Erik Forsberg

Abnormal mast cells in mice deficient in a heparin-synthesizing enzyme

Heparin is a sulphated polysaccharide, synthesized exclusively by connective-tissue-type mast cells and stored in the secretory granules in complex with histamine and various mast-cell proteases. Although heparin has long been used as an antithrombotic drug, endogenous heparin is not present in the blood, so it cannot have a physiological role in regulating blood coagulation. The biosynthesis of heparin involves a series of enzymatic reactions, including sulphation at various positions,. The initial modification step, catalysed by the enzyme glucosaminyl N -deacetylase/N -sulphotransferase-2, NDST-2 (refs 4–7), is essential for the subsequent reactions. Here we report that mice carrying a targeted disruption of the gene encoding NDST-2 are unable to synthesize sulphated heparin. These NDST-2-deficient mice are viable and fertile but have fewer connective-tissue-type mast cells; these cells have an altered morphology and contain severely reduced amounts of histamine and mast-cell proteases. Our results indicate that one site of physiological action for heparin could be inside connective-tissue-type mast cells, where its absence results in severe defects in the secretory granules.

Heparan sulfate: lessons from knockout mice

Kidney agenesis, “broken heart,” abnormal mast cells, somatic overgrowth, lung dysfunction, and chondrodysplasia are some phenotypes of mice where different genes important for heparan sulfate (HS) expression have been knocked out. What can we learn about HS function by studying these mice? In this Perspective we focus on data obtained so far from mouse models where the targets for the genetic modifications are the various enzymes taking part in HS biosynthesis. In addition, we will briefly discuss some mouse models in which genes for HS proteoglycan core proteins have been knocked out.

Heparan sulfate and development: differential roles of the N-acetylglucosamine N-deacetylase/N-sulfotransferase isozymes.

Heparan sulfates (HSs) are N- and O-sulfated polysaccharide components of proteoglycans, which are important constituents of the cell surface as well as the extracellular matrix. Heparin, with extensive clinical application as an anticoagulant, is a highly sulfated form of HS present within the granules of connective tissue type mast cells. The diverse functions of HS, which include the modulation of growth factor/cytokine activity, interaction with matrix proteins and binding of enzymes to cell surfaces, depend greatly on the presence of specific, high affinity regions on the chains. N-acetylglucosamine N-deacetylase/N-sulfotransferases, NDSTs, are an important group of enzymes in HS biosynthesis, initiating the sulfation of the polysaccharide chains and thus determining the generation of the high affinity sites. Here, we review the role of the four vertebrate NDSTs in HS biosynthesis as well as their regulated expression. The main emphasis is the phenotypes of mice lacking one or more of the NDSTs.

Restricted distribution of laminin α1 chain in normal adult mouse tissues

Abstract The distribution of laminin α1 chain in adult mouse tissue was determined by immunofluorescence using monoclonal antibody 200, reacting with the globular carboxyterminus E3 fragment of α1 chain. Strong reactivity was noted only in a few tissues. Reactivity was restricted to epithelial basement membranes. Expression was noted in several epithelial basement membranes of the urinary tract, and male and female reproductive organs. In addition, expression was seen in some parts of the nervous system. Expression was seen in pia mater which surrounds the brain, and in the extracellular matrices covering the vitreous chamber and the lens of the eye. Staining was seen in the adrenal gland cortex, with strongest staining in the zona glomerulosa. Staining was negative in all other studied epithelial basement membranes, such as the lung (trachea or lung epithelium), epidermis, and all parts of the gastrointestinal tract (liver, gut) except for weak staining in the ventricle and Brunner’s glands. No expression was seen in basement membranes of fat, Schwann, or endothelial cells in any studied parts of the body. Both small- and large-size vessel walls were negative both in endothelial basement membranes and blood vessel walls, with the exception of some larger brain blood vessels in locations where epithelial cells have invaginated. Neither smooth muscle, myocardium or striated muscle expressed α1 chain. We conclude that α1-containing heterotrimers including laminin-1 (α1β1γ1) have a very restricted tissue distribution.

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

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.

Separation of fibronectin from a plasma gelatinase using immobilized metal affinity chromatography

Conventional preparations of plasma fibronectin are known to contain a co‐purifying gelatinase [1986, J. Biol. Chem. 261, 4363–4366], but so far useful methods to remove the protease have not been available. In this study a number of different methods were tested in order to achieve separation of the two proteins. Immobilized metal affinity chromatography was found to be efficient for this purpose, and a convenient procedure to separate the two proteins under nondenaturing conditions on chelating Sepharose charged with Co2+, Ni2+, or Zn2+ is described. An alternative method employing pH gradient elution of an Fe3+ gel also resolved fibronectin from the gelatinase. The Fe3+ gel bound both proteins at pH 6.0 but not at pH 7.4, suggesting that the two proteins were phosphorylated. The described procedures will now allow studies of the functions of fibronectin in the absence of the contaminating protease.

Disturbed Ca2+ kinetics in N-deacetylase/N-sulfotransferase-1 defective myotubes.

The biosynthesis of heparan sulfate, present on the cell surface and in the basal lamina surrounding cells, is a multistep process in which each step is mediated by a specific enzyme. The initial modification of the precursor polysaccharide, N-deacetylation followed by N-sulfation of selected N-acetyl-D-glucosamine residues, is catalyzed by the enzyme glucosaminyl N-deacetylase/N-sulfotransferase (NDST). This event is a key step that regulates the overall sulfate content of the polysaccharide. Here, we report on the effects of NDST deficiency on Ca2+ kinetics in myotubes from NDST-1- and NDST-2-deficient mice, indicating a novel role for heparan sulfate in skeletal muscle physiology. Immunostaining for specific heparan sulfate epitopes showed major changes in the heparan sulfate composition in skeletal muscle tissue derived from NDST-1–/– mice and NDST–/– cultured myotubes. Biochemical analysis indicates a relative decrease in both N-sulfation and 2-O-sulfation of skeletal muscle heparan sulfate. The core protein of heparan sulfate proteoglycan perlecan was not affected, as judged by immunohistochemistry. Also, acetylcholine receptor clustering and the occurrence of other ion channels involved in excitation-contraction coupling were not altered. In NDST-2–/– mice and heterozygous mice no changes in heparan sulfate composition were observed. Using high-speed UV confocal laser scanning microscopy, aberrant Ca2+ kinetics were observed in NDST-1–/– myotubes, but not in NDST-2–/– or heterozygous myotubes. Electrically induced Ca2+ spikes had significantly lower amplitudes, and a reduced removal rate of cytosolic Ca2+, indicating the importance of heparan sulfate in muscle Ca2+ kinetics.

Affinity of integrin α1β1 from liver sinusoidal membranes for type IV collagen

Hepatic sinusoidal membanes isolated from adult rats were extracted with detergent and fractionated on a wheat germ agglutinin affinity column. Bound glycoproteins were eluted with N‐acetyl glucosamine and chromatographed on a type IV collagen affinity column. Recovery of the bound fraction by EDTA and analysis by SDS‐PAGE revealed two glycoproteins with apparent molecular weights of 180 000 and 117 000. These were identified immunologically by Western blotting as the α and β subunits of integrin α1β1.