Glycoproteins and Glycolipids in Disease Processes - ACS Publications

19 Carcinoembryonic Antigen—A Marker of Human Colonic Cancer

Downloaded by PURDUE UNIV on October 25, 2016 | http://pubs.acs.org Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0080.ch019

CHARLES W. TODD and JOHN E. SHIVELY Division of Immunology, City of Hope National Medical Center, Duarte, CA 91010

The current literature has numerous examples of qualitative and quantitative changes in antigens associated with human malignancy. Despite the potential that these antigens offer for the control of cancer through detection, therapy, or prevention by immunization, in most cases the antigens are poorly defined and practical applications are lacking. One antigen which has achieved considerable application in the diagnosis and monitoring of cancer therapy is the carcinoembryonic antigen (CEA). Our study of the chemical nature of this complex molecule, or perhaps more accurately stated molecular complex, was undertaken with the goal of improving its clinical utility in the management of cancer. The initial reports by Gold and Freedman (1,2) suggested that CEA was synthesized by the rudimentary digestive system in the human fetus, that synthesis ceased prior to birth, but that it resumed with the onset of colonic adenocarcinoma in the adult. This sequence lead them to name the substance the carcinoembryonic antigen. Their development of a radioimmunoassay for CEA (3) and the potential importance of such a tumor marker stimulated studies of the diagnostic value of CEA assays in a variety of pathological conditions, both benign and malignant. The l i t erature on these clinical studies has been recently reviewed (4). These studies demonstrated that CEA-reactivity was not universally elevated in adenocarcinoma of the digestive system, that it was often elevated in other malignancies notably of breast, bladder, and lung, and that moderate elevations are often present in nonmalignant inflammatory diseases. Moreover, normal sera showed variable low levels of CEA-reactivity. Clearly the situation was more complex than originally visualized. Since radioimmunoassays are not completely specific (5), more precise chemical knowledge of the substances being detected was necessary. Most of the studies done on CEA have employed material isolated from hepatic metastases of colonic adenocarcinoma using essentially the method originally described by Krupey et al. (6), although the final block electrophoresis step is usually omitted. The CEA employed in most of our studies was obtained by homogeni0-8412-0452-7/78/47-080-342$05.00/0 © 1978 American Chemical Society Walborg; Glycoproteins and Glycolipids in Disease Processes ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

19.

