Enzymatically Active N-Deacetylase/N-Sulfotransferase-2 Is Present in Liver but Does Not Contribute to Heparan Sulfate N-Sulfation

Abstract

Heparan sulfate (HS) proteoglycans influence embryonic development through interactions with growth factors and morphogens. The interactions depend on HS structure, which is largely determined during biosynthesis by Golgi enzymes. NDST (glucosaminyl N-deacetylase/N-sulfotransferase), responsible for HS N-sulfation, is a key enzyme directing further modifications including O-sulfation. To elucidate the roles of the different NDST isoforms in HS biosynthesis, we took advantage of mice with targeted mutations in NDST1 and NDST2 and used liver as our model organ. Of the four NDST isoforms, only NDST1 and NDST2 transcripts were shown to be expressed in control liver. The absence of NDST1 or NDST2 in the knock-out mice did not affect transcript levels of other NDST isoforms or other HS modification enzymes. Although the sulfation level of HS synthesized in NDST1 ؊/؊ mice was drastically lowered, liver HS from wild-type mice, from NDST1 ؉/؊ , NDST2 ؊/؊ , and NDST1 ؉/؊ /NDST2 ؊/؊ mice all had the same structure despite greatly reduced NDST enzyme activity (30% of control levels in NDST1 ؉/؊ /NDST2 ؊/؊ embryonic day 18.5 embryos). Enzymatically active NDST2 was shown to be present in similar amounts in wild-type, NDST1؊/؊ , and NDST1 ؉/؊ embryonic day 18.5 liver. Despite the substantial contribution of NDST2 to total NDST enzyme activity in embryonic day 18.5 liver (≈40%), its presence did not appear to affect HS structure as long as NDST1 was also present. In NDST1 ؊/؊ embryonic day 18.5 liver, in contrast, NDST2 was responsible for N-sulfation of the low sulfated HS. A tentative model to explain these results is presented.

Lowered Expression of Heparan Sulfate/Heparin Biosynthesis Enzyme N-Deacetylase/N-Sulfotransferase 1 Results in Increased Sulfation of Mast Cell Heparin

“…Detailed analyses of a number of different organs of NDST2 Ϫ/Ϫ mice failed to identify any changes in HS structure (20). In contrast, NDST1 deficiency resulted in a systemic effect on HS biosynthesis (17,21). As discussed above, NDST1 appears to have a vital role for HS synthesis in most cells whereas NDST2 is crucial for heparin sulfation in MCs.…”

“…Finally, the inability of ES cells lacking both NDST1 and NDST2 to form MCs is probably unrelated to GAGosome composition but may instead reflect a defect in mesodermal differ- Ϫ/Ϫ cells and WT cells both have identical NDST1-containing GAGosomes, they synthesize HS with identical structure (21). A few of the GAGosomes in NDST1…”

“…We also know that the degree of N-sulfation depends both on isoform and the level of enzyme expressed; overexpression of NDST1 and NDST2 in 293 cells both result in increased HS N-sulfation, but the level of N-sulfation reached is higher in NDST2-overexpressing cells (40,43). Investigating the roles of NDST1 and NDST2 in liver HS biosynthesis, we previously could conclude that in the absence of NDST1 (and NDST3 and 4, not expressed in the liver), NDST2 was responsible for HS N-sulfation (21). In NDST2 Ϫ/Ϫ liver, only NDST1 was active.…”

“…Mouse strains deficient in NDST1 (17)(18)(19) have been characterized extensively (17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31). Complete lack of NDST1 results in perinatal lethality, forebrain defects, skeletal malformation, and lung hypoplasia.…”

Drosophila Heparan Sulfate, a Novel Design

“…an extended NS-domain (43,44). Conversely, in cells that express more than one NDST isoform, HS domain structure appears to depend on the relative amounts of the enzymes (45,46). On the other hand, genetic ablation of NDST2 from mouse liver cells, which normally express both NDST1 (dominant) and NDST2, had a marginal effect on HS domain structure (46).…”