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Originally published In Press as doi:10.1074/jbc.M501728200 on April 20, 2005

J. Biol. Chem., Vol. 280, Issue 25, 23876-23883, June 24, 2005
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A Non-sulfated Form of the HNK-1 Carbohydrate Is Expressed in Mouse Kidney*

Hideki Tagawa{ddagger}§, Yasuhiko Kizuka{ddagger}§, Tomoko Ikeda{ddagger}, Satsuki Itoh¶, Nana Kawasaki¶, Hidetake Kurihara||, Maristela Lika Onozato**, Akihiro Tojo**, Tatsuo Sakai||, Toshisuke Kawasaki{ddagger}, and Shogo Oka{ddagger}§{ddagger}{ddagger}

From the {ddagger}Department of Biological Chemistry and §Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan, the Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, Tokyo 158-8501, Japan, the ||Department of Anatomy, Juntendo University School of Medicine, Tokyo 113-8421, Japan, and the **Division of Nephrology and Endocrinology, University of Tokyo, Tokyo 113-8655, Japan

Received for publication, February 15, 2005 , and in revised form, April 20, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The HNK-1 carbohydrate, which is recognized by anti-HNK-1 antibody, is well known to be expressed predominantly in the nervous system. The characteristic structural feature of the HNK-1 carbohydrate is 3-sulfo-glucuronyl residues attached to lactosamine structures (Gal{beta}1-4GlcNAc) on glycoproteins and glycolipids. The biosynthesis of the HNK-1 carbohydrate is regulated mainly by two glucuronyltransferases (GlcAT-P and GlcAT-S) and a sulfotransferase. In this study, we found that GlcAT-S mRNA was expressed at higher levels in the kidney than in the brain, but that both GlcAT-P and HNK-1 sulfotransferase mRNAs, which were expressed at high levels in the brain, were not detected in the kidney. These results suggested that the HNK-1 carbohydrate without sulfate (non-sulfated HNK-1 carbohydrate) is expressed in the kidney. We substantiated this hypothesis using two different monoclonal antibodies: one (anti-HNK-1 antibody) requires sulfate on glucuronyl residues for its binding, and the other (antibody M6749) does not. Western blot analyses of mouse kidney revealed that two major bands (80 and 140 kDa) were detected with antibody M6749, but not with anti-HNK-1 antibody. The 80- and 140-kDa band materials were identified as meprin {alpha} and CD13/aminopeptidase N, respectively. We also confirmed the presence of the non-sulfated HNK-1 carbohydrate on N-linked oligosaccharides by multistage tandem mass spectrometry. Immunofluorescence staining with antibody M6749 revealed that the non-sulfated HNK-1 carbohydrate was expressed predominantly on the apical membranes of the proximal tubules in the cortex and was also detected in the thin ascending limb in the inner medulla. This is the first study indicating the presence of the non-sulfated HNK-1 carbohydrate being synthesized by GlcAT-S in the kidney. The results presented here constitute novel knowledge concerning the function of the HNK-1 carbohydrate.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Glycosylation is one of the major post-translational protein modifications that play important roles in a variety of cellular functions, including recognition and adhesion. We have been interested in the HNK-1 (human natural killer-1) carbohydrate, which is recognized by anti-HNK-1 monoclonal antibody (1). The carbohydrate is expressed predominantly in the nervous system in a wide range of species, and its expression is spatially and temporally regulated during development of the nervous system (2, 3). The characteristic structural feature of the carbohydrate is a sulfated trisaccharide, HSO3-3GlcA{beta}1-3Gal{beta}1-4GlcNAc1 (4, 5), and the inner structure, Gal{beta}1-4GlcNAc, is commonly found on various glycoproteins and glycolipids. Therefore, glucuronyltransferase(s) and sulfotransferase(s) are supposed to be key enzymes for the biosynthesis of this carbohydrate (6). Recently, we cloned two glucuronyltransferases (GlcAT-P and GlcAT-S) that are involved in the biosynthesis of the HNK-1 carbohydrate from rat, mouse, and human (7-11). To elucidate the function of the HNK-1 carbohydrate, we generated mice with a targeted deletion of the GlcAT-P gene (12). The HNK-1 carbohydrate has disappeared almost completely in the nervous system of GlcAT-P gene-deficient mice, and these mice exhibit reduced long-term potentiation at the Schaffer collateral CA1 synapses and defects in spatial memory formation. However, a trace of anti-HNK-1 antibody immunoreactivity remains on the surfaces of the soma and proximal dendrites of a subset of neurons in some limited regions. The remaining HNK-1 carbohydrate in GlcAT-P gene-deficient mice is assumed to be synthesized by GlcAT-S. More recently, we characterized the acceptor specificities of the two glucuronyltransferases using various oligosaccharides (13). The results suggested the possibility that the two glucuronyltransferases synthesize structurally and functionally different HNK-1 carbohydrates.

To elucidate the function of the HNK-1 carbohydrate synthesized by GlcAT-S, we examined GlcAT-S mRNA expression in various mouse tissues, including brain. To our surprise, Glc-AT-S mRNA was expressed at higher levels in the kidney than in the brain. In this study, we provide the first evidence that the HNK-1 carbohydrate is synthesized by GlcAT-S as the non-sulfated form in mouse kidney. The non-sulfated form of HNK-1 carbohydrate was expressed predominantly on meprin {alpha} and CD13/aminopeptidase N. We also examined in detail the distribution of the carbohydrate in mouse kidney. The results are important for understanding the functions of the HNK-1 carbohydrate other than those in the nervous system.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Monoclonal antibody (mAb) M6749 was a generous gift from Dr. H. Tanaka (Kumamoto University) (14). Anti-HNK-1 mAb was purchased from American Type Culture Collection. Rat anti-mouse CD13 mAb was purchased from Research Diagnostics Inc. Rabbit anti-rat aquaporin (AQP)-2 polyclonal antibody (pAb) was kindly provided by Dr. S. Sasaki (Tokyo Medical and Dental University, Tokyo, Japan). Rabbit anti-Tamm-Horsfall glycoprotein pAb was purchased from Bio-medical Technologies, Inc. (Stoughton, MA). Rabbit anti-CLC-K (chloride channel K) pAb was purchased from Alomone Laboratories (Jerusalem, Israel). Rabbit anti-AQP-1 pAb was purchased from Chemicon International, Inc. (Temecula, CA). Rabbit anti-megalin pAb was a gift from Dr. M. G. Farquhar (University of California-San Diego, La Jolla, CA). TRITC-conjugated donkey anti-mouse IgM F(ab')2 fragment and fluorescein isothiocyanate-conjugated donkey anti-rabbit IgG F(ab')2 fragment were from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Fluorescein isothiocyanate-conjugated goat anti-mouse IgM- and rat anti-mouse IgM-Sepharose 4B were purchased from Zymed Laboratories Inc.. Paragloboside, glucuronylparagloboside, and sulfoglucuronylparagloboside were from Wako Pure Chemical Industries (Osaka, Japan). Protein G-Sepharose TM4 Fast Flow was from Amersham Biosciences. {alpha}-35S-dUTP was purchased from PerkinElmer Life Sciences, and [{alpha}-32P]dCTP was from Amersham Biosciences. N-Glycosidase F was obtained from Roche Applied Science. Autoradiography NTB2 emulsion and D-19 developer were from Eastman Kodak Co.

