Cassader, Maurizio

Gambino, Roberto

Pagano, Gian Franco

Ruiu, G.

English

Institutional Research Information System University of Turin

Journal of Protein Chemistry, Vol. 18, No. 2, 1999Apolipoprotein H Is Not Affected by In Vitro GlycosylationRoberto Gambino,1 Gianluca Ruiu,2 Gianfranco Pagano,1 and Maurizio Cassader1,3Received June 19, 1998Increased nonenzymatic glycosylation of all major classes of apolipoproteins has been demonstratedin diabetes. In this work we deal with the in vitro nonenzymatic glycosylation of apolipoprotein H,whose role in lipid metabolism is still poorly understood and whose levels increase in diabetes.Apolipoprotein H was isolated from human plasma and purified through a combination of affinitychromatography and continuous elution electrophoresis. The in vitro glycosylation was performedby incubating purified apolipoprotein H with high concentration of glucose. Our results indicate thatthe in vitro nonenzymatic glycosylation has no effect on the physical properties of apolipoproteinH, despite the fact that this apolipoprotein contains a high number of lysine residues. Since thein vitro concentration of glucose was far higher than the levels normally found in diabetic subjects,it is unlikely for apolipoprotein H to become glycosylated in diabetes.1. INTRODUCTIONNonenzymatic glycosylation of proteins is a process thatoccurs in diabetes mellitus, and is related to the onsetseveral complications, including cataract formation(Monnier and Cerami, 1981; Monnier et al., 1979), con-nective tissue abnormalities (Yue et al., 1983; Kentet al., 1985), and macrovascular disease (Witztum et al.,1982; Gonen et al., 1981), the last being one of the majorcauses of morbidity and mortality in diabetes (Kanneland McGee, 1979).Moreover, increased nonenzymatic glycosylation ofall major classes of apolipoproteins (apo)4 has beendemonstrated in diabetes (Curtiss and Witztum, 1985).The functional consequences of this posttranslationalprocess are related to the abnormalities occurring in lipidmetabolism in diabetic subjects (Saudek and Eder, 1979;Gabor et al., 1980). Abnormalities in lipid metabolismand transport are among the main factors contributing tothe development of atherosclerosis in diabetic patients.In fact glycosylated apoB renders low-density lipopro-teins (LDL) functionally abnormal (Lopes-Virella et al.,1988) as well as immunogenic. They are more suscep-tible to oxidative modifications than nonglycosylatedlipoproteins (Bowie et al., 1993). Recognition of LDL bythe LDL receptor can be inhibited by various chemicalmodifications of the lysine or arginine residues of apoB(Steinbrecher and Witztum, 1984). Many studies havehighlighted the role of the lysine residues of apolipopro-teins for various functional activities, such as receptorrecognition (Weisgraber et al., 1978; Mahley et al.,1979), enzyme interaction and activation (Vainio et al.,1983; Musliner et al., 1979), and lipid binding (Sparrowand Gotto, 1982). Thus, nonenzymatic glycosylation ofspecific lysine residues of the various apolipoproteinscould have a profound influence on the function of thatapolipoprotein.Although the physical and metabolic properties ofin vitro glycosylated apolipoproteins have been studiedat length, similar investigations of apoH have not been1 Dipartimento di Medicina Interna, Universita' di Torino, 10126Torino, Italy.2 Laboratorio Centrale "Baldi e Riberi," Azienda Ospedaliera S. Gio-vanni Battista, Torino, Italy.3 To whom correspondence should be addressed.4 Abbreviations: apo, apolipoprotein; DEAE, diethylaminoethyl;EDTA, ethylenediaminetetraacetic-acid; HPLC, high-pressure liquidchromatography; IEC, ion exchange chromatography; IEF, isoelectro-focusing; LDL, low-density lipoproteins; SDS-PAGE, sodium dode-cyl sulfate-polyacrylamide gel electrophoresis; TBS, tris-buffer saline.1730277-8033/99/0200-0173$16.00/0 C 1999 Plenum Publishing CorporationKEY WORDS: Apolipoprotein H; nonenzymatic glycosylation; affinity chromatography; diabetes mellitus.174 Gambino, Ruiu, Pagano, and Cassaderperformed. In this work we deal with the in vitro non-enzymatic glycosylation of apoH, whose role in lipidmetabolism is still poorly understood and whose levelsincrease in diabetes (Cassader et al., 1997).In the present study apoH