Identification of the Plasticity-Relevant Fucose-R(1-2)-Galactose Proteome from the Mouse Olfactory Bulb

Fucose-R(1-2)-galactose [FucR(1-2)Gal] sugars have been implicated in the molecular mechanisms that underlie neuronal development, learning, andmemory. However, an understanding of their precise roles has been hampered by a lack of information regarding FucR(1-2)Gal glycoproteins. Here, we report the first proteomic studies of this plasticity-relevant epitope. We identify five classes of putative FucR(1-2)Gal glycoproteins: cell adhesion molecules, ion channels and solute carriers/transporters, ATP-binding proteins, synaptic vesicle-associated proteins, and mitochondrial proteins. In addition, we show that FucR(1-2)Gal glycoproteins are enriched in the developing mouse olfactory bulb (OB) and exhibit a distinct spatiotemporal expression that is consistent with the presence of a “glycocode” to help direct olfactory sensory neuron (OSN) axonal pathfinding. We find that expression of FucR(1-2)Gal sugars in the OB is regulated by the R(1-2)fucosyltransferase FUT1. FUT1-deficient mice exhibit developmental defects, including fewer and smaller glomeruli and a thinner olfactory nerve layer, suggesting that fucosylation contributes to OB development. Our findings significantly expand the number of FucR(1-2)Gal glycoproteins and provide new insights into the molecular mechanisms by which fucosyl sugars contribute to neuronal processes. Fucose-R(1-2)-galactose [FucR(1-2)Gal], a terminal sugar found on Nand O-linked glycoproteins, has been implicated in cognitive processes such as learning and memory. Both taskspecific learning and long-term potentiation (LTP), an electrophysiologicalmodel of learning andmemory, have been shown to induce protein fucosylation in hippocampal neurons (1). Moreover, injection of exogenous L-fucose (L-Fuc) or 20-fucosyllactose enhanced LTP in hippocampal slices and in vivo (2, 3). Conversely, inhibition of FucR(1-2)Gal linkages on proteins by incorporation of 2-deoxy-D-galactose (2-dGal) into glycans caused reversible amnesia in animals and interfered with the maintenance of LTP (4-7). In addition to modulating synaptic plasticity, FucR(1-2)Gal may play important roles in regulating neurite outgrowth and neuronal morphology. Delayed synapse formation and stunted neurite outgrowthwere observed in hippocampal cultures treated with 2-dGal (8, 9). FucR(1-2)Gal sugars displayed on multivalent polyacrylamide polymers were found to stimulate the outgrowth of hippocampal neurons, whereas other sugars such as L-Fuc, D-galactose (D-Gal), and fucose-R(1-3)-N-acetylglucosamine [FucR(1-3)GlcNAc] had no effect (8). Furthermore, addition of exogenous lectin Ulex europaeus agglutinin I (UEAI) or that of Lotus tetragonolobus, each of which binds to FucR(1-2) Gal moieties, also enhanced hippocampal neurite outgrowth (8). Recent reports suggest that FucR(1-2)Gal glycoproteins are expressed in the adult OB and developing OB (10-13) and may help direct the targeting of OSNs to discrete areas (10, 11). The differential expression of specific glycan structures that serve as chemotactic agents would provide a largely uncharacterized mechanism for OSN pathfinding. Previous work has revealed that FucR(1-2)Gal and N-acetylgalactosamine glycoconjugates show distinct patterns of expression in OSNs of the adult OB, consistent with the notion that a glycocodemay contribute to the complex topographical arrangement of OSNs (10). Moreover, mice lacking the R(1-2)fucosyltransferase (FUT1) gene displayed impaired development of the olfactory nerve and glomerular layers of the OB (11). Despite the involvement of FucR(1-2)Gal sugars in synaptic plasticity, neurite outgrowth, and development, only one FucR(1-2)Gal glycoprotein has been well-characterized from the mammalian brain. Synapsin I, a presynaptic phosphoprotein involved in neurotransmitter release and synaptogenesis (14), was identified as a prominent FucR(1-2)Gal glycoprotein in the This research was supported byNational Institutes of Health Grants RO1 GM084724-05 (L.C.H.