Further studies of glycoproteins from a cardiovascular connective tissue

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 1 0 4 , 1 9 - 2 6

(1964)

Further Studies of Glycoproteins from a Cardiovascular Connective TissueI B. R A D H A K R I S H N A M U R T H Y , A. F. FISHKIN, G. J. H U B B E L L AND G. S. BERENSON From the Departments of Medicine and Biochemistry, School of Medicine, Louisiana State University, New Orleans Received July 1, 1963 Glycoprotein material containing sialic acid was isolated from bovine aorta at O~176 by 0.15 M NaC1 extraction, (NH4)2SO4 fractionation, and zone electrophoresis on starch. Although the material was essentially homogeneous by free solution electrophoresis in several buffers at various pH values, electrophoresis on polyacrylamide gel resulted in a resolution into 4 fractions. Chemical analyses performed on 3 fractions indicated certain differences, for example, carbohydrate content. The compounds were capable of stimulating antibody formation, and immunologic studies further suggested that the number of antigenic components paralleled the resolution found on the gel. It is concluded that the isolation of a family of immunologically active aorta glycoproteins provides additional information in evaluating the complex physiology and biologic specificity of cardiovascular connective tissue. INTRODUCTION

Evidence that the "ground substance" of cardiovascular connective tissue has an intricate and complex molecular organization has been provided by the isolation of most of the known acid mucopolysaecharides and glycoprotein material from the aorta (1-3). These macromolecules which occur within the matrix of cardiovascular tissue are presumably important in preserving the integrity of the tissue. Yet, their precise role in maintaining vitality of such connective tissue has defied critical appraisal. It may be assumed, however, that some portion of the biologic specificity of cardiovascular tissue resides in substances comprising its matrix and that the glycoprotein components likely contribute to this specificity. The resolution, characterization of the glycoproteins, and determination of their specificity are necessary steps to unveil the physiologic roles of these complex chemical 1 Supported by the United States Public Health Service (H2942) and the Strickland Memorial Fund.

units found in cardiovascular connective tissue. The earlier studies to isolate a glycoprotein fraction involved methods which were admittedly harsh (4); therefore, milder procedures were adapted for extending those observations. An interesting finding of a number of glycoproteins was observed among which may be examples of genetically determined polymorphism that is suggested by chemical characterizations and immunologic studies. MATERIALS AND METHODS ISOLATION OF CRUDE GLYCOPROTEIN Fresh bovine aortas were obtained from a local abatoir and packed in ice for transport to the laboratory. All subsequent isolation procedures were performed at 0~176 The aortas were dissected free of extraneous tissue and then ground. Glycoprotein material was isolated from batches of 200 g. of the aortic tissue. The general procedure for isolation of the crude glyeoprotein is seen in Table I and differs considerably from that previously used (4). The essential steps after extraction consist of dialysis against distilled water to rid 19

20

RADHAKRISHNAMURTHY ET AL. TABLE 1 ISOLATION OF CRUDE GLYCOPROTEIN FROM BOVINE AORTA Aorta Extracted; 15 vol. 0.15 M NaC1, 48 hours Centrifuge Supernatant

Sediment (discard)

$

Dialyze-dist. H:O Centrifuge

i

Sediment (discard)

Supernatant Filter-celite P

Residue (discard)

Filtrate pH 4; acetate 0.01 M Centrifuge

I Sediment

Supernatant (NH4)2 SO~ 40% sat.; 16 hours Centrifuge f--

i

Supernatant

Sediment

$

(NH02 SO4 60% sat.; 16 hours)

Centrifuge

Sediment

Supernatant (NH4)~ SO4 sat.; 48 hours Centrifuge

I Crude glycoprotein of insoluble components at low ionic strength, adjustment to pH 4.0, and fractional precipitation with (NH0~SO4 . Glycoprotein material obtained at full saturation of (NH4):SO~ was dissolved in distilled water and dialyzed free from salts. The crude glycoprotein material was concentrated by electroconvection~ in a borate-sulfate buffer (pH 8.6, ~ = 0.1) for further study.

2 E-C 25 electrocoavection apparatus, E C Apparatus Corp., Swarthmore, Pennsylvania.

Supernatant

PURIFICATION BY ZONE ELECTROPHORESIS Purification of the crude glycoprotein was achieved by zone electrophoresis on starch using the borate-sulfate buffer. Details of this procedure are the same as described previously (4, 5). Fractions were displaced from centimeter sections of the starch and were analyzed for sialic acid by the diphenylamine reaction (6) and for polypeptide by biuret determinations (7). Selected samples were pooled, concentrated, subjected to repeat electrophoresis, and reisolated.

