scholarly journals Structure of the keratan sulphate chains attached to fibromodulin isolated from bovine tracheal cartilage. Oligosaccharides generated by keratanase digestion

1994 ◽  
Vol 302 (2) ◽  
pp. 417-423 ◽  
Author(s):  
R M Lauder ◽  
T N Huckerby ◽  
I A Nieduszynski
Keyword(s):  
High Field ◽  
General Structure ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Repeat Region ◽  
Keratan Sulphate ◽  
Tracheal Cartilage ◽  
Acetylneuraminic Acid ◽  
Strong Anion Exchange ◽  
Structure Formula

The structure of the repeat region and chain caps of the N-linked keratan sulphate chains attached to bovine tracheal cartilage fibromodulin has been examined. The chains were fragmented by keratanase digestion, the resultant oligosaccharides isolated by strong anion-exchange chromatography, and their structures determined using high-field 1H-n.m.r. spectroscopy. The chains were found to possess the following general structure: [formula: see text] All of the capping oligosaccharides isolated terminate with alpha(2-3)-linked N-acetylneuraminic acid. No alpha(2-6)-linked N-acetylneuraminic acid chain terminators, nor any fucose, alpha (1-3)-linked to N-acetylglucosamine along the repeat region, were detected. This work demonstrates that the structure of the repeat region and chain caps of N-linked keratan sulphate attached to fibromodulin isolated from bovine tracheal cartilage is identical with that of O-linked keratan sulphate chains attached to aggrecan derived from non-articular cartilage.

scholarly journals Age-related changes in the structure of the keratan sulphate chains attached to fibromodulin isolated from articular cartilage

1998 ◽  
Vol 330 (2) ◽  
pp. 753-757 ◽  
Author(s):  
M. Robert LAUDER ◽  
N. Thomas HUCKERBY ◽  
A. Ian NIEDUSZYNSKI ◽  
H. K. Anna PLAAS
Keyword(s):  
Articular Cartilage ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Keratan Sulphate ◽  
Bovine Articular Cartilage ◽  
Acetylneuraminic Acid ◽  
Age Related ◽  
Keratanase Ii ◽  
Age Related Changes ◽  
Related Increase

Bovine articular cartilage fibromodulin has been isolated from animals aged 3 months to 8 years, and the attached keratan sulphate (KS) chains digested with keratanase II. The oligosaccharides generated have been reduced, examined by high-pH anion-exchange chromatography and their structures identified by comparison with standards. It has been shown that in fibromodulin from young articular cartilage, the KS chains do not possess either non-reducing terminal (α2-6)-linked N-acetylneuraminic acid or fucose (α1-3)-linked to sulphated N-acetylglucosamine residues. However, an age-related increase has been observed in the abundance of both (α2-6)-linked N-acetylneuraminic acid and (α1-3)-linked fucose, neither of which is found in KS isolated from non-articular cartilage, irrespective of the age of the source. Interestingly, the KS chain length remains constant as a function of age, which possibly relates to a role in collagen fibril assembly. In addition, no significant age-related changes were identified in levels of galactose sulphation.

scholarly journals Specific Non-Reducing Ends in Heparins from Different Animal Origins: Building Blocks Analysis Using Reductive Amination Tagging by Sulfanilic Acid

Molecules ◽  
2020 ◽  
Vol 25 (23) ◽  
pp. 5553
Author(s):  
Pierre A. J. Mourier
Keyword(s):  
Building Block ◽  
Reductive Amination ◽  
Building Blocks ◽  
Anion Exchange Chromatography ◽  
Sulfanilic Acid ◽  
Exchange Chromatography ◽  
Sulfated Polysaccharides ◽  
Liquid Chromatography Mass Spectrometry ◽  
Anticoagulant Drugs ◽  
Strong Anion Exchange

Heparins are linear sulfated polysaccharides widely used as anticoagulant drugs. Their nonreducing-end (NRE) has been little investigated due to challenges in their characterization, but is known to be partly generated by enzymatic cleavage with heparanases, resulting in N-sulfated glucosamines at the NRE. Uronic NRE (specifically glucuronic acids) have been isolated from porcine heparin, with GlcA-GlcNS,3S,6S identified as a porcine-specific NRE marker. To further characterize NRE in heparinoids, a building block analysis involving exhaustive heparinase digestion and subsequent reductive amination with sulfanilic acid was performed. This study describes a new method for identifying heparin classical building blocks and novel NRE building blocks using strong anion exchange chromatography on AS11 columns for the assay, and ion-pair liquid chromatography-mass spectrometry for building block identification. Porcine, ovine, and bovine intestine heparins were analyzed. Generally, NRE on these three heparins are highly sulfated moieties, particularly with 3-O sulfates, and the observed composition of the NRE is highly dependent on heparin origin. At the highest level of specificity, the isolated marker was only detected in porcine heparin. However, the proportion of glucosamines in the NRE and the proportion of glucuronic/iduronic configurations in the NRE uronic moieties greatly varied between heparin types.

