Sialoforms of dipeptidylpeptidaseIV from rat kidney and liver

Birgit Schmausera, Christiane Kilian, Werner Reutter and Rudolf Tauberb,c,4

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.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
Dipeptidylpeptidase IV (DPP IV, CD26), a serine-typeexo- and endopeptidase found in the cell surface membrane of manytissues, was employed as a model membrane glycoprotein to studythe expression of sialoforms on cell surface glycoproteins. Native,enzymatically active DPP IV was purified from plasma membranes ofkidney and liver by lectin affinity chromatography in conjunctionwith crown ether anion exchange chromatography. The enzyme was gradient-elutedin continuous fractions, all showing a single polypeptide band ofabout 100 kDa when separated by sodium dodecyl sulfate–polyacrylamidegel electrophoresis (SDS–PAGE) under reducing, denaturingconditions. Analysis of the purified DPP IV by isoelectric focusing (IEF)showed that it consists of several polypeptides of different isoelectricpoints (IP) ranging from 5.5 to 7.0. In vitro-desialylationof the enzyme and subsequent isoelectric focusing revealed thatthe differences in isoelectric points were due to differences inthe degree of sialylation. Differences in the degree of sialylationbetween the fractions were also demonstrated by SDS–PAGEunder nonreducing and nondenaturing conditions. Increased sialylationof the enzyme as demonstrated by isoelectric focusing resulted in increasedmigration velocity in nonreducing and nondenaturing SDS–polyacrylamidegels. In vitro-desialylation of the enzyme andits resialylation confirmed that sialylation was responsible forthis extraordinary migration behavior. The native enzyme was predominantlysialylated via {alpha} 2,6-linkage, as shownby lectin affinity blotting employing Sambucus nigra agglutinin(SNA) and Maackia amurensis agglutinin (MAA). Thesefindings demonstrate that a distinct membrane glycoprotein may existin various sialoforms, distinguished from each other by a differentnumber of sialic acid residues. Moreover, these sialoforms can be individuallypurified by crown ether anion exchange chromatography.


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
Sialylation has been shown to be fundamentally important in determiningvarious biological properties of glycoproteins and glycolipids.Thus sialylation may mask the D-galactose (Gal) andN-acetyl-D-galactosamine (GalNAc) residues involvedin the clearance of serum glycoproteins by asialoglycoprotein receptors(34GoKawasaki and Ashwell, 1977; 2GoAshwell and Harford, 1982; 26GoIi et al., 1990; 65GoSpiess,1990; 7GoChiu et al.,1994), and it determines the specific binding affinityof cell surface glycoconjugates for various lectins (72GoVarki,1992). Moreover, sialic acids may modulate the biologicalactivity of coagulation factors and coagulation inhibitors suchas fibrinogen (9GoDang et al.,1989), von Willebrand factor (4GoBerkowitzand Frederici, 1988), protein C (Hau and Salem, 1991),and plasminogen (66GoStack et al.,1992), and of hormones such as erythropoietin (27GoImai et al., 1990). Sialicacids are also involved in infectious diseases, e.g. by acting asbinding sites for viruses or by reducing the antigenicity of parasitessuch as Trypanosoma cruzi (8GoColli,1993). Changes in sialylation of cell surface glycoconjugatesoccur during development and in malignancy and have been shown toinfluence cellular functions such as growth, differentiation, adhesion,and invasiveness (62GoSaitoh etal., 1992; 30GoJorgensen et al., 1995; 37GoKopitz et al., 1996; 43GoLeMarer and Stehelin, 1995; 75GoWieser et al., 1995). The biological significanceof sialylation is reflected in the widespread occurrence of sialicacid residues on a large number of different soluble and membrane-bound glycoconjugates.

