Mouse development is not obviously affected by the ... - Oxford Journals

Glycobiology vol. 22 no. 7 pp. 1007–1016, 2012 doi:10.1093/glycob/cws065 Advance Access publication on April 10, 2012

Mouse development is not obviously affected by the absence of dermatan sulfate epimerase 2 in spite of a modified brain dermatan sulfate composition

Barbara Bartolini2, Martin A Thelin2, Uwe Rauch3, Ricardo Feinstein4, Åke Oldberg3, Anders Malmström2, and Marco Maccarana1,2 2

Department of Experimental Medical Science, Biomedical Center D12, and Department of Experimental Medical Science, Biomedical Center B12, Lund University, SE-221 84 Lund, Sweden; and 4Department of Pathology, The National Veterinary Institute (SVA), SE 75189 Uppsala, Sweden 3

Received on February 14, 2012; revised on March 19, 2012; accepted on March 24, 2012

Dermatan sulfate epimerase 2 (DS-epi2), together with its homolog DS-epi1, transform glucuronic acid into iduronic acid in DS polysaccharide chains. Iduronic acid gives DS increased chain flexibility and promotes protein binding. DS-epi2 is ubiquitously expressed and is the predominant epimerase in the brain. Here, we report the generation and initial characterization of DS-epi2 null mice. DS-epi2deficient mice showed no anatomical, histological or morphological abnormalities. The body weights and lengths of mutated and wild-type littermates were indistinguishable. They were fertile and had a normal lifespan. Chondroitin sulfate (CS)/DS isolated from the newborn mutated mouse brains had a 38% reduction in iduronic acid compared with wild-type littermates, and compositional analysis revealed a decrease in 4-O-sulfate and an increase in 6-O-sulfate containing structures. Despite the reduction in iduronic acid, the adult DS-epi2−/− brain showed normal extracellular matrix features by immunohistological stainings. We conclude that DS-epi1 compensates in vivo for the loss of DS-epi2. These results extend previous findings of the functional redundancy of brain extracellular matrix components. Keywords: brain extracellular matrix / dermatan sulfate / dermatan sulfate epimerase 1/2 / iduronic acid

Introduction Chondroitin/dermatan sulfate (CS/DS) chains are linear sulfated glycosaminoglycans composed of repeated disaccharide 1 To whom correspondence should be addressed: Tel: +46-46222-9665; Fax: +46-46211-3417; e-mail [email protected]

units containing N-acetyl-D-galactosamine (GalNAc) and acid (GlcA) or L-iduronic acid (IdoA) residues (Malmstrom and Fransson 1975; Silbert and Sugumaran 2002). CS/DS chains are covalently bound to various core proteins to form proteoglycans (PGs), which are widely spread in extracellular matrices and on cell surfaces. PGs play roles in several biological processes, such as extracellular matrix (ECM) organization, cell growth, migration, adhesion, differentiation and wound repair (Trowbridge and Gallo 2002; Handel et al. 2005; Yamada and Sugahara 2008). Noteworthy is the presence of CS/DS-PGs in the developing and adult brain of mammals (Sugahara and Mikami 2007), where they have been proposed to act on neuronal plasticity. Modifications in CS/DS, such as O-sulfation in different positions of either GalNAc, GlcA or IdoA [GalNAc(6S), GalNAc(4S), GalNAc(4S,6S), GlcA(2S) and IdoA(2S)] (Kusche-Gullberg and Kjellen 2003), provide a variety of disaccharide units that are responsible for diverse binding specificities (Trowbridge and Gallo 2002; Yamada and Sugahara 2008). For instance, it has been shown that the binding of CS/DS to growth factors, such as fibroblast growth factor (FGF)-2, FGF-7 and FGF-10, requires IdoA residues and 4-O-sulfation (Taylor et al. 2005; Radek et al. 2009). Moreover, CS C [GlcA-GalNAc(6S)] and E [GlcA-GalNAc (4S,6S)] structures have been shown to be enriched in an injured central nervous system (CNS; Gilbert et al. 2005; Properzi et al. 2005; Sugahara and Mikami 2007). DS containing structures from young pig (Bao et al. 2004) and mouse (Hikino et al. 2003) brains have recently been associated with neurite outgrowth, and sulfated IdoA containing disaccharides were found to be spatiotemporally regulated in post-natal mouse cerebellum (Mitsunaga et al. 2006). Two enzymes catalyze the formation of IdoA by the C5-epimerization of GlcA, known as DS epimerases 1 and 2 (DS-epi1 and DS-epi2; Maccarana et al. 2006; Pacheco et al. 2009), and these epimerases are encoded in mice by Dse and Dsel (Dse-like) genes (Maccarana et al. 2009). DS-epi1 and DS-epi2 are ubiquitously present in tissues, although with different levels of expression (Nakao et al. 2000; Goossens et al. 2003). DS-epi2 has the highest expression in the kidney and brain, which is the tissue with the lowest expression of DS-epi1, as indicated also by in situ hybridization in various regions of the developing mouse brain (Akatsu et al. 2011). The degree of epimerization in tissues is therefore variable, and it is also influenced by the type of the core protein of the PGs (Tiedemann et al. 2001; Seidler et al. D-glucuronic

