Institut für Molekularbiologie und Biochemie derFreien Universität Berlin, Arnimallee 22, D-14195 Berlin-Dahlem,Germany and 4Institut fürKlinische Chemie und Biochemie, Virchow-Klinikum, Medizinische Fakultätder Humboldt Universität zu Berlin, Augustenburger Platz1, D-13353 Berlin-Wedding, Germany
Received on January 27, 1999. revisedon June 23, 1999; accepted on June 23, 1999.
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Abstract |
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Introduction |
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Transfer of sialic acid residues to newly synthesized glycoproteinsand glycolipids during biosynthesis has been shown to be preciselycontrolled by the cellular activity of various sialyltransferases(52Paulson et al., 1989). In addition, cell surface glycoproteins may bede- and resialylated during endocytosis and recycling, representinga mechanism of postbiosynthetic adaptation (73
Volz et al., 1995). Structural analysis ofglycoprotein glycans has shown that the oligosaccharides of a given glycoproteinmay differ in their extent of sialylation. This is true not onlyfor the oligosaccharides of the different glycosylation sites, butalso for the oligosaccharides bound to one distinct glycosylationsite (38
Kornfeld and Kornfeld, 1985; 51
Paulson and Colley, 1989; 63
Schachter,1995), giving rise to a microheterogeneity in glycan sialylation.Whether a different degree of sialylation of oligosaccharides may,furthermore, lead to the generation of differently charged isoformsof a glycoprotein distinguished by the number of sialicacid residues has so far been studied almost exclusivelyfor soluble glycoproteins, including serum transferrin (de Jongand van Eijk, 1988;) thyroxin-binding globulin (41
Lasne et al., 1982), thyrotropin (50
Papandreou et al., 1993; 68
Szkudlinski et al., 1993), prolactin (55
Price et al., 1995), lithostatine (56
De Reggi et al., 1995),and human chorionic gonadotropin (1
Amano et al., 1989; 48
Nemansky et al., 1995). As has been elegantly shown forserum transferrin (29
de Jong and van Eijk,1988) sialylation apart from genetic polymorphism andother forms of postbiosynthetic modification, like phosphorylationand sulfatation, may significantly influence the isoelectric pointof serum glycoproteins. Studies on the sialylation of membrane glycoproteinshave, however, examined samples of purified glycoproteins that werenot separated according to charge differences beforehand. Thesestudies could, hence, not address the issue, whether the observedmicroheterogeneity of glycan sialylation does result in the formationof differently charged isoforms of a membrane glycoprotein. It is,therefore, still largely unknown whether similar to serum glycoproteinsmembrane glycoproteins may exist in different isoforms in the same tissue,distinguished by the number of sialic acid residues per proteinmolecule.
In the present study we have addressed this question by studyingthe sialylation of dipeptidylpeptidase IV (DPP IV, CD 26). DPP IV,a serine-type exo- and endopeptidase, cleaves N-terminal dipeptidesfrom polypeptides with proline or alanine as the penultimate aminoacid (25Hopsu-Havu and Sarimo, 1967; 35
Kenny et al., 1976; 5
Bermpohl et al., 1998),e.g., substance P, ß-casomorphine andthe fibrin
-chain. DPP IV is expressedin all tissues so far investigated, in particular in the brush border membranesof small intestine, kidney and bile canaliculi (13
Gossrau,1979a; 15
Hartel et al.,1988). The cDNA for DPP IV was cloned in rat (23
Hong et al., 1989), mouse(44
Marguet et al., 1992),and human (45
Misumi et al.,1992; 69
Tanaka et al.,1992; 10
Darmoul et al.,1992). DPP IV is an integral type II membrane proteinanchored to the membrane by the signal peptide sequence (49
Ogata et al., 1989; 24
Hong and Doyle, 1990) and is present inthe plasma membrane as a homodimer (28
Jascur et al., 1991). DPP IV has eight consensussequences for N-glycosylation (22
Hong andDoyle, 1987) which are all N-glycosylated (54
Petell et al., 1987). Except for one oligosaccharidechain, which retains a high mannose structure, DPP IV N-glycans matureto complex-type structures on their way through the endomembranes(76
Yamashita et al., 1988; 16
Hartel-Schenk et al., 1991).The oligosaccharides of DPP IV from kidney exhibit extensive structuralheterogeneity (76
Yamashita etal., 1988).