F U R T H E R STUDIES OF GLYCOPROTEINS

21

ANALYSES

TABLE II

Purified glycoprotein material was studied by free-solution electrophoretic analysis in a model 38A Perkin-Elmer apparatus using 2 ml. cells. A sedimentation analysis was performed in a model E Spinco ultracentrifuge. Chemical analyses were based on dry weight at 105~ and consisted of polypeptide determinations by a biuret method, sialic acid, hexosamine, total neutral carbohydrates, fucose, nitrogen, phosphorus, and uronic acid. The methods were the same as used before (4). Glucose was determined by a glucose oxidase method s on an acid hydrolysate of the glycoproteins. An amino acid composition for one fraction was determined in an automatic amino acid analyzer after hydrolysis with 6 N HC1 for 6 hours. Tryptophan and tyrosine were determined by ultraviolet absorption (8). Amino acid compositions were also studied by paper chromatography. Paper chromatography on Whatman No. 52 paper was used for qualitative identification of neutral sugars with ethyl acetate-pyridine-water (2:1:2) as the solvent system. The hexosamine composition was studied by ninhydrin-pyridine degradation (9) and by silicated glass paper chromatography using benzene-pyridine-borate buffer, pH 8.6, ~ = 0.05 (10:9:1) as the solvent system

ELECTROPHORETIC STUDIES IN VARIOUS BUFFERS

(10). ]~LECTROPHORESIS ON POLYACRYLAMIDE GEL 4

Even after attempts for final purification by repeated zone electrophoresis on starch blocks, a further fractionation was achieved by gel electrophoresis on polyacrylamide. 5 The same boratesulfate buffer at pH 8.6 was used, although it was diluted to u = 0.03. A similar resolution was found in phosphate buffer at pH 7.8, u = 0.03. The runs at 300 v. and 150 ma. were for approximately 105 minutes. In order to obtain fractionated material with this technique for further study, marker strips were sectioned longitudinally from the center and the lateral margins and were stained with amidoblack. The background of the marker strips was decolorized in methyl alcohol-acetic acidwater (5:1:5). These strips, with blue bands designating the position of the protein, were approximated to the unstained gel which was then 3 Glueostat, Worthington Biochemicals Corp., Freehold, New Jersey. 4 E-C 470 vertical gel electrophoresis cell, E-C Apparatus Corp. Cyanogum 41 gelling agent of American Cyanamid Co.

(/~ = 0.1) Buffer

pH

Descending mobility X 10~sq.cm./v./sec.

Veronal Borate-sulfate Phosphate-NaC1 Acetate

8.6 8.6 7.5 4.0

-7.3 -8.3 -6.4 -0.2

sectioned into selected horizontal segments. Protein fractions were isolated from the gel sections by extraction with buffer in a tissue grinder, centrifugation, dialysis, and pervaporation. Adequate material was obtained for 3 major fractions, although a trace of an additional fraction was demonstrated by the stain. (Further studies, omitting the starch electrophoresis, indicated the possibility of 6 fractions.) IMMUNOLOGIC STUDIES

Three materials at various stages of isolation were studied for antibody producing properties. These consisted of the dialyzed crude extract prior to (NH4)2SO4 fractionation, after starch block electrophoresis, and the major fraction obtained from the acrylamide gel. Rabbits were injected subcutaneously twice weekly for 4 weeks with approximately 2.5 mg. of the glycoprotein (based on polypeptide content) with and without adjuvants. Sequestered calcium alginate ~ (11) as an adjuvant resulted in much higher titers than injections without adjuvants or with Freind's adjuvant 7 (12). Sera were studied for antibody responses by means of precipitation in capillary tubes at various dilutions of antigen, diffusion from wells in agar plates, and by immunoelectrophoresis in agar on glass slides. RESULTS T h e results are i l l u s t r a t e d in T a b l e s I I a n d I I I a n d Figs. 1-5. S ev er al c a r b o h y d r a t e - p r o t e i n fractions c o n t a i n i n g uronic a n d sialic acids were o b s e r v e d a t c e r t a i n isolation steps, i.e., after dialysis w i t h H20, a n d w h e n t h e e x t r a c t s were b r o u g h t to p H 4.0 w i t h a c e t a t e buffer. T h e p r e c i p i t a t e s o b t a i n e d a t 40 a n d 60 % (NH4)2SO4 s a t u r a t i o n were essentially free Colab Laboratories, Inc., Chicago Heights, Illinois. Colorado Serum Co., Denver, Colorado.