Northern Blot Analysis—Total RNA was extracted from the brains, kidneys, hearts, thymuses, spleens, and livers of 4-week-old C57BL/6 mice by the acid guanidine/phenol/chloroform method (15). Equal amounts of total RNA (10 µg in each lane) from these tissues were separated on a formaldehyde-containing 1.0% agarose gel and transferred to a nylon membrane (Hybond-N+, Amersham Biosciences), and then the RNA was cross-linked to the membrane by UV irradiation. To determine the integrity and quantities of RNAs, the membrane was stained with methylene blue. To prepare cDNA probes for Northern blot analysis, mouse GlcAT-P and GlcAT-S cDNAs were amplified by PCR using primers 5'-TAGGGAGTACTGCATGTCCG-3' and 5'-TATAGTTGCGTGGTGTCTCT-3' and primers 5'-ACGCGCAGCGAGCTGGTGAG-3' and 5'-ACGCGCAGCGAGCTGGTGAG-3', respectively. The amplified PCR fragments (mouse GlcAT-P nucleotides 198-496 (299 bp) and mouse GlcAT-S nucleotides 364-780 (417 bp)) were subcloned into the pGEM-T easy vector, resulting in vectors pGEM-GlcAT-P and pGEM-GlcAT-S, respectively. The GlcAT-P and GlcAT-S cDNA probes for Northern blot analysis were prepared by digestion of pGEM-Glc-AT-P and pGEM-GlcAT-S with NcoI and SalI. Mouse HNK-1 sulfotransferase cDNA was amplified by PCR using primers 5'-CGGTACCCATCACGTTGACCTTTAAGGATCCGGA-3' and 5'-TGGTACCGTCTCTCTGTCCGGTTCTTCCGGTACT-3' (with KpnI sites underlined). The amplified PCR fragment (mouse HNK-1 sulfotransferase nucleotides 72-695 (624 bp)) was digested with KpnI and then inserted into pBluescript II SK+, which had been digested with KpnI. The HNK-1 sulfotransferase cDNA probe for Northern blot analysis was prepared by digestion of the resultant vector with KpnI. The blot was hybridized overnight with 32P-labeled random-primed probes in 0.5 M NaH2PO4 (pH 7.2) containing 1% bovine serum albumin, 7% SDS, and 1 mM EDTA at 65 °C (16). The membrane was washed at room temperature with 2x SSC and 1% SDS, followed by three times with 2x SSC and 0.1% SDS. It was then washed twice with 0.2x SSC and 0.1% SDS at 65 °C. Finally, the membrane was analyzed using a FujiFilm BAS-2500 image analyzer.

Thin-layer Chromatography—Glycolipids (paragloboside, glucuronylparagloboside, and sulfoglucuronylparagloboside) were separated on a high performance TLC Silica Gel 60 plate (Merck). The developing solvent system was 60:40:9 (v/v/v) chloroform, methanol, and 0.2% CaCl2. Glycolipids were visualized by spraying with 0.2% orcinol in 2 M H2SO4 (17). For TLC immunoblotting, the TLC plate was dipped in a blotting solvent (40:20:7 (v/v/v) 2-propanol, 0.2% CaCl2, and methanol) for 30 s. Glycolipids were then transferred to a polyvinylidene difluoride membrane (ATTO Technology Inc., Tokyo) by pressing the plate and membrane at 180 °C for 30 s with a TLC thermal blotter (ATTO Technology Inc.). The membrane was incubated for 1 h with blocking solution (phosphate-buffered saline (PBS) containing 0.05% Tween 20 and 5% nonfat dried milk), and the membrane was then incubated for 2 h with primary antibodies (M6749 or anti-HNK-1). After washing with PBS containing 0.05% Tween 20, the membrane was incubated for 1 h with horseradish peroxidase-conjugated anti-mouse IgM secondary antibodies. After washing with PBS containing 0.05% Tween 20 followed by washing with PBS, glycolipids were detected by ECL (Pierce) with a Luminocapture instrument (ATTO Technology, Inc.).

Preparation of Cytosolic and Membrane Fractions from Mouse Kidney and Brain—A whole brain or kidney from a 4-week-old mouse was homogenized using a Polytron homogenizer in 9 volumes of 20 mM Tris-HCl (pH 7.5) containing 150 mM NaCl, 1 mM EDTA, and protease inhibitors (Nakalai Tesque, Kyoto, Japan) at 4 °C. The homogenate was centrifuged at 1000 x g for 10 min at 4 °C to remove nuclei. The supernatant was centrifuged at 105,000 x g for 1 h at 4 °C. The resulting supernatant and precipitate were used in the following experiments as the cytosolic and membrane fractions, respectively.

Extraction from the Membrane Fraction with Triton X-100 or Urea—The membrane fraction prepared from mouse kidney was incubated for 1 h at 4 °C with 20 mM Tris-HCl (pH 7.5) containing 1% Triton X-100, 0.5% deoxycholate, 150 mM NaCl, 1 mM EDTA, and protease inhibitors and then centrifuged at 105,000 x g for 1 h at 4 °C. The resulting supernatant fraction served as the Triton extract fraction. The precipitate (Triton-insoluble fraction) was further incubated for 16 h at 4 °C with 20 mM Tris-HCl (pH 7.5) containing 7 M urea, 150 mM NaCl, and 1 mM EDTA, and then centrifuged at 105,000 x g for 1 h at 4 °C. The supernatant fraction was dialyzed overnight against 20 mM Tris-HCl (pH 7.5) containing 150 mM NaCl and 1 mM EDTA and then recentrifuged under the same conditions. The dialyzed supernatant was used as the urea extract fraction.

