The Lecticans of Mammalian Brain Perineural Net Are O-Mannosylated

Article pubs.acs.org/jpr

The Lecticans of Mammalian Brain Perineural Net Are O‑Mannosylated Sandra Pacharra,† Franz-Georg Hanisch,†,‡ Martina Mühlenhoff,§ Andreas Faissner,∥ Uwe Rauch,⊥ and Isabelle Breloy*,† †

Institute of Biochemistry II, Medical Faculty, University of Cologne, Köln, Germany Center for Molecular Medicine Cologne, University of Cologne, Köln, Germany § Institute of Cellular Chemistry, Hannover Medical School, Germany ∥ Department for Cell Morphology and Molecular Neurobiology, Ruhr-University Bochum, Bochum, Germany ⊥ Department of Experimental Medical Science, Biomedical Center B12, Lund University, Lund, Sweden ‡

S Supporting Information *

ABSTRACT: O-Mannosylation is an important protein modification in brain. During the last years, a few mammalian proteins have been identified as targets of the protein-Omannosyltransferases 1 and 2. However, these still cannot explain the high content of O-mannosyl glycans in brain and the strong brain involvement of congenital muscular dystrophies caused by POMT mutations (Walker-Warburg syndrome, dystroglycanopathies). By fractionating and analyzing the glycoproteome of mouse and calf brain lysates, we could show that proteins of the perineural net, the lecticans, are O-mannosylated, indicating that major components of neuronal extracellular matrix are O-mannosylated in mammalian brain. This finding corresponds with the high content of O-mannosyl glycans in brain as well as with the brain involvement of dystroglycanopathies. In contrast, the lectican neurocan is not O-mannosylated when recombinantly expressed in EBNA-293 cells, revealing the possibility of different control mechanisms for the initiation of O-mannosylation in different cell types. KEYWORDS: O-glycans, O-mannosylation, lecticans, ECM, perineural net, O-glycosylation, ESI-MS/MS, MALDI-MS/MS, glycoproteomics, dystroglycanopathies

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2) by immunohistochemistry and Western blotting.8,9 These two proteins have been shown to be modified with an Omannose linked HNK1-epitope which has been described before.1 Phosphacan, the secreted splice variant of RPTPζ, shares several characteristics with the lecticans, a group of secreted chondroitin sulfate proteoglycans. They both, phosphacan and lecticans interact with several ECM proteins (tenascin-C, tenascin-R, and heparin binding proteins)10−12 and cell adhesion molecules like NCAM and NCAM L113,14 and have been shown to inhibit cell adhesion and neurite outgrowth.15 The lecticans are composed of aggrecan, versican, neurocan and brevican which have all been identified in brain. While versican is expressed in a variety of tissues, aggrecan occurs mainly in cartilage and neurocan and brevican are specific for neuronal tissue.16 All four proteins interact strongly with hyaluronan and several proteins of the extracellular matrix forming the perineuronal net, which provides structural stability

INTRODUCTION Protein-O-mannosylation was regarded as a modification specific for yeast and fungi proteins until it was shown in 1999 that about 30% of all O-glycans derived from Pronasestable glycopeptides prepared from rabbit brain glycoproteins are O-mannosyl glycans1 and that nonfunctional protein-Omannosyltransferases are the cause of congenital muscular dystrophies with severe brain malformations. α-α-Dystroglycan was discovered as the first O-mannosylated protein in mammals.2,3 During the last 5 years, few other mammalian glycoproteins have been identified as O-mannosylated, namely the transmembrane proteins CD244 and a human monoclonal IgG2 light chain expressed in CHO cells.5 Tenascin-R has been reported to be O-mannosylated in 19926 but was not analyzed further in this context. Although O-mannosyl glycans have originally been found on unidentified proteoglycans in brain,7 only two of the proteins which have been identified as O-mannosylated belong to the proteoglycans. Receptor protein tyrosine phosphatase RPTPβ and RPTPζ/phosphacan have been reported to be substrates for the protein-O-mannosyltransferases -1 and -2 (POMT1 and © 2013 American Chemical Society

Received: November 23, 2012 Published: February 21, 2013 1764

dx.doi.org/10.1021/pr3011028 | J. Proteome Res. 2013, 12, 1764−1771

Journal of Proteome Research

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through was reapplied several times. After washing with HA buffer and HA buffer with 1 M NaCl, bound proteins were eluted by the addition of HA buffer with 4 M guanidine HCl.