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(52GoPaulson et al., 1989). In addition, cell surface glycoproteins may bede- and resialylated during endocytosis and recycling, representinga mechanism of postbiosynthetic adaptation (73GoVolz 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 (38GoKornfeld and Kornfeld, 1985; 51GoPaulson and Colley, 1989; 63GoSchachter,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 (41GoLasne et al., 1982), thyrotropin (50GoPapandreou et al., 1993; 68GoSzkudlinski et al., 1993), prolactin (55GoPrice et al., 1995), lithostatine (56GoDe Reggi et al., 1995),and human chorionic gonadotropin (1GoAmano et al., 1989; 48GoNemansky et al., 1995). As has been elegantly shown forserum transferrin (29Gode 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 (25GoHopsu-Havu and Sarimo, 1967; 35GoKenny et al., 1976; 5GoBermpohl et al., 1998),e.g., substance P, ß-casomorphine andthe fibrin {alpha}-chain. DPP IV is expressedin all tissues so far investigated, in particular in the brush border membranesof small intestine, kidney and bile canaliculi (13GoGossrau,1979a; 15GoHartel et al.,1988). The cDNA for DPP IV was cloned in rat (23GoHong et al., 1989), mouse(44GoMarguet et al., 1992),and human (45GoMisumi et al.,1992; 69GoTanaka et al.,1992; 10GoDarmoul et al.,1992). DPP IV is an integral type II membrane proteinanchored to the membrane by the signal peptide sequence (49GoOgata et al., 1989; 24GoHong and Doyle, 1990) and is present inthe plasma membrane as a homodimer (28GoJascur et al., 1991). DPP IV has eight consensussequences for N-glycosylation (22GoHong andDoyle, 1987) which are all N-glycosylated (54GoPetell 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(76GoYamashita et al., 1988; 16GoHartel-Schenk et al., 1991).The oligosaccharides of DPP IV from kidney exhibit extensive structuralheterogeneity (76GoYamashita 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.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
Native, enzymatically active DPP IV was purified from plasma membranesof rat kidney and liver by lectin affinity chromatography in conjunctionwith ion exchange chromatography on crown ether-silica gel columns(Figure 1A,B). As a first step, isolatedbrush border membranes of kidney and liver were solubilized withthe nonionic detergent Triton X-100, which has been shown to besuitable for the solubilization of enzymatically active DPP IV (31GoJosic et al., 1985). TritonX-100 extracts were sequentially separated first on concanavalinA (Con A)-Sepharose and then on wheat germ agglutinin (WGA)-agarose. ConA has binding specificity for glycoproteins with oligosaccharidesof the oligomannosidic or the biantennary complex type (3GoBaenziger and Fiete, 1979; 47GoNarasimhan et al., 1979), whereas WGA binds oligosaccharideshaving chitobiose sequences and terminal N-acetyl-D-glucosamine (GlcNAc) residues(11GoDebray et al., 1981).Moreover, terminal sialic acid can also account for affinity toWGA due to the common structural element of an acetamido group (46GoMonsigny et al., 1980).



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Fig. 1. Purification of native, enzymaticallyactive DPP IV from rat kidney plasma membranes. (A)DPP IV was purified from isolated rat kidney plasma membranes asdetailed in Materials and methods. (B)Aliquots were taken at the following steps of the purification procedure,separated in a 7.5% SDS–polyacrylamide gel andsilver stained: 1, plasma membranes; 2, supernatant after freezingand thawing and centrifugation; 3, Triton X-100 extract; 4, methyl-{alpha}-D-mannopyranosideeluate from Con A-Sepharose; 5, proteins eluted from WGA-agarosewith N-acetyl-D-glucosamine; 6, eluate from crownether-silica gel.

 
DPP IV from both kidney and liver membranes was almost completelybound to Con A and eluted with 0.2 M methyl–{alpha}-D-mannopyranoside, resulting in 8-fold and 6-foldenrichment of kidney DPP IV and liver DPP IV, respectively.

