HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 277, Issue 2, 1443-1450, January 11, 2002

This Article Abstract Full Text (PDF) All Versions of this Article: 277/2/1443    most recent M104719200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Uchimura, K. Articles by Muramatsu, T. Search for Related Content PubMed PubMed Citation Articles by Uchimura, K. Articles by Muramatsu, T. Social Bookmarking                      What's this?

Functional Analysis of the Chondroitin 6-Sulfotransferase Gene in Relation to Lymphocyte Subpopulations, Brain Development, and Oversulfated Chondroitin Sulfates^*

Kenji Uchimura^§, Kenji Kadomatsu, Hitoshi Nishimura^¶, Hisako Muramatsu, Eishin Nakamura, Nobuyuki Kurosawa, Osami Habuchi^**, Fathy M. El-Fasakhany, Yasunobu Yoshikai^¶, and Takashi Muramatsu

From the  Department of Biochemistry, the ^¶ Laboratory of Host Defense & Germfree Life, Research Institute for Disease Mechanism and Control, and the  Department of Internal Medicine, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550 and the ^** Department of Life Science, Aichi University of Education, Kariya, Aichi 448-8542, Japan

Received for publication, May 23, 2001, and in revised form, October 30, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Chondroitin 6-sulfotransferase (C6ST) catalyzes the transfer of sulfate to position 6 of the N-acetylgalactosamine residue of chondroitin. To obtain direct evidence regarding the function of C6ST and its product, chondroitin 6-sulfate, in vivo, we isolated the mouse C6ST gene (C6st) and generated mice deficient in this gene (C6st^/) by embryonic stem cell technology. C6st^/ mice were born at approximately the expected frequency and were viable through adulthood. In the spleen of C6st^/ mice, the level of chondroitin 6-sulfate became almost undetectable. Analyses of these knockout mice provided insights into the biosynthesis of oversulfated chondroitin sulfates in mice; chondroitin sulfate D in the brain of null mice and the cartilage and telencephalon of null embryos disappeared, whereas the chondroitin sulfate E level in the spleen and brain of the null mice was unchanged. Despite the disappearance of chondroitin sulfate D structure, brain development was normal in the C6st^/ mice. Further analysis revealed that the number of CD62L⁺CD44^low T lymphocytes corresponding to naive T lymphocytes in the spleen of 5-6-week-old C6st^/ mice was significantly decreased, whereas those in other secondary lymphoid organs were unchanged. This finding suggested that chondroitin 6-sulfate plays a role in the maintenance of naive T lymphocytes in the spleen of young mice.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Chondroitin sulfate is a family of glycosaminoglycans (GAGs)¹ consisting of glucuronic acid (GlcA), N-acetylgalactosamine (GalNAc), and sulfate. The building block is a repeating disaccharide unit with GlcA1-3GalNAc, and its GalNAc residue is usually monosulfated. When sulfation occurs mainly at C-6 of the internal GalNAc residue, it is called chondroitin 6-sulfate, whereas the C-4 sulfated form is called chondroitin 4-sulfate. A variant of chondroitin 4-sulfate containing some iduronic acid residues instead of GlcA is known as dermatan sulfate, and its disaccharide unit is designated as the chondroitin sulfate B unit. Chondroitin sulfate D and E have oversulfated structures, GlcA(2S)1-3GalNAc(6S) and GlcA1-3GalNAc(4S,6S), respectively (1).

The structural diversity including various pattern of sulfate group attachment in GAGs suggests the pathological and biological importance of proteoglycan molecules (2, 3). Chondroitin sulfate proteoglycans are the main components in the cartilage, and their sulfation profiles vary in relation to aging (4, 5). Chondroitin sulfates in chondroitin sulfate proteoglycans have been implicated in some aspects of neuronal functions such as modulation of neurite outgrowth (6-8) and axonal regeneration (9, 10). Chondroitin sulfate chains also play critical roles in lymphocyte-endothelial cell interactions (11) and enhancing stimulation of T cell responses (12). Furthermore, chondroitin sulfate proteoglycans bearing chondroitin sulfate D or E bind to a growth factor, midkine (13-16), and chemokines (17), and have been suggested to participate in signaling or modulation of the activities of these factors. However, the in vivo function of chondroitin sulfate has not been clarified directly.

