Isolation and characterization from porcine serum of a soluble sulfotransferase responsible for 6-O-sulfation of the galactose residue in 2'-fucosyllactose: implications in the synthesis of the ligand for L-selectin.
A soluble sulfotransferase from porcine serum which catalyzes the transfer of sulfate from adenosine 3'-phosphate 5'-phosphosulphate (PAPS) to 2'-fucosyllactose (2'-FL) was purified 36,333-fold using a combination of conventional and affinity chromatographic steps. The purified enzyme preparation after non-denaturing discontinuous-PAGE exhibited a molecular mass of about 80 kDa by reducing SDS-PAGE. However, when a partially purified enzyme preparation was subjected to gel filtration on Sephacryl S-300, the enzyme activity eluted in the void volume, which indicated that the native enzyme existed as an oligomer. The purified enzyme showed Km values of 9.15 microM for PAPS and 15.38 mM for 2'-FL at the optimum pH value of 7.4. The substrate specificity of the purified enzyme was evaluated with various sugars that are structurally similar to sialyl LewisX (sLeX). Results indicated that 3'-sialyllactose and lactose were efficient acceptors of sulfation, whereas 6'-sialyllactose and 6'-sialyllactosamine were poor substrates for this sulfotransferase. Further, the reaction product analysis revealed that the sulfate substitution, when using 2'-FL as the substrate, was at the C-6 position of the galactose residue. Coincidentally, a similar enzyme activity was also found in porcine lymphoid tissues such as, lymph nodes (peripheral and mesenteric) and spleen. Collectively, these findings suggest that this enzyme might be involved in the synthesis of the ligand for L-selectin.
Huynh
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QK
Discovery Research, G. D. Searle and Company, Monsanto Company, St. Louis, MO 63167, USA. quang.k.huynh@monsanto.com
Shailubhai
K
K
Boddupalli
H
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Yu
H H
HH
Broschat
K O
KO
Jacob
G S
GS
D000818:Animals; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D005690:Galactose / Q000737:chemistry*; D006801:Humans; D007785:Lactose / Q000031:analogs & derivatives / Q000737:chemistry; D008970:Molecular Weight; D009844:Oligosaccharides / Q000737:chemistry; D010724:Phosphoadenosine Phosphosulfate / Q000737:chemistry; D013379:Substrate Specificity; D015238:Sulfotransferases / Q000097:blood / Q000737:chemistry* / Q000302:isolation & purification; D013552:Swine; D014312:Trisaccharides / Q000737:chemistry*
C403777:6'-sialyllactose; D009844:Oligosaccharides; D014312:Trisaccharides; D010724:Phosphoadenosine Phosphosulfate; C062923:N-acetylneuraminosyl(alpha2-6)lactosamine; D015238:Sulfotransferases; D007785:Lactose; D005690:Galactose; C031420:2'-fucosyllactose