Expression in Escherichia coli, purification and kinetic characterization of human heparan sulfate 3-O-sulfotransferase-1.
Heparan sulfate (HS) glycosaminoglycans are a structurally diverse class of complex biomolecules that modulate many important events at the cell surface and within the extracellular matrix and whose structural heterogeneity derives largely from the sequence-specific N- and O-sulfations catalyzed by an extensive repertoire of sulfating enzymes. We have expressed the human heparan sulfate 3-OST-1 isoform in Escherichia coli and subsequently purified a soluble, active enzyme. To assess its functionality, we determined the kinetic parameters for the recombinant 3-O-sulfotransferase-1 using a radiochemical assay that directly measures the 3-O-sulfation of unlabeled bovine kidney heparan sulfate in vitro using [(35)S]PAPS as the sulfate donor. The apparent K(m) values measured were in the low micromolar range (K(HS)(m) = 4.3 microM; K(PAPS)(m) = 38.6 microM); V(max) values of 18 and 21 pmol sulfate/min/pmol of enzyme for HS and PAPS, respectively. These values were compared with kinetic parameters likewise measured for recombinant 3-OST-1 purified from baculovirus-infected sf9 cells. The two enzymes appear to modify heparan sulfate in vitro to roughly the same extent and with comparable specificities. The expression of 3-OST-1 in E. coli represents an important step in subsequent structure-function studies.
Myette
James R
JR
Division of Bioengineering and Environmental Health, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Shriver
Zachary
Z
Liu
Jian
J
Venkataraman
Ganesh
G
Rosenberg
Robert
R
Sasisekharan
Ram
R
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002240:Carbohydrate Sequence; D002417:Cattle; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006497:Heparan Sulfate / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010724:Phosphoadenosine Phosphosulfate; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera; D015238:Sulfotransferases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism
D018076:DNA, Complementary; D006497:Heparan Sulfate; D007527:Isoenzymes; D010724:Phosphoadenosine Phosphosulfate; D011994:Recombinant Proteins; D015238:Sulfotransferases; C102180:heparan sulfate D-glucosaminyl 3-O-sulfotransferase