of the brain tissue, like collagens in the extracellular matrix of most other tissues. Although the expression of the lecticans in brain varies with their different splice forms, it can be stated that neurocan and versican are mainly expressed during early brain development and in later stages partly replaced by brevican and aggrecan.17 Like the O-mannosylated protein αdystroglycan, the lecticans are composed of an N-terminal and a C-terminal globular domain and a stretched, highly posttranslationally modified central domain. All lecticans can be modified with a variable number of chondroitin sulfate chains or with common N-glycan and mucin-type Oglycans.18−20 Keratin sulfate was described additionally on aggrecan and neurocan.18,21 Neurocan has also been shown to be modified with the HNK1-epitope22 which has been described as the terminal glycan epitope on the O-mannosyl glycans on RPTPβ and RPTPζ/phosphacan.8,9 In our search for other O-mannosylated proteins, we analyzed several high-mass protein fractions with a high content of O-mannosyl glycans23 and were able to identify neurocan, brevican or versican in some of these fractions. Therefore, we analyzed the lecticans further regarding their Oglycans and could show that the lecticans are indeed Omannosylated and that these proteins are the previously postulated O-mannosylated proteoglycans highly and predominantly expressed in mammalian brain.

Purification of TenascinR from Mouse Brain

TenascinR was purified by sequential immunoaffinity purification on a TenR monoclonal antibody column according to Czopa.25 Purification of NCAM1

NCAM was isolated from total brain of postnatal day 2−3 St8sia2−/− mice. Because of genetic ablation of the polysialyltransferase ST8SiaII, NCAM of these mice is only partially polysialylated.26 NCAM was immunoisolated essentially as described27 but using the anti-NCAM monoclonal antibody H2828 coupled to protein G sepharose as affinity column. Purification of Lecticans from Calf Brain

Brain Lysis. Calf brains were homogenized on ice in a glass tissue grinder in solubilization buffer (10 mM TrisHCl pH 7.5, 150 mM NaCl, 0.5% Chaps, protease inhibitor cocktail (cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail Tablets, Roche)). Crude lysate was sonicated on ice followed by centrifugation at 3900g for 30 min and ultracentrifugation of the supernatant at 28000g. The pellet was discarded and the supernatant was filtrated. WGA Lectin Chromatography. The supernatant was conveyed to an equilibrated WGA-agarose (Vector Laboratories) column in a circular pump driven system. On the next day, the column was washed with solubilization buffer and with washing buffer (10 mM TrisHCl pH 7.5, 0.5 M NaCl, 0.5% CHAPS) and WGA-bound proteins (mouse brain glycoproteins) were eluted from the lectin column with elution buffer 1 (10 mM TrisHCl pH 7.5, 150 mM NaCl, 0.5% CHAPS, 0.2 M GlcNAc) and elution buffer 2 (10 mM TrisHCl pH 3, 150 mM NaCl, 0.5% CHAPS, 0.2 M GlcNAc). Eluates were combined and the protein concentration was determined using the DC Protein Assay. An alternative elution was used prior to isoelectric focusing: WGA-bound glycoproteins were eluted with IEF solution (2% Chaps, 2 M Thiourea and 7 M Urea) containing 0.2 M GlcNAc. Isoelectric Focusing. Approximately 2 mg of glycoproteins was subjected to a preparative gel-free isoelectric focusing on the MicroRotofor Liquid-Phase IEF Cell (Bio-Rad) according to the manufacturer’s instructions in 3% BioLyte 3/10 (BioRad), 2% Chaps, 2 M thiourea and 7 M urea. Preparative SDS-PAGE. Glycoproteins were fractionated using preparative gel electrophoresis. Up to 5 mg of protein was mixed in equal volumes with sample buffer (100 mM TrisHCl pH 6.8, 45% (v/v) glycerin, 10% (v/v) mercaptoethanol, 1% (w/v) SDS, 0.05% (w/v) bromphenol blue), subjected to a gel column with a 3.5% stacking gel and a 5% separation gel (BioRad Miniprep Cell) and separated according to manufacturer’s protocol. Eluted proteins were fractionated into 30 fractions of 2.5 mL volume and concentrated using Amicon Ultra-15 Centrifugal Filter Units (Millipore). Salts and detergents were removed by several washes with 40% methanol. Digestion with Chondroitinase ABC. For chondroitinase ABC (Chase) digestion of glycosaminoglycan chains present on neurocan, proteins from HA-eluate were precipitated by methanol/chloroform precipitation, resuspended in Chase buffer (0.1 M TrisHCl pH 7.5, 30 mM NaAc, 2 mM EDTA) and incubated with Chase ABC (Sigma) at 37 °C overnight.

