Altered Heparan Sulfate Structure in Mice with Deleted NDST3 Gene Function
2008; Elsevier BV; Volume: 283; Issue: 24 Linguagem: Inglês
10.1074/jbc.m709774200
ISSN1083-351X
AutoresSrinivas Reddy Pallerla, Roger Lawrence, Lars Lewejohann, Yi Pan, Tobias M. Fischer, Uwe Schlomann, Xin Zhang, Jeffrey D. Esko, Kay Grobe,
Tópico(s)Fibroblast Growth Factor Research
ResumoWe report the generation and analysis of mutant mice bearing a targeted disruption of the heparan sulfate (HS)-modifying enzyme GlcNAc N-deacetylase/N-sulfotransferase 3 (NDST3). NDST3-/- mice develop normally, are fertile, and show only subtle hematological and behavioral abnormalities in agreement with only moderate HS undersulfation. Compound mutant mice made deficient in NDST2;NDST3 activities also develop normally, showing that both isoforms are not essential for development. In contrast, NDST1-/-;NDST3-/- compound mutant embryos display developmental defects caused by severe HS undersulfation, demonstrating NDST3 contribution to HS synthesis in the absence of NDST1. Moreover, analysis of HS composition in dissected NDST3 mutant adult brain revealed regional changes in HS sulfation, indicating restricted NDST3 activity on nascent HS in defined wild-type tissues. Taken together, we show that NDST3 function is not essential for development or adult homeostasis despite contributing to HS synthesis in a region-specific manner and that the loss of NDST3 function is compensated for by the other NDST isoforms to a varying degree. We report the generation and analysis of mutant mice bearing a targeted disruption of the heparan sulfate (HS)-modifying enzyme GlcNAc N-deacetylase/N-sulfotransferase 3 (NDST3). NDST3-/- mice develop normally, are fertile, and show only subtle hematological and behavioral abnormalities in agreement with only moderate HS undersulfation. Compound mutant mice made deficient in NDST2;NDST3 activities also develop normally, showing that both isoforms are not essential for development. In contrast, NDST1-/-;NDST3-/- compound mutant embryos display developmental defects caused by severe HS undersulfation, demonstrating NDST3 contribution to HS synthesis in the absence of NDST1. Moreover, analysis of HS composition in dissected NDST3 mutant adult brain revealed regional changes in HS sulfation, indicating restricted NDST3 activity on nascent HS in defined wild-type tissues. Taken together, we show that NDST3 function is not essential for development or adult homeostasis despite contributing to HS synthesis in a region-specific manner and that the loss of NDST3 function is compensated for by the other NDST isoforms to a varying degree. Heparan sulfate (HS) 2The abbreviations used are: HS, heparan sulfate; NDST, GlcNAc N-deacetylase/N-sulfotransferase; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; GAG, glycosaminoglycan; LC/MS, liquid chromatography/mass spectrometry; MAPK, mitogen-activated protein kinase; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; wt, wild type. 2The abbreviations used are: HS, heparan sulfate; NDST, GlcNAc N-deacetylase/N-sulfotransferase; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; GAG, glycosaminoglycan; LC/MS, liquid chromatography/mass spectrometry; MAPK, mitogen-activated protein kinase; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; wt, wild type. is produced by most mammalian cells as part of membrane and extracellular matrix proteoglycans (1Esko J.D. Lindahl U. J. Clin. Invest. 2001; 108: 169-173Crossref PubMed Scopus (782) Google Scholar). The chain grows by the copolymerization of GlcAβ1,4 and GlcNAcα1,4 residues and undergoes modification by one or more of the four NDST isozymes, which remove acetyl groups from subsets of GlcNAc residues and add sulfate to the free amino groups. In vertebrates, ndst1 and ndst2 mRNA are expressed in all embryonic and adult tissues examined, whereas ndst3 and ndst4 transcripts are predominantly expressed during embryonic development and in the adult brain (2Aikawa J. Grobe K. Tsujimoto M. Esko J.D. J. Biol. Chem. 2001; 276: 5876-5882Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). Most subsequent modifications of the HS chain by O-sulfotransferases and a GlcA C5-epimerase depend on the presence of GlcNS residues, making the NDSTs largely responsible for the generation of sulfated ligand binding sites in HS (3Lindahl U. Kusche-Gullberg M. Kjellén L. J. Biol. Chem. 1998; 273: 24979-24982Abstract Full Text Full Text PDF PubMed Scopus (569) Google Scholar, 4Esko J.D. 