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2002). DS-epi1 and DS-epi2 proteins share an N-terminal epimerase domain (Pacheco et al. 2009). In addition, DS-epi2 has a C-terminal domain, which is highly homologous to CS/DS O-sulfotransferases, whose specificity has not yet been unraveled. Here, we have functionally disrupted the Dsel gene in mice. The epimerase activity was affected in all the examined tissues, and in the brain, the iduronate content was reduced. The DS-epi2-deficient animals showed no apparent dysfunctions.

Results Generation of Dsel-deficient mice The disruption of the Dsel gene was achieved by the insertion of a neomycin resistance cassette replacing 400 bp of the gene, including the proximal promoter, the ATG translational start and the sequence encoding the signal peptide (Figure 1A). Southern blot analysis of ES clones confirmed the homologous recombination of the gene-interrupting targeting vector (Figure 1B). Targeted ES cells were injected into blastocysts, and genotyping of the resulting mice confirmed

Fig. 1. Targeted disruption of the Dsel gene. (A) First line, the schematic view of Dsel organization in the genome. Second line, targeted Dsel locus showing the position of the primers used for genotyping and the length of the amplified PCR product. Third line, targeting vector showing the neomycin cassette inserted in the reverse transcriptional orientation with respect to the Dsel gene, with the position of the primers used for genotypying and the length of the amplified PCR product. Fourth and fifth lines, expected fragments generated after the EcoRI digestion of wild-type (7.7 kb) and mutated (2.6 kb) alleles, respectively, and the position of the probe used to detect these fragments by Southern blotting. (B) Southern blotting from two isolated clones of ES cells transfected with the targeting vector. (C) PCR genotyping of mice. (D) Western blot analysis of DS-epi2 expression in wild-type and DS-epi2−/− tissue and cells. MEF homogenates were directly analyzed by western blot (30 μg of protein applied), or after the enrichment of DS-epi2 by Red-Sepharose, starting from 1000 or 200 μg of protein from the skin and MEF homogenates, respectively. Control and DS-epi2 overexpressing 293HEK (5 μg of protein applied) are shown.

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the presence of the Dsel mutated allele (Figure 1C). The protein encoded by Dsel, DS-epi2, was not visible by the western blot analysis of solubilized wild-type skin but appeared as a faint band in lysates from the wild-type mouse embryonic fibroblast (MEF; Figure 1D). When the extracts were enriched by Red-Sepharose affinity chromatography, DS-epi2 was visible as an expected 148-kDa band in wildtype skin and MEF, but absent in the DS-epi2−/− samples. In summary, Dsel−/− functional inactivation resulted in mice lacking the DS-epi2 enzyme.

DS-epi2−/− mice are vital and do not display any gross anatomical or histological defects Data presented in this report compare littermates born from the breeding of heterozygous DS-epi2+/− mice in a 129/SvJ// C57BL/6 mixed genetic background. At weaning of 155 mice, 24% of them were Dsel+/+, 55% Dsel+/− and 21% Dsel−/−, which is in agreement with the allelic Mendelian distribution. DS-epi2-deficient mice did not show any aberrant mortality. There was no significant difference in weight and length at weaning and at 9 weeks of age between wild-type and DS-epi2 −/− mice [one-way analysis of variance (ANOVA) with Dunnett’s post test; P < 0.05]. DS-epi2−/− mice were fertile and there was no difference in litter size obtained from DS-epi2−/− or DS-epi2+/− parents (average = 9 pups/litter; number of litters analyzed from each genotype of the parents ≥5). The brain, heart, kidney, liver, lungs, lymph nodes, muscles, pancreas, skin, small intestines (duodenum and cranial jejunum), spleen, stomach and thymus of 10-week-old DS-epi2-deficient mice were histologically examined and no differences were observed compared with wild-type littermates (data not shown).

