Pre-Beta High Density LipoproteinUnique Disposition of Apolipoprotein A-l Increases

Susceptibility to Proteolysis

Steven T. Kunitake, G. Chi Chen, Sue-Fang Kung, James W. Schilling,David A. Hardman, and John P. Kane

Apolipoprotein A-l-contalnlng llpoprotelns (high density llpoproteins, HDL) can beseparated Into two subtractions, which have pre-beta and alpha electrophoreticmobilities, respectively. These fractions differ In both composition and structure.Some preparations of pre-beta-mlgratlng HDL, but not alpha-migrating HDL, werefound to contain two polypeptldes with Mr of approximately 26 and 14 kDa, which arescission products of apolipoprotein (apo) A-l. They are recognized by monospeclflcantibodies to apo A-l and have ^-terminal sequences Identical to those of matureapo A-l. This proteolytic scission of apo A-l occurs primarily after venlpuncture.Immediate addition of protease Inhibitors minimized the appearance of the fragmentsIn plasma. To study the relative susceptibilities of pre-beta and alpha HDL toproteolysis, the llpoprotelns were Incubated In vitro with plasmln. The apo A-l Inpre-beta HDL was extensively degraded, but that In alpha-migrating HDL wasdegraded to a much lesser extent, Indicating that the appearance of apo A-l fragmentsIn pre-beta HDL was due to enhanced sensitivity to proteolysis. To varying degrees,thrombln, kallikreln, elastase, arglnlne C endoprotease, and chymotrypsin alsoappear to cleave pre-beta HDL faster than alpha HDL Most of the proteasesgenerated a 12 to 14 kDa peptlde fragment under conditions of limited cleavage.These results suggest that the conformatlonal state of apo A-l In pre-beta-mlgratlngHDL or Its spatial relationship to llplds Is significantly different from that of apo A-l Inalpha-migrating HDL. Furthermore, this conformation of apo A-l appears to expose aprotease-sensltive region near the midpoint of the sequence. Finally, when studies ofpre-beta HDL are undertaken, care should be taken to prevent proteolytic degradationof this particle. (Arteriosclerosis 10:25-30, January/February 1990)

H igh density lipoproteins (HDL) are a collection ofsubspecies with different compositions and proper-

ties. One such HDL subpopulation has pre-beta electro-phoretic mobility (pre-beta HDL).1 This group of lipopro-teins has a composition that is distinctly different from thebulk of alpha-migrating HDL.2 Pre-beta HDL also appearto possess structural properties that distinguish themfrom the bulk of HDL.2 The in vitro proteolytic degradationof apolipoprotein (apo) A-l has been demonstrated for theenzymes plasmin,34 thrombin,4 trypsin,5'6 and chymotryp-sin.5 The degradation of apo A-l on intact HDL has alsobeen studied,8-6 allowing the identification of exposed andprotected regions. But no apparent differences in the pro-teolytic degradation of HDL fractions HDLj, and HDU have

From the Cardiovascular Research Institute and the Depart-ment of Medicine, University of California, San Francisco, andCalifornia Biotechnology, Incorporated, Mountain View, California

This research was sponsored In part by the American HeartAssociation, California Affiliate, Sain Bernardino, Marin, andContra Costa Chapters; National Institutes of Health Grants HL31210 and HL 14237 Arteriosclerosis SCOR; and grants from theWine Institute and the Diabetes Research Foundation. DavidHardman is a recipient of an award from the ARCS (AchievementRewards for College Scientists) Foundation, Northern CaliforniaChapter.

Address for reprints: Steven T. Kunitake, Ph.D., Cardiovascu-lar Research Institute, University of California, San Francisco, CA94143.

Received March 6, 1989; revision accepted July 27, 1989.

been determined. We describe here the enhanced sensi-tivity of the apo A-l in pre-beta HDL to in vitro proteolyticcleavage when compared to alpha HDL

MethodsIsolation of Plasma

Plasma was isolated from freshly drawn venous blood bycentrifugation at 10 000 g for 30 minutes at 4°C in thepresence of preservatives and inhibitors, 0.04% ethylene-diaminetetraacetjc acid (EDTA), 0.05% NaN3, 1 ^g/mlgentamycin, 0.3 mg/ml benzamidine, 1 mM PMSF,0.13% eamino caproic acid, and 10 ^g/ml alpha2 macro-globulin, final concentrations.