SDS-PAGE and Western Blot Analysis—Proteins were separated by 3-10% gradient SDS-PAGE with the buffer system of Laemmli (18) and then transferred to nitrocellulose membranes. After blocking with 5% nonfat dried milk in PBS containing 0.05% Tween 20, the membranes were incubated with anti-HNK-1 mAb and mAb M6749, followed by horseradish peroxidase-conjugated goat anti-mouse IgM antibody, and then protein bands were visualized by ECL (Pierce) with a Luminocapture instrument.

N-Glycosidase F Digestion—Proteins (20 µg) were denatured with 20 mM sodium phosphate buffer (pH 7.2) containing 0.5% SDS, 1% 2-mer-captoethanol, and 4 mM EDTA, and then the protein solution was diluted with 4 volumes of 20 mM sodium phosphate buffer (pH 7.2) to reduce the concentration of SDS to 0.1%. After the addition of 0.5% Nonidet P-40 (final concentration), N-glycosidase F (2 units) was added to the solution, and the solution was incubated for 16 h at 37 °C.

Preparation of mAb M6749 Affinity Beads—Antibody M6749 (1 mg) was conjugated to rat anti-mouse IgM-Sepharose 4B (1 ml) according to the procedure described by Schneider et al. (19) with slight modifications. Briefly, Sepharose 4B conjugated with rat anti-mouse IgM antibody was incubated with mAb M6749 at 4 °C for 1 h with gently shaking. The Sepharose beads were washed with PBS and 0.2 M triethanolamine (pH 8.2) and then suspended in 60 mM dimethyl suberimidate dihydrochloride (Pierce) in 0.2 M triethanolamine (pH 8.2). The mixture was gently agitated at room temperature for 45 min and centrifuged at 500 x g for 1 min at 4 °C, and the beads were resuspended in 60 mM monoethanolamine (pH 8.2). The mAb M6749-conjugated beads were washed with 0.1 M borate buffer (pH 8.2) containing 0.02% NaN3 and kept at 4 °C until used.

Purification of Glycoproteins on an Affinity Column—The mAb M6749-conjugated beads were equilibrated with PBS containing 0.1% Triton X-100. The Triton or urea extract prepared as described above was applied to the column. After washing the column with an excess volume of PBS containing 0.1% Triton X-100, proteins specifically bound to the beads were eluted with 50 mM diethylamine (pH 11.5). The eluate was immediately neutralized by the addition of a 0.05 volume of 1 M NaH2PO4. The purified glycoproteins were concentrated, subjected to SDS-PAGE, and then stained with Coomassie Brilliant Blue. A piece of polyacrylamide gel containing the 80- or 140-kDa glycoprotein was excised. After carboxymethylation, the glycoprotein in the gel was digested with N-glycosidase F and trypsin. The peptides released from the gel were analyzed by liquid chromatography (LC)-tandem mass spectrometry (MS/MS).

Protein Identification—The peptides released from the gel were subjected to LC-MS/MS analysis with a hybrid quadrupole time-of-flight mass spectrometer (QSTAR Pulsar I, Applied Biosystems, Ontario, Canada) interfaced on-line with a capillary high performance liquid chromatography system (Magic 2002, Michrom Bioresources, Inc. Auburn, CA) equipped with a Magic C18 column (0.2 x 50 mm, 3 µm; Michrom Bioresources, Inc.). The eluents were water containing 2% CH3CN and 0.01% formic acid (pump A) and water containing 90% CH3CN and 0.01% formic acid (pump B), and the peptides were eluted with a 5-65% linear gradient of pump B over 20 min at a flow rate of 2 µl/min. Data-dependent MS/MS acquisitions were performed for pre-cursors with charge states of 2 or 3 over a survey mass range of 400-2000. Proteins were identified by searching the Mass Spectrometry Protein Sequence Database using the Mascot search engine (Matrix Science Ltd., London, United Kingdom).



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  		FIG. 1.
Northern blot analyses of GlcAT-P, GlcAT-S, and HNK-1 sulfotransferase mRNA expression in mouse tissues. Total RNA was isolated from the brains, kidneys, hearts, thymuses, spleens, and livers of 4-week-old C57BL/6 mice. Ten micrograms of total RNA was electrophoresed and hybridized with 32P-labeled GlcAT-S (A), HNK-1 sulfotransferase (ST) (B), and GlcAT-P (C) cDNAs as probes. The positions of marker RNAs are indicated on the left. The same blots were stained with methylene blue to assess the integrity of the RNAs (D). The positions of ribosomal RNAs are indicated on the right.

 
Release of N-Linked Oligosaccharides from the Gel-separated Glycoprotein—The gel containing the 80-kDa glycoprotein was excised and cut into pieces. The gel pieces were dehydrated with 50% acetonitrile after destaining, equilibrated with 50 mM sodium phosphate buffer (pH 7.2), and incubated with 5 units of N-glycosidase F at 37 °C for 18 h. N-Linked oligosaccharides were extracted three times from the gel pieces by intermittent sonication for 30 min in water. All extracts were combined and lyophilized. Released N-linked oligosaccharides were reduced with NaBH4.

LC-Multistage MS/MS (MSn) of N-Linked Oligosaccharides—LC-MS was carried out using a Magic 2002 system connected to quadrupole linear ion trap mass spectrometer (LTQ, Thermo Electron Corp., San Jose, CA). A 5-µm Hypercarb column (0.2 x 150 mm; Thermo Electron Corp.) was used. The eluents consisted of 5 mM ammonium acetate (pH 9.6) containing 2% CH3CN (pump A) and 5 mM ammonium acetate (pH 9.6) containing 80% CH3CN (pump B). The borohydride-reduced N-linked oligosaccharides were eluted with a 5-30% linear gradient of pump B over 60 min at a flow rate of 2 µl/min. A tube lens offset of 160 V, a capillary voltage of 2.0 kV, and a capillary temperature of 200 °C were the operating conditions for MS and MS/MS. A full scan mass spectrum (m/z 450-2000) and data-dependent collision-induced dissociation-MSn spectra for the most abundant ions were acquired in positive ion mode. The isolation window for precursor masses was set to ±5 Da. Collision energy was set to 35%, and the activation Q value was set to 0.250.