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; 58GoReusch,1988; 33GoJosic et al.,1989). Kidney and liver DPP IV activity were completelybound to the column, when applied in 10 mM Tris–HCl, pH7.2, 10 mM KCl, 0.1% Triton X-100. The column was thenwashed with 30 bed volumes of 10 mM Tris–HCl, 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 (0–750 mM NaCl) (Figure 2A). Elution of DPP IV activity started at 65mM NaCl and proceeded until 140 mM NaCl. Sodium dodecyl sulfate–polyacrylamidegel electrophoresis (SDS–PAGE) 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(39GoKreisel et al., 1982; 70GoTiruppathi et al., 1990).Purity of the eluted fractions was also shown by SDS–PAGEunder 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 SDS–PAGE 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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Fig. 2. Elution profiles of DPP IVeluted from crown ether-silica gel with a continuous (A)and discontinuous (B) NaCl gradient. GlcNAc eluatefrom WGA-agarose obtained from rat kidney plasma membranes as detailedin Figure 1 was applied to a 1,10-diaza-18-crown-6-silica gel column(120 mm x8 mm) (for details, see Materialand methods). Material bound to the column was eluted at aflow rate of 1 ml/min with either (A)a continuous (0–750 mM NaCl) or (B) a discontinuous(0–400 mM NaCl) NaCl gradient in 10 mM Tris–HCl,pH 7.2, 10 mM KCl, 0.1% (w/v) octylglucoside.(A) 0–150 mM NaCl for 40 min, 150–750 mMNaCl for 20 min, 750 mM NaCl for 30 min. (B) 0–80mM NaCl for 10 min, 80 mM NaCl for 15 min, 80–400 mM NaClfor 40 min, 400 mM NaCl for 20 min. The last 10 fractions were elutedwith NaCl in elution buffer containing 0.1% (v/v)of Triton X-100 to elute hydrophobic proteins also. Fractions of1 ml were collected and assayed for DPP IV activity (solid squares)or nucleotide pyrophosphatase activity (shaded circles).

 


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Fig. 3. Electrophoretic separationunder denaturing and nondenaturing conditions of DPP IV fractionseluted from crown ether-silica gel. DPP IV from kidney plasma membraneswas applied to crown ether-silica gel and eluted as described inthe legend to Figure 2. An aliquot of fraction 26 was desialylatedwith immobilized sialidase from Clostridium perfringens.Fractions eluted from crown ether-silica gel and a sample of desialylatedDPP IV were separated in 7.5% polyacrylamide gels eitherunder nondenaturing (A) or denaturing (B)conditions, and were silver-stained. Lanes: 1, 12 desialylated DPPIV; 2–10, fractions 17, 19, 21, 23, 25, 26, 27, 29, 31;11, fraction 17.

 
In general, elution of DPP IV from crown ether-silica gel dependedon the increasing concentration of NaCl. When a constantly increasingmolarity of NaCl in the elution buffer was applied, DPP IV elutedsteadily and was almost completely recovered from the column (Figure 2A). On the other hand, when a constant NaClconcentration was applied, only part of DPP IV activity was eluted,and DPP IV activity remaining bound to the column under these conditionscould only be eluted with increasing NaCl concentrations (Figure 2B). These results indicated that DPP IV occursin both liver and kidney plasma membranes in differently chargedforms which could be separately purified. As was demonstrated in repeatedexperiments, DPP IV from both kidney and liver always eluted atNaCl concentrations between 65 mM and 140 mM NaCl, and could beclearly separated from nucleotide pyrophosphatase and leucine aminopeptidaseunder these conditions. For subsequent analyses, fractions containingDPP IV were collected and were used either separately or after pooling, dependingon the purpose of investigation.

Previous studies had shown that DPP IV from rat kidney brushborder membranes is N-glycosylated with heterogenous sialylatedglycans of the complex type (76GoYamashita 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 {alpha}2,6- and {alpha}2,3-linked sialic acid residues, respectively (18GoHashimoto et al., 1981; 64GoSpellman et al., 1989).DPP IV from both liver and kidney showed distinct binding with SNAand lesser binding with MAA, indicating the presence of {alpha}2,6-as well as {alpha}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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Fig. 4. Lectin affinity blotting ofDPP IV with MAA and SNA. DPP IV from liver and kidney plasma membraneswas incubated with immobilized sialidase from Clostridiumperfringens or was mock-treated for control. DPP IV samplesand control proteins were dotted onto nitrocellulose membranes (1 µg of DPP IV from kidney, 0.5 µgof DPP IV from liver, 1 µg of fetuin,transferrin or carboxypeptidase Y, respectively). Filters were blocked,incubated first with digoxigenin-labeled MAA or SNA, then with sheepanti-digoxigenin Fab fragments conjugated to alkaline phosphatase,and were stained as detailed in Materials and methods. Glycoproteinsused as controls for positive and negative binding were fetuin,transferrin and carboxypeptidase Y.