Isolation of Pre-Beta- and Alpha-migrating HighDensity Llpoprotelns

Immunosorbed HDL were isolated by selected-affinityimmunosorption, as described previously.910 Briefly, apo A-l-containing lipoproteins (immunosorbed HDL) were recov-ered from plasma by retention on an anti-apo A-I-Sepharosecolumn and were eluted by 0.2 M acetic acid, pH 3.0. ThepH of the eluate was adjusted immediately to 7.0 with 2 MTris, pH 10.0. The antibodies used to make the selectedaffinity column were isolated through the use of anapo A-I-Sepharose column and the same elution buffer.After concentration and dialysis, the immunosorbed HDL

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B

Figure 1. Electrophoretic separation of apoproteins (apo) frompre-beta high density lipoprotein (HDL). A. Sodium dodecyl sutfate5% to 25% polyacryiamlde gel electrophoresls of two differentpre-beta HDL preparations compared to a set of molecular weightstandards and stained with Coomassie blue. B. Autoradiograph ofan immunoblot of the transferred proteins from a similar gel. Amonospecific anti-apo A-1 antisera was used for the identification ofprotein bands.

were passed through protein A-Sepharose and anti-human serum albumin-Sepharose columns to removecontaminating immunoglobulins and albumin. The pre-beta and alpha subpopulations of HDL were separated bystarch block electrophoresis, as previously described.1

Potato starch was hydrated in 50 mM barbital buffer, pH 8.6,and was formed into an 8x50 x 1 cm block. ImmunosorbedHDL were applied to the block 5 cm from the anodic end,and a constant current of 65 mA was applied through theblock for 14 hours. The block was then divided into 1 cmsegments in the direction of migration, and the apo A-lcontent of each fraction was determined by immunonephe-tometry. Appropriate fractions were pooled and eluted torecover pre-beta- and alpha-migrating HDL

Protease Incubation of HighDensity Upoproteln

Aliquots of 25 yjQ of pre-beta or alpha HDL protein in 80 /dof reaction buffer (10 mM Tris, pH 8) were incubated at 4°C inthe presence or absence of 14 mUnits purified plasmin(Boehringer-Mannheim, West Germany). After 1 or 12 hours,the incubation was terminated by the addition of decyt sutfateto a final concentration of 0.1 %, and the samples were boiledfor 1 minute. Protease digestions by 2 /Ag elastase(Boehringer-Mannheim, West Germany), 0.18 U kallikrein,1.2 U thrombin (Sigma, St Louis, MO), 10 tug chymotrypsin(Sigma), or 1.0 U arginine-C endoprotease (Sigma) wereperformed as described for plasmin, except that the incuba-tions were for 0.5 to 1 hours at room temperature.

92.5

66.2

45.0

31.0

21.5

14.4

BRgure 2. Plasmin digestion of pre-beta- and alpha-migrating high density lipoprotein (HDL). Alphaand pre-beta HDL were incubated for 1 hour at 4°C with or without plasmin. The apoproteins wereanalyzed by sodium dodecyl sutfate 5% to 25% potyacrytamide gel electrophoresls. A. Alpha HDLwithout plasmin. B. Alpha HDL with plasmin. C. Pre-beta HDL without plasmin. D. Pre-beta HDL withplasmin. An amount of sample equivalent to 5 fig of undigested protein was applied to each lane.

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PRE-BETA HIGH DENSITY LIPOPROTEIN Kunrtake et al. 27

92.5

66.2

45.0

31.0

BFigure 3. Extended plasmin digestion of pre-beta- and alpha-migrating high density llpoproteln(HDL). Alpha and pre-beta HDL were incubated for 12 hours at 4°C wtth or without plasmin. Afterincubation, the apoproteins were analyzed by sodium dodecyl sutfate 5% to 25% polyacrytamlde gelelectrophoresis. A. Alpha HDL without plasmin. B. Alpha HDL wtth plasmin. C. Pre-beta HDL withoutplasmin. D. Pre-beta HDL with plasmin. An amount of sample equivalent to 5 /ig of undigested proteinwas applied to each lane.

Analysis of ProteinsThe apoproteins were analyzed by electrophoresis in

discontinuous 5% to 25% gradient gels in the presence of0.1% sodium dodecyl sulfate (SDS), 0.025 M Tris, 0.192 Mglycine (pH 8.9) buffer.11 The samples were prepared byaddition of an equal volume of solubilizing buffer containing4% SDS, 0.125 M Tris, and 20% glycerol (pH 6.8) and wereboiled for 30 seconds. In all gels, each lane contained anamount of protein equivalent to 5 /ig based on the proteincontent measured before incubation with proteases. Afterelectrophoresis, the gels were either stained with Coomassieblue R 250 or electrophoreticalry transferred to nitrocellu-lose sheets.12 The transferred proteins were analyzed byreaction with anti-apo A-l antisera and 126l-labeled protein A.

