RESEARCH 15; j73-579 Press Ltd.1979. Printed

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THE EFFECT OF PLASMIN ON HUMAN PLASMA LOW DENSITY LIPOPROTEIN J.

C.

H.

Steele,

Jr.

Departmentof Biochemistry, Medical Center Research Institute,

Duke University and Whitehead Medical Durham, N. C. 27710 U.S.A.

(Received 2.10.197&; in revised form 2.4.1979 Accepted by Editor N. Niewiarowski)

INTRODUCTION Human serum contains a number of potential proteolytic activities, the primary roles of which have been well documented in blood coagulation, comHowever, the possibility that polyplement activation, and other systems. peptides not directly involved in such processes might also be affected by these proteases has not been extensively investigated previously. In the present work, the effect of human plasmin [EC 3.4.4.141 on the polypeptide moiety of human plasma low density lipoprotein (LDLl) has been examined as part of a study of the stability of this lipoprotein. LDL is a water-soluble complex consisting of protein, esterified and free cholesterol, phospholipid, triglyceride, and other minor lipids (1). The protein component, termed apolipoprotein B (apoB), consists of two polypeptides of approximate molecular weight 250,000 (2) and contains about 5% It is known to be susceptible by weight covalently-linked carbohydrate (3). to cleavage by several non-serum proteases including trypsin (4.5,6,7). Plasmin, like trypsin, is a serine protease that cleaves after basic amino acid residues but exhibits a greater substrate specificity (8). An inhibitory interaction between plasmin and LDL was suggested by some early reports However, protein cleavage with peptide release has purportedly been (9910). shown not to occur (11,12), and the interaction itself has been disputed (13). In view of the importance of the question of apoB proteolysis by serum proteases either physiologically or during --in vitro manipulation, the effect of plasmin on the protein moiety of intact LDL has been studied using sodium 1

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.

:

LDL,

low density

573

lipoprotein:

apoB apolipopro

ji$

PLAS?IIS

X&D PL.JISXA LDL

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dodecyl sulfate polyacrylamide gel electrophoresis to monitor the appearance of cleaved peptides during incubation of the actis-e prorease with the lipoprotein.

METHODS LDL was prepared from fresh human plasma of healthy adult volunteers by standard sequential ultracentrifugation techniques (14). The lipoprotein was isolated between the densities of 1.020 and 1.050 g per ml in NaBr/l.3 mM Na2EDTAf4 mMNaN3, pH 7.0 solutions. A single polypeptide, apoB, was present in the lipoprotein as evidenced by gel electrophoresis in sodium dodecyl sulfate. Human plasminogen. pure by gel electrophoretic criteria, was generously provided by Dr. J. Sodetr, while urokinase (B grade) was a Calbiochem product; each was separately dissolved in plasmin buffer (50 U&ITris HCl/ 10 mM L-lysine, pH 8.0) at concentrations of 6 mg per ml and 6000 Plough units per ml, respectively. Plasminogen was completely converted to plasmin just before use by adding 100 unfts urokinase per mg plasminogen and incubating at 37’C for 25 minutes. LDL in plasmin buffer was incubated at 37°C and appropriate aliquots of plasmin were added to achieve final enzyme concentrations of 0.2 to 1200 pg per ml. The final LDL concentration was 0.7 mg protein per ml, its normal serum concentration (15). At various times of incubation at 37”C, 80 ~1 aliquots were removed, added to 45 pl of a sodium dodecyl sulfate/2-mercaptoethanol solution (9.0 g sodium dodecyl sulfate and 11 ml 2-mercaptoethanol per 100 ml solution), and immediately heated to 100°C for one minute to inactivate the plasmin. After sixty minutes the incubation was ended and all samples were run on sodium dodecyl sulfate gels. As a control, LDL was incubated under identical conditions, including urokinase, exPlasmin samples treated under these concept that plasminogen was omitted. ditions showed only the plasmin heavy chain of approximate molecular weight The plasmin light chain of molecular weight 65.000 on gel electrophoresis. 25,000 was not seen, due to its autolysis during the incubation, nor were any plasminogen or other digestion products of other polypeptides evident--e.g., it. For gel chromatographic experiments LDL (0.9 mg protein in 500 ~1 plasmin buffer) was mixed with 1.5 mg urokinase-activated plasmin in 275 ~1 Then 100 ~1 of 4.4 mM diisoprobuffer and incubated at 37°C for 60 minutes. pyl-fluorophosphate (Sigma) in column buffer (50 mM Tris HC1/5 &I Na2EDTA/ 1 mM NaN3, ptl 8.0) was added to inactivate the protease. After adding two a 500 ~1 aliquot was applied to drops of glycerol to increase sample density, a 1.5 x 85 cm Sepharose 4B (Pharmacia) column and eluted in 1.5 ml fractions The absorbancy at 280 nm was monitored and samples from with column buffer. In a peak tubes were used for gel electrophoresis in sodium dodecyl sulfate. control experiment, LDL treated identically except for the omission of plasThe column’s void and internal min was chromatographed on the same column. volumes were determined using solutions of calf thymus deoxyribonucleic acid (Sigma) and Z-mercaptoethanol, respectively. In all experiments, protein concentrations were determined by the Lowry procedure using bovine serum albumin (Nutritional Biochemicals) as a standard (16); the assay was modified by adding sodium dodecyl sulfate to all samples at a final concentration of 70 mM to abolish interference due to lipid. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate was performed as detailed elsewhere (17) using 3.3% ac’rylamide gels. Protein bands were deAll chemicals without specified tected using Coomassie blue staining. sources were reagent grade products.

