Studies on elephant aortic elastic tissue Part II. Amino acid analysis, structural glycoproteins and antigenicity

EXPERIMENTAL.

AND

MOLECULAR

Studies Part II. Amino

PATHOLOGY

on Elephant

Acid Analysis, K. G.

MCCULLAGH,~

( 1973)

IS,202213

Aortic

Structural

Elastic

Tissue

Glycoproteins

and Antigenicity

S. DEROUETTE AND L. ROBERT

Research Division, Cleveland Clinic Foundation, Clevet!and, Ohio 44106 and Laboratoire de Biochimie du Ti.ssu Conjunctif., Facultk de M&icine, Universit6 de Paris, Val-de-Marne, 6 rue du Gal-San&l,94-Crdteil, France Received April 12, 1972 The aortic tissue of the African elephant was separated into a series of soluble fractions by a recently described method. Elastin was isolated as the residue remaining after solubilization of all other components with CaClacitrate buffer, trichloroacetic acid, urea, and mercaptoethanol. Elephant elastin was found to possess the typical amino acid compositionof this protein but in comparison with bovine elastin was slightly richer in acidic and polar amino acids, particularly in the abdominal aorta. Elephant arterial elastin was found to be closely associated with another protein, solubilized from elastic tissue only after disuKde bond reduction with mercaptoethanol. This material had a microfibrillar appearance under the electron microscope and a high hexose content. It was considered to consist of structural glycoproteins similar to those isolated from other connective tissues. The amounts of this elastic tissue glycoprotein were shown to be higher in the abdominal aorta than in the thoracic, while the amount of elastin in the two segments followed the reverse pattern. Structural glycoproteins, isolated from elephant arterial tissue, were shown to cross react with antibodies to human aortic glycoproteins but not with antibodies to calf cornea glycoproteins, suggesting a similarity between glycoproteins isolated from similar tissue. It is suggested that the comparatively large amounts of fibrillar glycoprotein in the elephant elastic tissue interferes with the staining of elastin and might encourage the deposition of calcium salts.

It has been shown that elephant (Loxodonta africana) arterial elastic fibers are not stained by orcein or the resorcin dyes and that relatively large amounts of fibrillar material may come to surround the central core of amorphous elastin ( McCullagh, 1973b, Paper I). In addition, disruption and calcification of the arterial elastic fibers is particularly common in the elephant and appears to be an important factor in the scenario of medial sclerosis ( McCullagh and Lewis, 1967). It is conceivable that the susceptibility of elephant elastic fibers to disruption and calcification comes about as a result of a basic difference in chemical structure from the elastic fibers of other species. It has already been demonstrated that elephant elastin 2 differs from human elastin in its rate of alkaline hydrolysis 1 This work was completed while working of Cambridge and M. R. C., England. 2 For a definition of “elastin” and “elastic article).

at the fiber”

202 Copyright All rights

0 1973 by Academic Press, Inc. of reproduction in any form reserved.

Dunn as used

Nutritional herein,

Laboratory, see Paper

University I

(preceding

ELEPHANT

AORTIC

ELASTIC

TISSUE

203

and in its ultraviolet absorption and fluorescence spectra (McCullagh, 197313, Paper I). This investigation is particularly concerned with properties imparted to elastic tissue by the non-elastin components of the fiber. METHODS Fresh elephant aortic material was obtained from animals shot in East Africa (Laws and Parker, 1968). It was frozen in the field, transported in the frozen state, and stored at temperature of -20°C until required for analysis. Elastin was isolated from the other connective tissues of the arterial wall by the method of Robert, Robert, and Moczar (1968a). This method involved the repeated extraction of dry defatted aorta with a 1M CaCle-citrate buffer, trichloroacetic acid, and urea. The isolation procedure simultaneously provided a method for the separation of the other constituents of the arterial wall in a series of soluble fractions. When the soluble fractions were dialyzed against water and freezedried, their contribution to the total weight of tissue could be calculated. This arterial fractionation scheme is described diagrammatically in Fig. 1. Amino Acid Analysis Compositional analyses were carried out on samples of th, clastin residue and of the protein extracted after disulfide reduction of elastic tissue. Ten milligram samples of dry protein, derived from both the thoracic and abdominal aorta, were hydrolyzed with 6 ml of 6 N hydrochloric acid in sealed Pyrex tubes at 100°C for 40 hours. After cooling, the hydrolysates were filtered into 50 ml round-bottomed flasks and the acid removed under reduced pressure in a rotary evaporator. The samples were then applied to a Technicon amino acid analyzer. The column measured I40 cm x 0.6 cm and was filled with Technicon “type A” chrome beads. It was eluted with the conventional citrate buffer gradient system at a flow rate of 30 ml/hour. The peaks were identified by reference to standard chromatograms and their unit areas calculated by multiplying their height by their width at half height, which was measured by the dot counting technique described by Spa&man, Stein, and Moore (1958). The unit areas of the peaks were multiplied by constants derived from the chromatograms of standard mixtures to give the number of pmoles of each amino acid present in the sample. Electron

