

Bacterial capsular polysaccharides play an important role in virulence and survival. The Escherichia coli K5 capsule consists of a repeat structure of -4)GlcA-beta(1,4)-GlcNAc alpha(1-, identical to N-acetylheparosan. A 60-kDa protein, KfiC, has been identified as a bifunctional glycosyltransferase, responsible for the alternating alpha and beta addition of each UDP-sugar to the nonreducing end of the polysaccharide chain. Using hydrophobic cluster analysis, a conserved secondary structure motif characteristic of beta-glycosyltransferases was identified along with two highly conserved aspartic acid residues at positions 301 and 352 within the KfiC protein. Site-directed mutagenesis was used to identify catalytically active amino acids within domain A of the KfiC protein. The conserved aspartic acid residues at 301 and 352 were shown to be critical for the beta addition of UDP-GlcA (uridine diphosphoglucuronic acid) to defined nonreducing end oligosaccharide acceptors, suggesting that these conserved aspartic acid residues are catalytically important for beta-glycosyltransferase activity. A deleted derivative of the kfiC gene was generated, which encoded for a truncated KfiC (kfiC') protein. This protein lacked 139 amino acids at the C terminus. This enzyme had no UDP-GlcA transferase activity but still retained UDP-GlcNAc transferase activity, indicating that two separate active sites are present within the KfiC protein.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-133866 (URN)9565598 (PubMedID)
Available from: 2010-11-16 Created: 2010-11-16 Last updated: 2018-05-24
Bourgeois, C., Bour, J., Lidholt, K., Gauthray, C. & Pothier, P. (1998). Heparin-like structures on respiratory syncytial virus are involved in its infectivity in vitro.. Journal of Virology, 72(9), 7221-7227
Open this publication in new window or tab >>Heparin-like structures on respiratory syncytial virus are involved in its infectivity in vitro.
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1998 (English)In: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 72, no 9, p. 7221-7227Article in journal (Refereed) Published
Abstract [en]

Addition of heparin to the virus culture inhibited syncytial plaque formation due to respiratory syncytial virus (RSV). Moreover, pretreatment of the virus with heparinase or an inhibitor of heparin, protamine, greatly reduced virus infectivity. Two anti-heparan sulfate antibodies stained RSV-infected cells, but not noninfected cells, by immunofluorescence. One of the antibodies was capable of neutralizing RSV infection in vitro. These results prove that heparin-like structures identified on RSV play a major role in early stages of infection. The RSV G protein is the attachment protein. Both anti-heparan sulfate antibodies specifically bound to this protein. Enzymatic digestion of polysaccharides in the G protein reduced the binding, which indicates that heparin-like structures are on the G protein. Such oligosaccharides may therefore participate in the attachment of the virus.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-52532 (URN)000075328600029 ()9696816 (PubMedID)
Available from: 2008-10-17 Created: 2008-10-17 Last updated: 2017-12-04Bibliographically approved
Lind, T., Tufaro, F., McCormick, C., Lindahl, U. & Lidholt, K. (1998). The putative tumor suppressors EXT1 and EXT2 are glycosyltransferases required for the biosynthesis of heparan sulfate. Journal of Biological Chemistry, 273(41), 26265-26268
Open this publication in new window or tab >>The putative tumor suppressors EXT1 and EXT2 are glycosyltransferases required for the biosynthesis of heparan sulfate
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1998 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 273, no 41, p. 26265-26268Article in journal (Refereed) Published
Abstract [en]

