Systematic identification of <i>N</i>‐acetylheparosan oligosaccharides by tandem mass spectrometric fragmentation

Minamisawa, Toshikazu; Suzuki, Kiyoshi; Hirabayashi, Jun

doi:10.1002/rcm.2310

Cited by 12 publications

(6 citation statements)

References 26 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Some fractions contained HPR heptasaccharide (HPR-7). The structures of the oligosaccharides were determined by MALDI-TOF mass spectrometry and electrospray ionization-mass spectrometry analyses (17). The amount of HPR oligo-and polysaccharides was determined by carbazole assay method with GlcUA monosaccharide as standard (18).…”

Section: Methodsmentioning

confidence: 99%

Glucuronyltransferase Activity of KfiC from Escherichia coli Strain K5 Requires Association of KfiA

Sugiura

Baba

Kawaguchi

et al. 2010

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Heparan sulfate is a ubiquitous glycosaminoglycan in the extracellular matrix of most animals. It interacts with various molecules and exhibits important biological functions. K5 antigen produced by Escherichia coli strain K5 is a linear polysaccharide N-acetylheparosan consisting of GlcUA ␤1-4 and GlcNAc ␣1-4 repeating disaccharide, which forms the backbone of heparan sulfate. Region 2, located in the center of the K5-specific gene cluster, encodes four proteins, KfiA, KfiB, KfiC, and KfiD, for the biosynthesis of the K5 polysaccharide. Here, we expressed and purified the recombinant KfiA and KfiC proteins and then characterized these enzymes. Whereas the recombinant KfiC alone exhibited no GlcUA transferase activity, it did exhibit GlcUA transferase and polymerization activities in the presence of KfiA. In contrast, KfiA had GlcNAc transferase activity itself, which was unaffected by the presence of KfiC. The GlcNAc and GlcUA transferase activities were analyzed with various truncated and point mutants of KfiA and KfiC. The point mutants replacing aspartic acid of a DXD motif and lysine and glutamic acid of an ionic amino acid cluster, and the truncated mutants deleting the C-terminal and N-terminal sites, revealed the essential regions for GlcNAc and GlcUA transferase activity of KfiC and KfiA, respectively. The interaction of KfiC with KfiA is necessary for the GlcUA transferase activity of KfiC but not for the enzyme activity of KfiA. Together, these results indicate that the complex of KfiA and KfiC has polymerase activity to synthesize N-acetylheparosan, providing a useful tool toward bioengineering of defined heparan sulfate chains. Heparan sulfate (HS)3 is a linear polysaccharide of alternating hexuronic acid (D-glucuronic acid (GlcUA) or L-iduronic acid (IdoUA)) and D-glucosamine (GlcN) residues carrying sulfogroups at various sites of sugar residues. Usually, HS chains are covalently attached to a core protein in the form of proteoglycans and are present ubiquitously on the cell surface and in the extracellular matrices of animals. HS chains interact with cytokines, growth factors, coagulation factors, proteases and their inhibitors, and other molecules and contribute to several biological processes, including development, morphogenesis, cell proliferation and differentiation, and cancer cell invasion (1, 2).The biosynthesis of HS begins with the synthesis of the linkage tetrasaccharide, GlcUA-Gal-Gal-Xyl, on the serine residues of core proteins. The backbone of HS polysaccharide is then synthesized onto the linkage tetrasaccharide by alternating addition of monosaccharide units of GlcNAc and GlcUA with ␣1-4 and ␤1-4 bonds, respectively, using UDP-sugar donors with desired GlcNAc transferase (GlcNAc-T) and GlcUA transferase (GlcA-T). The elongation reaction is performed by HS co-polymerases, EXT1 and EXT2, in the Golgi apparatus (3, 4). The backbone is modified by several reactions, including N-deacetylation and N-sulfonation of GlcN by N-deacetylase/ N-sulfotransferase, C5-epimerization of GlcUA to f...

show abstract

Section: Methodsmentioning

confidence: 99%

Glucuronyltransferase Activity of KfiC from Escherichia coli Strain K5 Requires Association of KfiA

Sugiura

Baba

Kawaguchi

et al. 2010

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…The number "−2" designates deprotonation (−2H) accompanied with cleavage of the glycosidic bond because proton transfer occurs at Z and B fragment formation in the negative ion mode (mass shift rule) [29]. The m/z values observed for fragment ions between C and Y fragments having the same sugar length from saturated free oligosaccharide of nonsulfated GAG and for fragment ions between C and Z fragments from unsaturated free oligosaccharides were the same [27,30]. All the observed fragments from PA-CH oligosaccharides were distinguished from each other due to the presence of aglycon PA at their reducing ends.…”