was glycosylated in vitroin an attempt to explain why apoH levels were found tobe elevated in diabetic patients. Glycosylated apoH wassubjected to isoelectric focusing (IEF) and ion exchangechromatography (IEC) to prove the nonenzymaticprocess. Native apoH served as control.2. MATERIALS AND METHODS2.1. MaterialsTris, urea, Tween 20, acrylamide, and N,N'-methyl-ene-bis-acrylamide, analytical grade, were purchased fromBio-Rad (Milan, Italy). Gel Bond and ampholytes (pH4 -6.5 and pH 6.5-9) were obtained from Amersham Phar-macia (Milan, Italy); nitrocellulose sheets were from Bio-Rad. Rabbit polyclonal antibody to apoH was kindly sup-plied by Behring (Scoppito, Italy). Goat anti-rabbit IgG(alkaline phosphatase conjugated), the alkaline phos-phatase conjugate substrate kit, and trinitrobenzensulfonicacid were purchased from Sigma (Milan, Italy). Molecu-lar weight standards (low MW) were from Bio-Rad.2.3. Glycosylation of apoHGlycosylation of apoH was performed by incubat-ing purified apoH (0.1 mg/ml) with 0.2 M glucose insterile PBS containing 1 mM EDTA (pH 7.4) at 37°C for7 days (Makino et al., 1995).2.4. SDS-PAGE and Western Blot of Nativeand Glycosylated apoHThe apolipoprotein purity and the increased elec-trophoretic mobility due to nonenzymatic glycosylationwere determined by sodium dodecyl sulfate-polyacryl-amide gel electrophoresis (SDS-PAGE) on 12% poly-acrylamide gels under nonreducing conditions. Elec-trophoresis buffer was 25 mM Tris, 0.192 M glycine,0.1% SDS, pH 8.3.ApoH was then blotted after SDS-PAGE in 25 mMTris, 0.192 M Glycine, and 20% methanol, pH 8.3, at295 mA for 100 min. Nitrocellulose membrane wasblocked with 4% bovin serum albumin in 10 mM Tris,0.15 M NaCl, 0.1% Thimerosal, pH 7.6, and subse-quently probed with the specific antibody anti-apoHraised in the rabbit and with the secondary anti-rabbitimmunoglobulin G antibody conjugated with alkalinephosphatase. The blot was developed with the alkalinephosphatase conjugate substrate kit.2.5. IEF and Western Blot of Nativeand Glycosylated apoHIEF was carried out in a Bio-Phoresis TM horizon-tal electrophoresis cell (Bio-Rad), using a Model3000/300 power unit. The polyacrylamide gel, T-5%,C-3%, contained 2% ampholytes (pH 4-6.5 and 6.5-9;3.75:1 v/v) and 3 M urea. Cathode and anode solutionswere 100 mM NaOH and 40 mM glutamic acid, respec-tively. After prefocusing at 500 constant V for 35 min,1 (ug of apoH was applied onto each lane. Electrophore-sis was performed at 10 constant W for 3 hr. After IEFwas performed, the proteins were transferred overnightat room temperature onto 0.45-(um-pore-size nitrocellu-lose membrane by simple diffusion. After transfer, thenitrocellulose was incubated with specific antibody anti-apoH as described above.2.6. Assay of Chemical ModificationThe number of lysine residues modified in the apo-lipoprotein upon glycosylation was estimated with thetrinitrobenzensulfonic acid assay (Habeed, 1966). The re-2.2. Isolation and Purification of apoHApoH was isolated from human plasma and purifiedthrough a combination of affinity chromatography andcontinuous elution electrophoresis as previously de-scribed (Gambino et al., 1996). Briefly, rabbit anti-apoHantibodies (22 mg) were immobilized on cyanogenbromide-activated Sepharose 6B. Normal serum sampleswere diluted into 40 ml of buffer and applied to the col-umn. ApoH bound to the column was eluted with 0.1 Mglycine, 0.05% NaN3, pH 2.5. Sample fractions wereconcentrated with Centriprep-10 concentrators (AmiconInc., Beverly, MA) at 3000 rpm and 20°C in a Beckman-J6B centrifuge (Beckman Instruments, Palo Alto, CA) toa final volume of about 1 ml and subjected to continuouselution electrophoresis.Continuous elution electrophoresis of the proteins(about 2.5 mg) was then performed with a Prep-Cell(Bio-Rad) through a cylindrical gel (gel tube inside di-ameter 37 mm; gel length 50 mm) composed of 8.5%acrylamide, 2.7% N,N'-methylene-bis-acrylamide, and0.1% SDS. The buffers were 0.025 M Tris, 0.192 Mglycine, and 0.1% SDS, pH 8.4. Separation was achievedat 40 mA in about 8 hr.Apolipoprotein H is Not Affected by In Vitro Glycosylation 175action was carried out in the presence of either potassiumthiocyanate or sodium lauryl sulfate in sodium bicarbon-ate buffer at pH 8.5. The absorbance was read