-W.), T32 GM08501 (H.E.M.), and 5T32 GM07616-30S1 (C.K.). *To whom correspondence should be addressed. E-mail: lhw@ caltech.edu. Telephone: (626) 395-6101. Fax: (626) 564-9297. D ow nl oa de d by C A L T E C H o n A ug us t 3 , 2 00 9 Pu bl is he d on J un e 15 , 2 00 9 on h ttp :// pu bs .a cs .o rg | do i: 10 .1 02 1/ bi 90 06 40 x 7262 Biochemistry, Vol. 48, No. 30, 2009 Murrey et al. adult hippocampus (9). Fucosylation was shown to regulate the stability and turnover of synapsin I, protecting it fromproteolytic degradation by the calcium-activated protease calpain. Inhibition of protein fucosylation using 2-dGal reduced synapsin expression and delayed synapse formation in hippocampal cultures (9). The neural cell adhesion molecule (NCAM) has also been suggested to be fucosylated (15-17), although no functional studies have been performed. Here, we report the first proteomic studies of the plasticityrelevant FucR(1-2)Gal epitope. Using lectin affinity chromatography (LAC) coupled to mass spectrometry (MS), we identified five classes of putative FucR(1-2)Gal glycoproteins: cell adhesion molecules, ion channels and solute transporters/carriers, ATP-binding proteins, synaptic vesicle-associated proteins, and mitochondrial proteins. In addition, we provide evidence that fucosylation of NCAM and other glycoproteins by the R(1-2)fucosyltransferase FUT1 contributes to OB development, consistent with the notion of a glycocode for the regulation of OSN targeting. Together, our studies significantly expand the number of FucR(1-2)Gal glycoproteins from two to more than 30 and suggest new roles for protein fucosylation in mediating neuronal communication and development. MATERIALS AND METHODS Lectin Blotting of Brain Regions. Adult male mice (3-4 months of age) and postnatal day 3 (P3) pups were anesthetized with CO2 and dissected to remove the cerebellum, cortex, hippocampus, hypothalamus, olfactory bulb, striatum, and thalamus. For lectin blotting, dissected tissues were cut into small pieces and placed immediately on ice and then lysed in boiling 1% SDS (5 volumes/weight) with sonication until the mixture was homogeneous. Proteins were resolved via 10% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF, Fisher) membranes. The membranes were blocked with 3% periodated BSA in PBS (9) and then incubated with horseradish peroxidase (HRP)-conjugated UEAI [Sigma; 50 μg/mL in Tris-buffered saline (pH 7.4) containing 0.1% Tween 20 (TBST)] for 2 h at room temperature (RT).After beingwashed (3 10min inTBST), the membranes were developed as described previously (9). LAC. UEAI lectin conjugated to agarose (Vector Laboratories) or control Protein A conjugated to agarose (Vector Laboratories) was packed into three separate minicolumns [∼333 μL bed volume each (Bio-Rad)] and run in parallel. The resin was equilibrated with 10 column volumes (CV) of lectin binding buffer [100 mM Tris (pH 7.5), 150 mM NaCl, 1 mM CaCl2, 1 mMMgCl2, 0.5%NP-40, and 0.2% sodium deoxycholate supplemented with EDTA-free Complete protease inhibitors (Roche)]. Cell lysate was prepared as follows. The OBs from 3050 P3 pups were isolated and