Immunoprecipitation—A Triton extract fraction prepared as described above was incubated with anti-CD13 mAb for 1 h. The mixture was additively incubated with protein G-Sepharose TM4 Fast Flow for 2 h with gently shaking. The beads were precipitated by centrifugation at 500 x g for 3 min, and then washed three times with an excess volume of PBS containing 0.1% Triton X-100. Proteins bound to the Sepharose beads were eluted by boiling in Laemmli sample buffer.

Histochemical Staining—C57BL/6 mice (4 weeks old) were deeply anesthetized by diethyl ether inhalation and perfused with PBS containing 0.1% heparin and then with 4% paraformaldehyde in PBS. Their kidneys were post-fixed overnight, followed by dipping in 30% sucrose solution. For immunofluorescence staining, sections (5-10 µm thick) were prepared, incubated with the primary antibodies, and then incubated with the secondary antibodies conjugated with fluorescein isothiocyanate or rhodamine. These sections were visualized and digitized with a Fluoview laser confocal microscope system (Olympus Corp., Tokyo) or an LSM510 confocal laser scanning microscope (Carl Zeiss AG, Oberkochen, Germany).

In Situ Hybridization Analysis—C57BL/6 mice (4-weeks-old) were killed by cervical dislocation. Sections (14 µm thick) of their kidneys were prepared and placed onto glass slides precoated with 3-aminopropyltriethoxysilane (Sigma) and then stored at -80 °C. Antisense and sense RNA probes for GlcAT-P and GlcAT-S were prepared using pGEM-GlcAT-P and pGEM-GlcAT-S as described above. Transcription from the T7 or SP6 promoter with 35S-UTP yielded a 299-bp GlcAT-P antisense probe or a 417-bp GlcAT-S antisense probe, respectively. Similarly, transcription from the SP6 or T7 promoter generated sense probes of the same size. The sections were fixed in 4% formaldehyde, treated with 10 µg/ml protease K, and acetylated with 24 mM acetic anhydride. The sections were then hybridized overnight with 35S-labeled RNA probes in hybridization buffer (20 mM Tris-HCl (pH 8.0) containing 0.1 g/ml dextran sulfate sodium, 300 mM NaCl, 50% formamide, 0.002% (w/v) N-lauroylsarcosine, 1x Denhardt's solution (Nakalai Tesque), 2 mM dithiothreitol, 12.5 mM EDTA, 0.5 mg/ml brewers' yeast tRNA (Roche Applied Science), and 0.2 mg/ml salmon testis DNA (Wako Pure Chemical Industries)) at 55 °C. After hybridization, the sections were washed with 5x SSC containing 5 mM dithiothreitol and then with high stringency buffer (5x SSC and 50% formamide containing 5 mM dithiothreitol) at 68 °C for 30 min each. After the sections had been washed three times with RNase buffer (10 mM Tris-HCl (pH 7.4) containing 50 mM NaCl and 5 mM EDTA), they were incubated in RNase buffer containing RNase A (2 µg/ml) at 37 °C for 10 min, followed by washing with RNase buffer at 37 °C for 10 min. The sections were then washed with high stringency buffer at 68 °C for 30 min and with 2x SSC and then with 0.2x SSC at room temperature for 10 min each. Finally, the sections were dehydrated and analyzed with the BAS-2500 image analyzer.

Microautoradiography—The sections were dipped in autoradiography NTB2 emulsion at 42 °C in a dark room and then stored for 3 weeks at 4 °C in the dark. The sections were dipped in developing solution (31.3 g of D-19 developer/200 ml of H2O) for 3 min, in stop solution (1% acetic acid) for 30 s, and in fixing solution (24% sodium thiosulfate) at 19 °C for 8 min each in a dark room. They were then washed several times with H2O, followed by dehydration. Finally, dark-field microscopic examination was performed.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Northern Blot Analysis of Mouse GlcAT-P, GlcAT-S, and HNK-1 Sulfotransferase—To determine whether or not the HNK-1 carbohydrate is expressed in other locations besides the nervous system, we investigated the expression of mouse GlcAT-P, GlcAT-S, and HNK-1 sulfotransferase mRNAs in various mouse tissues by Northern blot analysis. As shown in Fig. 1C, GlcAT-P mRNA of 4.0 kb was specifically expressed in the brain (12). In contrast, GlcAT-S mRNAs of 2.5 kb (major) and 6.2 kb (minor) were detected not only in the brain, but also in the kidney, with the expression level of GlcAT-S mRNA in the kidney being higher than that in the brain (Fig. 1A). HNK-1 sulfotransferase mRNAs of 3.3 kb (major) and 5.4 kb (minor) were expressed in the brain, thymus, and spleen, but not in the kidney, suggesting that the HNK-1 carbohydrate, which is not sulfated, is expressed in the kidney.

Reactivity of Anti-HNK-1 mAb and mAb M6749—To examine the presence of the non-sulfated HNK-1 carbohydrate in mouse kidney, we used two monoclonal antibodies (anti-HNK-1 and M6749) that have similar specificities (14). Anti-HNK-1 antibody requires sulfate on the GlcA residue for its binding (20), but antibody M6749 can react with terminal non-sulfated GlcA residues as well as sulfated GlcA residues (7). The specificity of the two monoclonal antibodies for the HNK-1 carbohydrate was confirmed by TLC using three glycolipids: paragloboside, glucuronylparagloboside, and sulfoglucuronylparagloboside. Almost the same amounts of glycolipids were subjected to TLC (Fig. 2A). The glycolipids were then transferred to a polyvinylidene difluoride membrane and blotted with antibody M6749 (Fig. 2B) or anti-HNK-1 antibody (Fig. 2C). As expected, anti-HNK-1 antibody reacted only with sulfoglucuronylparagloboside (Fig. 2C, lane 3). In contrast, antibody M6749 reacted with glucuronylparagloboside as well as with sulfoglucuronylparagloboside (Fig. 2B, lanes 2 and 3).