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Cell Biol. 2007; 177: 539-549Crossref PubMed Scopus (100) Google Scholar, 26MacArthur J.M. Bishop J.R. Stanford K.I. Wang L. Bensadoun A. Witztum J.L. Esko J.D. J. Clin. Invest. 2007; 117: 153-164Crossref PubMed Scopus (167) Google Scholar).In this report, we asked to what extent HS function during development and in the adult vertebrate depends on NDST3 function and to what extent NDST3 contributes to the generation of sulfated HS. Moreover, we wished to examine whether the formation of free amino groups present on heparan sulfate is related to NDST3 activity. We describe that NDST3-deficient mice are born at slightly sub-mendelian ratio, are fertile, and show subtle changes in some hematological parameters and in their behavior. No significant overall changes in HS sulfation could be detected in those mice, but microdissection of the adult brain revealed a region-specific activity of NDST3, leading to changes in HS sulfation in the mutant brain. Mice made deficient in both NDST3 and NDST1 function revealed a role of NDST3 in the proper sulfation of nascent HS, resulting in the complete lack of one disulfated disaccharide product. We thus conclude that, although NDST3 is expressed in various tissues and contributes to HS synthesis, its activity can be substituted by the other NDSTs.EXPERIMENTAL PROCEDURESTargeted Recombination of the ndst3 Gene—The thymidine-kinase/neomycin-containing targeting vector was constructed by insertion of loxP sites in intron sequences surrounding exon 2 (the first coding exon) of ndst3, including 327 of 873 amino acids of the open reading frame. The final targeting vector was linearized using SalI before transfection of ES cells. R1 ES cells were grown, transfected, and subjected to neomycin G418 selection. Homologous recombinants were identified by Southern blotting and PCR and transfected with a cre-expressing vector, followed by gancyclovir selection. Four type II recombinants were chosen and injected in C57Bl/6J blastocysts. The mouse line obtained was backcrossed into a C57Bl/6 background for >10 generations. The primers employed for genotyping were: 5′-P1: 5′-ggtacccggggatcaattcg-3′; P2: 5′-ccagaaggctaacactgtaaag-3′; P3: 5′-gaaagtgaagtctctgggcgg-3′; and P4: 5′-gcttggatgatttggtcacact-3′. Assessment of the significance of the deviation from mendelian inheritance was performed using the Chi-square test. Compound mutant mice were derived from matings with NDST1 (18Grobe K. Inatani M. Pallerla S.R. Castagnola J. Yamaguchi Y. Esko J.D. Development. 2005; 132: 3777-3786Crossref PubMed Scopus (159) Google Scholar)- and NDST2 (22Forsberg E. Pejler G. Ringvall M. Lunderius C. Tomasini-Johansson B. Kusche-Gullberg M. Eriksson I. Ledin J. Hellman L. Kjellén L. Nature. 1999; 400: 773-776Crossref PubMed Scopus (403) Google Scholar)-deficient mice.Reverse Transcription-PCR Analysis of mRNA Expression and Protein Detection—For reverse transcription-PCR analysis in human tissues, normalized cDNA was obtained from Clontech (Human MTC Panels I+II). PCR was performed by running 38 cycles for hndst1-4. For the specific amplification of each hndst, eight specific primers were used as follows: hndst1-F (5′-ctggagccctcggcggatgc-3′) and hndst1-R(5′-ccagggtactcgttgtagaag-3′), hndst2-F (5′-aggaacccttgcccctgccc-3′) and hndst2-R (5′-gattgtgtgagtgaagaggc-3′), hndst3-F (5′-tgtgtttcctgtgagtccagatgtgtg-3′) and hndst3-R (5′-attgtcctcctcacttccatcagcctg-3′), hndst4-F (5′-aacaggaaatgacacttattgaaacc-3′), and hndst4-R (5′-actttggggcctttggtaatatg-3′).Histology