It was the aim of the present study to examinewhether cellular membrane-bound DPP IV does in vivo existin differently charged isoforms distinguished from each other bythe number of sialic acid residues per protein molecule. This wasinvestigated in that native, enzymatically active DPP IV was purified fromplasma membranes of rat liver and kidney employing a purificationprotocol mainly based on crown ether ion exchange chromatography.By this method, indeed, differently charged forms of native DPPIV could be separately purified. Further analysis showed that theydiffer in their degree of sialylation.
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Results |
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DPP IV from liver membranes showed high affinity for WGA. BoundDPP IV activity could be eluted with 0.2 M GlcNAc resulting in 2-foldenrichment. By contrast, only 60% of the total activityof kidney DPP IV bound to WGA. Therefore, WGA affinity chromatographywas finally omitted in the purification of kidney DPP IV.
The essential step in the purification of native DPP IV from bothtissues was anion exchange chromatography on crown ether-silicagel (Pedersen and Frensdorff, 1972; 58Reusch,1988; 33
Josic et al.,1989). Kidney and liver DPP IV activity were completelybound to the column, when applied in 10 mM TrisHCl, pH7.2, 10 mM KCl, 0.1% Triton X-100. The column was thenwashed with 30 bed volumes of 10 mM TrisHCl, pH 7.2, 10mM KCl, containing 0.1% octylglucoside instead of Triton X-100,in order to replace the Triton X-100. Bound glycoproteinswere eluted with a NaCl gradient (0750 mM NaCl) (Figure 2A). Elution of DPP IV activity started at 65mM NaCl and proceeded until 140 mM NaCl. Sodium dodecyl sulfatepolyacrylamidegel electrophoresis (SDSPAGE) and silver staining of theeluted fractions containing DPP IV activity showed that only DPPIV was eluted under these conditions (Figure 3).Under denaturing conditions, DPP IV migrated with a relative molecularmass (Mr) of
100 kDa forthe kidney enzyme (Figure 3B) and 110 kDafor the liver enzyme (not shown), in accordance with previous reports(39
Kreisel et al., 1982; 70
Tiruppathi et al., 1990).Purity of the eluted fractions was also shown by SDSPAGEunder nonreducing and nondenaturing conditions (Figure 3A). At a NaCl concentration of 120 mM and higher,additional glycoproteins including nucleotide pyrophosphatase (EC3.6.1.9) (Figure 2) and leucine aminopeptidase(EC 3.4.11.2) (not shown) were eluted. Both nucleotide pyrophosphataseand leucine aminopeptidase were identified by functional assay oftheir enzymatic activities, by their Mr as determinedby SDSPAGE and by use of specific antibodies. When TritonX-100 was added to the final elution buffer (750 mM NaCl, Figure 2A; 400 mM NaCl, Figure 2B), nucleotidepyrophosphatase (Figure 2) and leucine aminopeptidase(not shown) eluted as a sharp peak. No additional DPP IV was recoveredfrom the column in the presence of Triton X-100.
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Previous studies had shown that DPP IV from rat kidney brushborder membranes is N-glycosylated with heterogenous sialylatedglycans of the complex type (76Yamashita et al., 1988). With respect to these findings,sialylation of liver and kidney DPP IV was examined comparatively.The enzyme purified from either liver or kidney plasma membraneswas probed with Sambucus nigra agglutinin (SNA)and Maackia amurensis agglutinin (MAA) which havebinding specificities for
2,6- and
2,3-linked sialic acid residues, respectively (18
Hashimoto et al., 1981; 64
Spellman et al., 1989).DPP IV from both liver and kidney showed distinct binding with SNAand lesser binding with MAA, indicating the presence of
2,6-as well as
2,3-linked sialic acids.Desialylation of purified DPP IV from liver and kidney DPP IV bysialidase caused loss of binding reactivity with both SNA and MAA(Figure 4).