Microscopy

The freeze-dried material obtained from the 8M urea + O.lM mercaptoethanol extract was homogenized with distilled water and the suspension subjected to ultrasonic vibration at 50 kilocycles/second for 15 minutes. The suspension was allowed to stand for 2 hours and the top 3 ml removed into another tube and the ultrasonic treatment repeated, After allowing a further 2 hours for the sedimentation of coarse particles, a drop of the suspension was applied to a collodion film, negatively stained, and examined with an AEI EM 6B electron microscope. Protein and Hexose Determinations The protein micro-Kjeldahl

content method

of the freeze-dried fractions was determined by the using sodium selenate as the catalyst and calculating

204

McCULLAGH,

DEROUETTE,

AND ROBERT

protein as nitrogen x 6.25. Hexoses were determined in the freeze-dried, TCA, urea, and mercaptoethanol soluble fractions after mild hydrolysis in 0.5 N HCl at 100°C for 4 hours. Hexoses were determined in the hydrolysate by the anthrone method of Trevelyan and Harrison ( 1952).

Immunological

Methods

The antigenicities of the various freeze-dried extracts were determined from the amount of antibody produced by rabbits following multiple injection of the extracts. For this purpose 5 mgm samples of the freeze-dried fractions were dissolved in 0.9% sodium chloride, mixed with Freund’s complete adjuvant and injected intradermally in rabbits. This procedure was repeated twice a week for 4 weeks. There followed a period of 4 weeks during which no further injections were made. At the end of 8 weeks an injection was made intravenously, and 2 to 3 days later the rabbits were killed, the serum collected, and serial dilutions made. The strength of the antibody titer was determined by the passive hemagglutination test, the homologous antigen being fixed on the surface of gluteraldehyde-treated sheep red blood cells with the aide of tetraazotised benzidine (Robert et al., 1971). Cross reacting antibodies were detected by the use of immunodiffusion on thin agarose layers, according to the micromethod of Manski et al. ( 1961). RESULTS

Connective

Tissue Proteins of the Arterial Wall

The concentration of elastin and other proteins in the thoracic and abdominal aortic wall of the elephant is shown in Table I. Included in the table for comparison are the results of similar analyses of pig aortic wall and human aortic wall taken from Moczar and Robert (1970) and Robert et al. ( 1968b). The CaC12-Tris-citrate (CTC) buffer removes practically all the freely diffusable proteins of the arterial wall. These include trapped serum protein, mucqpolysaccharides, soluble cellular proteins, and soluble collagen and glycoproteins. Most of the collagen in the extract precipitates out on dialysis, and the fraction labeled CTC precipitate is therefore an indication of the amount of crude soluble collagen in the aortic wall. The amount of this fraction in the elephant aorta was consistently lower than that of the human or pig aorta. The CTC-insoluble residue contains the major structural proteins of the artery. As described above, we first extracted polymeric collagen by repeated hot trichloroacetic acid extraction. Fitch et al. (1955) have shown that hot TCA selectively cleaves collagen from elastin, and we have shown previously that structural glycoproteins resist the TCA treatment (Robert and Dische, 1963). We have also shown that the TCA extraction procedure removes most of the nucleic acids contained in the aortic wall (Moczar and Robert, 1970). However, it leaves behind a group of structural glycoproteins which are only removed when the TCA residue is subsequently extracted with 8 M urea. The TCA and urea fractions, taken together, account for the major part of the arterial tissue. They vary in amount from site to site and from species to species, depending upon the fibrous nature of the artery. It will be seen from Table I that the abdominal aorta of the elephant contains considerably more