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MATERIALS AND METHODS All chemicals were purchased from Sigma-Aldrich and were of analytical grade. Exceptions are marked in the text. Proteins were handled at +4 °C, long time storage was at −20 °C. Cells and Cell Culture

Cell culture work was performed with media and plasticware from Biochrom (Berlin, Germany). The human embryonic kidney cell line EBNA-293 (Invitrogen, Karlsruhe, Germany) was cultivated at 37 °C and 7.5% CO2 in Dulbecco’s minimal essential medium (DMEM), supplemented with 5% fetal calf serum, 100 U/mL penicillin and 100 μg/mL streptomycin. Transfected cells were cultivated under selection pressure by adding 5 μg/mL puromycin (Sigma, Munich, Germany) to the media. Recombinant Expression of Neurocan in EBNA-293 Cells

A detailed description of the expression construct can be found elsewhere.24 Secreted full length neurocan from rat was expressed in EBNA-293 cells and enriched in serum-free cell culture supernatant by ultracentrifugation and subsequently purified by hyaluronic acid chromatography. Hyaluronic Acid Affinity Chromatography

EAH-sepharose 4B (GE Healthcare) was equilibrated in ddH2O pH 4.5 (with HCl) and hyaluronic acid (Sigma, sodium salt from Streptococcus equi) dissolved in ddH2O pH 4.5 was added. The coupling reaction was started by the addition of solid N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide (EDC) and incubation at room temperature (RT) for 2 h under regular pH adjustment. After incubation at 4 °C overnight, the reaction was stopped by the addition of 1 M acetic acid and incubation at RT for 6 h. The HA-sepharose was extensively washed with Na acetate buffer and TrisHCl buffer and equilibrated with HA buffer (20 mM TrisHCl pH 8.0, 0.5 M NaCl, 10 mM EDTA, 0.25% CHAPS). A 3-mL Mobicol column was packed with 600 μL of a 50% HA-sepharose slurry and washed with HA buffer. Proteins in HA buffer were applied to the column and the flow 1765



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Gel Electrophoresis and Western Blotting

exclude isobaric species. Keratins and trypsin are typical contaminants and are not assigned. Oligosaccharide Analysis. Glycan chains were released from the protein by reductive β-elimination. For this purpose, the glycoproteins were incubated with 0.5 M NaBH4 in 50 mM NaOH for 18 h at 50 °C. The reaction was stopped by adding 1 μL of acetic acid. Salt was removed with 50 μL of Dowex 50WX8 (Bio-Rad) in a batch procedure. Excessive borate was evaporated in a stream of nitrogen by adding several 0.1 mL aliquots of 1% acetic acid in methanol. Permethylation of the glycan chains, released by reductive β-elimination, was performed as described.29 Glycan Analysis by MALDI-TOF/TOF Mass Spectrometry. Mass spectrometric data were acquired on a MALDITOF/TOF (UltrafleXtreme, Bruker) with a 1-kHz SmartbeamII laser and positive ion detection on a FlashDetector combined with a 4 GHz digitizer in the reflection mode. As matrix, αcyano-4-hydroxycinnamic acid (Bruker) was used (40 mg/mL in acetonitrile/H2O 2:1) on a MTP 384 stainless steel T F target (Bruker). Spectra were analyzed with the Flex Analysis software (version 3.3). The precursor mass of all glycan alditol survey mass spectra was identical to the theoretical mass/ charge with an accuracy of one digit after the decimal point if not stated otherwise.