22

RADHAKRISHNAMURTHY ET AL.

of sialic acid. All of these fractions are of considerable interest; however, the present studies were limited to material obtained at full saturation. The latter material was

FIG. 1. Moving-boundary electrophoretic studies in various buffers of bovine aorta gtycoprotein. The glycoprotein material was isolated by means of zone electrophoresis on starch. The illustrations represent runs at approximately 70 minutes at 10 ma., except for the acetate buffer shown at 46 minutes. The mobilities are listed in Table II.

negative for uronic acid and was readily soluble in water or buffers. The glycoprotein material obtained as precipitate at full saturation of (NH4)2SO4 represented 0.3 % of the original wet weight. After isolation b y repeated zone electrophoresis it was indicated to be homogeneous b y free-solution electrophoresis in borate-sulfate, phosphate, and acetate buffer systems. However, in veronal, p H 8.6, traces of a slower moving component were detected. Reproductions of these studies are shown in Fig. 1, with mobilities shown in Table II. An isoelectric point for the m a j o r material was estimated at p H 3.9. Sedimentation studies at 56,000 r p m at 25~ of a 1% concentration of the glycoprotein in borate-sulfate buffer showed a single peak until 56 minutes of centrifugation when a trace of a heavier component became apparent (Fig. 2). A sedimentation coefficient of 4.6 was observed for the major peak. Proteins with similar coefficients fall within a molecular weight range of 60,00070,000 (13). Since there were evidences of more than one fraction in the glycoprotein material, the problem of finer resolution was approaehed b y the technique of vertical gel electrophoresis on the acrylamide gel. This method has been found useful in resolving other proteins (14-16), and can be performed in a very simplified apparatus (17). The technique was employed as a preparative

FIG. 2. Ultracentrifuge studies of bovine aorta glycoprotein material which was isolated by repeated starch block eleetrophoresis in borate-sulfate buffer (t~ = 0.1). Each frame was recorded at 8-minute intervals.

FURTHER STUDIES OF GLYCOPROTEtNS

23

TABLE III ANALYSES OF GLYCOPROTEIN FRACTIONS FROM BOVINE AORTA Electrophoretic fractionation (Dry wt. %) Determination Zone (starca)

Polyacrylamide gel fractions I

FIG. 3. Polyacrylamide gel electrophoretic studies of aorta glycoprotein material. The material was isolated first by starch block zone electrophoresis. A sample containing 40 rag. of material was placed in a wide slot and nfigrated for 105 minutes in a borate-sulfate buffer, pH 8.6, = 0.03. The marker strips are shown at the center and lateral margins. Fractions I, II, and I I I were obtained from adjacent, unstained gel sections.

procedure as described above and is illustrated in Fig. 3. The chemical analyses (averages of replicate determinations) of the 3 more rapidly moving fractions are shown in Table I I I in comparison with the material isolated from starch. Analytical differences in the individual fractions are indicated. Nitrogen determinations from blank gel extracts gave evidence of residual N contamination, and, therefore, these determinations of glycoprotein from the gels are not reported. The major component which represented approximately two-thirds of the total materials isolated from the gel is shown as Fraction I I I . F r o m this fraction, some 18 amino acids could be demonstrated by paper chromatography and with an automatic amino acid analyzer. Chromatographic studies revealed the presence of only glucosamine as the only hexosamine in the 3 fractions obtained for study, as well as in the material isolated on starch. The constituent neutral sugars of the individual fractions, however, showed certain variations, for example, manuose was not found in Fraction I (Fig. 4). Glucose, accounting for approximately 10% of the total neutral sugars, was observed in material isolated by gel electrophoresis, omitting starch electrophoresis. These

78.6 Polypeptide (biuret) 2.1 Sialic acid (diphenylamine) 5.7 Total carbohydrate (phenol-H2SO4) Glucose Fucose 3.4 Hexosamine 13.8 Nitrogen 0.04 Phosphorus