ELEPHANT

AORTIC

ELASTIC

205

TISSUE

Dried, delipidated, aortic tissue Extracted with CTC buffer at 4” 5 X 24 hr. CTC

1

4

extract

Insolubl! residue Extracted with 5% TC.4 at 90’ 2 X 30 min.

dialyzed

4-l

Supernatant

r-----i

Precipitate CRUDE SOLUBLE COLLAGEN

SOLUBLE PROTEINS

Isidue extract

Urea

Elastic Tissue Extracted with 8M urea + 0.1M mercaptoethanol 3 X 24 hrs.

CRUDE STRUCTURAL GLYCOPROTEINS

1 Residue

4 + mercaptoethanol extract

Urea

Residue Extracted with 8M urea at 20” 4 X 24 hr.

TCA extract INSOLUBLE FIBROUS COLLAGEN

GLYCOPROTEINS

OF

EL4STIN

ELASTIC

FIBERS FIG. scheme.

1.

Diagrammatic

representation

of

the

elastin

isolation

and

arterial

fractionation

collagenous tissue than does the thoracic aorta. This difference is also reflected in the amount of elastic tissue in the two sites. It can be seen from the table that the proportion of total elastic tissue in the elephant’s thoracic aorta was 51%, but that in the abdominal aorta it fell dramatically to 16.370, which was in keeping with the observed histological structure of the aorta in the two regions ( McCullagh, 1970). A similar variation in elastic tissue content is seen TABLE PROTEIN

Fraction

Major

CTC(

extract

soluble

precipitate

crude

TCA

extract

fibrous

extract

structural

Urea Urea

CONSTITUENTS

+ SH extract

component proteins

soluble

collageu

collagen glycoproteins ,microfibrils

,

I OF THK

AORTIC

W.ILL*

Elephant

aorta

Thoracic

Abdominal

Human aorta (thoracio)

3.6

4.0

6.3

Pig aorta (thorark) 4.-i

0.6

0.7

2.4

1 .9

33.2

61.9

19.0

50.9

11.6

17.1

40.5

23.0

5.3

8.0

2.6

20.7

45.7

8.3

39 .-,

“0.7

elastic tissue Residue & mg/lOO

’ mg dry defatted

‘e1ast.m weight.

206

McCULLAGH,

DEROUETTE,

AND

ROBERT

in the pig aorta (Grant, 1967), although only the results of the analysis of the thoracic aorta have been included in Table I. It will be seen from Table I that a further component of the elastic tissue is solubilised by 8 M urea in the presence of 0.1 M mercaptoethanol. This fraction we have called the microfibrillar fraction, due to its electron microscopic appearance (see Fig. 2), and the similarity of its amino acid composition to the similarly named fraction obtained from calf elastic tissue by Ross and Bornstein ( 1969). The residue which is left after this last extraction may be regarded as true elastin. In the elephant’s thoracic aorta the microfibrillar component represented 10% of the fiber which was similar to its proportion in the human aorta (9% ). In the abdominal aorta, however, it represented 49% of the whole. Thus, not only is the abdominal aortic elastic tissue in the elephant severely diminished in quantity in comparison with the thoracic aorta and the human aorta, but also it is different in quality, almost half of its weight being made up of a nonelastin component. The appearance of the freeze-dried urea + mercaptoethanol extract under the electron microscope is shown in Fig. 2. With negative staining this fraction appeared to be composed of clumps of fine microfibrils, very similar to those SIXrounding the amorphous core of the elastic fiber seen in viva and depicted in Paper I. Table II gives the hexose and hydroxyproline content of some of the extracts. All had a relatively high hexose content, without any major differences being noticeable between thoracic and abdominal aortas. However, the hexose content

FIG. staining,

2.

Electron magnification

micrograph X 45 000.

of the

freeze-dried

urea + mercaptoethanol

extract.

