Structural Characterization of N-Glycans of Cauxin

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Thus the separated glycopeptide ... glycopeptide, a protonated doubly charged molecular related ion ..... rides using a two-dimensional mapping technique. Anal ...
Biosci. Biotechnol. Biochem., 71 (3), 811–816, 2007

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Structural Characterization of N-Glycans of Cauxin by MALDI-TOF Mass Spectrometry and Nano LC-ESI-Mass Spectrometry Yusuke SUZUKI,1; * Masao M IYAZAKI,1; * Emi I TO,2 Minoru S UZUKI,1;2 Tetsuro Y AMASHITA,3 Hideharu T AIRA,3; y and Akemi S UZUKI1 1

Sphingolipid Expression Laboratory, Supra-Biomolecular System Research Group, RIKEN Frontier Research System, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan 2 Applied Mass Spectrometry Laboratory, Center for Intellectural Property Strategies, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan 3 Department of Agro-Bioscience, Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka 020-8550, Japan Received October 26, 2006; Accepted December 6, 2006; Online Publication, March 7, 2007 [doi:10.1271/bbb.60599]

Cauxin is a carboxylesterase-like glycoprotein excreted as a major component of cat urine. Cauxin contains four putative N-glycosylation sites. We characterized the structure of an N-linked oligosaccharide of cauxin using nano liquid chromatography (LC)-electrospray ionization (ESI) and matrix-assisted laser desorption/ ionization quadrupole ion trap time-of-flight mass spectrometry (MALDI-QIT-TOF MS) and MS/MS, and high-performance liquid chromatography (HPLC) with an octadecylsilica (ODS) column. The structure of the N-linked oligosaccharide of cauxin attached to 83 Asn was a bisecting complex type, Gal 1-4GlcNAc 12Man 1-3(Gal 1-4GlcNAc 1-2Man 1-6)(GlcNAc 1-4)Man 1-4GlcNAc 1-4(Fuc 1-6)GlcNAc. Key words:

cauxin; glycoproteins; N-glycan; mass spectrometry

Cauxin, a member of the mammalian carboxylesterases, is a glycoprotein excreted as a major component of the urine of domestic cats (Felis catus).1) Cauxin excretion is species-, sex-, and age-dependent.2) A large amount of cauxin is excreted only in closely related members of the Felidae lineage, including the domestic cat, bobcat (Lynx rufus), and Siberian lynx (Lynx lynx). Although the cauxin gene is conserved in several mammalian species, including human, mouse, rat, dog, and cattle, these species do not excrete cauxin. In cats at least six months of age, cauxin excretion is significantly higher in intact males than in castrated males or mature females. The excretion of cauxin in immature cats begins about three months after birth and increases with *

age. Recently, we found that the biological role of cauxin is to regulate the production of 2-amino-7hydroxy-5,5-dimethyl-4-thiaheptanoic acid, termed felinine, in a species-, age-, and sex-dependent manner.3) Felinine is a putative precursor of pheromones used for conspecific recognition and reproductive status by mature cats.4) Cauxin hydrolyzes the peptide bond of the felinine precursor 3-methylbutanol cysteinylglycine, producing felinine and glycine in cat urine. Cauxin is expressed in a tissue-specific manner in the kidney proximal straight tubular cells and is secreted from the apical compartment of the cells into the urine.1,2) The high level of cauxin excretion in male cats (50–100 mg/d) suggests that cauxin is efficiently assembled in and secreted from these cells. Asparagine (N)-linked oligosaccharides have a crucial quality-control mechanism in protein production. Multiple roles have been assigned to N-linked oligosaccharides, including folding of proteins in the endoplasmic reticulum (ER), protection from degradation due to proteolysis, intracellular trafficking, secretion, maintenance of protein conformation, and modulation of biological activities.5–7) The amino acid sequence of cauxin contains four putative N-glycosylation sites of the sequence motif Asn-X-Ser/Thr. It is possible that the N-glycans of cauxin are involved in the efficient assembly and apical sorting of cauxin in the cells. In this paper, we report the first structural analysis, to our knowledge, of the N-glycans of cauxin using mass spectrometric techniques. Cauxin was purified from cat urine as described previously.1) All solvents used for matrix-assisted laser

These authors contributed equally to this work. To whom correspondence should be addressed. Tel: +81-19-621-6154; Fax: +81-19-621-6157; E-mail: [email protected] Abbreviations: MALDI, matrix-assisted laser desorption/ionization; TOF, time-of-flight; ESI, electrospray ionization; HPLC, high-performance liquid chromatography; MS, mass spectrometry; QIT, quadrupole ion trap y