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Discussion |
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First, separation of a glycoprotein fraction of rat liver and kidneymembranes by crown ether affinity chromatography and elution withincreasing concentrations of NaCl resulted in the elution of electrophoreticallyhomogenous fractions of DPP IV at NaCl concentrations ranging from65 mM to 140 mM. Immobilized crown ethers function as anion exchangers (Pedersenand Frensdorff, 1972) and can be used for ion exchange separationsof proteins (58Reusch, 1988; 33
Josic et al., 1989).
Employing this method DPP IV could be clearly separated fromother membrane glycoproteins such as nucleotide pyrophosphataseand leucine aminopeptidase. The observation that DPP IV eluted inbuffer containing octylglucoside as detergent, whereas effectiveelution of nucleotide pyrophosphatase and leucine aminopeptidaserequired the presence of Triton X-100, which has a lower criticalmicelle concentration (CMC) than octylglucoside, indicates thatthe other membrane glycoproteins were retained on the column alsoby hydrophobic interactions.
The differently charged DPP IV fractions from the crown ethercolumn were further analyzed by isoelectric focusing. Less chargedfractions eluting at low NaCl concentrations migrated in the morebasic part of the gel, while the more highly charged fractions migratedin the more acidic part. Enzymatic desialylation with sialidasefrom Clostridium perfringens converted chargedDPP IV fractions into a form that comigrated with the DPP IV fractionthat was eluted at low NaCl concentration. This indicates that theobserved charge differences are due to different extents of sialylation.
The presence of sialic acid residues in purified DPP IV could beconfirmed by lectin affinity blotting using SNA and MAA. In accordancewith previous reports (76Yamashita et al., 1988), the results showed that the sialicacid residues are predominantly
2,6-linkedto Gal with only traces of
2,3-linkedsialic acid residues. Whereas sialylated and desialylated DPP IV couldbe clearly distinguished from each other by lectin affinity blotting,no clear distinction between the sialylated and the desialylatedform could be made by SDSPAGE under reducing, denaturingconditions. While DPP IV from liver exhibited a discrete shift fromapparently 110 kDa to 100 kDa after desialylation, no differencein Mr was detectable for the sialylated and the desialylatedform of kidney DPP IV. The molecular mass determination in SDSPAGE,however, is valid only for linear polypeptides, and deviations havebeen described for polypeptides that carry nonpolypeptide componentssuch as glycans (42
Leach et al.,1980). Since the glycan moiety may influence the apparentMr, changes within the glycan moiety such as desialylationmay also modify the latter. Hence, deglycosylation cannot be usedfor the characterization of Mr values within a systemthat does not take into account these sources of deviation.
On the other hand, sialylation influenced the migration of DPPIV in SDSpolyacrylamide gels under nonreducing and nondenaturingconditions, in that desialylated forms migrated more slowly thansialylated DPP IV. Hence, sialylated forms having a higher molecularmass exhibit a lower apparent Mr when separated by SDSPAGEunder nonreducing, nondenaturing conditions. This is explained bythe fact that nonreduced and nondenatured DPP IV does not fullybind SDS and does not fulfil the standards of SDSPAGE(Reynolds and Tanford, 1970a,b). As charge does influence the migrationof the DPP IV molecules not associated with SDS, the migration reflects thecharge and in our case the extent of sialylation. This is very effectivelydemonstrated by SDSPAGE under nonreducing and nondenaturingconditions of the DPP IV fractions eluted from the crown ether column.DPP IV fractions eluting at low NaCl concentrations, and thereforepossessing a low charge, migrated at the lowest velocity, DPP IVfractions eluting at high NaCl concentrations, and therefore carryinga higher charge, migrated at the highest velocity. The graduallyincreasing charge of the DPP IV fractions eluting at gradually increasingNaCl concentrations resulted in a gradually increasing migrationvelocity (Figure 3A). Enzymatic desialylationof charged DPP IV to produce a less charged form that comigratedwith the early eluting forms of DPP IV indicates that the chargeshift during elution from crown ethers is inversely related to thecharge shift resulting from the loss of sialic acids. The changein migration velocity between the early eluting DPP IV fractionsand the late eluting DPP IV fractions can therefore be ascribedentirely to charge differences conferred by the variable contentof sialic acid residues. Further proof is given by enzymatic de-and resialylation of DPP IV. Whereas enzymatic desialylation ofcharged DPP IV results in a distinct reduction of migration velocityin SDSPAGE under nonreducing and nondenaturing conditions,the enzymatic resialylation of desialylated DPP IV with sialyltransferasefrom rat liver leads to an increase in migration velocity exactlyto the level of the originally charged form. DPP IV therefore existsin differently charged forms that can be explained by different extentsof sialylation.