Sulfate groups attached to GAG chains are transferred from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) by sulfotransferases with strict acceptor specificities (2). Chondroitin 6-sulfotransferase (C6ST) catalyzes the last step of synthesis of chondroitin 6-sulfate, and transfers sulfate to the C-6 position of the GalNAc residue of chondroitin. Recent studies have shown that C6ST can sulfate the Gal residues of keratan sulfate (18) as well as sialyl lactosamine oligosaccharides (19). C6ST cDNAs have been cloned in the chicken (20) and subsequently in the mouse (21) and human (22). To determine the roles of C6ST and its product, chondroitin 6-sulfate, in vivo, we utilized gene knockout technology deleting the mouse C6ST gene, C6st. In the present study, we have shown that the number of naive T lymphocytes in the spleen of C6st^/ mice was significantly decreased.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Molecular Cloning of the C6st Gene and Construction of Targeting Vector-- To obtain mouse C6ST genomic DNA, a 129SV/J mouse genomic library (Stratagene) was screened using mouse C6ST cDNA (ml1; Ref. 21) as a probe. Plaque hybridization was carried out as described previously (21). Two positive clones (termed 8-6 and 3-1), containing a 15-kb DNA fragment that included the C6ST coding exons, were isolated (Fig. 1A). After sequencing the insert DNA fragments, the localization of the exons was determined by comparison of the sequences between the inserted genomic DNA and several cDNAs obtained by 5'-rapid amplification of cDNA ends PCR as described previously (21) and Southern blot analysis using the cDNA as a probe (21). The C6st targeting vector was constructed from a basic targeting vector with MC1neo (polyoma virus thymidine kinase gene promoter and neomycin resistance gene) and DTA (diphtheria toxin fragment A gene) (23) and C6st fragments. To delete a 1.2-kb portion of C6st exon II (KpnI-SmaI sites in Fig. 1B), a 4.7-kb SacI/KpnI fragment and 2.3-kb SmaI fragment were used as the 5'-arm and the 3'-arm, respectively (Fig. 1B).

Generation of Targeted ES Cells and Mice-- Aliquots of 18 µg of NotI-linearized targeting vector DNA were electroporated into 1 × 10⁷ D3 ES cells. The cells were plated on mitomycin C-treated G418-resistant SL-10 cell feeder layers. G418 (250 µg/ml; Sigma) was added 24 h after plating. G418-resistant colonies were picked up after 7-8 days and then propagated to be stored and examined for homologous recombination by Southern blot analysis as described below. Approximately 15 ES cells of the targeted clones were injected into blastocysts derived from naturally mated C57BL/6J females. The injected embryos were transferred to the uteri of pseudopregnant ICR mice. To yield null mutants of 129SV/J background, chimeric male mice were mated with 129SV/J mice. Then, F1 progeny were back-crossed four times to 129SV/J mice and mated with each other.

Southern and Northern Blot Analyses-- Southern blot analysis was performed as described previously (21) for DNA samples digested with EcoRI. The membrane was hybridized with a 0.7-kb HincII-NcoI fragment corresponding to the genomic DNA downstream of the 3'-arm of the targeting vector (Fig. 1B). The homologously recombined DNA gave a 7.8-kb band, whereas the wild-type DNA gave a 14.2-kb band (Fig. 1C). Total RNA prepared from the mouse spleen was electrophoresed and transferred onto a nylon membrane, and then hybridized with the radioactive probes as described previously (21). Hybridization was performed using a 400-bp DNA fragment corresponding to part of the C6st gene (PCR product described below) as a probe.

PCR-- Aliquots of 0.5 µg of DNA were mixed with 20 µl of 1 × AmpliTaq^® buffer containing 0.2 mM each dNTP, 1.5 mM MgCl₂, 10 pmol of each primer, and 0.5 unit of AmpliTaq^® DNA polymerase (Applied Biosystems). PCR amplification was carried out at 94 °C for 3 min, with 35 cycles of 94 °C for 0.5 min, 56 °C for 0.5 min, and 72 °C for 1 min. To screen for homologously recombined DNA, C6st primers were used: 5'-ATGCATCTCTCTTGTCCCTGA-3' (6ST-F) and 5'-CACATACAGGTCGCATAGCAA-3' (6ST-R). The wild-type allele gave a 400-bp band, whereas the mutated allele gave no band. Neo primers were also used: 5'-CAGCGTCTTGTCATTGGCGA-3' (Neo-F) and 5'-GCTCTTCGTCCAGATCATCC-3' (Neo-R). The wild-type allele gave no band, whereas the mutated allele gave a 600-bp band.

Assaying of C6ST and Chondroitin 4-Sulfotransferase (C4ST)-- Mouse tissues were removed and then homogenized with a Dounce homogenizer in 1.5 ml of 0.25 M sucrose, 10 mM Tris-HCl, pH 7.2, and 0.5% Triton X-100. The homogenates were centrifuged at 10,000 × g for 15 min, and the activities of C6ST and C4ST in the supernatant fractions were measured as described previously (24) with slight modifications. After the standard reaction, ³⁵S-labeled chondroitin substrates were purified using a Fast Desalting Column (Amersham Biosciences, Inc.) and were collected as described previously (24). The collected ³⁵S-labeled products were dissolved in 50 µl of a buffer containing 0.05 M Tris acetate, pH 7.5, and 10 milliunits of chondroitinase ABC (Proteus vulgaris; Seikagaku Kogyo, Tokyo, Japan). After incubation for 2 h at 37 °C, the digested materials were applied to a Partisil 10-SAX column (4.6 cm × 25 cm, Whatman), and fractions were collected at 30 s intervals as described previously (20). C6ST activity and C4ST activity were determined according to the incorporation into Di-6S and Di-4S, respectively.