at 335 nmagainst blank.2.7. HPLC Ion-Exchange ChromatographyIEC of the apoH was performed by using an anion-exchange column HEMA-IEC Bio 1000 DEAE (7.5x75mm) (Alltech, Milan, Italy) connected to a BeckmanHPLC system. ApoH was applied to the anion column in20 mM Tris, 0.01% NaN3, pH 8.0. The protein waseluted with a linear gradient consisting of 20 mM Tris,0.01% NaN3 pH 8.0, and 20 mM Tris, 0.01% NaN3 1 MNaCl, pH 8.0, in 30 min. The absorbance was monitoredat 280 nm.2.8. Dot Blot of FractionsRapid screening of HPLC fractions was conductedin the Bio-Dot microfiltration apparatus (Bio-Rad). Asheet of nitrocellulose is clamped between the gasket andthe 96-well sample template. Fifty ul of sample is al-lowed to filter through the membrane. After the antigenis immobilized, the nitrocellulose is incubated in 4% al-bumin blocking solution. Nitrocellulose is subsequentlyincubated with rabbit IgG anti-apoH at a 1/2000 dilutionat room temperature for 1.5 hr and then with goat IgGanti-rabbit IgG labeled with alkaline phosphatase at a1/6000 dilution. Dots are visualized with the procedurecontained in the Immun-Blot Assay kit (Bio-Rad).Fig. 1. SDS-PAGE gel electrophoresis of Apo H after incubation ofglucose. Lanes 1 and 10: molecular mass markers are (from top tobottom) 97.4, 66.2, 42.7, 31.0, 21.5, 14.4 kDa. Lane 3 contains a freshsample of apoH; lane 4 contains apoH incubated in the presence ofglucose; lane 5 contains apoH incubated in the absence of glucose.These samples were run in the presence of B-mercaptoethanol. Lanes7-9 contain the same samples run in nonreducing conditions.ApoH incubated in the presence of glucose had no in-crease in electrophoretic mobility when compared withapoH incubated in the absence of glucose. From the pic-tures it is also evident that no proteolytic cleavage hadoccurred during the incubation at 37°C for 7 days. Wethought SDS-PAGE was unable to detect slight differ-ences in molecular weight even when the samples wererun under reducing conditions.3. RESULTSGlucose incorporation into apoH was performed byincubating 0.1 mg/ml of apoH with 0.2 M glucose inPBS, pH 7.4, at 37°C for 7 days. After this incubation,apoH was run through an SDS-polyacrylamide slab gelin order to detect any variation in molecular weight.Figure 1 shows that the molecular weight of glyco-sylated apoH did not differ from that of apoH incubatedunder the same conditions in the absence of glucose.Lane 3 contains a fresh sample of apoH; lane 4 containsapoH incubated in the presence of glucose; lane 5 con-tains apoH incubated in the absence of glucose. Thesesamples were run in the presence of B-mercaptoethanol.Lanes 7-9 contain the same samples run in nonreducingconditions.Figure 2 illustrates the Western blot of the gel dis-played in Fig. 1 and probed with the specific antibodies.Fig. 2. Western blot of apoH probed with the specific antibody. Fromleft to right: Lanes 1 and 10: prestained molecular mass markersmarked with black points are (from top to bottom) 106.0, 80.0, 49.5,32.5, 27.5, 18.5 kDa. Lane 3 contains a fresh sample of apoH; lane 4contains apoH incubated in the presence of glucose; lane 5 containsapoH incubated in the absence of glucose. These samples were run inthe presence of B-mercaptoethanol. Lanes 7-9 contain the samesamples run in nonreducing conditions.176 Gambino, Ruiu, Pagano, and CassaderThe presence of modified amino groups was as-sessed with the trinitrobenzensulfonic acid assay. Thetrinitrobenzensulfonic reagent reacts rapidly with theamino groups of proteins. The reaction was performed inthe presence of either potassium thiocyanate or sodiumlauryl sulfate in sodium bicarbonate buffer at pH 8.5. Ineither case there was no difference in absorbance be-tween apoH incubated with glucose and apoH incubatedwithout glucose.Then, apoH incubated with glucose and apoH incu-bated in the absence of glucose were analyzed throughIEF followed by Western blot. The IEF result is shownin Fig. 3. Lane 1 contains a fresh sample of apoH; lane2 contains apoH incubated in the presence of glucose;lane 3 contains apoH incubated in the absence of glu-cose. All the samples show the same number and type ofisoforms. The isoelectric point of apoH isoforms was notaltered after the incubation with 0.2 M glucose. If thenonenzymatic