Expression of the Non-sulfated HNK-1 Carbohydrate on N-Linked Oligosaccharides—To confirm that the non-sulfated form of the HNK-1 carbohydrate is expressed in mouse kidney, we performed immunoblot analysis with anti-HNK-1 antibody and mAb M6749. Two major bands (80 and 140 kDa; indicated by arrows in Fig. 3A) were detected with antibody M6749, but not with anti-HNK-1 antibody, for the membrane fraction prepared from mouse kidney (Fig. 3, A and B, lanes 3). No reactivity was detected for the cytosolic fraction (Fig. 3, A and B, lanes 2), indicating that the non-sulfated form of the HNK-1 carbohydrate is expressed on membrane-associated glycoproteins in mouse kidney. As a control, we used the membrane fraction prepared from mouse brain. Similar bands were detected with both mAb M6749 and anti-HNK-1 antibody (Fig. 3, A and B, lanes 1), suggesting that almost all of the HNK-1 carbohydrate is sulfated in the brain. To confirm the specificity of antibody M6749, we also performed immunoblot analysis using various mouse tissues. As shown in Supplemental Fig. 1, the immunoreactive bands with antibody M6749 were observed only in the kidney, which expressed GlcAT-S, but not in the other tissues examined (heart, thymus, spleen, and liver). These results indicate that antibody M6749 is highly specific for the HNK-1 carbohydrate, which is synthesized by GlcAT-P and/or GlcAT-S.



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  		FIG. 2.
Reactivities of anti-HNK-1 mAb and mAb M6749. TLC was carried out with paragloboside (lanes 1), glucuronylparagloboside (lanes 2), and sulfoglucuronylparagloboside (lanes 3), the migration positions of which were visualized with orcinol/H2SO4 reagent (A). These glycolipids were blotted onto a polyvinylidene difluoride membrane as described under "Experimental Procedures" and detected with mAb M6749 (B) and anti-HNK-1 antibody (C).

 



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  		FIG. 3.
Western blot analyses of mouse kidney using anti-HNK-1 antibody and mAb M6749. A and B, cytosolic (lanes 2) and membrane (lanes 3) fractions were prepared from 4-week-old C57BL/6 mouse kidneys as described under "Experimental Procedures." As a control, a membrane fraction of mouse whole brain (lanes 1) was also prepared. Twenty micrograms of protein from each fraction was subjected to SDS-PAGE (3-10% gel) under reducing conditions, transferred to nitrocellulose membranes, and then probed with mAb M6749 (A) and anti-HNK-1 antibody (B). Arrows indicate major antibody M6749-reactive bands (80 and 140 kDa) for mouse kidney. In C, a membrane fraction prepared from C57BL/6 mouse kidney was incubated in the absence (lane 1) or presence (lane 2)of N-glycosidase F for 16 h at 37 °C and then subjected SDS-PAGE (3-10% gel) under reducing conditions. Western blot analysis with antibody M6749 revealed that almost all the immunoreactivity of antibody M6749 was abolished upon N-glycosidase F digestion.

 
Next, to determine whether or not the non-sulfated HNK-1 carbohydrate is attached to N-linked oligosaccharides, we performed N-glycosidase F digestion. As shown in Fig. 3C, the reactivity of both the 80- and 140-kDa proteins with antibody M6749 completely disappeared upon N-glycosidase F digestion, indicating that the non-sulfated HNK-1 carbohydrate is expressed on the glycoproteins as an N-linked form. These results also indicate that antibody M6749 does not cross-react with O-linked forms of carbohydrates such as glycosaminoglycans.

Purification of Glycoproteins Bearing the Non-sulfated HNK-1 Carbohydrate—To identify the two major glycoproteins bearing the non-sulfated HNK-1 carbohydrate, each glycoprotein was extracted from the membrane fraction of kidney. As shown in Fig. 4 (lane 2), the 140-kDa protein was extracted with 1% Triton X-100 (Triton extract), whereas the 80-kDa protein was not. The Triton-insoluble fraction was then further extracted with 7 M urea (urea extract). Almost all of the 80-kDa protein was extracted in the urea extract (Fig. 4, lane 3) because there were no reactive bands for the Triton- and urea-insoluble fractions (lane 4). For purification, urea was removed from the urea extract by dialysis against an excess volume of PBS. The Triton extract fraction containing the 140-kDa glycoprotein and the urea extract containing the 80-kDa glycoprotein were applied to a antibody M6749-conjugated Sepharose column. Glycoproteins specifically bound to the column were eluted with 50 mM diethylamine (pH 11.5). The purified glycoproteins were concentrated, subjected to SDS-PAGE, and then stained with Coomassie Brilliant Blue (data not shown). A piece of polyacrylamide gel containing the 80- or 140-kDa glycoprotein was excised, digested with trypsin, and then analyzed by LC-MS/MS. The 80- and 140-kDa band materials were identified as meprin {alpha} and CD13/aminopeptidase N, respectively (data not shown).



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  		FIG. 4.
Extraction of glycoproteins bearing the non-sulfated HNK-1 carbohydrate. Glycoproteins bearing the non-sulfated HNK-1 carbohydrate were extracted with 1% Triton X-100 and 0.5% deoxycholate (Triton extract) (lane 2) from a membrane fraction (lane 1) prepared from C57BL/6 mouse kidney. Triton-insoluble glycoproteins were further extracted with 7 M urea (lane 3). The urea-insoluble glycoproteins were extracted with SDS sample buffer (lane 4). Samples were subjected to SDS-PAGE (3-10% gel) under reducing conditions and then transferred to nitrocellulose membranes. Glycoproteins bearing the non-sulfated HNK-1 carbohydrate were detected with antibody M6749.