How can such a variety of charged forms develop within one typeof tissue? Heterogeneity of sialylation may occur for various reasons.
First, during biosynthesis, a different extent of branching willresult in a different extent of sialylation. The microheterogeneityof oligosaccharides at a single amino acid site may be explainedby competition between the glycosyltransferases on the endomembraneassembly line (for review, see Schachter, 1995). Thus, the extentof branching originates from competition of the different glucosaminyltransferases.Variety in their activities will produce various antennae. Sincebranching is terminated by glucosaminyltransferase III, strong expression ofthe latter in kidney tissues (36Kobata, 1992)further contributes to the variety in branching.
Sialylation may be varied at the level of activity of sialyltransferases(52Paulson et al., 1989).If the expression of sialyltransferase activities is regulated onthe cellular level, the extent of sialylation on oligosaccharidestructures may be varied further.
Finally, postbiosynthetic processes may also contribute to a differentdegree of sialylation. Thus, during endocytosis and recycling, sialylatedstructures are subject to enzymatic de- and resialylation (12Duncan and Kornfeld, 1988; 39
Kreisel et al., 1982; 57
Reichner et al., 1988; 73
Volz et al., 1995). Studies by Volz etal. on sialylated and desialylated cell surface DPP IV showedthat on the other hand DPP IV is not desialylated in cell homogenates in vitro. This result rules out that partiallydesialylated forms of DPP IV observed in the present study weregenerated during the purification process. In vivo desialylationmost likely occurs on the cell surface. Cell surface sialidaseshave been reported to desialylate oligosaccharide structures, namely toselectively desialylate gangliosides in the plasma membrane of neuroblastomacells (37
Kopitz et al., 1996).Moreover, a neuraminidase has also been reported on the cell surfaceas part of the elastin/laminin receptor complex (21
Hinek, 1996), where it maybe involved in the postbiosynthetic generation of desialylated structures.
The ability to separate and to purify various isoforms of DPP IVdistinguished from each other by the extent of sialylation willform the basis for future studies on the role of sialylation indetermining the biological properties and behavior of DPP IV. Bearingin mind the various biological roles of DPP IV, this may be of particularsignificance for sialic acid functions in the immune system, incell adhesion and for brush border functions.
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Materials and methods |
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Molecular mass standards, reagents for SDSPAGE, N-octylglucoside,concanavalin A-Sepharose, methyl--D-mannopyranoside, N-acetyl-D-glucosamine,leucine p-nitroanilide, thymidine monophosphate p-nitrophenyl ester and sialidase from Clostridiumperfringens (EC 3.2.1.18), attached to beaded agarose, werefrom Sigma (Deisenhofen, Germany). Triton X-100 was from Aldrich(Steinheim, Germany). The sialic acid binding lectins of Maackiaamurensis and Sambucus nigra,
-2,6N-acetylneuraminyltransferase from rat liver (EC 2.4.99.1) and cytidine5'-monophosphate (CMP)-N-acetylneuraminicacid were obtained from Boehringer Mannheim (Mannheim, Germany).
Tosyl-Gly-Pro p-nitroanilide was from Bachem(Bubendorf, Switzerland), Servalyte (211 and 49T),and DL-dithiothreitol (DTT) was from Serva (Heidelberg,Germany).
Nitrocellulose membranes (BA85, 0.45 µm)were obtained from Schleicher & Schuell (Dassel, Germany).