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desorption/ionization time-of-flight (MALDI-TOF), MALDI-quadrupole ion trap (QIT)-TOF mass spectrometry (MS), and nano liquid chromatography-electrospray ionization (nano LC-ESI) MS were of HPLC grade. MALDI-TOF and MALDI-QIT-TOF MS analyses of cauxin were performed using AXIMA-CFR and AXIMA-QIT mass spectrometers (both from Shimadzu Corporation, Kyoto, Japan) in positive ion mode.8–11) Ions formed by a pulsed nitrogen UV laser ( ¼ 337 nm) were accelerated to a kinetic energy of 20 kV. 2,5Dihydroxybenzoic acid (DHB, Wako Pure Chemicals Industries, Osaka, Japan) and were used as the matrix at a concentration of 10 mg/ml in 10% ethanol (aqueous). A cauxin, 250 pmol in 2.5 ml, was digested with peptide-N4-(N-acetyl--glucosaminyl)-asparagine amidase (Glycopeptidase F, Takara Bio, Otsu, Japan) according to the manufacturer’s protocol. Pre- and post-digestion solutions of cauxin (1 ml) were mixed with 1 ml of the matrix solution, and the resulting mixtures were placed onto a MALDI target. The solvent was removed with a gentle stream of air, and the solid sample/matrix mixture was then analyzed by MALDI-TOF MS. Trypsin (Sigma-Aldrich Japan, Tokyo) digestion of cauxin (1.1 nmol) was carried out at 37  C for 16 h in 100 ml of Tris-buffered saline (100 mM Tris–HCl, pH 8.5) at an enzyme/protein ratio of 1:35 (wt/wt). The tryptic digests of cauxin (100 pmol) were separated by nano liquid chromatography (LC) with a KYA Model DiNa-M nano high-performance liquid chromatograph (HPLC) (KYA Technologies, Tokyo, Japan) equipped with a reversed-phase octadecylsilica (ODS) column (0.3 mm i.d.  30 mm) and a trapping ODS column (0.3 mm i.d.  30 mm). The separations were performed on a programmed gradient from 3% to 80% CH3 CN in water containing 0.1% formic acid for 60 min at a flow rate of 200 nl/min and a column temperature of 40  C. Nano-LC electron spray ionization (ESI)-MS and MS/ MS were performed using a quadrupole time-of-flight mass spectrometer (QSTAR Pulsar i, MDS Sciex, Toronto, Canada) equipped with an electrospray ion source at 2,000 V. MS/MS measurements were carried out by setting the collision energy to 50 V.12,13) The tryptic digests of cauxin (1 nmol) were separated by reversed phase HPLC with a C30 column (2.0 mm i.d.  250 mm, Nomura Chemical, Aichi, Japan). The separation was performed at a flow rate of 1.2 ml/min at 40  C by a programmed gradient system consisting of 0.1% trifluoroacetic acid (TFA) in water (solvent A) and acetonitrile (solvent B), and the gradient: 5% B for 5 min, 5 to 50% B for 30 min, 50 to 100% B for 10 min, and 100% B for 45 min. Thus the separated glycopeptide was recovered and digested (10 pmol) with glycopeptidase F and the released oligosaccharides were pyridylaminated (PA), as described previously.14) The PAoligosaccharides were applied to a Sepharose CL4B column (20 ml packed volume) in 20 ml of an organic solvent of 1-butanol/ethanol/H2 O (4:1:1, v/v), and eluted with 100 ml of ethanol/H2 O (1:1, v/v) after the