Extraction of Chondroitin/Dermatan Sulfates from Mouse Tissues-- Mouse spleens (1 g) and brains (4.5 g) were removed from 8 mice (5-6-week-old males) and 20 mice (over 10-week-old males), respectively. Tissues were homogenized in 10 ml of ice-cold 10 mM Tris-HCl, pH 7.5, containing 1 mM phenylmethanesulfonyl fluoride for 3 min. The homogenate was mixed with an equal volume of 10 mM Tris-HCl, pH 7.5, containing 0.5 M sucrose, 0.1 M KCl, 10 mM MgCl₂, and 2 mM CaCl₂, and then centrifuged at 8000 × g for 10 min. The pellet was suspended in 10 ml of 10 mM Tris-HCl, pH 7.5, containing 0.1 M NaCl, 1 mM EDTA, 8 mM CHAPS, and 2.5 mg of proteinase K, and then incubated at 50 °C for 4 h. After further incubation with 2.5 mg of proteinase K at 50 °C for 12 h, 50 µg of RNase A (Sigma) and 50 µg of DNase I (Takara, Tokyo) were added, and then the reaction mixture was stored at 37 °C for 1 h. The extract was centrifuged at 10,000 × g for 10 min. The supernatant was applied to a column containing 4 ml of DEAE-Sephacel following by adjusting its concentration of NaCl to 0.5 M. The column was washed with five column volumes of 10 mM Tris-HCl, pH 7.5, containing 0.5 M NaCl and 8 mM CHAPS. Chondroitin/dermatan sulfate fractions were then eluted with two column volumes of 10 mM Tris-HCl, pH 7.5, containing 1.5 M NaCl and 8 mM CHAPS with monitoring at 210 nm. The collected fractions were dialyzed against distilled water.

Disaccharide Analysis on HPLC-- Aliquots of 10 µg of chondroitin/dermatan sulfates extracted from the mouse spleen and brain were digested with chondroitinase ABC as described above. Samples before or after chondro-6-sulfatase (P. vulgaris; Seikagaku Kogyo) treatment (10 milliunits, at 37 °C for 12 h) were then analyzed by HPLC (Partisil-10 SAX, 4.6 cm × 25 cm, Whatman) as described previously (20).

Flow Cytometric Analysis-- After mincing the spleen, thymus, peripheral lymph nodes, mesenteric lymph nodes, and Peyer's patches in Hanks' balanced salt solution, single-cell suspensions were prepared in the same ice-cold solution. Cells were incubated at 4 °C for 15 min in 10 µg/ml Fc blocker (anti-CD32/16, 2.4G2; PharMingen) before staining with various antibodies. Cells were stained for 30 min at 4 °C in Hanks' balanced salt solution containing 400-fold diluted antibody, and then washed with the same buffer. FACSCalibur flow cytometer and CellQuest soft ware (Becton Dickinson) were used to analyze the stained cells. The antibodies against the following antigens were from PharMingen: CD3 (2C11), CD4 (RM4-5), CD8 (53-6.7), CD11b (M1-70), CD11c (HL3), CD21 (7G6), CD22 (Cy34.1), CD23 (B3B4), CD24 (M1/69), CD43 (S7), CD44 (IM7), CD62L (MEL-14), mouse IgM (R6-60.2), mouse IgD (11-26c.2a), Ly-6G (Gr-1), B220 (RA3-6B2), TER-119 (Ly-76), and I-A^b (Afb-120.1).

Immunohistochemistry and Staining-- Mouse tissues were frozen in Tissue-Tek (Sakura Finetek). Cryostat sections (5 µm thick) were fixed in acetone for 5 min and then reacted with the appropriate antibody for 1 h at room temperature. The sections were washed in phosphate-buffered saline and subsequently incubated with secondary antibody for 30 min at room temperature. After washing in phosphate-buffered saline , slides were developed with 3,3'-diaminobenzidine. For detection of carbohydrate epitopes, a Vectastain^® ABC kit (Vector Laboratories) was used instead of secondary antibody. The antibodies used were biotin-conjugated anti-chondroitin 6-sulfate (MC21C), anti-chondroitin sulfate D (MO-225), and anti-keratan sulfate (5D4), all from Seikagaku Kogyo. Subsequently, spleen sections were counterstained by methyl green solution. For Nissl's staining, brains were embedded in paraffin and serially sectioned in the coronal plane.

Lymphocyte Trafficking-- Lymphocyte trafficking in vivo was carried out according to the published procedures with slight modifications (25, 26). Mesenteric and axillary lymphocytes from 5-6-week-old C6st^+/+ mice were isolated and then labeled with 5 µM 5-chloromethylfluorescein diacetate (CMFDA; Molecular Probes). After washing with Hanks' balanced salt solution, the cells (2.5 × 10⁷ in 250 µl of the washing solution) were injected into the tail veins of age-matched C6st^+/+ and C6st^/ mice. After 1.5 h, the animals were sacrificed and secondary lymphoid organs were removed. CMFDA-positive lymphocytes in the organs were analyzed by flow cytometry. For detection of the trafficked cells in the spleen, the organ was immediately frozen and then cryostat sections were prepared as described above. Biotin-conjugated anti-CD3 antibody and PE-conjugated streptavidin (PharMingen) were used to detect the T cell zones.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Genomic Organization of the Mouse C6st Gene-- Two overlapping clones covering the mouse C6st gene were isolated by screening a 129SV/J mouse genomic library (Stratagene) using mouse C6ST cDNA as a probe. Both clones, termed clone 8-6 and 3-1 (Fig. 1A), contained the whole protein-coding region of the C6ST cDNA, and the region appeared to be encoded by two exons divided by a 1.2-kb intron (Fig. 1A). Exon II contained the translation initiation codon. The sequences of the intron-exon splice junctions obeyed the GT-AG rule (27). The split site of exon II and exon III was between nucleotides 375 and 376 of mouse C6ST cDNA described previously (see Fig. 1A in Ref. 21). To determine the transcription initiation site of the gene, several cDNAs encoding 5'-flanking region of mouse C6st mRNAs in the spleen were isolated by 5'-rapid amplification of cDNA ends PCR analysis as described previously (21) and then sequenced. Sequence comparison between the cloned cDNAs and the genomic clones revealed that three unique DNA sequences, types a-c as exon I, were present in the 5' region upstream of C6st translation initiation codon (Fig. 1A). Furthermore, alternative transcription initiation sites and alternative usage of exon Ia-Ic were identified (Fig. 1A), suggesting that tissue-specific expression of the mouse C6st gene might be regulated by multiple promoters. Thus, the mouse C6st gene spans over 20 kb (Fig. 1A).