DS-epi2-deficient mice showed a reduced epimerase activity There are two DS-epimerases, DS-epi1 ( product of the Dse gene) and DS-epi2 [ product of the Dsel (-like) gene]. The enzymatic assay does not distinguish the two enzymes, and therefore, the contribution in the activity of each of the two enzymes cannot be estimated. The epimerase-specific activity varied in the wild-type tissues; the skin had 730 dpm/h/mg protein, spleen 713, lung 380, liver 78, kidney 63 and brain (16 dpm/h/mg) had by far the lowest activity. The total epimerase activity decreased in all DS-epi2-deficient tissues: skin had 24%, lung 34%, liver 38%, spleen 44%, kidney 55% and brain 89% reduction (Figure 2A). The assay was also conducted on DS-epi1−/− and DS-epi2−/− MEFs (Figure 2B). DS-epi1−/− and DS-epi2−/− MEFs had a reduction of 88 and 32%, respectively, when compared with wild type. The results showed that DS-epi2, in agreement with the findings in DS-epi1−/− mice (Maccarana et al. 2009), contributes less than DS-epi1 to the total epimerase activity in vivo, but not in the kidney and brain. In the latter tissue, DS-epi2 was by far the predominant epimerase, in agreement with in situ hybridization results of the developing brain (Akatsu et al. 2011). DS-epi1 mRNA was not significantly up-regulated in the DS-epi2−/− 3-day-old pup brain and kidney (quantitative real-time polymerase chain reaction [qRT–PCR] in Figure 2C).

Fig. 2. DS epimerase activity decreases in DS-epi2−/− mice. (A) Extracts from 5-week-old mouse tissues were assayed for epimerase activity. For each tissue, the same amount of protein was assayed, regardless of genotype; values are relative to the ones of wild-type mice, set to 100% in each tissue (*P < 0.05 and *** P < 0.001 obtained by Student’s t-tests comparing wild-type and DS-epi2−/− tissues). (B) Epimerase activity was assayed in lysates of MEFs from DS-epi1- and DS-epi2-deficient mice. (C) qRT-PCR comparing DS-epi1 mRNA isolated from the brain and kidney of 3-day-old wild-type (set to 1) and DS-epi2−/− mice. Values are the mean ± SD of triplicates.

DS-epi2 inactivation reduces iduronic acid content in brain and kidney CS/DS Three days old mice were injected with 35S-sulfate, and the labeled CS/DS chains were extracted and purified from the brain and kidney. No difference in the amount of recovered 35 S-CS/DS was found comparing wild-type and DS-epi2−/− tissues (dpm/mg of tissue), and no major differences in the chain length were observed (Figure 3A). CS/DS chains were degraded by chondroitinase B, which cleaves only GalNAcIdoA linkages, to investigate the amount and the distribution of iduronic acid residues. Alternatively, samples were digested with a mixture of chondroitinase AC-I + AC-II, which cleave only GalNAcGlcA linkages. The digestion 1009

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Fig. 3. DS-epi2 deficient brain and kidney CS/DS has 38 and 13%, respectively, less iduronic acid compared with wild type. Three days old pups were 35 S-labeled in vivo, CS/DS chains were purified from the brain and kidney and, following HS degradation, were recovered after size permeation on a Superose 6 column (A). Heparins of different molecular weights were used as markers. Recovered CS/DS were cleaved with chondroitinase B (B and E) or a mixture of AC-I plus AC-II (D and G), and size-separated on Superdex Peptide column. Elution positions of di-, tetra- and hexasaccharides are indicated with arrows; free sulfate peaks, derived from 4-sulfatase contaminating the lyases’ preparations, are indicated by stars. Empty circles, DS-epi2+/+ brain; filled circles, DS-epi2−/− brain; empty squares, DS-epi2+/+ kidney; filled squares, DS-epi2−/− kidney. Lyase’s degradations, followed by the size separation of the products, were performed twice. (C and F) The proportions of iduronic acid residues in brain CS/DS chains as calculated from di, tetra and hexa structures depicted in (B) and (E), respectively.