 
In order to examine whether the differently charged DPP IV fractionsas separated by crown ether anion exchange chromatography representisoforms of the enzyme, distinguished by their extent of sialylation,the fractions eluted with increasing NaCl concentrations were analyzedby SDS–PAGE under nonreducing and nondenaturing conditionsand by isoelectric focusing, and compared with enzymatically desialylatedDPP IV. When the various DPP IV fractions, as obtained by crown etherchromatography, were separated by isoelectric focusing (IEF) andwere compared with an aliquot of enzymatically desialylated DPPIV, a pattern of DPP IV isoforms exhibiting a stepwise decreasein their isoelectric points from approximately 6.8 to 5.5 was obtained(Figure 5). Whereas DPP IV isoforms elutedat low NaCl concentration (lanes 2, 12) comigrated with the desialylatedform (lane 13), DPP IV isoforms eluted at higher NaCl concentrationmigrated to the anionic part of the gel. This result demonstratesthat the charge difference between DPP IV isoforms separated byelution with increasing NaCl concentrations is due to a differentdegree of sialylation. The different degree of sialylation alsoinfluenced the migration of DPP IV in SDS–PAGE under reducing,denaturing conditions, as well as under nonreducing, nondenaturing conditions(Figure 6). As shown for pooled samplesof DPP IV from both kidney and liver membranes, enzymatically desialylatedDPP IV (lanes 2, 4) migrated slightly faster than sialylated DPPIV (lanes 1, 3) when separated under reducing, denaturing conditions.The effect of desialylation on migration was reversed under nonreducing,nondenaturing conditions (lanes 5–8). Under these conditionsdesialylated DPP IV (lanes 6, 8) migrated more slowly than sialylatedDPP IV (lanes 5, 7). DPP IV separated under these conditions retainedenzymatic activity as demonstrated by enzyme activity staining (Figure 7). The different electrophoretic behavior ofsialylated and desialylated DPP IV under these conditions most probably reflectsthe fact that in the presence of higher concentrations of SDS chargedifferences due to sialic acid are masked by SDS molecules tightlybound to denatured DPP IV. Faster migration of sialylated DPP IVunder nonreducing and nondenaturing conditions is caused by thehigher anionic charge of the molecules at pH 8.8. Under these conditionsnative DPP IV binds SDS to a lesser extent than does denatured DPPIV. The stepwise differences in the sialylation of the DPP IV fractions separatedby crown ether chromatography could be also shown by SDS–PAGEunder nonreducing and nondenaturing conditions (Figure 3A). Isoforms of DPP IV eluted at low NaCl concentration(lanes 2, 11) comigrated with desialylated DPP IV (lanes 1, 12).DPP IV fractions eluted at higher NaCl concentrations (lanes 3–10)showed increased migration in the gel.



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Fig. 5. Isoelectric focusing of DPPIV fractions eluted from crown ether-silica gel. DPP IV from kidneyplasma membranes was applied to a crown ether-silica gel columnand eluted as described in the Figure 2 caption. An aliquot of fraction26 was desialylated with immobilized sialidase from Clostridiumperfringens. Fractions from crown ether-silica gel and an aliquotof desialylated DPP IV were submitted to isoelectric focusing. Thegel was silver stained (as detailed in Materials andmethods). Lanes: 1, sample buffer; 2–11, fractions17, 19, 21, 23, 25, 26, 27, 29, 31, 33; 12, fraction 17; 13, desialylatedDPP IV. In lanes 11 and 12 DPP IV fractions eluting at low (fraction17, lane 12) and high (fraction 33, lane 11) NaCl concentrationswere separated side by side.