View larger version (32K): [in this window] [in a new window]   Fig. 1.   Genomic organization and targeting strategy of the mouse C6st locus. The protein-coding region of the C6st is distributed in two exons (A). C6st transcripts are thought to be produced by alternative usage of multiple exons 1 encoding the 5'-UTR of C6st mRNA. Boxes denote exons. Solid and open boxes represent protein-coding and untranslated regions, respectively. The top shows isolated genomic clones, 8-6 and 3-1. The bottom shows the putative splicing patterns for each form of C6st mRNA and transcription initiation sites (arrows) (A). The targeting construct was designed to replace the exon encoding the C6ST catalytic domain with the neomycin resistance gene. Restriction enzyme sites indicated are: E, EcoRI; S, SacI; K, KpnI; Sm, SmaI; H, HincII; N, NcoI. Boxes denote exons (B). In homozygous mice, C6st/, Southern blot analysis confirmed homologous recombination (C). No expression of the mRNA was detected by Northern blot analysis. The bottom panel shows ribosomal RNAs, indicating the equal loading of the RNA samples (D). E and F, C6ST (E) and C4ST (F) activities in the organs of C6st+/+ and C6st/ mice. C6ST activities in various tissues of C6st/ mice were less than 10 cpm/µg of protein/h (ND, not detected). PLN, peripheral lymph node; MLN, mesenteric lymph node; PP, Peyer's patch.

Gene Targeting of Mouse C6st Locus-- We designed a targeting construct to delete part of the catalytic region in exon III. The deletion resulted in loss of the whole 5'-PAPS binding domain and a portion of the 3'-PAPS binding domain (28), so that any resulting translated enzyme would not have the ability to catalyze sulfation. By standard gene knockout technology, mice with deletion C6st (C6st^/) were produced. By mating of C6st^+/ mice, C6st^/ mice were born in the Mendelian ratio. There were no apparent differences between wild-type and the knockout mice in their gross morphology or results of histological examination. No significant differences were observed in body weight of C6st^/ mice as compared with C6st^+/+ mice 1, 2, 4, 6, 8, or 10 weeks after birth. The knockout mice reproduced normally and also survived for 2 years without any abnormalities as compared with wild-type controls. Southern blot analysis confirmed that the C6st gene was deleted in the knockout mice (Fig. 1C), and no C6st mRNA was detected in these mice (Fig. 1D). C6ST activity was not detected in the spleen, lung peripheral lymph nodes, mesenteric lymph nodes, or Peyer's patches of C6st^/ mice (Fig. 1E). Chondroitin 4-sulfotransferase activity in such tissues was also assayed and appeared to be normal in C6st^/ mice (Fig. 1F).

Participation of C6st in Formation of Chondroitin Sulfate Structures-- We chemically analyzed chondroitin/dermatan sulfates extracted from the spleen. After chondroitinase ABC digestion, the major disaccharide released from the sample of C6st^+/+ was Di-4S (Fig. 2A, peak 3). In addition, a small peak appeared at the position of Di-6S (peak 2) and Di-diS_E or Di-diS_B (peaks 5 and 6). Chondroitin 6-sulfatase digestion caused disappearance of Di-6S and Di-diS_E peaks (Fig. 2B). In C6st^/ mice, Di-6S level was decreased to less than 10% of that in the wild-type control (Fig. 2C), whereas amounts of Di-4S and Di-diS_E were not changed (Fig. 2D). Thus, C6st is not involved in formation of the E unit, GlcA1-3GalNAc(4S,6S), in the spleen.