and in Situ Detection of RNA—Embryos were fixed in 4% paraformaldehyde overnight, dehydrated, embedded in paraffin, and sectioned. Sections were stained with hematoxylin and eosin for histological analysis. For in situ hybridization, 700 base probes against the most variable N-terminal region of ndst1 and ndst3 and a 500-base probe against the ndst2 3′-untranslated region were employed (DIG RNA Labeling Kit, Roche Applied Science). Quantitation of apoptosis was performed on paraffin sections of two mutant and two wild-type E12.5 embryos, using the TUNEL Assay Kit (Roche Applied Science). Patched expression was detected using anti-PTC1 antiserum (Acris Antibodies, Hiddenhausen, Germany) and secondary fluorescein isothiocyanate-labeled goat anti-rabbit antibodies (Dianova, Hamburg, Germany) on three mutant and wild-type embryos.Adult Mouse Brain Immunohistochemistry—Bielschowsky stain, Gallays stain, anti-MAC-3, anti-PCNA, and anti-GFAP were employed to detect possible cellular abnormalities in NDST3 mutant brain. Images were taken on a Zeiss Axiophot microscope employing a 10×/0.3, a 20×/0.5, and a 63×/1.25 Zeiss objective and a Leica DFC280 camera. Leica software was used for image capturing and Photoshop 7 software run on Macintosh computers for the generation of figures. Contrast and brightness were adjusted for whole images during figure assembly.Preparation of HS—Mutant and wild-type tissues were pooled, homogenized using an Ultra-Turrax homogenizer (IKA, Germany), digested with 2 mg/ml Pronase in 320 mm NaCl, 100 mm sodium acetate (pH 5.5) overnight at 40 °C, diluted 1:3 in water, and applied to a 2.5-ml column of DEAE-Sephacel. After washing the column with 0.3 m NaCl, the glycosaminoglycans were eluted with 1 m NaCl. For disaccharide analysis, the GAG pool was β-eliminated overnight at 4 °C (0.5 m NaOH, 1 m NaBH4), neutralized with acetic acid until the pH was ∼6 and applied to a PD-10 (Sephadex G-25) column (Amersham Biosciences). Glycosaminoglycans eluting in the void volume were lyophilized, purified on DEAE as described above, again applied to a PD-10 column, and lyophilized. 100 mg to 1 g of tissue, depending on the source, typically yielded 40-140 μg of GAGs. 10 μg of GAG samples were digested using heparin lyases I, II, and III (1.5 milliunits of each in 100-μl reactions, IBEX, Montreal, Canada) at 37 °C for 1 h, and the resulting disaccharides were separated from undigested chondroitin sulfate using a 3-kDa spin column (Centricon, Bedford, MA). Compositional disaccharide analysis of wild-type and NDST3 E15.5 embryos was then carried out by high-performance liquid chromatography analysis using Carbopac PA1 columns (Dionex). Compositional disaccharide analysis of compound mutant embryos and defined adult brain areas was carried out by liquid chromatography/mass spectrometry (LC/MS). First, disaccharides were separated on a C18 reverse phase column (0.46 × 25 cm, Vydac) with the ion pairing reagent dibutylamine (Sigma), and eluted species were evaluated using a quantitative mass spectrometric method. Analysis of the disaccharide composition by post-column derivatization with 2-cyanoacetamide (27Toyoda H. Nagashima T. Hirata R. Toida T. Imanari T. J. Chromatogr. B Biomed Sci. Appl. 1997; 704: 19-24Crossref PubMed Scopus (50) Google Scholar) or by the LC/MS method gave comparable results. 3R. Lawrence, R. Cummings, and J. E. Esko, submitted for publication. A comparison of the two methods using 0.5 μg of commercial porcine heparin showed an error of 2% for abundant disaccharides to 20% for minor species.Cell Proliferation—Cell proliferation was measured using fibroblasts derived from E14.5 embryos. Cells were labeled using 100 mm bromodeoxyuridine in medium for 5 h, fixed with 4% paraformaldehyde in phosphate-buffered saline, and detected using anti-bromodeoxyuridine antibodies (Zymed Laboratories Inc.). Analysis of FGF2-dependent MAPK pathway activation was performed using anti-ERK1/2 and anti-phospho-ERK1/2 polyclonal antibodies (Promega, Madison, WI). Fibroblasts derived from the heads of E14.5 wild-type and mutant embryos (n = 4) were cultured in DMEM plus 10% FBS, starved for 20 h in DMEM without FBS, incubated in complete medium, DMEM without FBS, or 10 ng/ml FGF2 in DMEM without FBS for 5 min, and lysed. Analyses were done in duplicates.Behavior—For behavioral tests, 13 male and 13 female NDST3 mutant mice were compared with 12 male and 10 female wt controls. Mice were kept under a 12-h/12-h light dark cycle for 3 weeks before testing began. Food and water was available ad libitum. All procedures and protocols met the guidelines for animal care and experiments in accordance with national and European (86/609/EEC) legislation. General health and neurological status were assessed using a protocol, including tests as described elsewhere (28Rogers D.C. Peters J. Martin J.E. Ball S. Nicholson S.J. Witherden A.S. Hafezparast M. Latcham J. Robinson T.L. Quilter C.A. Fisher E.M. Neurosci. Lett. 2001; 306: 89-92Crossref PubMed Scopus (148) Google Scholar). Animals were inspected for physical appearance and underwent neurological testing, including acoustic startle, visual placing, grip strength, and reflex functions to ensure that behavioral findings were not the result of deteriorating physical conditions of the animals. The Barrier test was employed to assess spontaneous exploratory behavior, the Open-field test to assess exploration and fear of open spaces, and the Elevated plus-maze, consisting of elevated open stages that mice are reluctant to enter, to assess anxietyrelated behavior. All tests were conducted as blind studies. For a detailed description of behavioral tests see Ref. 29Lewejohann L. Reinhard C. Schrewe A. Brandewiede J. Haemisch A. Gortz N. Schachner M. Sachser N. Genes Brain Behav. 2006; 5: 64-72Crossref PubMed Scopus (100) Google Scholar. Data analysis was conducted using the statistical software “R” (The R Project for Statistical Computing, available on the web) using non-parametric statistics. Comparison of two samples was done using the two-sample Wilcoxon test (Mann-Whitney U test).Hematology—Hematological assays involved the analysis of white blood cell count, numbers of neutrophils, lymphocytes, monocytes, platelets, eosinophils, basophils, and red blood cells and assessment of hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, red cell distribution width, mean platelet volume, glucose, protein C, CO2, aspartate transaminase levels, alanine aminotransferase levels, alkaline phosphatase levels, urea levels, and potassium levels. ApiZym assays (BioMerieux) were used to assess the presence of alkaline phosphatase, esterase (C 4), lipase (C 8 and C 14), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, α-chymotrypsin, acid phosphatase, α-galactosidase, β-galactosidase, β-glucuronidase α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase, and α-fucosidase.RESULTSTargeted Disruption of ndst3—To study the function of the HS biosynthetic GlcNAc N-deacetylase/N-sulfotransferase (NDST) isozymes in mammalian biology, conditional knockout mice for Ndst3 were generated using the cre-loxP system and homologous recombination in embryonic stem cells (Fig. 1). In the targeting vector, CRE-recombination sequences (loxP sites) were positioned in intron sequences surrounding the second exon of ndst3, which included most of the 5′-untranslated region and 327 of 873 amino acids of the open reading frame, containing the signal peptide, cytoplasmic tail, transmembrane region, and part of the catalytic domain (Fig. 1A). Chimeric mice were generated by blastocyst injections of four embryonic stem cell clones. The resulting type II ndst3 mouse line was crossed with ZP3-cre mice, deleting the “floxed” allele in the oocyte and generating mice with a systemic deletion of ndst3 (Type I, Fig. 1B). Type I Ndst3 mice showed only an insignificant deviation from the expected Mendelian distribution (28% NDST3+/+, 50% NDST3+/-, and 22% NDST3-/-, n = 283), were fertile, and appeared normal.Expression of