7F~

04.9

nI

79.0

4.7

2.3

2.4

9.1

5.7

5.2

0.54 0.51 5.6

0.57 0.54 4.4

0.35 0.48 3.5

observations, along with those by chemical analysis, are a good indication t h a t the carbohydrate moieties are not uniform among the several fractions isolated. The antigenic response of the glycoprotein from both starch and gel electrophoresis enhanced by calcium alginate resulted in antigen titers detectable to a dilution of 1:1024. Crude extracts appeared to be less reactive. I n agar diffusion 3 to 4 precipitin bands could be detected against material obtained after electrophoresis on starch; however, the major fraction isolated following gel electrophoresis demonstrated one prominent band in agar diffusion with a trace of an additional band (Fig. 5). These results correlated essentially with the resolution achieved by gel electrophoresis. (Immunoelectrophoresis in agar of preparations after only (NH4)2SO4 fractionation with rabbit antisera to bovine serum resulted in approximately 13 bands, s) The important observation, irrespective of the number of bands shown by techniques in agar, is the ease of demonstrating antibody formation to connective tissue glycoproteins. 8 Observations by Dr. Pierre Arquembourg in this laboratory.

24

RADHAKRISHNAMURTHY ET AL.

FIG. 4. Composite illustration of paper chromatographic studies of neutral sugars of three glycoprotein fractions isolated after gel electrophoresis. Hydrolysis for 6 hours in 4 N HC1, ethyl acetate-pyridine-water (2:1:2), AgNO3 stain. Note the absence of mannose in Fraction I. Certain variations of individual sugar content for the three fractions were suggested by intensities of chromatographic spots for both neutral and amino sugars. DIscussION Methods of isolation which are mild, yet have a high degree of resolution, are necessary prerequisites to the study of the functional role of glycoproteins. Because of the apparent resistance of glycoprotein fractions to a variety of conditions, many of the early studies of such compounds ignored principles accepted for the isolation of proteins. A very simple technique of extraction at low temperature and precipitation near the isoelectric point with (NH4)2SO4 allowed isolation of materials with considerably larger molecular weight and somewhat different chemical analyses, particularly of an increased polypeptide

content and less sialic acid (3). It is expected that further improvement could be accomplished with the development of some type of assay correlative to the native state of these substances. The differentiation of various fractions by the gel electrophoresis introduces the problem of relationship of the fractions. Are the fractions fragments of a larger material? Certain variations in the chemical analyses, for example, the neutral sugar compositions, coupled with relative antigenic specificity indicate the possibility of a polymorphism of glycoproteins in the matrix of this cardiovascular connective tissue. Such was expected by earher observa-

FURTHER STUDIES OF GLYCOPROTEINS

25

F:(~. 5. Immtmodif'tusion s t u d y of b()vble aorta glye~)prolei~. The eeJ)ter well coi~tained rabbit antiserum to a glycoprotei~ Fraction III isolated alter a single gel electrophoretic procedure. Wells 1 and 2 contained crude extract at 20.8 and 3 rag. per milliliter, respec tively, wells 3 and 4 contained starch block-isolated material at 15,2 and 3 rag. per milliliter; wells 5 and 6 contained Fraction III at 9.5 and 3 rag. per milliliter. 1)iffusion proceeded for 48 hours at 37~

tions using a variety of isolation methods (18). Heretofore, many frustrating attempts have failed to demonstrate antigenic activity of components of connective tissue. The acid mucopoJysaccharides have been shown to be antigenically unreactive (19). Chondroitin sulfate-protein complex from bovine nasal septum has been shown to be only mildly reactive (20). Still, studies of tissue or organ transplantation, and skin grafting, which initiate a connective tissue inflammatory reaction, suggest that certain components of connective tissue can be immunologically reactive and take part in host rejection. Furthermore, antigenic activity has been demonstrated by a variety of immunologic methods for crude

blood vessel preparations (21 23). Untbrtunately, in those studies, since the chemistry of the antigens was not the immediate subject of interest the specific antigenic materials were not pinpointed. The ease of elieiting antibody response by extensively fractionated glycoprotein materials in these studies suggests that iramunologic reactions involving connective tissue may in large part be due to the glycoproteins. Further characterization of these components in cardiovascular tissue as well as other connective tissue would be of inestimable value. Since there is virtually no current information which allows correlation of chemical and immunologic observations on the tissue glycoproteins [comparable to

26

RADHAKRISHNAMURTHY ET AL.