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Structural evaluation of chondroitin/dermatan sulfates as revealed by disaccharide analysis. Chondroitin/dermatan sulfates were prepared from the spleen of 5-6-week-old C6st+/+ (A and B) and C6st/ mice (C and D), and from the brain of over 10-week-old C6st+/+ (E and F) and C6st/ mice (G and H). Chondroitinase ABC-digested (A, C, E, and G) and additional chondro 6-sulfatase-digested (B, D, F, and H) samples were subjected to HPLC. In the spleen of mice, chondroitin 4-sulfate was the main component (peak 3 in A) and a small amount of chondroitin 6-sulfate was present (peak 2 in A). Also, the chondroitin 4 and 6 disulfate structure was detected (peak 5 in A). In the spleen of C6st/ mice, a very small peak of 6-sulfate was detected (peak 2 in C). In the brain of mice, the major disaccharides component was Di-4S (peak 3 in E). Small amounts of Di-6S (peak 2 in E) and Di-diSD (peak 4 in E) shown in the sample from C6st+/+ brain were mostly disappeared in that of C6st/ brain (peak 2 in G), whereas the amount of Di-diSE was not changed (peak 5 in G). Elution profiles around the peak positions of Di-diSD, Di-diSE, and Di-diSB are magnified as insets (E and G). Standard substances were eluted at the peak positions described below: Di-0S, peak 1; Di-6S, peak 2; Di-4S, peak 3; Di-diSD, peak 4; Di-diSE, peak 5; Di-diSB, peak 6.

In chondroitin/dermatan sulfates extracted from the brain of adult mouse, the major disaccharides component was also Di-4S (Fig. 2, E-H, peak 3). As in the case of the sample from the spleen, a small amount of Di-6S present in the sample from C6st^+/+ brain (Fig. 2E, peak 2) mostly disappeared in that of C6st^/ brain (Fig. 2G, peak 2), whereas the amount of Di-diS_E was not changed (Fig. 2, E and G). Interestingly, a small amount of Di-diS_D detected in the sample from C6st^+/+ mice (Fig. 2E, peak 4) was absent in the sample from C6st^/ (Fig. 2G). Therefore, C6st is involved in formation of D unit, GlcA(2S)1-3GalNAc(6S), in the adult brain.

Expression of chondroitin sulfate was also examined by immunohistochemical staining. When cryostat sections of the spleen were stained with MC21C antibody, which reacts with chondroitin 6-sulfate, reticular fibers and trabeculae including arteries were positive in the C6st^+/+ mice (Fig. 3A), whereas those from C6st^/ mice were negative except in the arteries (Fig. 3B). Cartilage tissues in C6st^+/+ day 13.5 embryos strongly expressed chondroitin sulfate D as determined by staining with MO225 antibody (Fig. 3C). MO225-positive signals completely disappeared in the C6st^/ embryos (Fig. 3D). Thus, C6st is also involved in formation of D unit in the embryo cartilage. C6ST also sulfates the C-6 position of Gal residues in keratan sulfate in vitro (18, 21). 5D4 monoclonal antibody that recognizes keratan sulfate was used to evaluate whether biosynthesis of keratan sulfate in C6st^/ mice was affected. 5D4-positive signals were observed in the thalamus of C6st^+/+ day 15.5 embryos (Fig. 3E). The intensity of 5D4-positive signals was unchanged in the thalamus of the C6st^/ embryos (Fig. 3F). Therefore, C6st gene is not involved in synthesis of keratan sulfate in the embryonic thalamus.

View larger version (120K): [in this window] [in a new window]   Fig. 3.   Structural evaluation of chondroitin/dermatan sulfates and keratan sulfate by immunohistochemical study. Cryostat sections of the spleen (A and B) and the day 13.5 (C and D) and 15.5 (E and F) embryos were prepared from C6st+/+ (A, C, and E) and C6st/ (B, D, and F) mice. They were stained with MC21C antibody that detects chondroitin 6-sulfate (A and B) or MO225 antibody that detects chondroitin sulfate D (C and D) or 5D4 antibody that detects keratan sulfate (E and F). Cartilage tissues (C and D) and dorsal thalamus (E and F) are shown. Arrowheads and arrows indicate reticular fibers and trabeculae including arteries, respectively. Original magnifications in A and B are ×400, and in C-F are ×100.

Brain Development in C6st^/ Mice Was Normal-- Because the chondroitin sulfate D motif, which disappeared from the brain of C6st^/ mice, was shown to promote neurite outgrowth (7), we compared brain structure between C6st^+/+ and C6st^/ mice by histological analysis, and found no differences. In particular, normal organization of the cerebral cortex and orientation of processes for pyramidal cells in C6st^/ brain were revealed by Nissl's staining method (Fig. 4, A-D). Then, we investigated whether the chondroitin sulfate D structure also disappeared from the brain of the null embryos. By using MO225 monoclonal antibody, chondroitin sulfate D structure was detected in the neocortical neuroepithelium in the telencephalon of C6st^+/+ day 15.5 embryos, whereas the signals in that of C6st^/ day 15.5 embryos were not observed (Fig. 4, E and F).

View larger version (142K): [in this window] [in a new window]   Fig. 4.   Normal organization of the cerebral cortex in the C6st/ brain. Whole brains of C6st+/+ (A and C) and C6st/ (B and D) mice were embedded in paraffin. Then, coronal sections were prepared and stained by Nissl's method. Cerebral cortex (A and B) and pyramidal cells in the cortex (C and D) were shown. Cryostat sagittal sections of the brain in C6st+/+ (E) and C6st/ (F) day 15.5 embryos were stained with MO225 antibody that detects chondroitin sulfate D. Neocortical neuroepithelium in the telencephalon are shown (E and F). Original magnifications in A and B are ×40, and in C-F are ×400.