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products were separated according to their size. Brain CS/DS displayed a small amount of chondroitinase B cleaved material in the wild type, suggesting that iduronic acid content in the brain is low and is further reduced in DS-epi2−/− CS/DS chains (Figure 3B). Iduronic acid content was 2.0% in the wild-type and 1.25% in the DS-epi2 deficient brain, with a reduction by 38% (Figure 3C). Treatment with AC-I + AC-II degraded most of the CS/DS to disaccharides (91% in wild type and 92% in DS-epi2−/−), confirming that a small amount of iduronic acid is present in the brain (Figure 3D). Tetrasaccharide structures from GlcA-GalNAc-IdoAGalNAcGlcA sequences in the native chains were reduced by 33%, from 4.8% in wild type to 3.2% in DS-epi2−/−. The saccharides eluted in the void volume represented sequences with adjacent iduronic acid (IdoA-GalNAc)>6 units, called iduronic acid blocks, and were 1.9% in the wild-type and 1.8% in the DS-epi2−/− brain. These structural differences indicate that DS-epi2 in the brain is mainly responsible for the biosynthesis of isolated or short stretches of iduronic acid. In contrast to the brain, kidney CS/DS contains a substantial amount of iduronic acid. Quantitation based on the chondroitinase B digestion pattern (Figure 3E) indicated 28.9% iduronic acid in wild type compared with 25% in DS-epi2−/−, representing a 13% relative reduction (Figure 3F). This reduction was mainly due to a decrease in iduronic acid blocks, which were 26.6% in wild-type and 23.3% in DS-epi2−/− chains (Figure 3G). Degradation profiles of brain and kidney DS-epi2+/− CS/ DS, both after chondroitinase B and AC-I + AC-II, were indistinguishable from the wild type (data not shown). DS-epi2 deletion results in a reduction in 4-Oand an increase in 6-O-sulfate containing structures Biological activities of CS/DS depend on the fine structure of protein-binding domains. The total compositional analysis of brain and kidney chains was obtained by degradation with chondroitinase ABC, which cleaves both GalNAcGlcA and GalNAcIdoA linkages. Brain DS-epi2−/− CS/DS had a 2.1% reduction in the predominant monosulfated 4-O-sulfated disaccharides (ΔA) and 2.2% increase in the monosulfated 6-O-sulfated ΔC (Table I). The three known disulfated disaccharides were all present, and the two 4-O-sulfated ΔB and

ΔE were slightly decreased. Chondroitinase AC-I + AC-II degradation, which cleaves GalNAcGlcA linkages and therefore excludes from the analysis IdoA containing disaccharides and the one located at their non-reducing terminus, showed similar differences. The exceptions were, as expected, ΔB disaccharides, which are not present in the disaccharide fraction generated from the chains of both genotypes. Similarly, kidney DS-epi2−/− CS/DS had a 1.4% reduction in the predominant monosulfated 4-O-sulfated disaccharides (ΔA) and a 2% increase in the 6-O-sulfated disaccharides. The 4-O-sulfated containing disulfated disaccharides decreased as well.

Brain ECM architecture did not change in DS-epi2−/− mice In DS-epi2−/− mice, the brain is the tissue with the highest relative reduction in epimerase activity and in iduronic content, and therefore, the brain was chosen to be further investigated. Coronal sections of the brains from DS-epi2-deficient mice were evaluated by plain histology, and no deviations from the wild-type littermates were found (Figure 4A1–4). Furthermore, fluorescence histochemical stainings for chondroitinase ABC-sensitive carbohydrate structures were performed. The CS epitope detected by the CS56 mouse monoclonal antibody was observed in the DS-epi2 deficient brain as well as in the wild-type brain at matching locations (Figure 4B1–4). After chondroitinase ABC treatment of the brain slices from both genotypes, the CS56 epitope was no longer detectable (Figure 4B5–8). N-Acetyl-galactosamine containing carbohydrate structures were detected by the Wisteria floribunda agglutinin (WFA) lectin in the deep cerebellar nuclei and the cerebral cortex of both DS-epi2+/+ and DS-epi2−/− brains (Figure 4C1–4). This staining also disappeared after chondroitinase ABC treatment (results not shown). The carbohydrate structure detected by WFA is reported to depend on the presence of aggrecan (Giamanco et al. 2010). We also investigated the presence of aggrecan with an antiserum prepared against an unglycosylated polypeptide. Aggrecan was detected at the same locations stained by the WFA lectin, and the staining with the peptide antibody was enhanced after chondroitinase ABC digestion (Figure 4D3, 4, 7 and 8). The aggrecan antibody also stained distinct areas of the hippocampal formation (Figure 4D1, 2, 5

Table I. Disaccharide composition of brain and kidney CS/DS chains Disaccharide unit