The apo A-l fragments were separated by high-performance liquid chromatography (HPLC) on a Mono Qion-exchange column (Pharmacia, Uppsala, Sweden). Thepeptides were eluted with a 0.0 to 0.3 M NaCI gradient in thepresence of 6 M urea and 0.005 M Tris (pH 7.4). Theisolated proteins were then subjected to amino acidsequence analysis on an Applied Biosystems model 470 Aprotein microsequencer.13

ResultsThe protein patterns observed in several preparations of

pre-beta HDL obtained from several donors at differenttimes fell into two groups. Some preparations contained

predominantly intact apo A-l, while others also containedproteins of approximately 26 kDa and 14 kDa, along withtraces of other bands (Figure 1 A). These three bands weredetected by immunoblotting with anti-apo A-l antisera (Fig-ure 1B), indicating that the 26 kDa and 14 kDa bands werefragments of apo A-l. Indeed, after isolation of the 28, 26,and 14 kDa bands by HPLC, it was determined that the/V-terminal sequence (4 amino acids) of each band wasidentical to that reported for mature apo A-l. At the sametime, no 26 or 14 kDa fragments were found in concurrentpreparations of alpha HDL

The degradation of apo A-l in pre-beta HDL appearedto occur after blood drawing. We found that the appear-ance of the proteolytic fragments in pre-beta HDL couldbe minimized by the addition of protease inhibitors,especially alpha2 macroglobulin, to blood immediatelyafter phlebotomy. The degree of degradation did notappear to be dependent upon a subject's plasma lipid orlipoprotein contents. In fact, the degradation of pre-betaHDL from a single subject was variable, depending uponthe length of time the sample was unprotected by prote-ase inhibitors. The progression of degradation of a singlesample was found to be a function of storage time withoutprotection by protease inhibitors.

To investigate this enhanced sensitivity of pre-beta HDL toproteolytic degradation, we performed a series of in vitroincubations of the isolated HDL subtractions with plasmin.

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Figure 4. Limited digestion of alpha and pre-beta high density llpoprotein (HDL) by variousproteases. Sodium dodecyl surtate 5% to 25% polyacryiamide gel electrophoresis of alpha (Lanes 2,4,6,8,10, and 12) and pre-beta (Lanes 3,5, 7,9,11, and 13) HDL apoproteins applied in tandem afterlimited incubation: 0 Alone (2, 3). T. With thrombln (4, 5). K. With kallikrein (6, 7). E. With elastase (8, 9).A. With arginine-C endoprotease (10,11). C. With chymotrypsin (12,13). The migration of the proteins inLanes 12 and 13 was slightly impeded due to the presence of Ca++ in the reaction mixture. Theapproximately 40 kDa band found in Lanes 4 and 5 was thrombin or a protein found in the thrombinpreparation used. A standard mixture of proteins was applied (1) for comparison. An amount of sampleequivalent to 5 ̂ g of undigested protein was applied to each lane.

After an incubation of 1 hour, plasmin had a modest effect onthe apo A-l of alpha HDL, creating a light band at approxi-mately 14 kDa However, the apo A-l of pre-beta HDL wasdegraded, broadening the band at 28 kDa and forming aprominent band at approximately 14 kDa (Figure 2). Belowthe lower band, a faint zone of staining material appeared,probably representing the degradation products of theC-terminal portion of apo A-l. After 12 hours, the 28 kDa apoA-l of alpha HDL did not appear to undergo further degrada-tion, whereas intact apo A-l had disappeared from pre-betaHDL, leaving only faint bands (Figure 3). Since equalamounts of protein were anaryzed, the decrease in theintensity of the apo A-l band truly reflects degradation of thisprotein. Interestingly, prolonged incubation of alpha HDLwith plasmin did cause the disappearance of apo A-ll and the14 kDa fragment of apo A-l, yet no further degradation of apoA-l was observed. This suggests that in alpha HDL, only aportion of the total apo A-l is susceptible to plasmin degra-dation under the conditions used. As demonstrated by theseresults, the apo A-l of pre-beta HDL appears to be moresensitive to proteorysis by plasmin when compared to theapo A-l of alpha HDL

The small differences in mobility of undegraded apo A-lobserved in Figures 2, 3, and 4 appear to be due tovariations in interlane migration. The /V-terminalsequences of the apo A-l in both alpha HDL and pre-betaHDL were found to be identical. When the proteins ofalpha and pre-beta HDL were mixed and subjected to

electrophoresis, there was only one apo A-l band visibleand no broadening of this band was detected.