column was washed twice with 50 ml of the organic solvent. The eluted PA-oligosaccharides were dried with a Speedvac. Standard PA-oligosaccharides were purchased from Glyence (Nagoya, Japan). The PA-oligosaccharides were separated by reversed phase HPLC with an ODS-silica column (4.6 mm i.d.  250 mm, Takara Bio). The PA-oligosaccharides were detected at an excitation wavelength of 320 nm and an emission wavelength of 400 nm. Separation was performed at a flow rate of 1.0 ml/min at 50  C using a gradient elution system with solvent A (10 mM sodium phosphate buffer, pH 3.8), solvent B (solvent A containing 0.5% 1-butanol), and a linear gradient: from 20% to 50% B for 60 min and 50% B for 25 min.15,16) The isolated PAoligosaccharides were desalted by Sepharose CL4B, as described above, and analyzed by MALDI-QIT-TOF MS and MS/MS. The molecular mass of cauxin, as determined by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS–PAGE), was 70 kDa, and that of deglycosylated cauxin resulting from treatment with glycopeptidase F was 60 kDa.1) In the MALDI mass spectrum of cauxin, a molecular associated ion was observed at m=z 67,614. After digestion with glycopeptidase F, the molecular related ion of deglycosylated cauxin was detected at m=z 57,981 (see Supplemental Fig. 1 online). These results show that cauxin is a glycoprotein, and that the total molecular mass of the glycan moieties of cauxin is about 9.6 kDa. The glycopeptides derived from trypsinized cauxin were characterized using nano LC-ESI-MS and MS/MS. Four peaks were detected by monitoring the fragment of m=z 204 (N-acetylhexosamine) (Fig. 1A). The mass spectra of these peaks showed that one peak migrating at 4.1 min was a glycopeptide. In the mass spectrum of this glycopeptide, a protonated doubly charged molecular related ion (½M þ 2H2þ ) was observed with strong intensity at m=z 1,376.5 (Fig. 1B), indicating that the molecular weight of the glycopeptide was 2,751. A number of peaks detected in the MS spectrum might be glycopeptides due to the heterogeneity of oligosaccharides, but these peaks were very minor, and further structural analysis was not possible. To obtain further information on the structure of the glycopeptide, MS/MS analysis was carried out by selecting the protonated molecular ion at 1,376.5 as the precursor ion. In the MS/MS spectra, the ion formed by elimination of one hexose (Hex) was detected at m=z 1,713.6, two Hex at m=z 1,551.6, one Hex and one Nacetylhexosamine (HexNAc) at m=z 1,510.6, two Hex and one HexNAc at m=z 1,348.5, three Hex and one HexNAc at m=z 1,186.5, three Hex and two HexNAc at m=z 983.5, and another due to the elimination of three Hex and three HexNAc at m=z 780.4 (Fig. 1C). The ion at m=z 780.4 is probably that of the core peptide produced by the elimination of glycan moieties from the glycopeptide. The determined molecular weight of the peptide corresponds to the peptide Asn-

Structural Characterization of N-Glycans of Cauxin by Mass Spectrometry

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-Hex

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-HexNAc

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Fig. 1. Nano HPLC-ESI-MS of Tryptic Digests of Cauxin. A, MS/MS chromatogram of tryptic digests of cauxin monitoring m=z 204, which is generated by the elimination of N-acetylhexosamine. B, Mass spectrum of the glycopeptide indicated with an asterisk in A. C, MS/MS spectrum selecting m=z 1,376.5 as a precursor ion.

A

Potential N-glycosylation site

Asn83 Asn131

Asn360

Asn440 545

83

NATSYPK 131

LEASEDCLYLNIYAPAHADNGSNLPVMVWFPGGAFK 360

AFNSVPSIIGVNNHECAFLLSTEFSEILGGSNR 440

YHRDAGAPVYFYEFQHPPQCLNDTRPAFVK

B Galβ1-4GlcNAcβ1-2Manα1

Fucα1

6 6 GlcNAcβ1-4Manβ1-4GlcNAcβ1-4GlcNAc 3 Galβ1-4GlcNAcβ1-2Manα1

Fig. 2. Possible Glycan Attachment Sites of Cauxin (A), and the Proposed Structure of Cauxin Glycan Attached to

83

Asn (B).

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Fig. 3. MALDI-QIT-TOF MS and MS/MS Spectra of Standard and Cauxin PA-Oligosaccharides. A, MALDI-QIT-TOF MS spectrum of the standard PA-oligosaccharide, Gal1-4GlcNAc1-2Man1-3(Gal1-4GlcNAc1-2Man1-6)(GlcNAc1-4)Man1-4GlcNAc1-4(Fuc1-6)GlcNAc-PA, and B, cauxin PA-oligosaccharide. C, MS/MS spectrum selecting m=z 2,090.90 as a precursor ion in A, and that D, selecting m=z 2,090.84 as a precursor ion in B.