WGA was immobilized on Fractogel (Tosohaas, Yamaguchi, Japan)as described previously (32Josic et al., 1987).
The 1,10-diaza-18-crown-6 ligands were immobilized on epoxy-activatedsilica gel (particle size 7 µm and poresize 30 nm; Eurochrom, Saeulentechnik Knauer). A semipreparative columnwith the dimension of 120 mm x 8.0 mmwas produced and packed by Saeulentechnik Knauer (Berlin, Germany).
Determination of enzyme activities
DPP IV activity was measured according to 39Kreisel et al. (1982) with tosyl-Gly-Pro p-nitroanilideas substrate. Leucine aminopeptidase activity was determined accordingto Roman and Hubbard (1983) using leucine p-nitroanilideas substrate. Enzyme activities were calculated from a standardcurve of p-nitroanilide at 405 nm. For assayingnucleotide pyrophosphatase activity 290 µlof buffer (150 mM TrisHCl, pH 9.0, 10 mM MgCl2,0.1% (v/v) Triton X-100), 10 µlof the sample and 100 µl of thymidinemonophosphate p-nitrophenyl ester (2 mM in H2O)were incubated for 30 min in a water bath at 37°C. Thereaction was stopped by adding 600 µlof 0.1 mM NaOH. Enzyme activity was calculated from a standard curveof p-nitrophenol at 405 nm.
Detection of DPP IV activity on nitrocellulosemembranes
DPP IV separated on SDSpolyacrylamide gels under non-denaturingconditions was blotted onto nitrocellulose membranes and stainedaccording to 74Walborg et al.(1985). Briefly, the membranes were incubated with 0.2M TrisHCl, pH 7.8, 1 mM glycyl-L-proline-4-methoxy-ß-naphthylamide for 15 min at 37°C, followed by an incubation in 1 Msodium-acetate, pH 4.2, 0.05% (w/v) Fast GarnetGBC. Enzymatic activity is indicated by the red azo dye. Membraneswere rinsed in 0.2 M TrisHCl, pH 7.8, and dried.
Isolation of plasma membranes
Plasma membranes from liver and kidney were prepared as describedpreviously (14Harms and Reutter, 1974; 67
Stewart and Kenny, 1984) and frozen at 80°C. Extraction of plasma membranes wascarried out according to 31
Josic et al. (1985). Briefly, isolated plasma membraneswere thawed, resuspended in buffer L (1 mM NaHCO3, pH7.0, 0.5 mM CaCl2) as for liver membranes or in bufferK (2 mM HEPES, pH 7.2, 100 mM mannitol) as for kidney membranesand homogenized with five strokes in a loose fitting Dounce homogenizerand centrifuged for 30 min at 60,000 x g. The supernatant was decanted and the pelletwas resuspended in buffer A (10 mM TrisHCl, pH 7.8, 150mM NaCl, 1 mM CaCl2, 1% (v/v) TritonX-100) at a concentration of 2 mg protein/ml buffer with20 strokes in a Dounce homogenizer. After 6 h on ice the mixturewas centrifuged at 50,000 x g for30 min. The supernatant containing the solubilized proteins wasstored at 80°C.
Lectin affinity chromatography
Plasma membrane proteins (100 mg protein/column) from liveror kidney solubilized in buffer A were applied to a concanavalinASepharose column (20 x 160mm) equilibrated with buffer A. The column was washed with fivebed volumes of buffer A. Material bound to the column was elutedwith 200 ml of 0.2 M methyl--D-mannopyranosidein buffer A at a flow rate of 1 ml/min. Fractions of 10ml were collected and assayed for DPP IV activity. Fractions containingDPP IV activity were pooled and exhaustively dialyzed against bufferA. The dialysed eluate was applied to a WGA-Toyoperl column equilibatedwith buffer A. The column was washed with five bed volumes of bufferA. Material bound to the column was eluted in 10 ml fractions with200 ml 0.2 M N-acetyl-D-glucosamine in buffer Aat a flow rate of 1 ml/min. Fractions (10 ml) containingDPP IV activity were pooled and exhaustively dialyzed against bufferB (10 mM TrisHCl, pH 7.2, 10 mM KCl).