SDS-PAGE on a discontinuous gel (3% stacking gel and 10% or 3.5% to 10% gradient running gel) was done on a Mini-Protean II-gelelectrophoreses system (Bio-Rad, Munich, Germany). The separated proteins were either stained with coomassie or silver or blotted onto a nitrocellulose membrane (Protan BA 83, Schleicher & Schuell, Einbeck, Germany) in a semi-dry transfer cell (Trans-Blot SD, Bio-Rad) or a tank blot cell (BioRad) according to the manufacturer’s protocol. Neurocan was detected with an anti-neurocan rabbit antibody (Sigma) and peroxidase conjugated swine anti-rabbit IgG-HRP (DAKO). Protein-antibody conjugates were detected by enhanced chemiluminescence (Roche, Grenzach-Wyhlen, Germany). Protein Identification

Proteins were dissolved in 20 μL of 8 M urea in 100 mM TrisHCl (pH 8.0) and reduced by adding 10 μL of 20 mM dithiothreitol overnight at 4 °C. Alkylation was done for 30 min with 10 μL of 50 mM iodoacetamide in the dark at 20 °C. After adding 50 μL of H2O, proteins were digested with 1 μg of trypsin (sequencing grade, Promega) overnight at 37 °C. The digest was stopped by the addition of 90 μL of 10% formic acid (FA). Peptides were extracted by C18 Zip Tip and dried in a Speed Vac. Liquid chromatography (LC)−MS data were acquired in positive mode on a HCT ETD II ion trap mass spectrometer (Bruker Daltoniks, Bremen, Germany) equipped with a nano ESI source (Bruker Daltonics, Bremen, Germany) operated at 4200 V with nebulizer gas (N2) pressure set to 8 psi and a dry gas (N2 at 250 °C) flow of 4 L/min. Samples in 10% FA were introduced by an easy nano LC system (Proxeon, Odense, Dennmark) using a vented column setup comprising a 0.1-mmby-20-mm trapping column and a 0.075-by-150-mm analytical column, both self-packed with ReproSil-Pur C18-AQ, 5 μm (Dr. Maisch, Ammerbuch, Germany). Five micriliters to 18 μL sample was aspirated into the sample loop and a total of 25 μL was loaded onto the trap column using a flow rate of 6 μL/min. Loading pump buffer was 0.1% FA. Peptides were eluted with a gradient of 0−35% acetonitrile (ACN) in 0.1% FA over 20 min at 300 nL/min column flow rate. Subsequently, the ACN content was raised to 100% over 2 min and the column was regenerated in 100% ACN for additional 8 min. SPS parameter settings on the ion trap were tuned for a target mass of 700 m/ z, compound stability 100% and a Trap Drive Level of 100. Ions were scanned with 8100 amu/s in a range from m/z 300 to 2500 in MS mode and m/z 200 to 3000 in MS/MS mode. MS/ MS spectra were generated by CID fragmentation. Spectra were acquired with the Compass 1.3 for esquire/HCT software. Proteins were identified by using a local installation of MASCOT 2.2 (Matrix Science Ltd., London, U.K.), bovine proteins were searched in NCBI (National Center for Biotechnology Information, release 20090531) (298 531 entries bovine; 56 329 318 entries total) and Swiss-Prot (version 2012_05-09) (94 602 entries bovine; 26 617 536 entries total) databases. For mouse proteins, the Swiss-Prot database was used (160 918 entries mouse). Searches were submitted via Proteinscape 2.1 (Bruker Daltoniks, Bremen, Germany) with the following parameter settings: enzyme “trypsin”, species “human”, fixed modifications “carbamidomethyl”, optional modifications “Methionine oxidation” and missed cleavages “1”. The mass tolerance was set to 0.4 Da for peptide and fragment spectra. Proteins with a score above 90 and with more than one identified peptide were taken into consideration to

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RESULTS AND DISCUSSION

Novel O-Mannosylated Proteins in Brain Lysates

We have previously identified neurofascin as an O-mannosylated protein in mouse brain.31 In a global search for other Omannosylated proteins in this organ, we fractionated WGAenriched glycoproteins from mouse brain lysate by preparative 1D gel electrophoreses. We analyzed the O-glycan profile of several fractions containing proteins with an apparent molecular mass above 150 kDa. All glycans were detected and annotated as monoisotopic sodium adducts (Na+, M + 23 Da) by MALDI-MS/MS in positive ion mode. Oligosaccharide structures were always verified by MS/MS. The identity of the O-mannosyl structure was proofed by the observation of a signal at m/z 724 (HexHexNAcHex-ol + Na+) in the fragmentation pattern of the O-glycan signal at m/z 1099.6 (Supporting Information Figure 1) Some of these fractions contained highly O-mannosylated proteins with apparent masses that do not match the masses of known O-mannosylated proteins. O-Glycoprofiling of the highmass fractions revealed additionally to the typical O-mannosyl tetrasaccharide at m/z 1099.8 a branched structure at m/z 1910.3 (Figure 1 and Table 1). The ion at 1344.7 can be assigned to an O-man glycan as well as to a mucin-type Oglycan. A differentiation based on MS/MS-data can be done only on the basis of minor signals and therefor the identification of O-mannosylated proteins is based only on the appearance of signals at m/z 1099.6 or m/z 1910.0. The lecticans are expressed in high amounts in brain and contribute to the formation of the perineuronal net (PNN). An O-mannosylation of this group of highly abundant proteins would explain earlier results, showing that about 30% of all Oglycans in brain are O-mannosylated.1,23 To obtain more information about these proteins, we analyzed the eluate from a tenascin-R affinity purification. Tenascin-R is also strongly expressed in the PNN and was reported to be O-mannosylated earlier.6 As the lecticans bind firmly to tenascin-R, they coelute together with tenascin-R. O1766



