Mice Deficient in Heparan Sulfate 6-O-Sulfotransferase-1 Exhibit Defective Heparan Sulfate Biosynthesis, Abnormal Placentation, and Late Embryonic Lethality

Abstract

Heparan sulfate (HS) plays critical roles in a variety of developmental, physiological, and pathogenic processes due to its ability to interact in a structure-dependent manner with numerous growth factors that participate in cellular signaling. The divergent structures of HS glycosaminoglycans are the result of the coordinate actions of several N-and O-sulfotransferases, C5-epimerase, and 6-O-endosulfatases. We have shown that 6-O-sulfation of the glucosamine residues in HS are catalyzed by the sulfotransferases HS6ST-1, -2, and -3. To determine the biological and physiological importance of HS6ST-1, we now describe the creation of transgenic mice that lack this sulfotransferase. Most of our HS6ST-1-null mice died between embryonic day 15.5 and the perinatal stage, and those mice that survived were considerably smaller than their wild-type littermates. Some of these HS6ST-1-null mice exhibited development abnormalities, and histochemical and molecular analyses of these mice revealed an ϳ50% reduction in the number of fetal microvessels in the labyrinthine zone of the placenta relative to that in the wild-type mice. Because we observed a modest reduction in VEGF-A mRNA and protein in the tissues of HS6ST-1-null mice, an HS-dependent defect in cytokine signaling probably contributes to increased embryonic lethality and decreased growth. Biochemical studies of the HS chains isolated from various organs of our HS6ST-1-null mice revealed a marked reduction of GlcNAc(6SO 4 ) and HexA-GlcNSO 3 (6SO 4 ) levels and a reduced ability to bind Wnt2. Thus, despite the presence of three closely related 6-O-sulfotransferase genes in the mouse genome, HS6ST-1 is the primary one used in HS biosynthesis in most tissues. Heparan sulfate (HS)2 proteoglycans on the cell surfaces interact with numerous growth factors, morphogens, receptors, and extracellular matrix proteins. These complex interactions regulate the activity, gradient formation, and stability of many receptor-ligand interactions. Thus, HS has important functions in a variety of developmental, morphogenetic, and pathogenic processes (1-3). The specificities of the interactions between HS and ligands are thought to be due, at least in part, to the fine structure of HS characterized by the specific localized patterns of sulfates and the hexuronic acid isoform residues (4 -8). The fine and divergent structures of HS are generated in the Golgi apparatus through the coordinate actions of HS modification enzymes, namely N-deacetylase/N-sulfotransferases (NDSTs), Hsepi (C5-epimerase), and 2-O-, 6-O-, and 3-O-sulfotransferases, following the biosynthesis of the HS backbone structure comprising alternating GlcA and GlcNAc residues (9, 10). Further modification of HS occurs at the cell surface by the action of 6-O-endosulfatase (11-13). The localization of some of these HS modification enzymes to the Golgi may be facilitated by their tendency to form multienzyme complexes, as shown in the case of Hsepi and HS2ST (HS 2-O-sulfotransferase) (14, 15). These modification enzymes, with...

Systemic inactivation of Hs6st1 in mice is associated with late postnatal mortality without major defects in organogenesis

“…Histological analysis of the lungs from Hs6st1 2/2 adult mutants showed enlargement of alveolar spaces, in agreement with a previous independent report ( Fig. 6a,b; Habuchi et al, 2007). Airspace enlargement was associated with an abnormal pattern of elastin staining, which looked fragmented and irregularly deposited in the alveoli (Fig.…”

“…This was accompanied by a decrease in the litter size which averaged 5.6 6 0.5 pups/litter for the Hs6st11/2 3 Hs6st11/2 crosses and 5.5 6 0.8 pups/litter for the Hs6st11/2 3 Hs6st12/2 crosses. Together the data suggested that prenatal lethality occurred in some Hs6st1 2/2 mutants, consistent with a previous report (Habuchi et al, 2007). The cause of lethality was not determined, as we could not recover dead embryos for analysis.…”

“…In our experiments the Hs6st1 2/2 mice were analyzed on a mixed (C57BL/6J 3 129) genetic background. This may explain why features such as prenatal lethality and difference in size at birth appeared to be less prominent in our model than that described in another independent report (Habuchi et al, 2007). The higher survival rate of Hs6st12/2 animals in our study allowed us to uncover yet another manifestation of Hs6st1 deficiency, namely the severe inability to gain body weight that led to an increase in postnatal lethality compared to their heterozygous littermates.…”

“…This transcript, however, lacks the critically important PAPS binding site and thus cannot be functional. In support of that, a recent report (Habuchi et al, 2007) demonstrates that genetic deletion of exon 1 sequences containing the PAPS binding site results in dramatic decrease in the overall levels of HS 6-O-sulfation in various tissues.…”

“…Nonpenetrant prenatal lethality and a postnatal lung defect have been associated with altered HS 6-O sulfation in mice lacking not only Hs6st1 but also the 6-O endosulfatase Sulf2 genes (Habuchi et al, 2007;Lum et al, 2007).…”