ndst3 in the Mouse and in Human Tissues—To investigate ndst3 expression in adult human tissues, semiquantitative reverse transcription-PCR analysis using cDNA derived from various tissues was conducted (Fig. 1F). Only after 38 cycles of amplification, could ndst3 expression be detected in the brain, kidney, liver, pancreas, spleen, testis, and thymus. This expression pattern was more restricted than that of ndst1 and ndst2. Due to the lack of an isoform-specific anti-NDST3 antibody, ndst3 in situ hybridization was next conducted to detect areas of ndst3 transcription. Strongest ndst3 transcription was detected in cerebellar granule cells, the hippocampus, the brain stem, and the cortex/olfactory bulb (Fig. 2, C and G). ndst1 and ndst2 in situ analysis revealed non-overlapping expression of ndst1 restricted to cerebellar Purkinje cells (Fig. 2A and inset), whereas ndst2 showed overlapping expression in the granule cell layer (Fig. 2B). In the hippocampus, ndst1-3 were all strongly expressed (Fig. 2, E-G). ndst3 expression in the developing embryo was also analyzed at various stages. In the E10.5 embryonic head, ndst3 expression was detected in trigeminal (V) neural crest tissue (supplemental Fig. S1, A-C). In the E12.5 embryonic skull, ndst3 was still expressed in the trigeminal ganglion and additionally in restricted areas of the fourth ventricle, the metencephalic/myelencephalic part of the rhombencephalon, the developing telencephalon, and the spinal cord (supplemental Fig. S1, D-I). ndst3 expression was found to be more widespread in the E15.5 embryo (supplemental Fig. S1, J-L). Strongest expression was found in neural tissue such as the telencephalon (J), the spinal cord (K), as well as in hind brain (L). ndst3 was also strongly expressed in the developing lung and the frontonasal process that forms much of the face (supplemental Fig. S1, J and K).FIGURE 2Detection of ndst expression in the adult mouse brain. A-D, ndst in situ hybridization shows strong ndst1 expression (dark blue stain) in cerebellar Purkinje neurons (A, arrowhead and inset). Ndst2 and ndst3 show overlapping expression in cerebellar granule cells (B and C, arrow) but not in the molecular layer or Purkinje cell layer. D, ndst3 sense control. The arrowhead indicates the granule layer. E-H, ndst1, ndst2, and ndst3 are all expressed in the CA1-3 and dentate gyrus of the hippocampus (E-G, arrows) and the cortex. H, ndst3 sense control. The arrowheads indicate the dentate gyrus and CA1-3. m, molecular layer; w, white matter; g, granule cell layer; dg, dentate gyrus; co, cortex; CA, pyramidal cell layer.View Large Image Figure ViewerDownload Hi-res image Download (PPT)HS Composition in Total Adult Mouse Brain and Embryo—Heparan sulfate can be depolymerized to constituent disaccharides using a combination of three heparin lyases. The individual disaccharides containing one, two, or three sulfate groups can then be separated and quantitated using high-performance liquid chromatography analysis or by mass spectrometry. Disaccharide analysis of HS derived from E15.5 embryos by high-performance liquid chromatography revealed a slight increase in the amount of non-sulfated UA-GlcNAc and monosulfated UA-GlcNAc6S, whereas the amount of UA-GlcNS and UA2S-GlcNS6S was decreased, demonstrating some NDST3 activity in the embryo (Fig. 3). We next analyzed disaccharide composition of purified HS from mutant and wild-type P50 mouse brain by quantitative LC/MS. As shown in Fig. 4A and in Table 1, the amount of sulfation varied in different regions of wild-type mouse brain. Most notably, the cerebellum had reduced levels of all sulfated disaccharides (69 sulfates per 100 disaccharides). The highest overall sulfation was detected in hippocampus, cortex, and brain stem (112, 103, and 96 sulfates per 100 disaccharides, respectively). Generally, the difference in overall sulfation of wild-type tissues was due to parallel differences in N-, 6-O-, and 2-O-sulfation (Table 1).FIGURE 3NDST3-/- mutant heparan sulfate is undersulfated. HS was isolated from E15.5 NDST3-/- embryos and wild-type littermates, and samples were digested with heparin lyases. The resulting disaccharides were analyzed by fast protein liquid chromatography. Values denote the percent of total disaccharide. HS from NDST3-/- embryos showed a slight increase in the amount of non-sulfated UA-GlcNAc and monosulfated UA-GlcNAc6S and reduced amounts of UA-GlcNS and UA2S-GlcNS6S. The relative amounts of UA2S-GlcNH6S, UA-GlcNS6S, UA2S-GlcNS, and UA2S-GlcNAc6S remained unchanged.