title: Selective impairment of the synthesis of basic fibroblast growth factor binding domains of heparan sulphate in a COS cell mutant defective in N-sulphotransferase.
abstract: N-Sulphation is a key step in the overall sulphation of heparan sulphate. We have isolated a COS cell-derived mutant, CM-15, that is impaired in its ability to bind to basic fibroblast growth factor (bFGF) and has a 2- to 3-fold reduction in N-sulphotransferase activity [Ishihara et al., (1992a) Anal. Biochem., 206, 400-407]. We now provide structural evidence that CM-15 is selectively impaired in the synthesis of highly sulphated regions or 'blocks' that display high-affinity binding to bFGF; these are completely N-sulphated blocks of decasaccharide or greater length that are enriched in O-sulphate groups. The synthesis of sulphated blocks that did not show high affinity to the growth factor was relatively unimpaired in the mutant cells; this included fully N-sulphated octamer (or smaller) blocks and, unexpectedly, decasaccharide or larger blocks that were poorly O-sulphated. In the latter fraction, the failure to form high-affinity binding regions was the result of a failure to stimulate O-sulphation rather than N-sulphation in CM-15 cells. In agreement with other studies, disaccharide analysis of the wild-type-derived sulphated blocks suggested that 2-O-sulphation of iduronate residues in the polymer was a necessary element to produce a high-affinity binding sequence once N-sulphation was completed in the decasaccharide or larger fraction. These results suggest that a selective reduction in both N- and O-sulphation in the larger blocks produced by CM-15 cells is a consequence of the reduction of N-sulphotransferase activity.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Glycomed Inc., Alameda, CA 94501.
authors.forename: M
authors.initials: M
authors.lastname: Ishihara
authors.forename: Y
authors.initials: Y
authors.lastname: Guo
authors.forename: S J
authors.initials: SJ
authors.lastname: Swiedler
mesh_terms: D001665:Binding Sites; D002240:Carbohydrate Sequence; D002460:Cell Line; D002846:Chromatography, Affinity; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism*; D006497:Heparan Sulfate / Q000096:biosynthesis* / Q000737:chemistry*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D013431:Sulfates / Q000032:analysis / Q000378:metabolism; D015238:Sulfotransferases; D013466:Sulfurtransferases / Q000172:deficiency* / Q000378:metabolism
chemical_list: D016222:Fibroblast Growth Factor 2; D006497:Heparan Sulfate; D010446:Peptide Fragments; D013431:Sulfates; D015238:Sulfotransferases; D013466:Sulfurtransferases; C021350:heparitin sulfotransferase