TODD A N D SHIVELY

Carcinoembryonic

z a t i o n of l i v e r metastases o f colon adenocarcinoma i n water, a d d i t i o n o f an equal volume o f 2M p e r c h l o r i c a c i d , c e n t r i f u g a t i o n , d i a l y s i s o f the supernatant, c o n c e n t r a t i o n , chromatography on Sepharose 4B, and rechromatography on Sephadex G200 ( 7 ) . The CEA a c t i v i t y during these i s o l a t i o n s was followed by a t r i p l e isotope double antibody radioimmunoassay (8.), which has been r e c e n t l y improved by the s u b s t i t u t i o n o f C o f o r N a as a v o l ume marker f o r the supernatant (9). CEA prepared i n t h i s way when examined by e l e c t r o n microscopy c o n s i s t e d o f twisted rod or c r u l l e r shaped p a r t i c l e s with dimensions o f 9 x 40 nm (10). Further p u r i f i c a t i o n o f CEA by a f f i n i t y chromatography on concanavalin A Sepharose removed a small amount (15%) of contaminants, which were not r e t a i n e d by the a f f i n i t y column. This m a t e r i a l was found to c o n t a i n mucopolysaccharides as i d e n t i f i e d by c e l l u l o s e acetate s t r i p e l e c t r o phoresis, depolymerization with bovine t e s t i c u l a r hyaluronidase, and i d e n t i f i c a t i o n o f g l u c u r o n i c a c i d as the t r i m e t h y l s i l y l d e r i v a t i v e o f i t s methyl g l y c o s i d e by gas chromatography-mass spectrometry (12). E a r l y s t u d i e s by Krupey et al. (13) revealed CEA to be a g l y c o p r o t e i n with a molecular weight about 180,000, comprising about 60% carbohydrate and H0% p r o t e i n . This high percentage o f carbohydrate may e x p l a i n the f a c t that the molecular dimensions as v i s u a l i z e d by e l e c t r o n microscopy i n d i c a t e a molecular volume about 16 times g r e a t e r than that c a l c u l a t e d f o r a c l o s e l y packed molecule o f 180,000 molecular weight. Presumably the frequency and s i z e o f the carbohydrate groupings i n t e r f e r e with compact f o l d i n g o f the chain. There appears to be only one polypeptide chain present i n CEA. Consistent with t h i s view i s the f i n d i n g that r e d u c t i o n and a l k y l a t i o n o f the d i s u l f i d e bonds p r i o r to e l e c t r o n microscopic examination o f CEA leads to the appearance of t h r e a d - l i k e s t r u c t u r e s . The maximum extended length observed, 220 nm, approaches that expected f o r a p r o t e i n o f the r e q u i s i t e s i z e a l l o w i n g 0.364 nm extension per amino a c i d r e s i d u e (14). C h a r a c t e r i z a t i o n o f CEA by i s o e l e c t r i c f o c u s i n g (15), i o n exchange chromatography (15,16) and a f f i n i t y chromatography on concanavalin A Sepharose ^11>17>_18) demonstrated considerable heterogeneity, as might be expected f o r a h i g h l y g l y c o s y l a t e d p r o t e i n . Indeed, v a r i a t i o n i s seen i n the r e l a t i v e amounts o f the carbohydrate components of CEA i s o l a t e s from d i f f e r e n t tumors (19). T y p i c a l r e s u l t s from a CEA p r e p a r a t i o n p u r i f i e d by a f f i n i t y chromatography on concanavalin A Sepharose (11_) are presented i n Table I . The carbohydrate compositions were determined by the method of Clamp (20) f o r n e u t r a l sugars, by the method o f Warren (21) f o r s i a l i c a c i d , and by the method o f L i u and Chang (22) with the amino a c i d a n a l y z e r f o r the amino sugars. The s i a l i c a c i d has been i d e n t i f i e d as N-acetylneuraminic a c i d (23)• These analyses r e q u i r e d samples o f approximately 250 yg. More r e c e n t l y we have developed a method approximately 2 orders o f magnitude more s e n s i t i v e employing methanolysis and t r i f l u o r o a c e t y l a t i o n to generate methyl N , 0 - t r i f l u o r o a c e t y l g l y c o s i d e s , which a r e separated by gas chromatography on 7m x 0.5 mm packed 5 7


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2 2

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c a p i l l a r y columns and q u a n t i t a t e d by e l e c t r o n capture d e t e c t i o n


(24). In c o n t r a s t to the v a r i a b i l i t y seen f o r the carbohydrate composition, the amino a c i d composition o f v a r i o u s CEA i s o l a t e s remains q u i t e constant (25). A t y p i c a l a n a l y s i s i s presented i n Table 1. Using the r e s u l t s i n Table 1 some deductions regarding the s t r u c t u r e o f CEA can be made. The t o t a l number o f amino a c i d residues i s 829. A s i n g l e polypeptide chain with 829 amino a c i d residues with a length of 0.364 nm per residue (14) would have a length of 302 nm, i f f u l l y extended. This compares f a v o r a b l y with the 220 maximum observed by e l e c t r o n microscopy c o n s i d e r i n g the approximations i n v o l v e d . The low content of N - a c e t y l galactosamine (1.5? of the sugar r e s i d u e s ) suggests that the o l i gosaccharide u n i t s are not bound to the p r o t e i n backbone through N-acetylgalactosamine l i n k e d to s e r i n e or threonine. The f a i l ure to accomplish 3 - e l i m i n a t i o n supports t h i s view (19). Altern a t i v e l y , i f the attachment i s through N-acetylglucosamine l i n k e d to asparagine, the t o t a l a s p a r t i c acid/asparagine content l i m i t s the number o f o l i g o s a c c h a r i d e u n i t s to 128 with an average s i z e of 4 r e s i d u e s . Subsequently we s h a l l see evidence that there are about h a l f as many u n i t s with an average of twice t h i s s i z e . The amount of N-acetyglucosamine i n d i c a t e s some o f these residues must be d i s t a l from the attachment s i t e s i n c e the r a t i o o f Nacetylglucosamine to a s p a r t i c acid/asparagine i s 1.5. The sum of s e r i n e (91 r e s i d u e s ) and threonine (74 r e s i d u e s ) i s 165. Thus there are ample residues to e s t a b l i s h the asp,x,ser/thr sequences i n the p r o t e i n chain, b e l i e v e d to code f o r N-acetylglucosamineasparagine attachment (26). More p r e c i s e information on the s t r u c t u r e o f the o l i g o saccharide u n i t s was obtained by methylation a n a l y s i s (27) employing gas chromatography and mass spectrometry as depicted i n Figure 1. Samples were methylated by the method of Hakomori (28_) i n which the m e t h y l s u l f i n y l anion (29) was used to generate the polysaccharide alkoxide before the a d d i t i o n o f methyl i o d i d e . A c e t o l y s i s , h y d r o l y s i s , r e d u c t i o n , and a c e t y l a t i o n o f the permethylated p o l y s a c c h a r i d e s were performed using the procedures described by S t e l l n e r et al. (30). I d e n t i f i c a t i o n o f the p a r t i a l l y methylated a l d i t o l acetates was performed by the method of Bjorndal et al. (31) f o r the n e u t r a l sugar d e r i v a t i v e s and by the method of S t e l l n e r et al. (30) f o r the amino sugar d e r i v a tives. The products are i d e n t i f i e d by t h e i r r e t e n t i o n times i n the gas chromatograph and t h e i r fragments as produced i n the mass spectrometer. There are c e r t a i n p r e f e r r e d cleavage p o i n t s that occur on e l e c t r o n impact i n the mass spectrometer. The order of the preference o f these cleavages i s i n d i c a t e d by the numbers 1, 2, and 3 (Figure 1). A d e t a i l e d procedure has been published (32). The r e s u l t s of these s t u d i e s (27) on 4 CEA samples are presented i n Table 2 together with the r e s u l t s on a sample analyzed by Hammarstrom et al. (33). I t i s apparent that considerable


