Glycanogenomics: a qPCR-approach to investigate biological glycan function.
As an indirect approach towards glycan structures, qRT-PCR analyses using the DeltaDeltaC(T) method were performed to investigate changes in expression levels of heparan sulfate-synthesising enzymes of stimulated and unstimulated HMVECs. We chose NDSTs as early enzymes initiating sulfation and 3OSTs which act late generating specific binding sites. Major changes in expression patterns were found for the NDST3 and 3OST1 isoforms. Both enzymes were down-regulated 7- and 6-fold, respectively, following TNF-alpha stimulation, and 3.5- and 7.6-fold following LPS-stimulation suggesting a common restructuring process of HS in inflammation leading to a less diverse sulfation pattern. Immunostaining of TNF-alpha-stimulated cells using a phage display-derived antibody specific for 3-O-sulfation and unsulfated regions of HS resulted in significant fluorescence changes between unstimulated and stimulated.
Krenn
Evelyn C
EC
Institute for Pharmaceutical Sciences, Department of Pharmaceutical Chemistry, University of Graz, Universitätsplatz 1, A-8010 Graz, Austria.
Wille
Iris
I
Gesslbauer
Bernd
B
Poteser
Michael
M
van Kuppevelt
Toin H
TH
Kungl
Andreas J
AJ
D002460:Cell Line; D004730:Endothelium, Vascular / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D023281:Genomics / Q000379:methods*; D006497:Heparan Sulfate / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D011134:Polysaccharides / Q000096:biosynthesis* / Q000235:genetics; D015238:Sulfotransferases / Q000235:genetics*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D006497:Heparan Sulfate; D011134:Polysaccharides; D015238:Sulfotransferases; D014409:Tumor Necrosis Factor-alpha; C021350:heparitin sulfotransferase