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 4Disaccharide analysis of mutant embryo and various regions of the mouse brain. HS was isolated from microdissected NDST3 mutant and wild-type brain or embryo after samples were digested with heparin lyases. The resulting disaccharides were analyzed by quantitative LC/MS. Values denote the mean % of total disaccharide. A, overall sulfation and relative amounts of disaccharides are highly variable among various wild-type brain regions and the wild-type embryo. Wild-type cortex and hippocampus show high levels of sulfation and wild-type cerebellum, and embryo showed lower levels. B-F, compositional disaccharide analysis in brain stem (pons and medulla, B), cerebellum (C), hippocampus (D), and cortex (E) derived from wild-type and NDST3 mutant mice. Relatively unchanged disaccharide composition in the brain stem and hippocampus (B and D) indicate low NDST3 activity in those tissues, or compensatory activity of other NDST isoforms. However, the relative amounts of mono-, di-, and trisulfated disaccharides in the cortex (E) were reduced, and the relative amount of non-sulfated UA-GlcNAc was strongly increased, indicating that NDST3 contributes to HS synthesis in that brain area. In contrast, an increase in the relative amount of trisulfated UA2S-GlcNS6S and decrease in non-sulfated UA-GlcNAc in the cerebellum (C) upon NDST3 deletion indicates overcompensation by another Ndst isoform, possibly by NDST2, which mediates synthesis of highly sulfated heparin in mast cells and is highly expressed in cerebellar granule cells. Results are presented as percent of total disaccharide. F, quantitative LC/MS results of B-E. Disaccharides UA2S-GlcNAc, UA-GlcNH6S, UA-GlcNH, and UA2S-GlcNH were not detected in any tissue investigated.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE 1Total amount of sulfates per 100 disaccharides in various brain regionsBrain stemCerebellumHippocampusCortexKOWTKOWTKOWTKOWTTotal amount of sulfate per disaccharideNS40483733515735536 S25262519313019252 S1722241726251325Total S8296866910811268103 Open table in a new tab Like in the embryo, NDST3 deletion did not lead to large changes in HS sulfation in various parts of the brain (Fig. 4, B-E), with two notable exceptions. In the cerebellum, the amount of trisulfated disaccharide UA2S-GlcNS6S increased ∼2-fold, whereas the amount of nonsulfated UA-GlcNAc decreased (Fig. 4C). In the cortex, all of the N-sulfated disaccharides decreased with the exception of UA-GlcNS6S, and UA-GlcNAc increased strongly (Fig. 4E). Detection of disaccharides containing free amino groups was also included in the analysis to determine the role of NDST3 in their generation. UA-GlcNH6S and UA2S-GlcNH were not detected in wild-type or mutant samples (data not shown). UA2S-GlcNH6S was detected in wild-type and mutant cerebellum and hippocampus, and no reduction was noted in NDST3-/- animals (Fig. 4F). These results indicate that deletion of NDST3 in the adult brain results in a variable and region-specific change in sulfation patterns.Histology and Immunohistochemistry of Mutant Tissues—Anti-HS HepSS1 and 10E4 antibody stainings were comparable on adult tissue sections and cultured embryonic fibroblasts. HepSS1 staining was detected in all mutant and wild-type tissues at all stages (supplemental Fig. S2, A and B). Despite high ndst3 expression in the embryo, but consistent with only moderate changes in HS sulfatio
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