 


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Fig. 6. Effect of desialylation onthe electrophoretic mobility of DPP IV under denaturing and nondenaturingconditions. DPP IV purified from kidney and liver plasma membraneswas desialylated with immobilized sialidase from Clostridiumperfringens under non-denaturing conditions, or was mock-treatedfor control. Samples for electrophoresis under denaturing (lanes1–4) or nondenaturing (lanes 5–8) conditions wereprepared from the aliquots, electrophoretically separated and silver-stainedas described in Materials and methods. Lanes: Sialylated(1, 5) and desialylated (2, 6) DPP IV from kidney; sialylated (3,7) and desialylated (4, 8) DPP IV from liver.

 


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Fig. 7. Enzymatic activity of DPP IVpurified from liver and kidney plasma membranes after SDS–PAGEunder nondenaturing conditions. DPP IV was purified from liver andfrom kidney plasma membranes and was submitted to SDS–PAGEunder nondenaturing conditions as detailed in Materialsand methods. Protein separated in the gel was transferred ontonitrocellulose membranes. Enzymatic activity of DPP IV was detectedwith glycyl-proline 4-methoxy-ß-naphthylamide andFast Garnet GBC as detailed in Materials and methods.1. DPP IV from liver, 2. DPP IV from kidney.

 
In order to examine whether the change in migration observedafter enzymatic desialylation is indeed caused by the removal ofsialic acid residues, desialylated DPP IV was resialylated, using {alpha}2,6-sialyltransferase (EC 2.4.99.1)from rat liver. When separated by SDS–PAGE under nondenaturing, nonreducingconditions resialylated DPP IV comigrated with the original nativesialylated DPP IV, showing that the different migration of sialylatedand desialylated DPP IV is solely due to the presence and absenceof sialic acid residues, respectively (Figure 8).



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Fig. 8. Enzymatic resialylation ofDPP IV after enzymatic desialylation under nondenaturing conditions.DPP IV purified from kidney plasma membranes was desialylated withimmobilized sialidase from Clostridium perfringens.Aliquots were then resialylated with {alpha} 2,6-sialyltransferasefrom rat liver for 24, 48, or 72 h. Samples were prepared and wereelectrophoretically separated under nondenaturing, nonreducing conditions,and were silver stained. Lanes: 1, sialylated DPP IV; 2, desialylatedDPP IV in acetate buffer; 3, desialylated DPP IV in cacodylate buffer;4, 5, 6, DPP IV resialylated with sialyltransferase for 24, 48 or72 h, respectively.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
The present paper shows that DPP IV from plasma membranes ofrat kidney and liver exists in differently charged isoforms distinguishedfrom each other by the extent of sialylation. This assumption isbased on the following evidence.

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 (58GoReusch, 1988; 33GoJosic 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 (76GoYamashita et al., 1988), the results showed that the sialicacid residues are predominantly {alpha}2,6-linkedto Gal with only traces of {alpha}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 SDS–PAGE 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 SDS–PAGE,however, is valid only for linear polypeptides, and deviations havebeen described for polypeptides that carry nonpolypeptide componentssuch as glycans (42GoLeach 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 SDS–polyacrylamide 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 SDS–PAGEunder 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 SDS–PAGE(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 SDS–PAGE 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 SDS–PAGE 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 (36GoKobata, 1992)further contributes to the variety in branching.

Sialylation may be varied at the level of activity of sialyltransferases(52GoPaulson 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 (12GoDuncan and Kornfeld, 1988; 39GoKreisel et al., 1982; 57GoReichner et al., 1988; 73GoVolz 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 (37GoKopitz et al., 1996).Moreover, a neuraminidase has also been reported on the cell surfaceas part of the elastin/laminin receptor complex (21GoHinek, 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.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
Animals and chemicals
Male Wistar rats and Buffalo rats, weighing about 160–180g, werebred in our laboratory and fed a commercial diet containing 18–20% (w/w)protein (Altromin R; Altromin, Lage/Lippe, Germany), andwater ad libitum. All chemicals were of analytical grade and wereobtained from Merck (Darmstadt, Germany), Roth (Karlsruhe, Germany),Serva (Heidelberg, Germany), or Sigma (Deisenhofen, Germany).