PLASMIN m

PLASMA

LDL

575

RESULTS Freshly prepared LDL, not shown, and urokinase-treatedLDL, Figure IA, appeared identicalon sodium dodecyl sulfate gels, showing one major band, with a few very minor bands of slightly faster mobility evident on overloaded gels. On the other hand, LDL Incubatedwith plasmin had a gel pattern indicating proteolysisof the apoB polypeptide. The normal serum concentration of plasmlnogenis about 200 )~gper ml; the extent to which it is converted to plasmin-in viva is not known, however (18). At plasmin concentrationsone hundred-foldless than this, some cleavage of apoB was observed (Figure 1B). As the incubationtime was lengthened (Figure X-D) or the plasmin concentration was increased (Figure 11)-G),the single major band seen in control LDL was cleaved first into two major bands; then as proteolysiscontinued,new bands of lower apparent molecular weight appeared while these higher apparent molecularweight bands disappearedfrom the gels. A minor band nearer the origin than uncleavedapoB was noted in gels of LDL samples treated with small amounts of plasmin (Figure lB-D); it was not seen in gels of control LDL or when higher plasmin concentrationswere used. Such aberrant behavior by a cleavage product could be due to aggregation,to abnormally low binding of detergent,or to an unusual shape--e.g.,a heavily glycosylatedpolypeptide (19). Gel-exclusionchromatography,Figure 2, of plasmin-treatedLDL showed that the modified lipoprotein,peak B-I, eluted at the same position as control LDL, peak A-I. On gel electrophoresisin sodium dodecyl sulfate, samples of peak B-I exhibited patterns identical to that in Figure lG, except that no plasmin heavy chain was present. This chain eluted as peak B-II, as shown by gel electrophoresis. Peak III, eluting in the internal volume of the column, was due to glycerol and/or the protease inhibitor,as it was not seen when these substanceswere omitted. The greater absorbancyof peak III in the plasmin-treatedsample suggests that It may also contain fragmentsof the light chain of plasmin and possiblyvery small peptides cleaved from apoB; however, gel electrophoresisof peak B-III samples showed no bands, ruling out the presence of larger polypeptides. DISCUSSION These results indicate that at least one serum protease possesses the potential to cleave the protein moiety of LDL at physiologicalconcentrations of each. This finding may help to explain the marked discrepancyregarding the number and size of the LDL polypeptidesreported by different laboratories. For instance,the two major bands of high apparent molecular weight seen in LDL samples treated with physiologicalconcentrationsof plasmln resemble the gel patterns described by some investigators(20,21),suggesting that limited degradationof the apoB polypeptldeby plasmin may have occurred during their lipoproteinisolationprocedures. A lack of precise experimental details precludedan exact repetitionof their isolation schemes to check this possibility. Such cleavage artifacts due to plasmin's action on other proteins have been reported (22). The observationthat plasmin can cleave peptide bonds of apoB in the intact LDL particle has additional implications. It indicates that such bonds are exposed at the lipoproteinsurface, as studies with other proteases and non-enzymaticmethods have shown (23). The fact that increasingconcentrationsof plasmin or longer incubationperiods produce peptides of decreasingmolecular weight suggests that there is a varying exposure of