Decrease of Naive T Lymphocytes in the Spleen of C6st/ Mice-- Although gross morphology of lymphoid organs was not different between C6st^+/+ mice and C6st^/ mice, the relatively strong expression of C6st mRNA in the spleen and bone marrow of mice led us to examine leukocyte populations in the spleen. We determined the numbers of free cells from the spleen, peripheral lymph nodes, mesenteric lymph nodes and Peyer's patches. The number of spleen cells in C6st^/ mice was similar to that of C6st^+/+ mice (Table I). Then, we analyzed lymphocyte subpopulations in C6st^+/+ and C6st^/ mice. Both the percentage and number of naive T lymphocytes were significantly decreased in the spleen of C6st^/ mice at 5-6 weeks after birth (Fig. 5, Table I). No such difference was observed in other lymphoid organs. The absolute number of naive T lymphocytes in the spleen of C6st^/ mice was ~68% of that in C6st^+/+ mice 5-6 weeks after birth. This result was reproducible in three independent sets of experiments. Neither chondroitin 6-sulfate nor chondroitin sulfate D was expressed on the surface of naive T lymphocytes as revealed by FACS using MC21C or MO225 antibody (data not shown). Thus, chondroitin 6-sulfate in the extracellular space (Fig. 3A) appears to be involved in the phenomenon

View this table: [in this window] [in a new window]   Table I FACS analysis of lymphocyte subpopulations in lymphoid organs of 5-6-week-old C6st+/+ and C6st/ mice Single cell suspensions were prepared from various tissues and quantified manually. The cells were labeled with the indicated antibodies as described under "Experimental Procedures" and analyzed by FACS. B220 was used as a marker of the B lineage. CD62L+CD44low and CD62LCD44high cells in CD3+ cells were considered to be naive and memory/effector T lymphocytes, respectively. Values represent the means ± S.D. of eight separate determinations. Statistical analysis was carried out using Student's t test. *, p  0.05; **, p  0.01.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   The number of naive T lymphocytes was decreased in the spleen of C6st/ mice. Cells were collected from the spleen (A and E), peripheral lymph nodes (B and F), mesenteric lymph nodes (C and G) and Peyer's patches (D and H) of 5-6-week-old C6st+/+ (A-D) and C6st/ (E-H) mice. The age of the C6st+/+ and C6st/ mice was identical. The cells were stained with fluorescein isothiocyanate-conjugated anti-CD44, PE-conjugated anti-CD62L, and biotin-conjugated anti-CD3 antibodies followed by Cy-Chrome®-streptavidin. Naive T lymphocytes (CD3+CD62L+CD44low) were determined by flow cytometry. The numbers indicate the percentages of CD62L+CD44low in the total CD3+ cell population. The results of one representative experiment among eight are shown.

We investigated the reason for the decrease in number of naive T lymphocytes in the spleen of young C6st^/ mice. FACS analysis did not reveal significant differences in the populations of lymphoid cell progenitors such as LinIL-7R⁺c-Kit^lowSca-1^low cells (29) in bone narrow (data not shown). Moreover, differentiation and maturation of thymocytes were normal in C6st^/ mice as revealed by FACS using anti-CD4 and anti-CD8 antibodies (data not shown). Additional analyses of splenic B lymphocytes in C6st^/ mice indicated normal cell-surface expression of CD21, CD22, CD23, CD24, CD44, I-A^b, IgM, IgD, and B220 (data not shown). With regard to the function of splenic lymphocytes, mitogenic responses to concanavalin A (for T cell response) and lipopolysaccharides (for B cell response) were not different between C6st^+/+ and C6st^/ (data not shown).

Lymphocyte Trafficking and Morphology of the Spleen in the C6st^/ Mice Were Normal-- Because we could not exclude the possibility that the decrease of naive T lymphocytes in the spleen of C6st^/ mice was caused by the abnormal recruitment of cells to the spleen, a lymphocyte homing study was performed as described under "Experimental Procedures." Lymphocyte trafficking of extrinsic labeled lymphocytes into secondary lymph nodes of C6st^/ mice was unaltered (Fig. 6A). Recruited cells were normally localized in the T cell area of the spleen of C6st^/ mice (Fig. 6B). The general architecture of the C6st^/ spleen was intact as revealed by hematoxylin and eosin staining (Fig. 6, C and D). Immunohistochemical analyses of spleen sections using anti-CD3, anti-B220, and anti-CD11c showed an overall normal architecture with a well organized T cell zone (Fig. 6B) and B cell zone (Fig. 6, E and F) in the white pulp and marginal zone (Fig. 6, E and F) in C6st^/ mice. These results indicated that C6st deficiency did not affect lymphocyte trafficking to secondary lymphoid organs, and the significant decrease in the number of naive T lymphocytes in the C6st^/ mice spleen might have been the result of abnormal maintenance of the cells in the spleen.