homogenized in lectin binding buffer by being passed through a 26-gauge needle five times and then sonicated to homogeneity. Samples were clarified by centrifugation at 12000g for 10 min. The total protein concentration of the lysate was determined using the BCA protein assay (Pierce). The lysate (3 mL per column, at 6-10 mg/mL) was bound batchwise with gentle end-over-end mixing at RT for 4 h. The agarose was then allowed to settle, and the flow-throughwas passed over the column three additional times. The columns were washed with 40 CV of lectin binding buffer, followed by 10CVof lectin binding buffer lacking detergent. Proteins were eluted in 10 CV of lectin binding buffer lacking detergent and supplemented with 200 mM L-Fuc and protease inhibitors. Protein eluates were concentrated to a volume of 100 μL in 10000 molecular weight cutoff (MWCO) Centricons (Millipore), followed by 10000 MWCO Microcons. Following concentration, samples were boiled with 35 μL of 4 SDS loading dye [200 mM Tris (pH 6.8), 400 mMDTT, 8% SDS, 0.2% bromophenol blue, and 40% glycerol] and loaded onto 10% SDS gels for electrophoresis as described previously (18). Silver Staining, In-Gel Digestion, and LC-MS Analysis. All silver staining reagents were prepared fresh before they were used. The staining and destaining, in-gel tryptic digests, and peptide extractions were performed as described previously (19). NanoLC-MS of in-gel tryptic digests was performed on a Thermo Fisher LTQ Orbitrap mass spectrometer using a modified vented column setup and data-dependent scanning (20). Samples were loaded onto a 360 μm 100 μm precolumn (2 cm, 5 μm Monitor C18) and desalted before the precolumn was placed in-line with the analytical column. Peptides were then elutedwith a linear gradient from0 to 40%Bover 30min (A, 0.1M aqueous HOAc; B, 0.1 M HOAc in CH3CN), with a flow rate of approximately 250 nL/min, and using a 360 μm 75 μm selfpacked column with an integrated electrospray emitter (10 cm, 5 μm Monitor C18). For data-dependent experiments, the mass spectrometer was programmed to record a full-scan ESI mass spectrum (m/z 650-2000, ions detected in the Orbitrap mass analyzer with a resolution set to 100000), followed by five data-dependent MS/MS scans in the ion trap (relative collision energy of 35%, 3.5 Da isolation window). Dynamic exclusion parameters were set as follows: repeat count= 1, repeat duration=15 s, and exclusion duration=30 s. MS/MS spectra were searched against a mouse subset of the European Bioinformatics Institute-International Protein Index (EBI-IPI) database (downloaded August 1, 2007), with an appended reversed database using Sequest 3.0. A fixed modification of Cys (þ57), a variable modification of Met (þ16), and trypsin cleavage were specified. Search results were compiled and filtered in Scaffold 1.0 (Proteome Software, Inc., Portland, OR). A protein identificationwas accepted if aminimumof five unique peptides matched to the protein, which corresponded to ag99% probability of a correct identification (see Table 1). Although a peptide tolerance of 3 Da was used in the search, the mass accuracy of the precursor ion scan for each identified peptide was manually verified as being within 20 ppm of the theoretical value. Proteins were considered nonspecific if any corresponding peptides were isolated from the control column or from FUT1deficient mice. Cytosolic proteins identified were removed from the data set because they are not likely to be glycosylated. As the specificity of FUT1 is not known, the list of identified proteins could not be filtered for the presence of a