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Figure 2. Analysis of a single fraction from preparative 1D gel electrophoreses of mouse brain glycoproteins. The MALDI-MS spectrum shows the permethylated glycan alditols, revealing high amounts of O-mannosyl glycans. Monoisotopic masses corresponding to O-mannosyl glycans are underlined. The left inset shows the silver stained SDS-PAGE of the fraction. The right inset shows the results of protein identification in the respective fraction by ESI-MS/MS. Glycoproteins are typed in bold.

Figure 1. MS/MS of the permethylated oligosaccharide with a precursor mass of 1910.3 Da, corresponding to the glycan composition Hex3HexNAc2NeuAc2 + Na+. The fragmentation pattern reveals a branched structure with a reduced core-hexose, indicating an Omannosylated glycan.

Table 1. Monoisotopic Masses of Permethylated O-Glycans Observed by MALDI-MS/MS and Their Corresponding Structures and Glycan Typesab m/z

composition

type

738.4

Hex2HexNAc-ol Na+

O-Man

912.5

FucHex2HexNAc-ol Na+

O-Man

1099.6

NeuAcHex2HexNAc-ol Na+

O-Man

1344.7

NeuAcHex2HexNAc2-ol Na+

O-Man

+

1361.7

dHexHex3HexNAc2-ol Na

1910.0

NeuAc2Hex3HexNAc2-ol Na+

O-Man O-Man

895.5 983.5 1069.6 1157.6 1256.6 1344.7 1518.7 1677.9 1705.9 1938.0

NeuAcHexHexNAc-ol Na+ Hex2HexNAc2-ol Na+ NeuAcdHexHexHexNAc-ol Na+ dHexHex2HexNAc2-ol Na+ NeuAc2HexHexNAc-ol Na+ NeuAcHex2HexNAc2-ol Na+ NeuAcdHexHex2HexNAc2-ol Na+ Hex3HexNAc4-ol Na+ NeuAc2Hex2HexNAc2-ol Na+ NeuAcFuc2Hex2HexNAc3-ol Na+

O-GalNAc O-GalNAc O-GalNAc O-GalNAc O-GalNAc O-GalNAc O-GalNAc O-GalNAc O-GalNAc O-GalNAc

Figure 3. O-Glycoprofiling of neurocan containing protein fraction. The MALDI mass spectrum of permethylated O-glycans from hyaluronan affinity-isolated mouse brain proteins is combined with the list of proteins identified by LC−MS/MS (inset). The only Oglycoprotein detected in this fraction was neurocan, which is characterized by a very high content of O-mannosyl glycans. Monoisotopic masses corresponding to O-mannosyl glycans are underlined. Glycan fragments without a reduced end are indicated with an asterisk; arrows indicate signals derived by a loss of a methyl group and sodium (−36 Da).

a

All glycans were detected as sodium adducts (Na+, M + 23 Da) by MALDI-MS/MS in positive ion mode. bWe identified the proteins of these highly O-mannosylated fractions by ESI-MS/MS and found the lecticans as candidates for novel O-mannosylated proteins in several distinct fractions (Figure 2, Supporting Information Figure 1 and Table 1).

We could confirm these results in another mammalian species. Calf brain lysates also showed O-mannosyl glycans in lectican-positive fractions. As some bovine proteins (e.g., neurocan) are not listed in the Swiss-Prot database, we additionally searched for bovine proteins in the NCBI database (see a detailed description in Materials and Methods).