Ala-Thr-Ser-Tyr-Pro-Lys (MW 780, Fig. 2A). The m=z difference between the glycopeptide (m=z 1,376.5, doubly charged) and the peptide (m=z 780.6) is equal to the molecular weight of the oligosaccharide, and hence the oligosaccharide of cauxin was identified as Hex5 HexNAc5 deoxyHex1 . Furthermore, the oligosaccharide structure was recognized to be bisected on the basis of detection of a bisected N-glycan diagnostic ion at m=z 1,551.6 (Hex1 HexNAc3 -peptide) in the MS/MS spectrum.17,18) The oligosaccharide database GALAXY15,16) was used to search the oligosaccharide structure attached at 83 Asn in the peptide, and one

candidate structure was selected, as shown in Fig. 2B. In order to confirm the oligosaccharide structure, we purified the glycopeptide by reversed phase HPLC with a C30 column, and the separated glycopeptide was confirmed by MALDI-TOF MS (data not shown). Then the separated glycopeptide (10 pmol) was digested with Glycopeptidase F, and the released oligosaccharides were pyridylaminated and analyzed by HPLC an ODS column. The cauxin PA-oligosaccharide was eluted at 64.6 min. It exhibited 20.1 glucose units, indicating that cauxin PA-oligosaccharide is identical to the standard PA-oligosaccharide, Gal1-4GlcNAc1-

Structural Characterization of N-Glycans of Cauxin by Mass Spectrometry

2Man1-3(Gal1-4GlcNAc1-2Man1-6)(GlcNAc1-4)Man1-4GlcNAc1-4(Fuc1-6)GlcNAc-PA (see Supplemental Fig. 2 online). In addition, the peak of cauxin PA-oligosaccharide (63–65 min) was recovered and analyzed by MALDI-QIT-TOF MS and MS/MS. The MS spectra of standard (Fig. 3A) and cauxin PAoligosaccharides (Fig. 3B) showed peaks at m=z 2,090.90 and 2,090.84 respectively. The MS/MS spectra of these two PA-oligosaccharides (Fig. 3C and D) provided the same fragment ions. These results together indicate that the oligosaccharide structure of cauxin is characterized as the bisecting complex type, as indicated in Fig. 2B. Our previous studies indicate that cauxin is secreted from the apical compartment of proximal straight tubular cells into the urine.1,2) It has been reported that the apical secretion of secretory proteins is mediated by N-linked oligosaccharides.19,20) N-glycans containing bisecting N-acetylglucosamine residue (Fig. 2B) have been reported in aminopeptidase N and dipeptidylpeptidase IV isolated from the brush-border membrane of rat proximal tubular cells21) and in megalin (gp330) from microsomes of the rat renal cortex.22) It has been suggested that the bisecting N-acetylglucosamine residue is involved in the apical sorting of proteins expressed in the proximal tubular cells. This study indicates that cauxin contains an N-linked oligosaccharide structure of the bisecting complex type. Based on this result, we suggest that the bisecting N-acetylglucosamine residue is a candidate targeting signal for apical transport of cauxin. We are now examining whether the bisecting N-acetylglucosamine residue is involved in apical secretion of cauxin. Our preliminary data show that cauxin expressed in COS-7 and MDCK cells accumulated in the Golgi complex and was not secreted from the cells. Further studies, such as coexpression of cauxin and N-acetylglucosaminyltransferase III that adds the bisecting N-acetylglucosamine residue to proteins in the cells, are warranted to elucidate the biological functions of N-glycans and the secretion mechanism of cauxin from cells. In this study, the structure of N-linked oligosaccharide and the glycosylation site of cauxin were characterized by nano LC–MS and MS/MS analyses of tryptic digests of cauxin, and the oligosaccharide structure was confirmed by the retention time of PA-oligosaccharide released from the glycopeptide of cauxin by HPLC with an ODS column, and by MALDI-QIT-TOF MS and MS/MS. There are four possible oligosaccharide attachment sites in the cauxin structure (Fig. 2A). We characterized one of the four glycopeptide structures. The other three have much larger molecular masses than the one that was characterized. The analysis of these three glycopeptides will require another peptidase digestion, such as endoprotease Glu-C, after triptic digestion to convert them to shortened peptide chains suitable for mass spectrometry.

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Acknowledgments This work was supported in part by the Biotechnology Foundation Research Program for Health Maintenance and Improvement in Japan; by the RR project of the Ministry of Education, Culture, Sports, Science and Technology of Japan; by the Core Research for Evolutional Science and Technology (CREST) program; and by the program for the Development of Systems and Technology for Advanced Measurement and Analysis of the Japan Science and Technology Agency (JST).

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