Total labeled sulfates (%) Brain CS/DS DS-epi2+/+

ΔA, HexA-GalNAc4S ΔC, HexA-GalNAc6S ΔB, HexA2S-GalNAc4S ΔE, HexA-GalNAc4S6S ΔD, HexA2S-GalNAc6S

Kidney CS/DS DS-epi2−/−

ABC

AC-I+AC-II

ABC

AC-I+AC-II

57.8 36.5 0.4 3.5 1.9

54.5 40.3 ≤0.1 3.9 1.4

55.7 38.7 0.3 3.3 2.0

50.8 43.8 ≤0.1 3.7 1.6

DS-epi2+/+ (ABC)

DS-epi2−/− (ABC)

67.6 22.7 1.6 7.0 1.0

66.2 24.7 1.4 6.7 1.0

Three days old pups were 35S-labeled in vivo, CS/DS chains were purified from the brain and kidney and cleaved with chondroitinase ABC or a mixture of AC-I plus AC-II. The resulting disaccharides were separated by anion-exchange high pressure liquid chromatography (HPLC).

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Fig. 4. Brain ECM architecture did not change in DS-epi2−/− mice. Coronal brain sections of two wild-type (uneven numbers) and three DS-epi2 KO (even numbers) mice were stained with hematoxylin/eosin (A1–4), the anti-CS CS56 monoclonal antibody (B1–8), the WFA, a lectin which recognizes N-acetyl-galactosamine (C1–4) and an anti-aggrecan polyclonal antiserum (D1–8). Sections B5–8 and D1–8 had been treated with chondroitinase ABC before staining. The treatment served as a negative control for CS56 (B5–8) and WFA staining (not shown) and as an enhancing step for aggrecan staining. CA1 + CA2, subdivisions of the hippocampus; Cx, cerebral cortex; DCN, deep cerebellar nuclei; DG, dentate gyrus; G, granular layer of cerebellar cortex; H, habenular nucleus; M, molecular layer of the cerebellar cortex; P, plexus choroideus; Bars in A1–A4 = 0.1 mm and in B1–D8 = 0.5 mm (shown in D8).

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and 6), without any obvious difference between the genotypes. Thus, we could not detect any structural differences, nor any alterations of chondroitinase-sensitive carbohydrate epitopes, in the brain of DS-epi2-deficient mice.

Discussion Epimerization of glucuronic acid to iduronic acid is an early modification step in DS biosynthesis, carried out by the two DS epimerases DS-epi1 and 2, which we previously cloned and characterized (Maccarana et al. 2006; Pacheco et al. 2009). Here, we report that mice deficient in DS-epi2 are viable, fertile and have no obvious phenotypic alterations. DS-epi2 appears to be redundant with respect to DS-epi1 in vivo. Both enzymes are expressed in all tissues. Comparison of the enzymatic assays in DS-epi1−/− and DS-epi2−/− tissues showed that DS-epi1 is the predominant epimerase in three of the five tissues examined, i.e. the skin, lung and spleen. The brain is the only tissue where most of the activity (89%) can be attributed to DS-epi2. Assay data indicated that DS-epi1 is also the predominant form in MEFs, cells which highly express both enzymes, which therefore could be detected at the endogenous level by western blots. Structural analyses of CS/DS extracted from DS-epi2−/− and DS-epi1−/− (Maccarana et al. 2009) organs show that a substantial decrease in epimerase activity is not proportionally reflected in DS modifications. In fact: (i) all DS-epi1+/− tissues, except the brain, had reduced activity (from 10 to 60% reduction, compared with wild type), but nevertheless the DS-epi1+/− DS structures were essentially unchanged, and (ii) the DS-epi2−/− kidney had 55% reduction in enzyme activity, reflected in 13% relative reduction in iduronic acid content. Surprisingly, also neonatal brain DS still retained 62% of the iduronic acid in DS-epi2-deficient animals. This indicates that DS-epi1 alone can fulfill most of the epimerization in the DS-epi2 deficient brain. The amount of DS-epi1 appears to be unchanged, since no up-regulation of DS-epi1 mRNA was detected by qRT–PCR of DS-epi2-deficient brain total mRNA. A similar observation was made in embryos with one deleted allele of the single heparan sulfate (HS) epimerase gene. These mice (HS-epi+/−) express 50% of the enzymatic activity but produce HS identical to wild-type littermates (Li et al. 2003). DS-epi2 is composed of two domains, an N-terminal epimerase domain and a C-terminal active O-sulfotransferase domain (unpublished observations). The substrate specificity of the latter activity remains to be established. In this report, we noticed a decrease in GalNAc 4-O-sulfated structures, both monosulfated (ΔA) or disulfated (ΔB and ΔE), indicating that DS-epi2 could be a 4-O-sulfotransferase. However, this conclusion is premature, since decreased iduronic acid could alter the substrates which O-sulfotransferase(s), other than DS-epi2, could act upon. In addition, the absence of DS-epi2 could disturb the postulated multienzymatic biosynthetic complexes, called GAGosomes, e.g. by preventing another 4-O-sulfotransferase to be included. Data from in vitro enzymatic reactions using recombinant purified DS-epi2 will be essential to describe the sulfotransferase activity.