information now available on blood group substances (24-26)], their role in the genetic m a k e - u p of an individual cannot be evaluated. If a genetically determined p o l y m o r p h i s m exists a m o n g glycoproteins, suggested b y the d a t a of chemical and immunologic specificity and individual variations (observations which are in progress), t h e n certain in vivo immunologic p h e n o m e n a which involve connective tissue, e.g., with tissue transplantation rejection, m a y be more easily understood when the nature of this family of proteins is clarified. T h e recent observations of a p o l y m o r p h i s m of serum al-acid glycoprotein (27) is particularly interesting with respect to the finding of a n u m b e r of glycoproteins in connective tissues (3, 4, 28). F u t u r e studies in this area are obviously necessary and m a y help answer some of the intriguing questions concerning the participation of cardiovascular tissue and other connective tissues in genetic and immunologic phenomena. ACKNOWLEDGMENTS The authors gratefully- acknowledge the suggestions and assistance of Drs. A. M. Altschul, J. M. Dechary, and W. J. Evans, and Mr. W. B. Carney of the Southern Regional Laboratories, U.S.D.A., in the Cyanogum gel studies and for performing sedimentation and quantitative amino acid determinations. REFERENCES 1. MEYER, K., DAVIDSON, E., LINKER, A., AND HOFFMAN,P., Biochim. Biophys. Acta 21,506 (1956). 2. BERENSON,G. S., Circulation Res. 7,889 (1959). 3. BERENSON, G. S., AND FISttKIN, A. F., Arch. Biochem. Biophys. 97, 18 (1962). 4. FISHKIN, A. F., AND BERENSON, G. S., Arch. Biochem. Biophys. 95, 130 (1961). 5. NEWMAN, J. K., BERENSON, G. S., MATHEWS, M. B., GOLDWASSER,E., AND DORFMAN, A., J. Biol. Chem. 217, 31 (1955). 6. ANDERSON,A. J., AND MACLAGAN,N. F., Biochem. J. 59,638 (1955). 7. MEHL, J. W., J. Biol. Chem. 157, 173 (1945).

8. BEAVEN, G. H., AND HOLIDAY,E. R., Advan. Protein Chem. 7,319 (1952). 9. STOFFYN,P. J., AND JEANLOZ, R. W., Arch. Biochem. Biophys. 52, 373 (1954). 10. RADHAKRISHNAMURTHY,B., KAYMAN,n . , AND BERENSON, G. S., Arch. Biochem. Biophys. 99, 534 (1962). 11. AMIES, C. R., J. Pathol. Bacteriol. 77, 435 (1959). 12. FREUND, J., in "The Nature and Significance of the Antibody Response" (A. M. Pappenheimer, Jr., ed.), p. 46. Columbia Univ. Press, New York, 1953. 13. EDSALL,J. W. in "The Proteins" (H. Neurath and K. Bailey, eds.), Vol. 1, part B, Chap. 7. Academic Press, New York, 1953. 14. RAYMOND,S., ANDWEINTRAUB,L., Science 130, 711 (1959). 15. RAYMOND, S., AND WANG, YI-Ju, Anal. Biochem. 1,391 (1960). 16. EVANS, W. J., CARNEY, W. B., DECttARY, J. M., AND ALTSCHUL,A. M., Arch. Biochem. Biophys. 96,233 (1962). 17. RADHAKRISHNAMURTHY, B., CHAPMAN, ]~., AND BERENSON, G. S., Biochim. Biophys. Acta, 75, 276 (1963). 18. BERENSON, G. S., J. Atherosclerosis Res. 1,386 (1961). 19. QU~NN, R. W., AND CERRONI, R., Proc. Soc. Exptl. Biol. Med. 96,268 (1957). 20. SAUNDERS, A. M., MATHEWS, M. B., AND DORFMAN, A., Federation Proc. 21, 26 (1962). 21. EBERT, J. D., Physiol. Zool. 24, 20 (1951). 22. PRESSMAN,D., SHERMAN,B., AND KORNGOLD, L., J. Immunol. 67, 493 (1951). 23. PIOMELLI, S., STEFANINI, M., AND MELE, R. H., J. Lab. Clin. Med. 54,241 (1959). 24. MORGAN, W. T. J., in "Ciba Foundution Symposium on the Chemistry and Biology of Mucopolysaccharides" (G. E. W. Wolstenholme and M. O'Conor, eds.), p. 200. Little, Brown and Co., Boston 1958. 25. MORGAN,W. W. J., AND PUSZTAI,A., Biochem. J. 81, 648, (1961). 26. KARAT, E. A., "Blood Group Substances." Academic Press, New York, 1956. 27. SCHMID, K., BINETTE, J. P., KAMIYAMA, S., PFISTER, V., AND TAKAHASI-II,S., Biochemistry 1, 959 (1962). 28. TURNER, R. W., FISI~KIN, A. F., AND BERNESON, G. S., Biochim. Biophys. Acta, 75, (1963).