Negative

ELEPHANT

AORTIC TABLE

HEXOSE

AND HYDROXYPROLINE

Extract

ELASTIC

TISSUE

207

II

CONCENTRATION

IN ELEPHANT

AORTIC

EXTRACTS”

Origin

Hexoses

Hydroxyproline

CTC

Thoracic Abdominal

10.6 8.0

7.5 10.6

TCA

Thoracic Abdomind

4.6 4.0

10.7 13.3

UREA

Thoracic Abdominal

4.9 3.9

UREA-SH

Thoracic Abdominal

6.5 6.3 _ __-..

0.10 0.15
6 mg/lOO mg protein.

was higher in the urea-mercaptoethanol extracts than in the urea extracts. In the pig, structural glycoproteins have previously been shown to be present in at least 3 distinguishable forms-urea extractable, urea-mercaptoethanol extractable, and insoluble (Moczar and Robert, 1970), and in this species all forms had a very similar composition both for amino acids and sugars. This is not the case in the elephant, for it appears that the more highly cross-linked forms, which were extractable only when mercaptoethanol was added to the urea, contained more sugar than the less highly cross-linked urea-extractable glycoproteins. The hydroxyproline content of the crude soluble collagen fraction (material which precipitates on dialysis of the CTC extracts) and of the TCA extracts was close to that found in other aortas (Robert et aZ., 1968; Moczar and Robert, 1970), and suggests the presence on average of about 36% noncollagenous proteins in the CTC fraction and about 18% in the TCA extract. The structural glycoprotein fractions (urea extracts) had no hydroxyproline or at least only very small amounts. Amino Acid Analysis Table III gives the composition of elephant aortic elastic tissue prepared by TCA and urea extraction. On the left hand side of the table the composition of the final elastin residue is shown, and on the right is the composition of the fraction removed by extraction with mercaptoethanol. In both cases, the first column is derived from the thoracic aorta and the second from the abdominal aorta. The composition of elephant aortic elastin was similar to that of ox aorta, ligamentum nuchae, and ear cartilage (Gotte et al., 1963), calf ligamentum nuchae (Ross and Bomstein, 1969), chicken aorta (Miller et al., 1964), and pig and human aorta (Grant, 1966); i.e., it possessedthe characteristic amino acid pattern of this protein. It had a high proportion of proline, glycine, alanine, and valine, but only small amounts of the hydroxy amino acids (hydroxyproline, threonine, and serine), and of the acidic and basic amino acids (aspartic acid, glutamic acid, lysine, and histidine). However, in comparison with bovine aortic elastin (Gotte ti aE., 1963) the elephant aortic elastin was slightly richer in aspartic acid, glutamic acid, threonine, and tyrosine while being slightly lower

McCULLAGH,

DEROUETTE,

AND

ROBERT

in glycine and valine. These differences were slight and did not significantly alter the pattern. In the thoracic aorta at least, they were not large enough to support the suggestion that the failure of elephant elastic fibers to stain with phenolic dyes ( McCullagh, 1972) was due to a different amino acid composition. Basic, acidic, and polar amino acids were more common in the elastin isolated from the abdominal aorta. Similar changes were found by Grant (1966) between thoracic and abdominal elastin samples from the pig, sheep, goat, and human. If elastin is regarded as a homogeneous protein, the difference in its composition in different sites may be only an apparent change caused by the presence of a contaminating protein which is less readily separable from elastin in the abdominal aorta than in the thoracic aorta. The increase in hydroxyproline in abdominal elastin samples suggests that this protein might be collagen, but the decrease in glycine and proline suggests that there could equally well be contamination with the protein separated from elastin by mercaptoethanol (SH extract). Possibly both could have been involved. The composition of the urea-SH extract shown in Table III was very similar to the composition of the microfibril sheath surrounding the amorphous core of the TABLE THF,

AMINO

ACID COMPOSITION FROM THE ELASTIC

OF ELASTIN AND TISSUE OF THE

ABDOMINAL

AORTA

(urea Amino

acid

Hydroxyproline Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Cystine Methionined Isoleucine Leucine Tyrosine Phenylalanine Isodesmosine Desmosine Lysine Histidine Arginine Amide Nz 8 Residues/1000 residues. b - = not present. 0 tr. = trace ( < 0.2). d Methionine includes methionine

III

Thoracic

OF THE

AN UNKNOWN GLYCOPROTEIN THORACIC AND

ELEPHANT”

Elastin + SH residue) Abdom.