Hereditary multiple exostoses, characterized by multiple cartilaginous tumors, is ascribed to mutations at three distinct loci, denoted EXT1-3. Here, we report the purification of a protein from bovine serum that harbored the D-glucuronyl (GlcA) and N-acetyl-D-glucosaminyl (GlcNAc) transferase activities required for biosynthesis of the glycosaminoglycan, heparan sulfate (HS). This protein was identified as EXT2. Expression of EXT2 yielded a protein with both glycosyltransferase activities. Moreover, EXT1, previously found to rescue defective HS biosynthesis (McCormick, C., Leduc, Y., Martindale, D., Mattison, K., Esford, L. E., Dyer, A. P., and Tufaro, F. (1998) Nat. Genet. 19, 158-161), was shown to elevate the low GlcA and GlcNAc transferase levels of mutant cells. Thus at least two members of the EXT family of tumor suppressors encode glycosyltransferases involved in the chain elongation step of HS biosynthesis.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-133862 (URN)9756849 (PubMedID)
Available from: 2010-11-16 Created: 2010-11-16 Last updated: 2018-05-24
Lidholt, K., Fjelstad, M., Lindahl, U., Goto, F., Ogawa, T., Kitagawa, H. & Sugahara, K. (1997). Assessment of glycosaminoglycan-protein linkage tetrasaccharides as acceptors for GalNAc- and GlcNAc-transferases from mouse mastocytoma.. Glycoconjugate Journal, 14(6), 737-742
Open this publication in new window or tab >>Assessment of glycosaminoglycan-protein linkage tetrasaccharides as acceptors for GalNAc- and GlcNAc-transferases from mouse mastocytoma.
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1997 (English)In: Glycoconjugate Journal, ISSN 0282-0080, E-ISSN 1573-4986, Vol. 14, no 6, p. 737-742Article in journal (Refereed) Published
Abstract [en]

Two glycosaminoglycan-protein linkage tetrasaccharide-serine compounds, GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser and GlcA beta 1-3Gal(4-O-sulfate)beta 1-3Gal beta 1-4Xyl beta 1-O-Ser, were tested as hexosamine accepters, using UDP-[H-3]GlcNAc and UDP-[H-3]GalNAc as sugar donors, and solubilized mouse mastocytoma microsomes as enzyme source. The nonsulfated Ser-tetrasaccharide was found to function as an acceptor for a GalNAc residue, whereas the Ser-tetrasaccharide containing a sulfated galactose unit was inactive. Characterization of the radio-labelled product by digestion with alpha-N-acetylgalactosaminidase and beta-N-acetylhexosaminidase revealed that the [H-3]GalNAc unit was alpha-linked, as in the product previously synthesized using serum enzymes, and not beta-linked as found in the chondroitin sulfate polymer. Heparan sulfate/heparin biosynthesis could not be primed by either of the two linkage Ser-tetrasaccharides, since no transfer of [H-3]GlcNAc from UDP-[H-3]GlcNAc could be detected. By contrast, transfer of a [H-3]GlcNAc unit to a [GlcA beta 1-4GlcNAca1-4](2)-GlcA beta 1-4-aMan hexasaccharide acceptor used to assay the GlcNAc transferase involved in chain elongation, was readily detected. These results are in agreement with the recent proposal that two different N-acetylglucosaminyl transferases catalyse the biosynthesis of heparan sulfate. Although the mastocytoma system contains both the heparan sulfate/heparin and chondroitin sulfate biosynthetic enzymes the Ser-tetrasaccharides do not seem to fulfil the requirements to serve as accepters for the first HexNAc transfer reactions involved in the formation of these polysaccharides.

Keywords
Biosynthesis, GalNAc transferase, GlcNAc transferase, glycosaminoglycan, proteoglycan
Identifiers
urn:nbn:se:uu:diva-52355 (URN)A1997XY64800006 ()9337087 (PubMedID)
Available from: 2008-10-17 Created: 2008-10-17 Last updated: 2017-12-04Bibliographically approved
Lidholt, K. (1997). Biosynthesis of glycosaminoglycans in mammalian cells and in bacteria [Review]. Biochemical Society Transactions, 25(3), 866-70
Open this publication in new window or tab >>Biosynthesis of glycosaminoglycans in mammalian cells and in bacteria
1997 (English)In: Biochemical Society Transactions, ISSN 0300-5127, E-ISSN 1470-8752, Vol. 25, no 3, p. 866-70Article, book review (Other academic) Published
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-60465 (URN)10.1042/bst025420s (DOI)9388563 (PubMedID)
Note
661st Meeting Bath Held at the University of Bath 9–11 April 1997Available from: 2008-10-17 Created: 2008-10-17 Last updated: 2017-12-01Bibliographically approved
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