576

PtiSHIX

X.VD PLAS.X& LDL

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FIG. 1 The gel electrophoreticpatterns of plasmin-treatedlow density lipoprotein are shown. A, control LDL. B-G, plasmin-treatedLDL incubated for 5 (C) or 60 (8, L+C) minutes with the following amounts of urokinase-activatedplasmin in a final volume of 600 ~1: B, 0.9 ~8: C-D, 9 pg; E, 91 yg; F 550 pg; and G, 740 )1g. H, calibrationstandards: xanthine oxidase (147,000daltons), phosphorylasea (94,000 daltons), bovine serum albumin (69,000daltons), ovalbumin (43,000daltons),%-chymotrypsinogen (25,700daltons), and lysotyme (14,300 daltons). Gels A-G each contain 60 pg of protein, gel H, 5 $g of each standard. The trackingdye location is marked with an India ink stab in each gel. An arrow indicates the plasmin heavy chain.

A

BCDEFG

H

A

QEO-

0.120 0.080 I

FIG. 2 The elution patterns on Sepharose 4B columns of control (A) or plasmintreated (B) lov density lipoprotein are shown as optical density at 280 nm versus grams eluate. Arrows mark the void and internalvolumes of the column.

1

0.040 %

0

I

;A

m

??

2

0.160-8 0.120 0.060 ;A c

1

0.040 I 0 40

60

nm

60 100 I20 140 g Eluote

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PLASMIN AND PLASm

LDL

577

susceptible peptide bonds, possibly the result of differing proximities of -the bonds to the lipoprotein surface or of masking due to constraints imposed by the intact polypeptide chain but released as proteolysis proceeds. The gel chromatographic experiment demonstrates that the major peptides produced by plasmin cleavage remain associated with the lipoprotein particle, as has been observed with trypsln-treated LDL (6,7); this implies that the apoB polypeptlde must be anchored at several sites along its length to the lipid matrix of the lipoprotein particle. The chromatographic experiments also provide an explanation for the negative conclusions regarding plasmin cleavage of apoB reported by others (11, 12), who used indirect methods dependent upon peptide release from the lipoprotein particle for detection of proteolysis. Thus conclusions (13) that LDL has no anti-fibrinolytic activity should be reexamined, for it appears that LDL can potentially act as a competing substrate for plasmin at physiological concentrations of each. Similarly, the proposal (9) that inhibition of fibrinolysis by increased serum concentrations of LDL or of other apoBcontaining lipoproteins contributes to the development and/or progression of atherosclerosis may possess some validity, in view of the demonstrated substrate status of apoB for plasmin. In attempts to elucidate the structure of LDL, some investigators have employed proteolytic digestion using trypsin (6,7) or subtilisin (24). Ultimately, such treatment of apoB may be useful in determining the amino acid It is of interest in this regard sequence of this very large polypeptide. that the gel patterns, and thus the polypeptides, shown in Figure 1 of plasmin-digested LDL are considerably different than those obtained using trypsin This observation may be of benefit in sequence anal(7) or subtilisin (24). ysis of apoB, especially since, given the more limited specificity of plasmin compared to trypsin (8). plasmin digestion may permit the isolation of overlap peptides to aid in aligning the products of a trypsin digest. Finally, the report (22) that many antiserum preparations contain plasmin raises questions regarding artifactual cleavage in any reported studies In which LDL was exposed to ixueunoglobulin preparations without proper precautions against proteolysis. This would include immunoprecipltation procedures for preparing LDL (25), ixnnunoelectrophoretic studies of LDL (26). and possibly immunoassay procedures for apoB-containing lipoproteins (27). Future ineeuuological studies of lipoproteins should therefore include addition of irreversible protease inhibitors, especially against plasmin, to all antiserum preparations before their use. ACRNOWLRD~NTS I wish to express my appreciation to Dr. J. A. Reynolds, in whose laboratory this work was performed, and to Dr. J. Sodetr., Dr. S. Pizza, and Dr. L. Fretto for the generous gift of plasminogen (J.S.) and for helpful discussions regarding serum proteases. This work was supported by Grant RL 14882 from the National Institutes of Health, J. A. Reynolds, Principal Investigator, .and by Predoctoral Fellowship S-TO-S-GM-01678 from the U.S. Public Health Service to J.C.H.S.