At postnatal day 7, the mouse brain contains 0.5–4% iduronic acid, and the cerebellum has the highest content (Akatsu et al. 2011). In agreement, 3-day-old pups contained 2% iduronic acid in the total brain, which was reduced to 1.25% in the DS-epi2−/− brains. IdoA containing oligosaccharides, most of them containing disulfated disaccharides, have been shown to interact with several neural growth factors, such as FGF-2, FGF-10, FGF-18, midkine and pleiotrophin. These oligosaccharides have also been implicated in axonal growth and neurite sprouting (Sugahara and Mikami 2007). However, iduronic acid is not always essential for this function (Li et al. 2010). The general view is that the specificity in the binding of CS/DS motifs to proteins is not dependent on a precise primary saccharide sequence but rather on a three-dimensional conformation in which the electrostatic potential distribution over the surface is crucial (Li et al. 2010; Purushothaman et al. 2012). The same concept of specificity was proposed for HS–protein interactions (Kreuger et al. 2006). Despite the reduction in iduronic acid and disulfated structures (ΔB and ΔE), the DS-epi2 deficient brain may still retain oligosaccharide domains necessary for binding to neural growth factors. Further, in the DS-epi2−/− brain, an iduronic containing structure was virtually unmodified, i.e. the iduronic acid blocks. These structures are known to interact with high affinity to neural growth factors (Nandini and Sugahara 2006) and could “rescue” the decreases in other IdoA containing structures. Therefore, given the importance in vivo of DS-epi1 in the biosynthesis of iduronic blocks (Maccarana et al. 2009), the role of DS-epi1 also appears central in brain CS/DS biosynthesis. The ECM of the brain is particularly rich in CS/DS-PGs, predominantly the hyalectan PGs including aggrecan, versican, brevican and neurocan. A specialized brain ECM is constituted by the perineural nets (PNNs), which cover the surface of the cell bodies and proximal dendrites of certain neurons in adult CNS and are thought to be important as inhibitors of synaptic contact, therefore playing a role in synaptic plasticity (Giamanco et al. 2010). PNNs, containing all four hyalectans, are defined structures detectable by the staining of some of their components, like aggrecan, CS and N-acetyl-galactosamine. The presence of PNNs was not affected by the absence of DS-epi2. The amount of brain CS/ DS was also not affected, as determined by the quantitation of the recovered 35S-labeled chains. The knock out of many brain CS/DS-PGs and other extracellular matrix components alone or even together, such as in neurocan, brevican, tenascin-C and tenascin-R quadruple KOs, give mild or undetected phenotypes (Rauch and Kappler 2006; Rauch 2007). Therefore, it appears that the fine structure of the matrix filling the interneuronal space has a minor impact on the gross structural appearance of the CNS. However, this does not exclude effects on its proper function. Further, in the two instances where a clear function of CS/DS chains in antagonizing the effect of HS has been demonstrated, this function does not appear to depend on the linkage of the CS/DS chains to a particular core protein (Kantor et al. 2004; Coles et al. 2011). Interestingly, DS-epi2 has been genetically linked to human type II bipolar disorder (Goossens et al. 2003). Also neurocan, a mouse brain dispensable CS/DS-PG, has recently been 1013

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linked to bipolar disorders (Cichon et al. 2011). However, human patients suffering from a congenital DS deficiency due to a defective dermatan 4-O-sulfotransferase 1 show normal cognitive development (Dundar et al. 2009). We cannot exclude that under more stringent behavioral tests, other than our general behavioral and motility observations, DS-epi2−/− mice would manifest alterations. It is not uncommon that two enzymes carry out similar reactions in glycosaminoglycan (GAG) biosynthesis. In some cases, such as the HS 6-O-endosulfatase, Sulf1 and Sulf2, the absence of a single gene is completely compensated for by the other gene, and only the double KO unravels the in vivo functions (Holst et al. 2007). We are currently establishing the DS-epi1 and 2 double KO. This mouse strain will presumably lack all iduronic acid and is expected to show a more severe phenotype than the single-gene deletions. It is not currently understood why two DS epimerases are present, but they may be crucial for the fine tuning of the DS structure. In conclusion, our report shows that DS-epi2 absence can be almost completely compensated for in vivo by DS-epi1.