 
To confirm the results of LC-MS/MS analysis, we carried out the following experiments. First, we used C3H/He mice (Fig. 5A), a meprin {alpha}-subunit-null mouse strain (21). The 80-kDa protein band detected in C57BL/6 mice (Fig. 5A, lane 1) was absent in the membrane fraction prepared from C3H/He mouse kidney (lane 2), although the 140-kDa protein band was detected in both strains (lanes 1 and 2), indicating that the 80-kDa glycoprotein carrying the non-sulfated HNK-1 carbohydrate is the meprin {alpha}-subunit. Next, we carried out immunoprecipitation analysis with anti-CD13/aminopeptidase N antibody. CD13/aminopeptidase N in the Triton extract fraction was immunoprecipitated with anti-CD13 mAb, and we then performed Western blot analysis with mAb M6749 (Fig. 5B) and anti-CD13 mAb (Fig. 5C). Antibody M6749 immunoreactivity was detected in immunoprecipitated CD13 (Fig. 5B, lane 2), indicating that the 140-kDa glycoprotein carrying the non-sulfated HNK-1 carbohydrate is CD13/aminopeptidase N. As shown in Fig. 5A, several minor bands (at ~90 and 180 kDa) were sometimes detected upon Western blot analysis, but were not identified in this study.



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  		FIG. 5.
Identification of carrier proteins bearing the non-sulfated HNK-1 carbohydrate. A, membrane fractions were prepared from C57BL/6 mouse (lane 1) and C3H/He mouse (lane 2) kidney. Samples were subjected to SDS-PAGE (3-10% gel) under reducing conditions, transferred to nitrocellulose membranes, and then probed with antibody M6749. B and C, a membrane fraction prepared from C57BL/6 mouse kidney (lanes 1) was extracted with 1% Triton X-100 and 0.5% deoxycholate, and CD13/aminopeptidase N was immunoprecipitated with anti-CD13 antibody (lanes 2). Samples were subjected to SDS-PAGE (3-10% gel) under reducing conditions, transferred to nitrocellulose membranes, and then probed with mAb M6749 (B) and anti-CD13 antibody (C).

 
LC-MSn of N-Linked Oligosaccharides—The gel containing the 80-kDa glycoprotein (meprin {alpha}) was excised and treated with N-glycosidase F. The released N-linked oligosaccharides were subjected to LC-MSn analysis. Fig. 6A shows the oligosaccharide profile obtained by a full mass scan (m/z 450-2000) of the N-linked oligosaccharides. Major molecular ions detected by the full mass scan were automatically subjected to data-dependent collision-induced dissociation-MS/MS. Oligosaccharides bearing the non-sulfated HNK-1 motif were sorted from the oligosaccharide profile using the product ion at m/z 542, GlcAGalGlcNAc, as a marker ion of the non-sulfated HNK-1 carbohydrate. Fig. 6B is the extracted ion chromatogram (m/z 542). The appearance of several peaks at ~35 min suggests the linkage of the non-sulfated HNK-1 motif to certain oligosaccharides in this protein.

The structures of the carbohydrates in peaks a and b in Fig. 6B were determined on the basis of their product ion spectra. The mass spectrum of peak a is shown in Fig. 6C. In addition to the intense ion at m/z 542, y ions produced by the loss of one and two non-sulfated HNK-1 motifs appeared at m/z 1267 and 1992, respectively. It was confirmed that the fragment at m/z 542 consists of hexonic acid, Hex, and HexNAc from the MS/MS/MS spectrum acquired by collision-induced dissociation-MS/MS of the product ion at m/z 542 (Fig. 6D). Based on these fragments together with the precursor ion at m/z 1026 (3+), this oligosaccharide was characterized as a fucosylated complex-type oligosaccharide bearing two non-sulfated HNK-1 motifs and is represented schematically in Fig. 6C (inset). Like-wise, the carbohydrate structure of peak b was characterized as a difucosylated complex-type form with a non-sulfated HNK-1 motif (Fig. 6E). These results clearly indicate that the non-sulfated HNK-1 carbohydrate is expressed on N-linked oligosaccharides of meprin {alpha}.

Localization of the Non-sulfated HNK-1 Carbohydrate in Mouse Kidney—To investigate the localization of the non-sulfated HNK-1 carbohydrate in mouse kidney, immunofluorescence staining with antibody M6749 was carried out. As shown in Fig. 7A, the non-sulfated HNK-1 carbohydrate was expressed predominantly in the cortex and was also detected in the inner medulla. Upon higher magnification of the positive areas, we found the non-sulfated HNK-1 carbohydrate was expressed in renal tubules and in Henle's loop (Fig. 6, B-E). As expected, anti-HNK-1 antibody immunoreactivity was almost negligible (data not shown), confirming that the sulfated form of the HNK-1 carbohydrate is not present in the kidney.

We examined the localization of the non-sulfated HNK-1 carbohydrate in detail by co-staining with antibodies for several marker proteins in the kidney. The immunoreactivity of antibody M6749 was co-localized with that of megalin (Fig. 8, A-C), one of the marker proteins for the proximal tubule (22), but not with that of AQP-2 (Fig. 8, D-F), a marker protein for the cortical collecting duct (23). However, the immunoreactivity of megalin observed in the early proximal tubules adjacent to the glomeruli (S1 segment) was not co-localized with that of antibody M6749 (Figs. 7 and 8A), indicating that the non-sulfated HNK-1 carbohydrate in the cortex is expressed predominantly on the apical membranes of the epithelial cells in the S2 and S3 segments of the proximal tubules. In the medullary region, the immunoreactivity of antibody M6749 corresponded well with that of CLC-K (Fig. 8, J-L), a marker protein for the thin ascending limb (24), but not with those of AQP-1 and Tamm-Horsfall glycoprotein, marker proteins for the thin descending limb and thick ascending limb, respectively (25, 26). The immunoreactivity of antibody M6749 in mouse kidney is summarized and schematically shown in Fig. 9.

In Situ Hybridization Analysis of GlcAT-S mRNA in Mouse Kidney—To investigate the topographical expression pattern of GlcAT-S mRNA, we performed in situ hybridization analysis on 14-µm mouse kidney sections. GlcAT-S mRNA was specifically detected in the kidney cortex (Fig. 10, A-C), which is consistent with the area in which the non-sulfated HNK-1 carbohydrate is expressed. In contrast, in the medullary region, the signal of the GlcAT-S mRNA was less than the detectable level (Fig. 10A). No detectable signal of GlcAT-P mRNA was observed in either the cortex or medullary region (Fig. 10D). These results reveal that the non-sulfated HNK-1 carbohydrate expressed in proximal tubules is synthesized by GlcAT-S.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The HNK-1 carbohydrate is expressed at high levels in the nervous system. The HNK-1 carbohydrate structure on P0 and the neural cell adhesion molecule, the major carrier glycoproteins in the peripheral and central nervous systems, respectively, has been determined (27-29). All of the HNK-1 carbohydrate structure determined was of the sulfated form, i.e. sulfoglucuronic acid attached to N-acetyllactosamine, the non-sulfated form of the HNK-1 carbohydrate not being found on these molecules. These results indicate that almost all of the HNK-1 carbohydrate in the nervous system is expressed as the sulfated form. This is the first report that the HNK-1 carbohydrate is expressed at high levels in mouse kidney as the non-sulfated form. We have also demonstrated that GlcAT-S (but not GlcAT-P) is involved mainly in the expression of the non-sulfated HNK-1 carbohydrate in mouse kidney.