(urea

Glycoprotein + SH extract)

Thoracic

12

26

-b

12 14 9 24 150 270 250 107 1 26 61 14 29 1.3 3.5 8 2

19 16 14 36 116 266 217 123 1 1 28 57 22 26 1.4 2.0 14 4

86 47 47 119 67 124 105 74 10 17 47 79 29 36 tr. tr. 51 18

(3:)

(&

sulfoxide.

Abdom. 94 46 46 121 78 160 100 51 9 11 44 74 21 31 tr. tr. 50 19 (12)

ELEPHANT

AORTIC

ELASTIC

TABLE PASSIVE

HEMAGGLUTINATION ANTISERA

TITERS AND

WITH

ELEPH.YNT

Rabbit

antisera

CTC extract calf tendon

to

TCA

extract

209

IV VARIOUS

THORKIC

Inverse

TISSUE

ANTI-CONNECTIVE AORTIC

TISSUK

AXTIGENS

of hemagglutination titers obtained with red cells coated with:

Urea

extract

Urea + SH extract

Proper titer with immunising antigena

of 32

32

1000

4 000

CTC extract of human aorta

8

16 000

:c2

2 000

Urea extract of human aorta

32

4 000

32

500

Urea extract calf cornea

16

8

8

a Proper indicat,ion

of

titers were obtained of the expected order

at a different date and are included of reaction between the antisera and

1000

in the table only their own antigens.

as an

elastic fiber in &o, recently reported by Ross and Bornstein (1969) and Robert et ~2. ( 1971). These investigators used disulfide bond reduction to separate the fibrils, confirming their solubilization by electron microscopy. The appearance of these fibrils before solubilization was remarkably similar to that of elephant elastic tissue microfibrils ( McCullagh, 1973, Paper I). It is reasonable to conclude from this, and from the actual appearance of this fraction under the electron microscope (Fig. 1 ), that material removed by the mercaptoethanol extraction procedure was actually the fibrillar component of the elastic fiber. immunological Studies Table IV gives the titers of antisera to calf tendon, human aorta, and pig aorta preparations necessary to cause hemagglutination of sheep red cells coated with the various elephant aortic extracts as antigens. It will be seen that significant titers were obtained between three of the antisera and fractions of the elephant aorta. Antisera to the CTC extracts of calf tendon agglutinated red cells coated with the microfibrillar glycoprotein preparation of elephant aorta at a dilution of 1 x lo-“. A higher titer of 16 x 10e3 was obtained between the CTC extracts of human aorta and the urea soluble glycoproteins from the elephant aorta. This cross reaction between CTC and urea extracts was anticipated (Robert et al., 1965). It must be borne in mind that the antigenic determinants in the CTC extracts are probably glycoproteins, some of which are similar to those contained in the urea extracts (Robert et al., 1971). Of more importance is the significant cross reaction between the urea extract of human aorta and the urea extract of elephant aorta, indicating an antigenic similarity between the structural glycoproteins present in this fraction in both species. In contrast, the antiserum to calf cornea structural glycoproteins did not react significantly with the elephant aortic structural glycoproteins.

210

McCULLAGH,

DEROUETTE,

AND ROBERT

AS-CTC-extr. calf

tendon AS-Urea-extr.

+

AS-K-Elastin

AS-Urea-extr. human

oorta

AS-CTC-extr. human

oorta

FIG. 3. Diagram of the precipitin lines formed on an immunodiffusion plate containing elephant aortic urea extract antigen in the center well and rabbit antisera to various connective tissue extracts in the peripheral wells. (For details see Robert et al., 1965).