Fractionation of DPP IV by crown ether HPAC
A 1,10-diaza-18-crown-6-silica gel column, equilibrated in water,was saturated with 10 ml 1M KCl, and then equilibrated in bufferB (10 mM TrisHCl, pH 7.2, 10 mM KCl, 0.1% (w/v)octylglucoside). The dialyzed eluate from WGA-agarose was appliedto the column at a flow rate of 1 ml/min. The column waswashed with buffer B until Triton X-100 was no longer detectablein the fluid phase (280 nm). Material bound to the column was elutedwith a continuous gradient (0750 mM NaCl) or a discontinuousgradient (0400 mM NaCl) in buffer B. Fractions of 1 mlwere collected and assayed for DPP IV activity. Hydrophobicallybound material that could not be eluted with a NaCl-gradient waseluted with 0.1% (v/v) Triton X-100 in bufferB containing either 750 mM NaCl (continuous gradient) or 400 mMNaCl (discontinuous gradient).
Sodium dodecyl sulfate polyacrylamidegelelectrophoresis (SDSPAGE)
Proteins were separated in 7.5% SDSpolyacrylamideslab gels according to the method of Laemmli (40Laemmli,1970). Samples were prepared in 62.5 mM TrisHCl,pH 6.8, 3% (w/v) SDS, 5% (v/v)mercaptoethanol, 10% (v/v) glycerol, and 0.001% (w/v)bromophenol blue and boiled for 3 min in a water bath.
For electrophoretic separation of native, enzymatically activeDPP IV under non-denaturing conditions SDSPAGE was performedin a modified manner. Samples were prepared in 62.5 mM TrisHCl,pH 6.8, 0.1% (w/v) SDS, 10% (v/v) glycerol,and 0.001% (w/v) bromophenol blue without being boiled,and were kept at room temperature.
Isoelectric focusing
Isoelectric focusing was performed in 0.75-mm-thick slab gels asdescribed by 71Van den Bosch etal. (1988) with the following modifications. Samplesof 35 µl were incubated with 50 µl of urea sample buffer (9.5 M urea,2% (v/v) Triton X-100, 2% (v/v)ampholines (40%, mixture of Servalyte 211 andServalyte 49T in a ratio of 1:1) 97 mM DL-dithiothreitol)at room temperature. Vertical 4% polyacrylamide gels containing9.0 M urea, 2% (v/v) Triton X-100, 6% (v/v)of a mixture of 40% ampholines (Servalyte 211and Servalyte 49T in a ratio of 1:1), 0.05% (v/v)TEMED and 0.02% (m/v) ammonium persulfate, wererun for 15 min at 200 V, for 30 min at 300 V, and for 1 h at 400V, using 20 mM H3PO4 as anodic buffer in the lowerchamber and 50 mM NaOH as cathodic buffer in the upper chamber.Thereafter, samples were applied to the gel, overlaid with 4.75M urea, 2% (v/v) Triton X-100, 1% (v/v) ampholines(40%; mixture of Servalyte 211 and Servalyte49T in a ratio of 1:1), 49 mM DL-dithiothreitol,and the gels were run for an additional 18 h at 400 V. Gels werethen silver stained according to Heukeshoven and Dernick (1988)after preincubation of the gels in 10% (w/v) trichloroaceticacid (TCA) for 2 h, and twice in 5% (w/v) sulfosalicylicacid for 1 h to remove ampholines.
The pH-scale was determined after focusing by slicing one laneof the gel that had been loaded with sample buffer from the topto the bottom into equal pieces and measuring the pH of each piecein 200 µl 10 mM KCl.
Staining of gels
Polyacrylamide gels were silver stained according to 20Heukeshoven and Dernick (1988) or accordingto 6
Blum et al. (1987).