Material and methods Materials Superdex Peptide 10/300 GL, Superose 6 10/30, PD-10 columns, Red-Sepharose gel and ECL-Plus reagent were from GE Healthcare (Uppsala, Sweden). DE52 anion-exchange resin was from Whatman (Uppsala, Sweden). 35|SO4 (1500 Ci/mmol) was from Perkin-Elmer. Chondroitinases ABC, B, AC-I and AC-II were from Seigakaku (Japan). Construction of the targeting vector and generation of chimeric mice A replacement targeting vector was generated from the Dsel 129/SvJ genomic sequence obtained from MGI database (Jackson Laboratories, Ban Harbor, ME). A 4-kb KpnI–ClaI and a 2-kb EcoRI–SalI fragment were ligated into pBluescript II KS (Stratagene, La Jolla, CA), which contains a phosphoglycerate kinase-neomycin resistance cassette inserted in the reverse transcriptional orientation, interrupting a 400-nucleotide sequence surrounding the initial ATG (Figure 1A). The targeting vector was linearized at a NotI site and used to transfect R1 mouse embryonic stem (ES) cells at the Biotech Research and Innovation Centre at University of Copenhagen. ES clones were analyzed by Southern blotting, after EcoRI digestion, with a 400-bp probe external to the targeting vector. This probe hybridized with the expected 7.7-kb wild-type allele and the 2.6-kb mutated allele (Figure 1B). Two targeted ES clones were injected into C57BL/6 blastocysts to generate chimeric mice. Chimeric males were obtained from both clones and mated with C57BL/6 females. Dsel+/− F1 mice, which have a mixed C57BL/6-129/SvJ genetic background and contain the mutated Dsel in the germ line, were intercrossed to produce mice with all genotypes. All experiments in this study were conducted with littermates of a C57BL/6-129/SvJ mixed genetic background. Mice derived from both ES cell clones were characterized for the Dsel allelic distribution at weaning. Mice from one ES clone were further characterized. Mice or cells were genotyped by 1014

PCR (forward primer for both alleles: 5′-ACGTGGTCAA ATGGC-3′; reverse primer for the wild-type allele: 5′GCTGTGAAATCCAGG-3′; reverse primer hybridizing to the neomycin cassette: 5′-ATTAAGGGCCAGCTC-3′). DS epimerase activity DS epimerase activity was assayed, measuring the release of C5-3H from the 3H-labeled chondroitin substrate to form 3 H2O, as described in Maccarana et al. (2009). Isolation of MEF cells Embryos from DS-epi2 +/+ and DS-epi2−/− mice were collected at embryonic day 13.5. After the removal of the head and the abdominal internal organs, the embryos were cut into pieces, trypsinized (15 min at 37°C), and outgrowing cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), 10% fetal bovine serum (FBS) and allowed to reach confluency. The genotype of MEF cells was established by PCR. MEF cells were immortalized by transformation with recombinant simian virus 40 large T antigen as described in Jia et al. (2009). Western blotting Skin and MEF were lysed in a lysis buffer containing 20 mM morpholinepropanesulfonic acid ( pH 6.5), 150 mM NaCl, 10% glycerol, 2 mM dithiothreitol, 1 mM ethylenediaminetetraacetic acid (EDTA), 1% Triton X-100 and protease inhibitors, i.e. 1 mM phenylmethylsulfonyl fluoride and aprotinin, leupeptin and pepstatin at 1 µg/ml each. Protein content was determined in cleared lysates by the Bradford method (Bio-Rad, Sweden), with bovine serum albumin as the standard. These extracts were directly applied to western blot or, alternatively, DS-epi2 was biochemically enriched before western blot analysis. In the latter cases, 1-mg protein of skin extracts or 0.2-mg protein of MEF lysates were incubated for 60 min with 20 or 5 µl of Red-Sepharose gel, respectively. The gel was extensively washed with lysis buffer, and bound material eluted with reducing Laemmli sample buffer. DS-epi2 was detected with 4 µg/ml of an immunopurified anti-DS-epi2 rabbit polyclonal antibody obtained after antigen peptide immunization ( peptide corresponding to amino acids 978–991 of the human/murine sequence; Innovagen, Sweden). ECL-Plus was used for detection. DS-epi1 qRT-PCR Total RNA was extracted from the brain and kidney with an RNeasy kit (Qiagen, Sweden) and DNase treated with a DNA-free kit (Applied Biosystems, Foster City, CA); cDNA synthesis was performed with a Maxima® First Strand cDNA Synthesis Kit for qRT–PCR (Fermentas, Sweden). The samples were mixed with primers and Maxima® SYBR Green/ROX qPCR Master Mix 2X (Fermentas) and amplified in an ABI Prism instrument (Applied Biosystems) starting with an initial 10-min heating at 95°C, followed by 40 cycles at 95°C for 15 s, 60°C for 30 s and 72°C for 30 s. The data were analyzed with sodium dodecyl sulfate (SDS) 2.1 software (Applied Biosystems). The calculated threshold cycle (CT) values were normalized to the CT value for ribosomal