These findings were confirmed by the immunodiffusion studies. Figure 3 shows an immunodiffusion plate, having in the center a urea extract of the abdominal elephant aorta and at the periphery several antisera to calf, pig, and human connective tissue extracts. The three lines given by the antielastin serum may be due to the presence of small amounts of structural glycoproteins in purified elastin as microfibrils, or alternatively to the presence of elastin as a contaminant in the urea extract. The antisera to the CTC extract and urea extract of human aorta produced several precipitation lines with the urea extract of elephant aorta, as might be expected from Table IV. The cross reaction between antisera to human aortic glycoproteins and the elephant aortic glycoprotein antigen indicates a species similarity in the structure of aortic glycoproteins. In contrast, no precipitin lines were observed with antisera to the CTC or urea extracts of calf tendon or to the urea extract of calf cornea. The cross reactions obtained appear to demonstrate an antigenic similarity among structural glycoproteins isolated from similar tissues, despite differences in the species of origin, and a dissimilarity among structural glycoproteins isolated from different tissues. DISCUSSION These studies indicate that the connective tissue composition of the elephant aorta is similar to that of other mammalian species investigated. The elephant, however, differs from most other species in having a dramatic change in aortic composition between thoracic and abdominal sections. For example, the elastic tissue content, which is approximately 50% of the total dry weight in the thoracic aorta, drops to a mere 16% in the abdominal aorta. In replacement of the elastic tissue, the abdominal aorta contains dramatically increased quantities of collagen and structural glycoproteins. The ratio of elastin to collagen drops from 1.4 in the thoracic aorta to 0.13 in the abdominal aorta. A shift of similar magnitude in the elastin-collagen ratio has been reported only in the pig (Grant, 1967). In most other species, including man, the difference between the elastin con-

ELEPHANT

AORTIC

ELASTIC

TISSUE

211

tent of the thoracic and abdominal aortas is considerably less. In man, for example, the elastin-collagen ratio varies from only 1.6 to 0.8. The structural glycoprotein content of the elephant aorta is of the same order of magnitude as that of the pig, although it is lower than that of the rabbit or human aorta. The structural glycoprotein fraction also varies in quantity some,what from thoracic to abdominal aorta, increasing as the proportion of collagen rises. If the total structural glycoprotein content is calculated from the weights of the urea and urea + mercaptoethanol extracts, it is found that the ratio of total structural glycoprotein to collagen remains constant in the elephant aorta at about 0.28. These differences in the chemical composition of the thoracic and abdominal aorta in the elephant are significant in view of the observation that only the abdominal aorta suffers from severe calcification, the thoracic aorta being largely devoid of medial calcific sclerosis (McCullagh and Lewis, 1967; McCullagh, 1970). In addition, atherosclerotic lesions are far more common in the elephant’s abdominal aorta than they are in the thoracic section. It is difficult to escape the conclusion that the absence of a significant amount of elastic tissue in the elephant’s abdominal aorta predisposes it to both medial sclerosis and intimal atherosclerosis. It is possible that the increased amounts of structural glycoproteins present in the elephant’s abdominal aorta may actually encourage calcification. Calcification in the arteries of the elephant was always seen first in and along side the internal elastic lamina ( McCullagh and Lewis, 1967). In man it has also been shown that calcification commences either in or very close to the elastic laminae (Yu and Blumenthal, 1963). The major difference in elastic tissue composition between the thoracic and abdominal aorta in the elephant remains the increase in the structural glycoprotein microfibrillar fraction. The amino acid analyses revealed that this nonelastin component contained large amounts of the dicarboxylic amino acids aspartic and glutamic. It has been suggested by Lansing et al. (1952) that the free carboxyl groups of these amino acids may bind calcium ions, while Yu and Blumenthal (1965) h ave suggested that calcium may chelate to the center of the C-terminal groups of these amino acids. Alternatively, the polysaccharide which is supposed to be present in this nonelastin sheath (Hall, 1967) may play an important part in encouraging calcification, as has been suggested by Yu and Blumenthal ( 1965), McLaughlin (KM%), and Schiffman et al. ( 1966). The hypothesis presented in Paper I that the poor staining of elephant elastic fibers by phenolic dyes is due to the protection of the elastin core of the fiber by a glycoprotein sheath appears to be supported by the present studies. The material soluhilized from elastic tissue by 8 &l urea + mercaptoethanol has been shown to be fibrillar in nature and similar in appearance to the material surrounding the central core of the elastic fiber in thin sections of elephant arteries. The amino acid composition of this microfibrillar component is remarkably similar to that reported by Ross and Bornstein (1969) and by Robert et al. (1971) for microfibrillar components of calf elastic tissue. We are in agreement with ROSS and Rornstein that the microfibrils represent a different connective tissue protein or proteins that is neither collagen nor elastin. The presence of a large quantity of hexose in this fraction suggests that it’s characterization as glycoprotein is justifi-