Pre-beta and alpha HDL were also subjected to degra-dation by thrombin, kallikrein, elastase, arginine C endopro-tease, and chymotrypsin (Figure 4). To varying degrees, theapo A-l in pre-beta HDL appears to be more sensitive tocleavage by most of these proteases than the apo A-l inalpha HDL, under conditions of limited digestion. Throm-bin and kallikrein both generated a low molecular weightfragment from pre-beta HDL apo A-l, whereas no degra-dation of alpha HDL was apparent. Elastase and chymo-trypsin extensively digested pre-beta HDLwith lesserdegrad-ation of alpha HDL In most cases, proteolytic degradationof pre-beta HDL led to the formation of a 12 to 14 kDafragment, suggesting that a region of hypersensitivity toproteolysis exists when apo A-l assumes the conformationor exposure that it possesses in pre-beta HDL

DiscussionWe have demonstrated that the apo A-l of pre-beta

HDL has a much greater sensitivity to proteolytic cleav-age by plasmin and several other endoproteases thandoes the apo A-l of alpha HDL. This implies that theapo A-l exists in a different environment or conformationon pre-beta HDL than on alpha HDL Pre-beta HDLcontain 90% protein, which is predominantly apo A-l.1

The unique composition of pre-beta HDL apparentlygives the particles a structure that is quite different from

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PRE-BETA HIGH DENSITY LIPOPROTEIN Kunitake et al. 29

that of the bulk of HDL. Specifically, the helicity of apo A-lin pre-beta HDL is significantfy less than that of apo A-l inalpha HDL.2 Also the lipid-poor environment found inpre-beta HDL may increase the exposure of a protease-sensitive region on apo A-l. Previous studies have shownthat isolated apo A-l is degraded by plasmin,34thrombin,4

trypsin,5'6 and chymotrypsin6 in vitro, consistent with thispossibility.

The proteotytic cleavage of apo A-l in pre-beta HDLthat we found appeared to occur after phlebotomy,because immediate addition of protease inhibitors mini-mized the formation of fragments. The original inhibitioncocktail contained the antibiotics, azide and gentamycin,which suggests that the cleavage is not due to microbialproteases but is caused by protease activity found inblood. Such an event would be analogous to the cleav-age of apo B-100 by kallikrein.141518 The exact proteaseresponsible is unknown; however, possible candidateproteases include an elastase-like metalloprotease thathas been described in association with ultracentrifugedHDL,171819 as well as the serine proteases involved in theclotting cascade and in thrombolysis.

While the proteolysis we described occurred during invitro incubations, the cleavage may have physiologicalimportance. Bachorik et al.20 have observed that whenHDL are exposed to cultured hepatocytes, the apo A-l isdegraded into fragments similar to those found in pre-betaHDL in this study. Furthermore, Gregg and associates21

have recently shown that apo A-l partially degraded toapproximately 26 kDa is removed from circulation morerapidly than nondegraded apo A-l. Since degradation ofpre-beta HDL appears to yield such fragments, it is possi-ble that pre-beta HDL might comprise a special kineticcompartment of apo A-l with increased turnover.

Initial cleavage of the apo A-l on pre-beta HDL, and tosome extent alpha HDL, by plasmin as well as severalother proteases generated apo A-l fragments of approx-imately 12 to 14 kDa. This region of cleavage spansamino acid residues 100 to 120 and may correspond tothe so-called "hinged domain" that has been hypothe-sized to explain observed quantized changes in HDLdiameter.22 The hypothesis suggests that this domainbecomes excluded from the surface monolayer of an HDLparticle as its diameter decreases. Such a conformationalchange might expose this region to proteolytic cleavage.

The finding that degradation of apo A-l in pre-beta HDLoccurs and can be minimized by the immediate additionof protease inhibitors indicates that future studies directedat the quantrtation or analysis of this lipoprotein shouldinvolve protection against proteolytic cleavage of apo A-l.

AcknowledgmentsWe gratefully thank Virginia H. Donaldson for her gift of

kallikrein and Ram Blau (Granny Goose, Oakland, CA) for thepotato starch.