Glycan analysis confirms that these proteins of the PNN are modified with O-mannosyl glycans (Supporting Information Figure 2 and Table 2). In an attempt to remove tenascin-R, we purified the hyaluronic acid-binding lecticans by hyaluronan affinity chromatography of mouse brain lysate. The eluate showed a very high content of O-mannosyl glycans and a protein composition containing neurocan as the only O-glycoprotein (Figure 3 and Supporting Information Table 3). This shows that neurocan from mouse brain is indeed O-mannosylated.

Enrichment of Lecticans from Calf Brain

We first enriched the glycoproteins by WGA-lectin chromatography. The efficiency of this enrichment was confirmed by Oglycan analysis of bound and unbound proteins by MALDIMS/MS (Supporting Information Figure 3) According to the 1767



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acidic nature of the attached glycosaminoglycan chains, we enriched the lecticans after WGA-lectin chromatography by preparative gel-free (or free-flow) isoelectric focusing. As expected, the majority of the glycosaminoglycans could be recovered in the first, most acidic fraction (IEF fraction 1). OGlycan analysis showed a very high content of O-mannosyl glycans in this fraction (Figure 4 and Supporting Information Table 4).

While neurocan, versican and brevican are the main components of this highly O-mannosylated fraction, there are still contaminating proteins, which have been reported to be Omannosylated (RPTPζ, 9; α-dystroglycan, 3, tenascin-R, 6), and glycoproteins, which have not yet been analyzed for potential O-mannosylation (Myelin-associated glycoprotein (MAG), oligodendrocyte-myelin glycoprotein and CD44). NCAM1 and MAG can be excluded from the list of possibly Omannosylated proteins. O-Glycan analysis of purified NCAM1 from mouse brain (postnatal day 2−3) revealed only core1 mucin-type O-glycans (Supporting Information Figure 4 and Table 5) and a MAG-containing fraction from calf brain lysate did not reveal the presence of O-mannosyl glycans (Supporting Information Figure 5 and Table 6). We fractionated the proteins from IEF fraction 1 further by preparative gel electrophoresis. To increase resolution, the proteins were digested with chondroitinase ABC before application (Figure 5). The neurocan containing fractions of the electrophoresis were identified by Western blotting, other proteins were located by their apparent mass on a silver stained SDS-gel. From all presumably lectican-positive fractions, the O-glycans were analyzed by MALDI-MS/MS and proteins were identified by LC-ESI-MS/MS. We could confirm the O-mannosylation of neurocan (Supporting Information Figure 6 and Table 7) in this species, showing that O-mannosylation of the lecticans is not restricted to mouse brain. We additionally tried to purify and analyze the remaining lecticans regarding O-mannosylation. Because of several splice variants, we were not able to separate brevican from versican, but we identified both in several O-mannosylated fractions

Figure 4. MALDI-MS of permethylated glycan alditols derived from preparative free-flow isoelectric focusing of calf brain proteins. Monoisotopic masses corresponding to O-mannosyl glycans are underlined. The left inset shows the silver stained SDS-PAGE of the respective fraction (IEF fraction 1) and the right inset lists results of protein identification by ESI-MS/MS.

Figure 5. (A) Silver stained SDS-PAGE of the preparative gel electrophoresis after isoelectric focusing. The boxed inset shows an anti-neurocan Western blot of the respective fractions from preparative gel electrophoresis identifying neurocan in fractions 1 and 2. (B) Western blot of the WGA eluate from calf brain lysate with anti-neurocan antibody before (-) and after (+) treatment with chondroitinase ABC. (C) Purification scheme for enrichment and analysis of the lecticans from calf brain. 1768



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(Supporting Information Figures 7 and 8 and Tables 8 and 9) revealing an O-mannosylation of at least one species. We identified versican undoubtedly as O-mannosylated (Figure 6

Figure 7. Analysis of recombinant neurocan. MALDI-MS of permethylated glycan alditols reveals only mucin-type O-glycans. The left inset shows a Western blot before and after chondroitinase digestion, which reveals a high degree of modification with chondroitin sulfate proteoglycans. The right inset refers to protein identification by LC−MS/MS, which demonstrated purity of the recombinant neurocan preparation. High signals derived from nonreduced Nglycan fragments (indicated with an asterisk) reveal a modification of the protein with N-glycans. Arrows indicate the loss of methyl and sodium. (Chase, chondroitinase ABC; GAG, glycosaminoglycan).