212

McCULLAGH,

DEROUETTE,

AND ROBERT

able. The significant differences in hexose content and antigenicity between the urea extracts and the urea + mercaptoethanol extracts of elephant aorta suggests that the glycoproteins of the microfibrillar sheath of the elastic tissue fiber may be different from the structural glycoproteins solubilized in 8 M urea. The immunochemical results confirm the presence of highly cross reactive antigenic sites in both urea soluble and insoluble glycoproteins, and lead us to propose that the glycoproteins are the major antigenic determinants in the connective tissues of the vascular wall. Until relatively recently, a view prevailed that connective tissue components, particularly polysaccharides and collagen were either nonantigenic or poorly so (Boake and Muir, 1955; Kirrane and Robertson, 1968). Recent studies in many laboratories have indicated that this is not so, and that antigenicity is conferred upon connective tissues by proteinpolysaccharide complexes. It is possible that the pattern of glycoproteins synthesized in a particular tissue may control the subsequent pattern of other connective tissue components ( Fitton-Jackson, 1967; Robert et al., 1965). If this were true, one might expect to find similar glycoproteins in the same tissues from different species. This, indeed, appears to be the case, for Sandson et al. (1966) found that the cartilage protein-polysaccharides from 3 different species cross react. The protein-polysaccharides or structural glycoproteins of the elephant aorta represent a similar picture, for antigenic cross reactions were observed with the glycoproteins of human aorta but not with the glycoproteins of calf cornea. ACKNOWLEDGMENTS This National in East Trustees

work was supported by the Medical Research Council, London, and the Centre de la Recherche Scientifique, (E.R. 53) and the DGRST, Paris. Material was collected Africa through the cooperation of Mr. I. S. C. Parker, Dr. R. M. Laws, and the of the Uganda National Parks.

REFERENCES W. C., and Mum, H. (1955). Th e non-antigenicity of chondroitin sulphate. Lancd ii, 1222-1223. Frrcrr, S. M., HARKNESS, M. L. R., and HARKNESS, R. D. ( 1955). Extraction of collagen from tissues. Nature (London) 176, 163. FI~ON-JACKSON, S. ( 1967). The morphogenesis of collagen. In “Treatise on Collagen,” (B.S. Gould, ed.), Vol. 2B, pp. l-66. Academic Press, London and New York. GOTTE, L., STERN, P. ELSDEN, D. F., and PARTRIDGE, S. M. (1963). The chemistry of connective tissues. The composition of elastin from three bovine tissues. Biochem. J. 87, 344351. GRANT, R. A. (1966) Variations in the amino acid composition of aortic elastin from different species. Brit. J. Exp. Path. 47, 163-167. GRANT, R. A. ( 1967). Content and distribution of aortic collagen, elastin and carbohydrate in different species. .I. Atherosder. Res. 7, 463-472. HALL, D. A. (1967). Elastic tissue alternations in vascular disease. In “Cowdry’s Arteriosclerosis,” (H. T. Blumenthal, ed. ), pp. 121-140. Thomas, Springfield, Illinois. KIRRANE, J. A., and ROBERTSON, W. VAN B. ( 1968). The antigenicity of native and tyrosylated neutral salt soluble rat collagen. Immunology 14, 139-148. LANSING, A. I., ROSENTHAL, T. B., ALEX, M., and DEMPSEY, E. W. (1952). The structure and chemical characterisation of elastic fibres as revealed by elastase and electronmicroscopy. And. Rec. 144, 555575. in LAWS, R. M., and PARKER, I. S. C. (1968). R ecent studies on elephant populations East Africa. Symp. Zool. Sot. Lord. 21, 319359. BOAKE,

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