References1. Kunitake ST, La Sala KJ, Kane JP. Apolipoproteln A4-

containing lipoproteins with pre-beta etectrophoretic mobility. JUpid Res 1985;26:549-555

2. Kunitake ST, La Sala KJ, Mendel CM, Chen GC, Kane JP.Some unique properties of apo A-l-containing lipoproteinswith pre-beta electrophoretlc mobility. In: Uppel K, ed.Proceedings of the workshop on lipoprotein heterogeneity.Washington, DC: NIH publication no 87-2646,1987:419-427

3. Ujnen HR, Collen D. Degradation of the apoprotein A-lpolypeptide chain of human high density lipoprotein byhuman plasmin. Thromb Res 1981;24:151-156

4. Bausaerman LL, Herbert PN. Degradation of serum amy-loid A and apolipoprotelns by serum proteases. Biochemis-try 1984;28:2241-2245

5. Shore VG, Sae AS-W, Shore B. Surface exposure ofapolipoproteins in high density lipoproteins. I. Reactivitieswith agarose-immobilized proteases. Biochim Biophys Ada1978;529:319-330

6. Byrne RE, Scanu AM. Soluble and immobilized trypsin asstructural probes of human plasma high-density lipopro-teins: enzyme properties and kinetics of proteotysls. Bio-chemistry 1983;22:2897-29O3

7. Jeng I, Steelman R, Rellly P, Jong Y, Schorrfeld G. Thedistinction between the exposed regions and the buriedregions of apoproteins in high density lipoproteins by theirreactivities with pronase. Blochem Biophys Res Commun1980:92:876-882

8. Swaney JB. Selective proteolytic digestion as a method forthe modification of human HDL3 structure. J Lipid Res1983:24:245-252

9. Kunitake ST, McVlcar JP, Hamilton RL, Kane JP. Isolationof human high density lipoproteins by immunoaffinity chro-matography. Circulation 1982;66(suppl ll):ll-240

10. McVlcar JP, Kunitake ST, Hamilton RL, Kane JP. Charac-teristics of human lipoproteins isolated by selected-affinityImmunosorption of apolipoprotein A-l. Proc Natl Acad SciUSA 1984:81:1356-1360

11. Laemmli UK. Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature 1970;227:680-685

12. Towbln H, Staehelln T, Gordon J. Electrophoretic transferof proteins from polyacrylamide gels to nitrocellulose sheets:procedure and some applications. Proc Natl Acad Sci USA1979:76:4350-4354

13. Hunkaplller MW, Hood LE. Analysis of phenylthiohydan-tions by ultrasensitive gradient high performance liquidchromatography. Meth Enzymol 1983:91:486-493

14. Cardin AD, Witt KR, Chan J, Margolis AS, Donaldson VH,Jackson RL Degradation of apolipoprotein B-100 of humanplasma LDL by tissue and plasma kallikreins. J Biol Chem1984;259:8522-8528

15. Hardman DA, Gustafson A, Schilling JW, Donaldson VH,Kane JP. Scission of human apolipoprotein B-100 by kal-likrein: Characterization of the cleavage site. Biochem Bio-phys Res Commun 1986;137:821-825

16. Gustafson A, Kane JP, Havel RJ. Determinants of kallikreinproteolysis of apolipoprotein B-100 in human blood plasma.Eur J Clin Invest 1988;18:75-80

17. Saklatvala J. Hydrolysis of the elastase substrate succlnyi-trialanlne nltroanilide by a metal-dependent enzyme in rheu-matoid synovial fluid. J Clin Invest 1977;59:794-801

18. Jacob MP, Bellow G, Robert L et al. Elastase-type activityassociated with high density lipoproteins in human serum.Biochem Biophys Res Commun 1981;103:311-318

19. Maeda H, Kobou S, Uzawa H. Hydrolysis of succlnyttriala-nine /P-nitroanilide by two enzymes associated with humanhigh-density lipoproteins. Arch Biochem Biophys 1983;226:629-635

20. Bachorik PS, Franklin FA Jr, Virgil DG, Kwrterovlch POJr. Reversible high affinity uptake of apo E-free high densitylipoproteins In cultured pig hepatocytes. Arteriosclerosis1985;5:142-152

21. Gregg R, Zech LA, Bojanovskl D, et al. Tangier disease:identification of normal and rapidly catabollzed forms ofplasma apolipoprotein A-l (abstract). Circulation 1985;72(suppl l

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22. Cheung MC, Segrest JP, Alters JJ, et al. Characterization single vertical spin urtracentrtfugatlon and Immunoaffinityof high density llpoprotein subspecies: structural studies by chromatography. J Upid Res 1987;28:913

Index Terms: pre-beta high density lipoproteins • apolipoprotein A-l • alpha high density lipoproteins •proteolysis

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S T Kunitake, G C Chen, S F Kung, J W Schilling, D A Hardman and J P Kanesusceptibility to proteolysis.

Pre-beta high density lipoprotein. Unique disposition of apolipoprotein A-I increases

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