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REFERENCES 1.

SCANU, A.X., EDELSTEIN,C., and KEIM. P. Serum Lipoproteins. In: The Plasma Proteins. 2nd Edit., Vol. 1. F.W. Putman (Ed.) New York - LG don: Academic Press, 1975, p. 317.

2.

SMITH, R., DAWSON, J.R., and TANFORD, C. The Size and Number of Polypeptide Chains in Human Serum Low Density Lipoprotein. J. -Biol. Chem. 247, 3376, 1972.

3.

AYRAULT-JARRIER,M., CHEFTEL, R.I., and POLONOVSKI,J. Les Clucides de lap-Lipoprotbine Sf 1,063 O-12 du Serum Sanguin Humain. &&. g. Chim. Biol. 43, 811, 1961. --

4.

BANASZAK,L.J., and MCDONALD, J.J. The Proteolysisof Human Serum r-lipoproteins. Biochemistry1, 344, 1962.

5.

MARGOLIS,s., and LANCDON, R.G. Studies on Human Serum Pl-Lipoprotein: III. EnzymaticModifications. J. -Biol. Chem. 241, 485, 1966.

6.

YEAGLE, P.L., LANGDON, R.G., and MARTIN. R.B. Phospholipid-Protein Interactionsin Human Low Density LipoproteinDetected by 31P Nuclear Magnetic Resonance. Biochemistry16, 3487, 1977.

7.

TRIPLETT,R.B., and FISHER, W.R. ProteolyticDigestion in the Elucidation of the Structureof Low Density Lipoprotein. 2. Lipid Res. 19. 478, 1978.

8.

WEINSTEIN,M.J., and DOGLITTLE,R.R. DifferentialSpecificitiesof Thrombin,Plasmin and Trypsin with Regard to Syntheticand Natural Substratesand Inhibitors. Biochim. Biophys. Acta 258, 577, 1972.

9.

RIDING, I.M., and ELLIS, D. AntiplasminActivity of ,m-Lipoprotein. 2. Atheroscler.e. 4, 189, 1964.