Treatment of purified DPP IV with sialidase
DPP IV purified from either kidney or liver plasma membranes wasdialyzed overnight against 50 mM sodium-acetate, pH 6.5, then incubatedwith sialidase immobilized to agarose (from Clostridiumperfringens, 0.61.0 U/ml gel, EC 3.2.1.18)at 37°C for 76 h under continuous agitation.Twenty units of DPP IV from kidney membranes or 1 U of DPP IV fromliver membranes were incubated with 0.3 U or 0.1 U of sialidase,respectively. Before use immobilized sialidase was thoroughly washedwith acetate buffer to remove the storage buffer. For control DPPIV was incubated under the same conditions without sialidase. Afterincubation, beaded neuraminidase was removed by centrifugation.Samples were dialysed against 25 mM TrisHCl,pH 7.5, 150 mM NaCl, 0.1% (w/v) octylglucoside,assayed for DPP IV activity, and prepared for further analysis.
Resialylation of desialylated DPP IV with sialyltransferase
Desialylated DPP IV was dialyzed overnight against sodium cacodylatebuffer (50 mM sodium cacodylate, pH 6.5, 50 mM NaCl, 0.1% (w/v)octylglucoside). Likewise, sialyltransferase from rat liver (EC2.4.99.1) (0.1U/50 µl) wasdialyzed against the same buffer. CMP-N-acetylneuraminic acid wasdissolved in sodium cacodylate buffer (1 mg/20 µl)and the pH was adjusted to 6.5. For resialylation, 0.2 U of desialylatedDPP IV were incubated with 1 mg of CMP-N-acetylneuraminic acid and0.02 U of sialyltransferase in a final volume of 150 µlat 37°C. After incubation for 24 h,48 h, and 76 h aliquots were withdrawn for analysis by SDSPAGE.
Lectin affinity blotting
Sialylation of different forms of DPP IV was analysed by lectin affinityblotting using the method of 17Haselbeck et al. (1990). Briefly, aliquots of purifiedDPP IV and of desialylated DPP IV from kidney (1 µg)or from liver (0.5 µg) in 25 mM TrisHCl,pH 7.5, 150 mM NaCl, 0.1% (w/v) octylglucosidewere heat denatured, then dotted onto nitrocellulose membranes (0.45 µm). 1 µgof human transferrin, having
2,6-linked sialicacid residues (18
Hashimoto etal., 1981), 1 µg of bovine fetuin,having
2,3-linked sialic acid residues(64
Spellman et al., 1989),and 1 µg of carboxypeptidase Y (11
Debray et al., 1981),lacking sialic acid residues, were dotted as positive and negativecontrols. Membranes were then blocked by incubation for 1 h in 0.5% (m/v)blocking reagent in buffer C (50 mM TrisHCl, pH 7.5, 150mM NaCl). After being washed twice with buffer C and once with bufferD (50 mM TrisHCl, pH 7.5, 150 mM NaCl, 1 mM MgCl2,1 mM MnCl2, 1 mM CaCl2), membranes were incubatedwith the digoxigenin-labeled lectins Sambucus nigra agglutinin(SNA) (1 mg/ml) or Maackia amurensis agglutinin(MAA) (5 mg/ml), each in buffer D, for 1 h at room temperature.Membranes were then washed again three times with buffer C and incubatedwith sheep anti-digoxigenin Fab fragments, conjugated to alkalinephosphatase, for 1 h. Bound lectin-digoxigenin conjugates were visualizedwith the alkaline phosphatase reaction using the 5-bromo-4-chloro-3-indolyl-phosphate/4-nitroblue tetrazolium chloride system by incubating the membranes in10 ml of the following solution: 37.5 µl5-bromo-4-chloro-3-indolyl-phosphate (50 mg/ml, in dimethylformamide)and 50 µl 4-nitro blue tetrazolium chloride(77 mg/ml in 70% dimethylformamide) in 10 ml 100mM TrisHCl, pH 9.5, 50 mM MgCl2, 100 mM NaCl.The membranes were rinsed with H2O to stop the reaction,and then dried.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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b Present address:Institut für Klinische Chemie und Pathobiochemie, UniversitätsklinikumBenjamin-Franklin, Fachbereich Humanmedizin der Freien UniversitätBerlin, Hindenburgdamm 30, D-12200 Berlin, Germany
c Towhom correspondence should be addressed.
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References |
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