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Table I.

Summary of disaccharide analysis


Heparan sulfate

O-[35S]–sulfated disaccharides (% of total O-[35S]–sulfated disaccharides)

 
GlcA-GlcNS6S (GMS)
 
IdoA-GlcNS6S (IMS)
 
IdoA2S-GlcNS (ISM)
 
IdoA2S-GlcNS6S (ISMS)
 
QSulf1 treatment 13.0 ± 1.0a 6.3 ± 1.2 50.7 ± 3.1a 30.0 ± 4.6a 
QSulf1(C-A) treatment 16.0 ± 2.0 5.0 ± 1.0 35.3 ± 4.5 43.7 ± 2.3 
Untreated control 19.0 ± 3.5 4.8 ± 1.8 33.0 ± 4.2 43.5 ± 0.7 
QSulf1-expressing cells 14.0 ± 1.0b 11.0 ± 3.6 58.0 ± 3.0a 17.0 ± 3.6a 
QSulf1(C-A)-expressing cells
 
16.3 ± 2.0
 
7.7 ± 2.5
 
38.0 ± 8.6
 
38.0 ± 10.1
 

Heparan sulfate

O-[35S]–sulfated disaccharides (% of total O-[35S]–sulfated disaccharides)

 
GlcA-GlcNS6S (GMS)
 
IdoA-GlcNS6S (IMS)
 
IdoA2S-GlcNS (ISM)
 
IdoA2S-GlcNS6S (ISMS)
 
QSulf1 treatment 13.0 ± 1.0a 6.3 ± 1.2 50.7 ± 3.1a 30.0 ± 4.6a 
QSulf1(C-A) treatment 16.0 ± 2.0 5.0 ± 1.0 35.3 ± 4.5 43.7 ± 2.3 
Untreated control 19.0 ± 3.5 4.8 ± 1.8 33.0 ± 4.2 43.5 ± 0.7 
QSulf1-expressing cells 14.0 ± 1.0b 11.0 ± 3.6 58.0 ± 3.0a 17.0 ± 3.6a 
QSulf1(C-A)-expressing cells
 
16.3 ± 2.0
 
7.7 ± 2.5
 
38.0 ± 8.6
 
38.0 ± 10.1
 
a

P < 0.05 (t test).

b

P < 0.1.

[35S]HS prepared from metabolically labeled 293T cells was reacted with Myc bead–purified QSulf1 or catalytic mutant QSulf1(C-A). Untreated control was [35S]HS without treatment. To test whether QSulf1 functions in vivo, [35S]HS was prepared from stable 293T cell lines expressing QSulf1 or QSulf1(C-A) by metabolic labeling with [35S]SO4. 35S-labeled disaccharides were generated by deaminative cleavage of HS, and reaction products were resolved by HPLC anion exchange chromatography. The radioactivity in each disaccharide product was quantified by scintillation counting. Results are presented as mol-percent of specific disaccharide products in three independent experiments. M in disaccharide abbreviations stands for the 2,5-anhydromannitol deamination products of GlcNS residues (see also Fig. 3).

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