Two glucuronyltransferases (GlcAT-P and GlcAT-S) have so far been identified as HNK-1 biosynthetic enzymes (7-11). We have generated GlcAT-P gene-deficient mice and demonstrated that almost all of the HNK-1 carbohydrate in the brain is synthesized by GlcAT-P because of the absence of the HNK-1 carbohydrate in GlcAT-P gene-deficient mouse brain (12). We have also demonstrated that the HNK-1 carbohydrate synthesized by GlcAT-P is involved in synaptic plasticity and in spatial learning (12). However, little is known about the function of the HNK-1 carbohydrate regulated by GlcAT-S. It should be noted that these two enzymes have significantly different acceptor specificities. Thus, GlcAT-P specifically recognizes N-acetyllactosamine (Gal{beta}1-4GlcNAc) at the nonreducing terminals of acceptor substrates. In contrast, GlcAT-S recognizes not only the terminal Gal{beta}1-4GlcNAc structure, but also the Gal{beta}1-3GlcNAc structure, and shows the highest activity to-ward triantennary N-linked oligosaccharides (13), suggesting that they may synthesize structurally and functionally different HNK-1 carbohydrates. To elucidate the function of the HNK-1 carbohydrate synthesized by GlcAT-S, it is important that GlcAT-S is expressed not only in the brain, but also in the kidney.



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  		FIG. 6.
LC-MSn of N-linked oligosaccharides. A, total ion chromatogram (TIC) (m/z 450-2000) obtained by a full mass scan of N-linked oligosaccharides extracted from the 80-kDa band (meprin {alpha}). B, extracted ion chromatogram (EIC) (m/z 542) obtained by data-dependent collision-induced dissociation-MS/MS of N-linked oligosaccharides extracted from the 80-kDa band (meprin {alpha}). C, MS/MS spectrum of peak a (precursor ion at m/z 1026). D, MS/MS/MS spectrum of peak a (precursor ion at m/z 542). E, MS/MS spectrum of peak b (precursor ion at m/z 1016).

 



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  		FIG. 7.
Localization of glycoproteins bearing the non-sulfated HNK-1 carbohydrate in mouse kidney sections. Frozen sections (10 µm) prepared from 4-week-old C57BL/6 mouse kidney were stained with antibody M6749. A, an overview image of antibody M6749 staining with fluorescein isothiocyanate-conjugated anti-mouse IgM antibody. Scale bar = 1 mm. B and D, high magnification fluorescent images of the areas indicated by boxes in A. C and E, the fluorescent images in B and D merged with the corresponding Nomarski differential interference contrast images, respectively. Scale bars = 40 µm.

 
Immunofluorescence staining of mouse kidney with antibody M6749 revealed that the non-sulfated HNK-1 carbohydrate was expressed predominantly on the apical membranes of the proximal tubules in the cortex, where the GlcAT-S mRNA was also detected (Figs. 7 and 10). The non-sulfated HNK-1 carbohydrate was expressed at high levels in the brush borders of the S2 and S3 segments of the proximal tubules. In comparison, no significant signal for antibody M6749 was observed in the S1 segment immediately after the glomerulus (Fig. 8). Protein kinases C{alpha} and C{beta}1 are another case of such segment-specific expression. The two isoforms of protein kinase C are localized only to the brush borders of the S2 and S3 segments of proximal tubules, whereas the S1 segment is negative (30). It is difficult to distinguish the segments of the proximal tubules with respect to protein absorption under normal conditions. However, lysosomal enzyme activities are increased in the S2 and S3 segments of proteinuric animals because of an increase in protein uptake (31). Therefore, the S2 and S3 segments might be more important under pathological conditions. In addition, antibody M6749 immunoreactivity was also detected in the thin ascending limb in the inner medulla, where no detectable level of either GlcAT-S or GlcAT-P mRNA was observed. These results suggest that the non-sulfated HNK-1 carbohydrate detected in the inner medulla could be synthesized by another glucuronyltransferase involved in the biosynthesis of the HNK-1 carbohydrate. The carbohydrate structure expressed in the thin ascending limb could also be different from those on CD13/aminopeptidase N and meprin {alpha} expressed in proximal tubules.



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  		FIG. 8.
Double immunofluorescence staining of mouse kidney with several antibodies against marker proteins. The expression of the non-sulfated HNK-1 carbohydrate in mouse kidney was evaluated using a double staining immunofluorescence technique with antibodies specific for each nephron segment (green; markers) and antibody M6749 (red; the non-sulfated HNK-1 carbohydrate). Image analysis revealed that antibody M6749 immunoreactivity was localized on the apical membranes of proximal tubular epithelial cells and the thin ascending limb. The signals for mAb M6749 coincided with those for megalin (marker for proximal tubules) (A-C) in the cortex and for CLC-K (marker for the thin ascending limb of Henle) (J-L) in the medulla. No co-localization of the non-sulfated HNK-1 carbohydrate with the signals for AQP-1 (marker for the thin descending limb) (G-I), AQP-2 (marker for the collecting duct) (D-F), and Tamm-Horsfall glycoprotein (THP; marker for the thick ascending limb) (M-O) can be seen. Interestingly, no signal for mAb M6749 can be seen in the S1 segment immediately after the glomerulus (C, arrowhead). Scale bars = 50 µm.

 



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  		FIG. 9.
Schematic representation of the non-sulfated HNK-1 carbohydrate in mouse kidney. The immunoreactivity of antibody M6749 is shown in red.