View larger version (34K): [in this window] [in a new window]   Fig. 6.   Lymphocyte trafficking and histological architecture of the spleen in C6st/ mice were normal. Lymphocytes extracted from 5-6-week-old C6st+/+ mice and labeled with a fluorescent dye (CMFDA; Molecular Probes) were injected into the tail vein of C6st+/+ or C6st/ mice. CMFDA-positive lymphocytes in the spleen, peripheral and mesenteric lymph nodes, and Peyer's patches of each mouse were analyzed by flow cytometry (A). Cryostat sections of the spleen in C6st+/+ (upper panel) and C6st/ (lower panel) mice were prepared 1.5 h after injection of the labeled cells and subsequently stained with biotin-conjugated anti-CD3 antibody followed by PE-conjugated streptavidin (red). Injected CMFDA-positive lymphocytes (green) were normally recruited to T cell zones in the C6st/ spleen (B). PLN, peripheral lymph node; MLN, mesenteric lymph node; P.P, Peyer's patch. Cryostat sections of the spleen of C6st+/+ (C and E) and C6st/ (D and F) mice were stained with hematoxylin and eosin (C and D), showing normal lymphocytic follicles. The splenic sections were immunostained with PE-conjugated anti-B220 (red) and biotin-conjugated anti-CD11c followed by Cy-Chrome®-streptavidin (blue), indicating that the histological architecture in the C6st/ spleen was intact (F). B220 and CD11c are markers for B lineage and dendritic cells, respectively.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The C6st gene was knocked out in the mouse, and C6ST activity was abolished almost completely in the spleen, lung, and lymph nodes of the null mutant mice. This result indicated that the C6st gene is responsible for the synthesis of chondroitin 6-sulfate, at least in these organs. Analysis of chondroitin sulfate chains in the spleen of the knockout mice gave further insight into the biosynthesis of chondroitin sulfate in this organ. The amount of chondroitin 6-sulfate was far less than that of chondroitin 4-sulfate. As expected, the peak of Di-6S was decreased to less than 10% of the wild-type level in the spleen of the knockout mice. The significance of the trace amounts of Di-6S in the knockout mice is not clear at present, because virtually no chondroitin 6-sulfotransferase activity was detected in the spleen of the knockout mice. However, other enzyme(s) that have chondroitin 6-sulfotransferase activity, e.g. N-acetylglucosamine-6-O-sulfotransferase-4/chondroitin 6-sulfotransferase-2 (30, 31), may partially compensate for the deficiency of C6st in the spleen. Interestingly, the level of Di-diS_E, which is sulfated on C-4 and C-6 positions of the GalNAc residue, did not differ between C6st^+/+ and C6st^/. This result suggested that the 4- and 6-disulfate structure is formed by 6-sulfation of 4-sulfated GalNAc in chondroitin 4-sulfate, and that the enzyme responsible for the 6-sulfation is different from C6ST. Indeed, 6-sulfotransferase forming the chondroitin 4,6-disulfate structure has been purified from squid cartilage, and shown to be devoid of chondroitin 6-sulfotransferase activity (32). Therefore, an enzyme similar to that in the squid appears to be also present in mammals. On the other hand, chondroitin sulfate D, which contains a different disulfated disaccharide unit, i.e. C-2 sulfated GlcA and C-6 sulfated GalNAc residue, completely disappeared in the brain of C6st^/ mice and in the telencephalon and cartilage tissues of C6st^/ embryos. Thus, C6ST is involved in synthesis of the chondroitin sulfate D structure. Furthermore, this result suggested that 6-O-sulfation of the GalNAc residue catalyzed by C6ST in vivo probably precedes the 2-O-sulfation of the GlcA residue, which is likely to be catalyzed by dermatan/chondroitin 2-sulfotransferase that requires the sulfated structure for its action (33).

Despite the disappearance of chondroitin sulfate D structure from the embryonic and adult brain, brain development was not impaired in C6st^/ mice. Both chondroitin sulfate D (7, 34) and E (34, 35) structures promote neurite outgrowth in vivo. It is possible that the persistence of the E structure in the deficient mice might compensate for the loss of the D structure.

T lymphocytes that have differentiated and matured in the thymus of young mice migrate to the T cell areas of secondary lymphoid organs, in which nonprimed T lymphocytes, i.e. naive T lymphocytes, encounter antigen-presenting cells followed by recirculation into the blood. Because C6ST is most prominently expressed in the spleen and bone marrow (21), we performed extensive analysis on lymphocyte subpopulations of C6st^/ mice and found that naive T lymphocytes were selectively decreased in the spleen of the knockout mice 5-6 weeks after birth. Because growth monitored by the gain of body weight was not different between C6st^+/+ and C6st^/ mice, this phenomenon is not because of retarded growth of C6st^/ mice. Most of peripheral naive T lymphocytes in young mice are composed of the cells emigrated from the thymus, and recirculated from peripheral lymphoid organs. C6st mRNA was not expressed in the thymus (21), and the population and number of CD4⁺CD8 and CD4CD8⁺ cells in the thymus of 5-6-week-old C6st^/ mice were unchanged (data not shown), indicating that null mutation of C6st^/ may have not affected the differentiation, maturation, and emigration of thymocytes in C6st^/ mice. It is likely that the selective paucity of naive T lymphocytes in the spleen of C6st^/ mice was caused by the functional change in the maintenance of lymphocytes in the spleen. Because recruitment of peripheral lymphocytes to the spleen was not impaired in C6st^/ mice (Fig. 6A), we considered that survival, retention, and/or emigration of naive T lymphocytes was affected in the spleen of these mice. Chemokines as well as the growth and survival factor midkine have strong affinity for a subpopulation of chondroitin sulfate (13, 17). Furthermore, a genetic defect in the expression of secondary lymphoid organ chemokine leads to a decrease in number of naive T lymphocytes in the white pulp of the spleen as well as peripheral lymph nodes (36, 37). It is possible that secondary lymphoid organ chemokine or another factor involved in survival, retention, and emigration of naive T lymphocytes binds to chondroitin 6-sulfate, and this binding plays a role in naive T lymphocytes' appropriate localization in the spleen. The decrease of naive T lymphocytes in the spleen of C6st^/ mice is expected to result in decreased immune response to bacteria and viruses in blood stream. One of the reasons of persistent existence of chondroitin 6-sulfate in the mouse might be their requirement for efficient defense to microorganisms.