glycosylation were successful, the posi-tive charge of lysine residues would have been neutral-ized by glucose. ApoH isoforms should have moved to-ward more-anodic isoelectric points. The missing shift inisoelectric point reveals that it is unlikely for apoH to be-come nonenzymatically glycosylated.The affinity for the anion-exchange column HEMA-IEC did not change after incubating apoH with glucose.Glycosylated apoH should have had greater affinity forthe anion-exchange column than native apoH. Both apoli-poproteins had symmetrical elution peaks and did not ex-hibit any chromatographic heterogeneity. Identificationof the peak related to apoH was performed through a dot-blot test (data not shown).4. DISCUSSIONOur results indicate that the in vitro nonenzymaticglycosylation has no effects on the physical properties ofapoH, despite the fact that this apolipoprotein contains ahigh number of lysine residues. The different chro-matography techniques reveal no apparent modificationoccurring on the apoH molecule. ApoH incubated in thepresence or in the absence of glucose had similar elec-trophoretic mobility when compared after SDS-PAGEunder nonreducing conditions. In the presence of reduc-ing agents such as (B-mercaptoethanol, the mobility ofboth samples was higher, but they did not differ fromeach other. Additionally, both samples had similar affin-ity for the anion-exchange column, which was due to theintegral unmodified lysine residues. The unchanged elec-trophoretic mobility and the similar affinity are in agree-Fig. 3. Western blot of apoH analyzed through IEF. From left to right:Lane 1: native apoH. Lane 2: ApoH incubated in the presence of glucose.Lane 3: ApoH incubated in the absence of glucose.ment with the result of IEF, where apoH incubated in thepresence of glucose shares the same pattern of isoformswith apoH incubated in the absence of glucose. Afterglycosylation of lysine residues apoH should lose its pos-itive charge and become more negative. As a result,apoH should move toward more-anodic regions and witha different pattern of isoforms. The failure for apoH toApolipoprotein H is Not Affected by In Vitro Glycosylation 177shift toward anodic regions means that apoH is not proneto becoming nonenzymatically glycosylated under theseconditions. Since the in vitro concentration of glucose(0.2 M) was far higher than the levels normally found indiabetic subjects, it is unlikely for apoH to become gly-cosylated in diabetes. This is probably caused by the factthat lysine residues lie in the inner side of apoH mole-cules and are not accessible for glucose.Many studies have demonstrated that in diabeticplasma, apoAI, All, B, CI, E, and albumin are glyco-sylated (Curtiss and Witztum, 1985; Garlick and Mazer,1983). It was demonstrated that the modification of lysine525 altered the conformation of albumin and in turn al-tered its ability to bind bilirubin and long-chain fattyacids (Shaklai et al., 1984). The glycosylation of proteinscould alter protein-lipid interactions. The important roleof lysine residues in apoB function and the fact that gly-cosylation can alter this function has been amply demon-strated (Witztum et al., 1982; Gonen et al., 1981; Makinoet al., 1995). On the contrary, apoH can retain its func-tions even if glucose levels are too high in plasma. Itsability to activate lipoprotein lipase both in vitro andin vivo could make apoH play a key role in modulation oftriglyceride metabolism in diabetes. Further studies areunder way to define more precisely the relationship be-tween plasma glucose concentration and the extent ofapoH glycosylation and as well as its functional role indiabetes.ACKNOWLEDGMENTSThis work was supported in part by grants fromthe Consiglio Nazionale delle Ricerche, Rome (Grantno. 95.01015.PF40, Progetto Finalizzato INVECCHI-AMENTO) and the Ministero dell'Universita' e dellaRicerca Scientifica e Tecnologica (Quota 60%), Rome,Italy.REFERENCESBowie, A., Owens, D., Collins, P., Johnson, A., and Tomkin, G. H.(1993). 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Apolipoprotein H is not affected by in vitro glycosylation.

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Apolipoprotein H is not affected by in vitro glycosylation.

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