protein S18. The primers used were as follows: for Dse 5′CCCACAGCTTCTCCTTCTTG-3′ and 5′- CTCCTCAAA AGGGACATCCA-3′, for ribosomal protein S18 5′-CCGC AGCTAGGAATAATGGA-3′ and 5′-CCCTCTTAATCATGGC CTCA-3′. Preparation of in vivo labeled CS/DS Three days old mice were intraperitoneally injected with 50 μl of phosphate buffered saline (PBS) containing 1 mCi 35SO4 and sacrificed after 4 h. To assess the structure of the CS/DS chains from the brain and kidney, organs from two wild-type and two DS-epi2−/− mice were isolated, cut into pieces and ground in 1 ml of 100 mM Tris ( pH 8.5 at 55°C) buffer, containing 200 mM NaCl and 5 mM EDTA. The mixture was made to contain 0.1% sodium dodecyl sulfate (SDS), and proteinase K at 100 μg/ml, and incubated at 55°C overnight. Further proteinase was added to cleared material, and the incubation was continued for an additional 4 h. The material was purified on DE-52 anion-exchange resin. HS chains were depolymerized by deamination reaction at pH 1.5 (Shively and Conrad 1976), followed by the re-isolation of CS/DS chains on Superose 6 run in 0.2 M NH4HCO3. Chondroitinase treatment of CS/DS and analysis of the cleavage products Cleavage of CS/DS chains by lyases and their analyses were done according to Pacheco et al. (2009). Immunohistochemical brain staining Formalin fixed tissues were routinely processed for histology and embedded in paraffin. Specimens were cut at 4 μm and were stained with hematoxylin and eosin. A Nikon Eclipse E600 microscope equipped with a Nikon DS-Fi1 digital camera was used. Brain sections were saturated 60 min with 5% normal goat serum in PBS. Mock or chondroitinase ABC digestion of the slices was performed for 60 min at 37°C in Tris 50 mM, Na-acetate 50 mM, pH 8.0, containing 20 mU/ml lyase. Chondroitinase ABC digestion served as a negative control for CS and N-acetyl-galactosamine staining, and as an enhancing step for aggrecan staining. Primary antibodies, or lectin, diluted 1:200 in saturation buffer and incubated overnight at 4°C, were: mouse anti-CS CS56 (C8035 Sigma, St. Louis, MO), biotinylated anti-N-acetyl-galactosamine WFA (Vector Laboratories, Burlingame, CA) and rabbit antiaggrecan (Millipore AB1031). Secondary reagents, diluted 1:200 in PBS, were: anti mouse IgG-Cy3 (Jackson ImmunoResearch, West Grove, PA), streptavidin-Cy3 (Sigma) and anti-rabbit IgG (Jackson ImmunoResearch), respectively. Statistical tests GraphPad Prism version 5.01 for Windows and GraphPad Software were used for statistical analysis. To determine the difference in body weight, one-way ANOVA with Dunnett’s post test was performed. Unpaired Student’s t-tests were applied for statistical analyses on epimerase activity of DS-epi2 −/− tissues. Probability values of P < 0.05 and P < 0.001 were considered statistically significant.

Funding This work was supported by grants from the Swedish Science Research Council, the Medical Faculty of Lund University, the Mizutani Foundation for Glycoscience to M.M., the Albert Österlund Foundation, the Greta and Johan Kock Foundation, the Tissue in Motion Medical Faculty Program to M.M., Polysackaridforskning AB to M.M., and the Anna-Greta Crafoord Foundation to B.B.

Conflict of interest None declared.

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