	ACKNOWLEDGEMENTS

We thank Drs. Keiichi Yoshida and Masakazu Fukuta for helpful suggestions.

	FOOTNOTES

^* This work was supported in part by Grants-in-aid for Scientific Research 12003898 and 12480187 from the Japan Society for the Promotion of Science and Grants-in-aid 09680616 and 10178102 for Scientific Research from the Ministry of Education, Science, Culture and Sports of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AB062107-AB062109.

^§ Research fellow of the Japan Society for the Promotion of Science.

To whom correspondence should be addressed. Tel.: 81-52-744-2059; Fax: 81-52-744-2065; E-mail: tmurama@med.nagoya-u.ac.jp.

Published, JBC Papers in Press, November 5, 2001, DOI 10.1074/jbc.M104719200

	ABBREVIATIONS

The abbreviations used are: GAG, glycosaminoglycan; CHAPS, 3-((3-cholamidopropyl)dimethylammonio)propanesulfonic acid; C4ST, chondroitin 4-sulfotransferase; C6ST, chondroitin 6-sulfotransferase; Di-6S, 2-acetamide-2-deoxy-3-O-(-D-gluco-4-enopyranosyluroic acid)-6-O-sulfo-D-galactose; Di-4S, 2-acetamide-2-deoxy-3-O-(-D-gluco-4-enopyranosyluroic acid)-4-O-sulfo-D-galactose; Di-0S, 2-acetamide-2-deoxy-3-O-(-D-gluco-4-enopyranosyluroic acid)-d-galactose; Di-diS_D, 2-acetamide-2-deoxy-3-O-(2-O-sulfo--D-gluco-4-enopyranosyluroic acid)-6-O-sulfo-D-galactose; Di-diS_B, 2-acetamide-2-deoxy-3-O-(2-O-sulfo--D-gluco-4-enopyranosyluroic acid)-4-O-sulfo-D-galactose; Di-diS_E, 2-acetamide-2-deoxy-3-O-(-D-gluco-4-enopyranosyluroic acid)-4,6-bis-O-sulfo-D-galactose; FACS, fluorescence-activated cell sorter; GlcA, D-glucuronic acid; PAPS, 3'-phosphoadenosine 5'-phosphosulfate; PE, phycoerythrin; HPLC, high performance liquid chromatography; CMFDA, 5-chloromethylfluorescein diacetate.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				K. Akita, A. von Holst, Y. Furukawa, T. Mikami, K. Sugahara, and A. Faissner Expression of Multiple Chondroitin/Dermatan Sulfotransferases in the Neurogenic Regions of the Embryonic and Adult Central Nervous System Implies That Complex Chondroitin Sulfates Have a Role in Neural Stem Cell Maintenance Stem Cells, March 1, 2008; 26(3): 798 - 809. [Abstract] [Full Text] [PDF]

				S. Oki, R. Hashimoto, Y. Okui, M. M. Shen, E. Mekada, H. Otani, Y. Saijoh, and H. Hamada Sulfated glycosaminoglycans are necessary for Nodal signal transmission from the node to the left lateral plate in the mouse embryo Development, November 1, 2007; 134(21): 3893 - 3904. [Abstract] [Full Text] [PDF]

				A. Yusa, K. Kitajima, and O. Habuchi N-Linked Oligosaccharides on Chondroitin 6-Sulfotransferase-1 Are Required for Production of the Active Enzyme, Golgi Localization, and Sulfotransferase Activity toward Keratan Sulfate J. Biol. Chem., July 21, 2006; 281(29): 20393 - 20403. [Abstract] [Full Text] [PDF]

				C. Mitsunaga, T. Mikami, S. Mizumoto, J. Fukuda, and K. Sugahara Chondroitin Sulfate/Dermatan Sulfate Hybrid Chains in the Development of Cerebellum: SPATIOTEMPORAL REGULATION OF THE EXPRESSION OF CRITICAL DISULFATED DISACCHARIDES BY SPECIFIC SULFOTRANSFERASES J. Biol. Chem., July 14, 2006; 281(28): 18942 - 18952. [Abstract] [Full Text] [PDF]

J. Liu, C.-H. Chau, H. Liu, B. R. Jang, X. Li, Y.-S. Chan, and D. K. Y. Shum Upregulation of chondroitin 6-sulphotransferase-1 facilitates Schwann cell migration during axonal growth. J. Cell Sci., March 1, 2006; 119(Pt 5): 933 - 942.