 
The meprin {alpha}-subunit and CD13/aminopeptidase N were identified as major carrier proteins of the non-sulfated HNK-1 carbohydrate. Meprins ({alpha} and {beta}) are members of the astacin family of metalloproteases (32). Meprins are capable of activation or degradation of bioactive peptides, growth factors, cytokines, hormones, and matrix proteins through limited proteolysis (33). Meprin {alpha} contains 10 potential N-linked glycosylation sites and is extensively glycosylated; ~25% of the total molecular mass is carbohydrate (34). CD13/aminopeptidase N is also an abundant metalloprotease in the brush borders of kidney proximal tubule cells (35) and is the enzyme responsible for the extracellular conversion of angiotensin III to angiotensin IV (36). CD13/aminopeptidase N also has 10 potential N-glycosylation sites. Thus, the non-sulfated HNK-1 carbohydrate is commonly expressed on two major metalloproteases that are extensively glycosylated in the kidney. It has been reported that N-linked carbohydrates on meprin {alpha} are required for optimal enzyme activity and for secretion, as found in a series of mutation analyses of N-glycosylation sites (37). These results suggest that the non-sulfated HNK-1 carbohydrate may be involved in the regulation of metalloprotease activity, which leads to the functional diversity of these two molecules.



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  		FIG. 10.
In situ hybridization analysis of GlcAT-S mRNA in mouse kidney. GlcAT-P and GlcAT-S mRNAs were detected with 35S-radiolabeled GlcAT-S and GlcAT-P RNA probes in 14-µm kidney sections prepared from 4-week-old C57BL/6 mice. A, B, and D, bright-field images of macroautoradiography of in situ hybridization with the GlcAT-S antisense, GlcAT-S sense, and GlcAT-P antisense RNA probes, respectively. C, dark-field image of microautoradiography of in situ hybridization with the GlcAT-S antisense RNA probe. Scale bar = 200 µm.

 
Meprin {alpha} and CD13/aminopeptidase N are expressed on the apical membranes of the proximal tubules in the kidney cortex, but not in the inner medulla (33, 35). We also confirmed that anti-CD13/aminopeptidase N antibody immunoreactivity is not detected in the inner medulla (data not shown). Therefore, the immunoreactivity of antibody M6749 in the inner medulla is not due to the non-sulfated HNK-1 carbohydrate expressed on meprin {alpha} and CD13/aminopeptidase N, but might be due to that expressed on minor glycoproteins (~90 and 180 kDa), which were detected upon Western blot analysis, as shown in Fig. 5A. Another possibility is that the non-sulfated HNK-1 carbohydrate is expressed on glycolipids.

The characteristic structural feature of the HNK-1 carbohydrate is sulfated glucuronic acid (4, 5). The important functional roles of the sulfate residue have been reported. Thus, the HNK-1 carbohydrate binds to laminin, but this binding is completely abolished upon desulfation of this carbohydrate (38, 39). HNK-1 sulfotransferase gene-deficient mice exhibit similar phenotypes compared with GlcAT-P gene-deficient mice, including a reduction in long-term potentiation and a defect in spatial learning (12, 40). Recently, interesting evidence has been reported concerning the functional role of glucuronic acid in the HNK-1 carbohydrate. Senn et al. (40) confirmed that the non-sulfated HNK-1 carbohydrate is expressed in HNK-1 sulfotransferase gene-deficient mice brain using two different monoclonal antibodies (anti-HNK-1 and 412). mAb 412 recognizes both non-sulfated and sulfated HNK-1 carbohydrates, as does antibody M6749. They analyzed electrophysiologically perisomatic inhibitory postsynaptic currents in hippocampal slices. Whereas application of anti-HNK-1 antibody to hippocampal slices from HNK-1 sulfotransferase gene-deficient mice did not change the perisomatic inhibitory postsynaptic currents, administration of antibody 412 decreased the perisomatic inhibitory postsynaptic currents to the same extent as did anti-HNK-1 antibody and mAb 412 in those from wild-type mice (40). The reduction of the perisomatic inhibitory postsynaptic currents after application of antibody 412 in the HNK-1 sulfotransferase gene-deficient mice indicates that the absence of sulfation does not completely abolish HNK-1 carbohydrate-induced modulation of perisomatic transmission. These experimental results indicate that the glucuronic acid residue, like the sulfate group, is an important functional constituent of the HNK-1 carbohydrate. These results suggest that the non-sulfated HNK-1 carbohydrate (glucuronic acid residue) expressed on the meprin {alpha}-subunit and CD13/aminopeptidase N also plays important roles in the kidney. We are now trying to produce GlcAT-S gene-deficient mice to demonstrate and investigate the functional role of the glucuronic acid residue of the HNK-1 carbohydrate, especially in the kidney.


   FOOTNOTES
 
* This work was supported in part by Grant-in-aid for Creative Scientific Research 16GS0313 (to S. O.) and Grant-in-aid for Scientific Research on Priority Areas A-14082203 (to T. K.) from the Ministry of Education, Culture, Sports, and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

The on-line version of this article (available at http://www.jbc.org) contains Supplemental Fig. 1. Back

{ddagger}{ddagger} To whom correspondence should be addressed: Dept. of Biological Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Shimoadachi-cho, Sankyo-ku, Kyoto 606-8501, Japan. Tel.: 81-75-753-4562; Fax: 81-75-753-4605; E-mail: shogo{at}pharm.kyoto-u.ac.jp.

1 The abbreviations used are: GlcA, glucuronic acid; GlcAT, glucuronyltransferase; mAb, monoclonal antibody; AQP, aquaporin; pAb, polyclonal antibody; TRITC, tetramethylrhodamine isothiocyanate; PBS, phosphate-buffered saline; LC, liquid chromatography; MS/MS, tandem mass spectrometry; MSn, multistage tandem mass spectrometry. Back



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TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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Glycobiology, February 1, 2008; 18(2): 152 - 157.
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Y. Kizuka, T. Matsui, H. Takematsu, Y. Kozutsumi, T. Kawasaki, and S. Oka
Physical and Functional Association of Glucuronyltransferases and Sulfotransferase Involved in HNK-1 Biosynthesis
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Sulfated HNK-1 Epitope in Developing and Mature Kidney: A New Marker for Thin Ascending Loop of Henle and Tubular Injury in Acute Tubular Necrosis
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