scholarly journals Heparin Isomeric Oligosaccharide Separation Using Volatile Salt Strong Anion Exchange Chromatography

2016 ◽  
Vol 88 (23) ◽  
pp. 11542-11550 ◽  
Author(s):  
Rebecca L. Miller ◽  
Scott E. Guimond ◽  
Maitreyi Shivkumar ◽  
Jemma Blocksidge ◽  
James A. Austin ◽  
...  
Keyword(s):  
Anion Exchange ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Strong Anion Exchange

scholarly journals Two linkage-region fragments isolated from skeletal keratan sulphate contain a sulphated N-acetylglucosamine residue

1990 ◽  
Vol 269 (1) ◽  
pp. 55-59 ◽  
Author(s):  
J M Dickenson ◽  
T N Huckerby ◽  
I A Nieduszynski
Keyword(s):  
Gel Permeation Chromatography ◽  
Anion Exchange Chromatography ◽  
Ester Group ◽  
Intervertebral Discs ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Keratan Sulphate ◽  
Linkage Region ◽  
Gel Permeation ◽  
Electron Micro Probe Analysis

Peptido-keratan sulphate fragments were isolated from the nucleus pulposus of bovine intervertebral discs (6-year-old animals) after chondroitin ABC lyase digestion followed by digestion of A1D1 proteoglycans by diphenylcarbamoyl chloride-treated trypsin and gel-permeation chromatography on Sepharose CL-6B. Treatment of these peptido-keratan sulphate fragments with alkaline NaB3H4 yielded keratan sulphate chains with [3H]galactosaminitol end-labels, and these chains were further purified by gel-permeation chromatography on Sephadex G-50 and ion-exchange chromatography on a Pharmacia Mono-Q column in order to exclude any contamination with O-linked oligosaccharides. The chains were then treated with keratanase, and the digest was chromatographed on a Bio-Gel P-4 column followed by anion-exchange chromatography on a Nucleosil 5 SB column. Two oligosaccharides, each representing 18% of the recovered radiolabel, were examined by 500 MHz 1H-n.m.r. spectroscopy, and shown to have the following structures: [formula: see text] The structure of oligosaccharide (I) confirms the N-acetylneuraminylgalactose substitution at position 3 of N-acetylgalactosamine in the keratan sulphate-protein linkage region found by Hopwood & Robinson [(1974) Biochem. J. 141, 57-69] but additionally shows the presence of a 6-sulphated N-acetylglucosamine. Electron micro-probe analysis specifically confirmed the presence of sulphur in this sample. This sulphate ester group differentiates the keratan sulphate linkage region from similar structures derived from O-linked oligosaccharides [Lohmander, De Luca, Nilsson, Hascall, Caputo, Kimura & Heinegård (1980) J. Biol. Chem. 255, 6084-6091].

scholarly journals Amplification and Purification of T4-Like Escherichia coli Phages for Phage Therapy: from Laboratory to Pilot Scale

2013 ◽  
Vol 80 (4) ◽  
pp. 1469-1476 ◽  
Author(s):  
Gilles Bourdin ◽  
Bertrand Schmitt ◽  
Laure Marvin Guy ◽  
Jacques-Edouard Germond ◽  
Sophie Zuber ◽  
...  
Keyword(s):  
High Speed ◽  
Production Systems ◽  
Anion Exchange Chromatography ◽  
Pilot Scale ◽  
Exchange Chromatography ◽  
Cold Chain ◽  
Contamination Level ◽  
Stirred Tank Reactors ◽  
Medium Speed ◽  
Strong Anion Exchange

ABSTRACTWe investigated the amplification and purification of phage preparations with respect to titer, contamination level, stability, and technical affordability. Using various production systems (wave bags, stirred-tank reactors, and Erlenmeyer flasks), we obtained peak titers of 109to 1010PFU/ml for T4-like coliphages. Phage lysates could be sterilized through 0.22-μm membrane filters without titer loss. Phages concentrated by differential centrifugation were not contaminated with cellular debris or bacterial proteins, as assessed by electron microscopy and mass spectrometry, respectively. Titer losses occurred by high-speed pelleting of phages but could be decreased by sedimentation through a sucrose cushion. Alternative phage concentration methods are prolonged medium-speed centrifugation, strong anion-exchange chromatography, and ultrafiltration, but the latter still allowed elevated lipopolysaccharide contamination. T4-like phages could not be pasteurized but maintained their infectivity titer in the cold chain. In the presence of 10 mM magnesium ions, phages showed no loss of titer over 1 month at 30°C.

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