10. SKRZYDLEWSKI,Z., and NIFWIAROWSKI,S. Influenceof Plasma Lipoproteins on the FibrinolyticActivity. -Thromb. Diath. Haemorrh. 17, 482, 1967. 11. SKBZYDLEWSKI,Z., NIEWIARGWSKI,S., and SKRZYDLEWSKA,J. Inhibitionof ProteolyticEnzymes by!-Lipoproteins. J. Atheroscler.&. 6, 273, 1966. 12. SMITH, E.B., MASSIE, I.B., and ALEXANDER, K.M. The Release of an Inunobilized LipoproteinFraction from AtheroscleroticLesions by Incubation with Plasmin. Atherosclerosis25, 71, 1976. 13. HERBERT, R.J., BENNETT, B., and GGSTON, D. Effect of Low-DensityLipoClin. Sci. Mol. Med. 45, 129, 1973. protein Preparationson Plasmin. --mm 14. HAVEL, R.J., EDER, B.A., and BRAGDGN, J.H. The Distributionand Chemical Compositionof UltracentrifugallySeparatedLipoproteinsin Human Serum. 2. Clin. Invest. 34, 1345, 1955. 15. LINDGRRN,F.T., and NICHOLS, A.V. Structureand Function of Human Serum Lipoproteins. In: The Plasma Proteins. 1st Edit., Vol. 2. F.W. PutPress, 1969, p. 1. man (Ed.) New York --Academic London:

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AXD PLASMA

LDL

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16. LOWRY, O.H., ROSEBROUGE,N.J., FARR, A.L., and RAiiALL, R.J. Protein Measurementwith the Folin Phenol Reagent. J. -Biol. Chem. 193, 265, 1951. 17. STEELE, J.C.H., Jr., and NIELSEN, T.B. Evaluationof Cross-Linked Polypeptidesin SDS Gel Electrophoresis. -Anal. Biochem. 84, 218, 1978. 18. ROBBINS, KC., and SUMMARIA, L. Human Plasminogenand Plasmin. In: Nethods in Enzymology. Vol. 19. G.E. Perlman and L. Lorand (Eds.) -YorkLondon: Academic Press, 1970, p. 184. 19. REYNOLDS,J.A., and TANFORD, C. The Gross Conformationof Protein-Sodium Dodecyl Sulfate Complexes. 2. -Biol. Chem. 245, 5161, 1970. 20. CHAPMAN, M.J., and KANE, J.P. Stability of the Apolipoproteinof Human Serum Low Density Lipoprotein: Absence of EndogenousEndopeptidase Res. Commun. 66, 1030, 1975. Activity. Biochem. Biophys.-21. SWANEY, J.B., and KIJEHL,K.S. Separationof Apolipoproteinsby an Acrylamide-GradientSodium Dodecyl Sulfate Gel ElectrophoresisSystem. Biochim. Biophys. Acta 446, 561, 1976. 22. BJERRUM, O.J., R&I&J, J., CLBNMRSEN,I., INGILD, A., and BPG-HANSEN, T.C. An Artefact in QuantitativeImmunoelectrophoresis of Spectrin Caused by ProteolyticActivity in Antibody Preparations. Stand. J. Immunol.4, Suppl. 2, 81, 1975. 23. LAGGNER, P. PhysicochemicalCharacterizationof Low Density Lipoproteins. In: Low Density Lipoproteins. C.E. Day and R.S. Levy (Eds.) New York - Loxn: Plenum Press, 1976, p. 49. 24. IKAI, A., and YAGISAWA, Ii. Proteolysisof Apoproteinsin Human Serum Low Density Lipoprotein. J. Biochem. (Tokyo) 81, 955, 1977. 25. KOSTNBR, G., and HOLASEK, A. Isolationof Human Serum Low-Density Lipoproteinswith the Aid of an Immune-SpecificAbsorber. Lipids 5, 501, 1970. 26. SALMON, S., AYRAULT-JARRIER,M., BEUCLER, I., and POLONOVSKI,J. Etude par Immuno&lectrophor&e Bidimensionnellede la DissociationSpontange des Lipoprodins de Basse Densitb. Biochimie 57, 1155, 1975. 27. SNIDERMAN,A., TENG, B., and JERRY, M. Determinationof B Protein of Low Density LipoproteinDirectly in Plasma. 2. Lipid Res. 16, 465. 1975.

The effect of plasmin on human plasma low density lipoprotein.

RESEARCH 15; j73-579 Press Ltd.1979. Printed THROMBOSIS @ Pergamon in Great Britain 004~-3848/7g/o~l5-Oj7~ BRIEF 802.00/O COMMUNICATION THE EF...
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