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

Hiroko Habuchi,Noriko Sugaya,Richard L Stevens,Koji Kimata,Naoko Nagai,Fukiko Atsumi

doi:10.1074/jbc.m607434200

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 approximately 50% 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.

Highlights

Roles of Heparan sulfate (HS) 6-O-Sulfation in Microvessel Formation can complexes in their secretory granules (19 –23)
We show that the HS chains in the liver, kidney, and lungs of these mice have a marked reduction in their 6-O-sulfate content
These results indicate that the expression of the HS6ST-1 gene was abolished in the homozygous mutant mice

Summary

EXPERIMENTAL PROCEDURES

Materials—Heparitinase I (Flavobacterium heparinum; EC 4.2.2.8), heparitinase II (F. heparinum; no number assigned), heparinase (F. heparinum; EC 4.2.2.7), and the unsaturated disaccharides kit for HS were purchased from Seikagaku Corp. The long arm in the targeting construct contained a 6-kb fragment that resided downstream of the deleted portion of the HS6ST-1 gene It was inserted upstream of the neo-cassette. Expression levels of Wnt, VEGF-A, HS6STs, and HS2ST mRNA—The placentas were dissected in cold phosphate-buffered saline from the wild-type, heterozygous, and homozygous embryos of the intercross between heterozygous mice. Expression Levels of VEGF-A Protein and HIF-1␣ in Placenta— Placental tissues from E15.5 embryos were isolated by removing most of the maternal deciduas and washed twice with cold phosphate-buffered saline and homogenized with a Polytron homogenizer in extract buffer containing 10 mM Tris-. The reaction was stopped by heating at 95 °C for 2 min, and the biotinylated HS was recovered by precipitation with 3 volumes of cold 95% (v/v) ethanol containing 1.3% potassium acetate and 1 mM EDTA. Sensorgrams were evaluated using BIAevaluation software (version 2) according to the manufacturer’s instructions

RESULTS

DISCUSSION

Koji Kimata