Figure 6. Analysis of a single, versican-containing fraction from preparative 1D gel electrophoresis of calf brain glycoproteins. The MALDI-MS spectrum shows the permethylated glycan alditols, revealing high amounts of O-mannosyl glycans. Monoisotopic masses corresponding to O-mannosyl glycans are underlined. The left inset shows the respective fraction on a silver-stained SDS gel. The right inset shows the results of protein identification in the respective fraction by ESI-MS/MS.

and Supporting Information Table 10) and we strongly assume the same modification for brevican. The fourth lectican, aggrecan, still has to be analyzed in this context, as this proteins is expressed only in later developmental stages and was not detected in our analysis. These results prove the O-mannosylation of mammalian lecticans, a group of proteins, which are highly abundant in brain, forming the PNN. This explains the high content of Omannosyl glycans in brain as well as the strong brain involvement in the Walker Warburg syndrome.

neurocan in kidney cells could potentially indicate the existence of distinct control mechanisms for O-mannosylation in brain. This may explain the lack of the previously described ciscontrolling peptide in all lecticans identified as O-mannosylated, a 40-meric upstream located peptide, which was shown to be necessary for the initiation of O-mannosylation on αdystroglycan.30

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CONCLUSION It has been puzzling for a long time that about 30% of all Oglycans in brain are O-mannosylated1,23 while α-dystroglycan was the only protein known to be O-mannosylated for most of the time. With the lecticans and the group of receptor tyrosine phosphatases being modified with O-mannosyl glycans, this high amount of O-mannosyl glycans can be explained as these proteins are strongly expressed in brain. The abnormally developed brain in congenital muscular dystrophies caused by O-mannosyltransferase deficiency could also not be explained completely. An O-mannosylation of the majority of the proteins responsible for formation of the perineuronal net in mammalian brain is in full accordance with these previous findings. While the lack of aggrecan or versican is embryonically or perinatally lethal,32,33 brevican- and neurocan knockout mice are viable and fertile.34,35 Even a quadruple knockout of brevican and neurocan together with TenascinR and -C did not show severe alterations in brain.36 This contrasts with the severity of symptoms caused by nonfunctional protein-Omannosyltransferases. Considering that several proteins of the PNN are O-mannosylated, it could be supposed that the existence of O-mannosyl glycans is absolutely necessary while the composition of O-mannosylated proteins can vary.

O-Glycan Analysis of Recombinantly Expressed Neurocan

Secreted rat neurocan was recombinantly expressed in EBNA293 cells and the protein was enriched from the serum-free cellculture supernatant by ultracentrifugation and purified by affinity chromatography. Approximately 30 μg of protein was precipitated and the O-glycans were analyzed by MALDI-MS of permethylated oligosaccharide alditols. Surprisingly, we detected no O-mannosyl glycans on recombinantly expressed fulllength neurocan, while the typical high modification with glycosaminoglycans could be observed as shown by Western blot before and after digestion with chondroitinase ABC. Additionally, N-glycosylation of the protein can be shown by the detection of N-glycan specific fragments of the nonreducing end (e.g., m/z 1444.7) by MALDI-MS of the permethylated Oglycans (Figure 7 and Supporting Information Table 11). The complete lack of O-mannosyl glycans on recombinant neurocan, expressed in EBNA-293-cells is unexpected. It is commonly known that recombinant glycoproteins, expressed in EBNA-293 cells, are less modified with O-mannosyl glycans than the respective endogenous proteins. Nonetheless, Omannosyl glycans could be observed applying the same technical strategies on neurofascin and α-dystroglycan when recombinantly expressed in the same cell line.30,31 The lack of O-mannosyl oligosaccharides on the brain-specific protein 1769



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S Supporting Information *

Additional material as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Address: Institute of Biochemistry II, Medical Faculty, University of Cologne, Joseph-Stelzmann-Str. 52, 50931 Köln, Germany. Tel: +49 221 478 7035. Fax: +49 221 478 7788. Email: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Stefan Mueller, Ursula Cullmann and Andrea Gruen from the Central Bioanalytics of the Center for Molecular Medicine Cologne for the performance of LC−MS analysis and protein identification on the QTOF2 and the PTMDiscovery system. We thank Sabine Kindermann for preparing tenascin-R. This work was supported by the DFG grant BR 3979/1-1 from the Deutsche Forschungsgemeinschaft (to I.B.).

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REFERENCES

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The Lecticans of Mammalian Brain Perineural Net Are O-Mannosylated

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