Molecular mass standards, reagents for SDS–PAGE, N-octylglucoside,concanavalin A-Sepharose, methyl-{alpha}-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, {alpha}-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 (2–11 and 4–9T),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 (32GoJosic 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 39GoKreisel 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 Tris–HCl, 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 SDS–polyacrylamide gels under non-denaturingconditions was blotted onto nitrocellulose membranes and stainedaccording to 74GoWalborg et al.(1985). Briefly, the membranes were incubated with 0.2M Tris–HCl, 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 Tris–HCl, pH 7.8, and dried.

Isolation of plasma membranes
Plasma membranes from liver and kidney were prepared as describedpreviously (14GoHarms and Reutter, 1974; 67GoStewart and Kenny, 1984) and frozen at –80°C. Extraction of plasma membranes wascarried out according to 31GoJosic 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 Tris–HCl, 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 concanavalinA–Sepharose 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-{alpha}-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 Tris–HCl, 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 Tris–HCl, 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 (0–750 mM NaCl) or a discontinuousgradient (0–400 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 polyacrylamide–gelelectrophoresis (SDS–PAGE)
Proteins were separated in 7.5% SDS–polyacrylamideslab gels according to the method of Laemmli (40GoLaemmli,1970). Samples were prepared in 62.5 mM Tris–HCl,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 SDS–PAGE was performedin a modified manner. Samples were prepared in 62.5 mM Tris–HCl,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 71GoVan 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 2–11 andServalyte 4–9T 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 2–11and Servalyte 4–9T 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 2–11 and Servalyte4–9T 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 20GoHeukeshoven and Dernick (1988) or accordingto 6GoBlum 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, 0.6–1.0 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 Tris–HCl,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 SDS–PAGE.

Lectin affinity blotting
Sialylation of different forms of DPP IV was analysed by lectin affinityblotting using the method of 17GoHaselbeck 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 Tris–HCl,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 {alpha} 2,6-linked sialicacid residues (18GoHashimoto etal., 1981), 1 µg of bovine fetuin,having {alpha} 2,3-linked sialic acid residues(64GoSpellman et al., 1989),and 1 µg of carboxypeptidase Y (11GoDebray 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 Tris–HCl, pH 7.5, 150mM NaCl). After being washed twice with buffer C and once with bufferD (50 mM Tris–HCl, 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 Tris–HCl, pH 9.5, 50 mM MgCl2, 100 mM NaCl.The membranes were rinsed with H2O to stop the reaction,and then dried.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
This research was supported by the Deutsche Forschungsgemeinschaft(SFB 366 and 312), the Bundesministerium für Bildung undForschung (BMBF), the Fonds der Chemischen Industrie and the SonnenfeldStiftung. Dedicated to Prof. Dr. Eckart Köttgen on theoccasion of his 60th birthday.


    Abbreviations
 
DPP IV, dipeptidylpeptidase IV (EC 3.4.14.5.); SDS–PAGE, sodiumdodecyl sulfate–polyacrylamide gel electrophoresis; Tx-100,Triton X-100; HPLC, high performance liquid chromatography; NPPase,nucleotide pyrophosphatase (EC 3.6.1.9.); Con A, concanavalin A;WGA, wheat germ agglutinin; MAA, Maackia amurensis agglutinin;SNA, Sambucus nigra agglutinin; TCA, trichloroaceticacid; kDa, kilodalton; Mr, relative molecular mass; CMP,cytidine 5-monophosphate; pI, isoelectric point; CMC,critical micelle concentration; Gal, D-galactose;GalNAc, N-acetyl-D-galactosamine; GlcNAc, N-acetyl-D-glucosamine.


    Footnotes
 
a Presentaddress: Deutsches Krebsforschungszentrum, Abteilung Molekulare Toxikologie,Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany Back

b Present address:Institut für Klinische Chemie und Pathobiochemie, UniversitätsklinikumBenjamin-Franklin, Fachbereich Humanmedizin der Freien UniversitätBerlin, Hindenburgdamm 30, D-12200 Berlin, Germany Back

c Towhom correspondence should be addressed. Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
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