FUT1 consensus sequence. Note that LAC may also result in the isolation of proteins that associate with FucR(1-2)Gal glycoproteins, which may also be of interest biologically but are themselves not directly glycosylated. Thus, further validation of individual proteins of interest by immunoprecipitation (see Figure 2B) and other methods is recommended to confirm the presence of the FucR(1-2)Gal epitope. Immunoblotting To Confirm Proteome Hits. To further validate putative proteome hits, proteins were isolated by lectin affinity chromatography as described above. Lysates from the UEAI column and the control column were resolved on Bis-Tris 4 to 12% NuPage gradient gels (Invitrogen) according to the manufacturer’s protocol in MOPS running buffer. Gels were transferred to PVDF, and the membranes were incubated D ow nl oa de d by C A L T E C H o n A ug us t 3 , 2 00 9 Pu bl is he d on J un e 15 , 2 00 9 on h ttp :// pu bs .a cs .o rg | do i: 10 .1 02 1/ bi 90 06 40 x Article Biochemistry, Vol. 48, No. 30, 2009 7263 Table 1: FucR(1-2)Gal Glycoproteins Identified from the Mammalian Olfactory Bulb protein function accession no. MW (Da) peptide no. sequence coverage (%) Cell Adhesion Molecules NCAM1 cell adhesion, axonal outgrowth, and fasciculation IPI00831465.1 93474 21 33.5 IgSF3 cell adhesion and neuronal fasciculation IPI00420589.2 134605 21 18.6 contactin-1 precursor cell adhesion and neuronal fasciculation IPI00123058.1 113372 10 10.2 NCAM L1 cell adhesion and neuronal fasciculation IPI00406778.3, IPI00785371.1, IPI00831568.1 140413 9 10.7 OCAM (NCAM2) cell adhesion and neuronal fasciculation IPI00127556.1, IPI00322617.1 93187 7 9.0 NRCAM cell adhesion and neuronal fasciculation IPI00120564.5, IPI00338880.3, IPI00395042.1, IPI00403536.1 122736 7 6.3 kirrel2 cell adhesion and neuronal fasciculation IPI00471420.1, IPI00623939.2 72754 5 10.3 Ion Channels and Solute Transporters/ Carrier Proteins slc12a2 chloride transport IPI00135324.2 130654 15 14.9 cacna2d1 calcium channel IPI00230013.3, IPI00319970.1, IPI00407868.1, IPI00410982.1, IPI00626793.3 122505 13 13.0 aralar1 Asp, Glu exchanger IPI00308162.3 74553 12 16.7 slc3a2 cationic amino acid transporter IPI00114641.2 58805 8 16.7 slc7a5 cationic amino acid transporter IPI00331577.3 55856 5 8.6 VDAC3 voltage-dependent anion channel IPI00122548.3, IPI00762642.1 30867 5 16.2 ATP-Binding Proteins/ ATP Synthase Naþ/Kþ-ATPase R1 maintenance of the electrochemical gradient IPI00311682.5 112967 34 35.4 ATP synthase R subunit ATP synthesis IPI00130280.1 59736 11 23.7 Naþ/Kþ-ATPase β1 maintenance of the electrochemical gradient IPI00121550.2 35771 6 25.2 vacuolar ATP synthase catalytic subunit A ATP synthesis IPI00407692.3 68309 6 13.1 ATP synthase γ chain ATP synthesis IPI00313475.1, IPI00750074.1, IPI00751391.1, IPI00775853.1, IPI00776084.1, IPI00776275.1 30239 6 24.1 ATP synthase B chain ATP synthesis IPI00341282.2 28931 6 19.9 Naþ/Kþ-ATPase R3 maintenance of the electrochemical gradient IPI00122048.2 111676 6 18.3 ATP-binding cassette, subfamily C, member 9 efflux of endogenous and xenobiotic molecules IPI00111686.5 148816 5 3.4 Synaptic Vesicle Proteins munc18 (syntaxin-binding protein 1) synaptic vesicle cycling IPI00415402.3 67553 13 23.6 synaptotagmin-1 synaptic vesicle cycling IPI00129618.1, IPI00750142.1 47400 9 24.0 NSF vesicle-fusing ATPase synaptic vesicle cycling IPI00656325.2 82598 8 11.7 Mitochondrial Proteins prohibitin-2 protein folding IPI00321718.4 33279 7 24.4 prohibitin protein folding IPI00133440.1 29802 7 29.4 ubiquinol-cytochrome c reductase complex core protein 2 enzyme IPI00119138.1 48218 6 19.0