Section: Maldi-lift-tof/tof Ms/ms Analysis Of the Pa-ch Oligosaccharidesmentioning

confidence: 80%

“…3). Ion-source decay (ISD) fragment ions that often appeared in the spectra of underivatized GAG oligosaccharides [27] were rarely observed in PA-CH oligosaccharides, since the laser power required for ionization of PA-CH oligosaccharides was substantially lower than that for ionization of the corresponding underivatized CH oligosaccharides,.…”

Section: Maldi-tof Ms Analysis Of the Pa-ch Oligosaccharidesmentioning

confidence: 99%

Sequential synthesis of chondroitin oligosaccharides by immobilized chondroitin polymerase mutants

et al. 2008

Self Cite

View full text Add to dashboard Cite

Escherichia coli strain K4 expresses a chondroitin (CH)-polymerizing enzyme (K4CP) that contains two glycosyltransferase active domains. K4CP alternately transfers glucuronic acid (GlcA) and N-acetyl-galactosamine (GalNAc) residues using UDP-GlcA and UDP-GalNAc donors to the nonreducing end of a CH chain acceptor. Here we generated two K4CP point mutants substituted at the UDP-sugar binding motif (DXD) in the glycosyltransferase active domains, which showed either glycosyltransferase activity of the intact domain and retained comparable activity after immobilization onto agarose beads. The mutant enzyme-immobilized beads exhibited an addition of GlcA or GalNAc to GalNAc or GlcA residue at the nonreducing end of CH oligosaccharides and sequentially elongated pyridylamine-conjugated CH (PA-CH) chain by the alternate use. The sequential elongation up to 16-mer was successfully achieved as assessed by fluorescent detection on a gel filtration chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and MALDI potential lift tandem TOF mass spectrometry (MALDI-LIFT-TOF/TOF MS/MS) analyses in the negative reflection mode. This method provides exactly defined CH oligosaccharide derivatives, which are useful for studies on glycosaminoglycan functions.

show abstract

“…Figure 3 and Table 3 show the results including that for NAH tetrasaccharide (DNAH4). 23) As in the case of saturated forms, C 3 ions (m/z 554) were produced for DCH4 and DHA4 most intensely. This demonstrated that this glycosidic linkage had been most fragile in the structure of the series.…”

Section: Unsaturated Tetrasaccharidesmentioning

confidence: 89%

“…22), 23) The fragmentation behavior observed was unique, reflecting the alternating HexA῍HexNAc sequence, as well as the glycosidic linkage position. 23) This prompted us to confirm MS potentiality to di#erentiate structural isomers of NAH oligosaccharides.…”

Section: Introductionmentioning

confidence: 97%

Characterization of Isomeric Unsulfated Glycosaminoglycan Oligosaccharides by Mass Spectrometry/Mass Spectrometry

Minamisawa

Suzuki

Maéda

et al. 2007

J. Mass Spectrom. Soc. Jpn.

Self Cite

View full text Add to dashboard Cite

Both saturated and unsaturated forms of isomeric unsulfated glycosaminoglycan (GAG) oligosaccharides, i.e., tetrasaccharides of chondroitin (CH) and hyaluronan (HA), were analyzed by electrospray ionization mass spectrometry/mass spectrometry. Although the only structural di#erence between them was the hydroxyl group at the C-4 position in N-acetylhexosamine (GalNAc or GlcNAc, respectively), given the same m/z value of precursor ions, these isomers in both their saturated and unsaturated forms could be separated by careful examination of diagnostic fragment ions in their product ion mass spectra when the relative abundances of these fragment ions were considered. In addition, the product ion mass spectrum of the unsaturated HA tetrasaccharide was compared with its linkage isomer, N-acetylheparosan tetrasaccharide. In this case, the isomers were more easily di#erentiated by comparing their characteristic spectral patterns. By adopting this approach, systematic di#erentiation of isomeric unsulfated GAG oligosaccharides should be achieved by means of fragmentation. It should also contribute widely to GAG-related biochemical and medicinal research in the future.

show abstract

Systematic identification of N‐acetylheparosan oligosaccharides by tandem mass spectrometric fragmentation

Cited by 12 publications

References 26 publications

Glucuronyltransferase Activity of KfiC from Escherichia coli Strain K5 Requires Association of KfiA

Glucuronyltransferase Activity of KfiC from Escherichia coli Strain K5 Requires Association of KfiA

Sequential synthesis of chondroitin oligosaccharides by immobilized chondroitin polymerase mutants

Characterization of Isomeric Unsulfated Glycosaminoglycan Oligosaccharides by Mass Spectrometry/Mass Spectrometry

Contact Info

Product

Resources

About