TODD A N D SHIVELY

Carcinoembryonic

Antigen

TABLE 1 COMPOSITION OF CEA

Moles per 180,000 MW

Components

Sugars N-Acetylgalactosamine

188.2

N-Ace tylglucosamine


N-Acetylneuraminic

7.5

acid

12.1

Fucose

102.4

Galactose

110.9

Mannose

82.4

Amino Acids Alanine

49.3

Arginine

20.1

A s p a r t i c acid/Asparagine

127.8

Cysteine

15.8

Glutamic acid/glutamine

84.6

Glycine

47.7

Histidine

20.1

Isoleucine

31.3

Leucine

67.8

Lysine

20.3

Methionine

Trace

Phenylalanine

18.9

Proline

58.6

Serine

91.2

Threonine

74.0

Tryptophan

11.9

Tyrosine

36.1

Valine

53.5

Carbohydrate

49.9?

Protein

50.1?

These v a l v e s were c a l c u l a t e d from the data f o r F r a c t i o n V, Table 2, Reference 11.



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v a r i a t i o n e x i s t s among these samples, but that a b a s i c p a t t e r n i s being followed. As expected from patterns p r e v i o u s l y observed i n mammalian g l y c o p r o t e i n s (34_), a l l the fucose i s t e r m i n a l . S i m i l a r l y a l l the N-acetylneuraminic a c i d i s t e r m i n a l . This f o l l o w s not from the methylation a n a l y s i s , but from the observ a t i o n that removal of the N-acetylneuraminic a c i d with neuraminidase exposed to a t t a c k by periodate a d d i t i o n a l moles of galactose e q u i v a l e n t to the moles o f N-acetylneuraminic a c i d removed (23). This observation together with the n e g l i g i b l e amounts of branching galactose observed by methylation a n a l y s i s (Table 2) a l s o f i x e s the p o i n t of attachment of the N - a c e t y l neuraminic a c i d at the 3 - p o s i t i o n of galactose (23). Threefourths of the mannose r e s i d u e s i n a l l the CEA samples were l i n k e d to 3 other sugar residues (branching mannose). Except f o r the small amounts of 3,4- and 2,3-linked g a l a c t o s e , the only other branching sugar i s N-acetylglucosamine of which 32-41? i s present a t branching p o i n t s . The data i n Table 2 can a l s o be used to c a l c u l a t e the number of o l i g o s a c c h a r i d e u n i t s l i n k e d to the p r o t e i n chain and t h e i r average s i z e (35). The number of o l i g o s a c c h a r i d e u n i t s equals the sum of the nonreducing terminal residues minus the sum o f the branching r e s i d u e s . From t h i s the average s i z e can be obtained by d i v i d i n g the t o t a l number of residues by the number of o l i g o s a c c h a r i d e u n i t s . Thus the numbers of o l i g o s a c c h a r i d e u n i t s f o r the CEA samples i n Table 2 are 51, 77, 124, 64, and 69 r e s p e c t i v e l y . On t h i s b a s i s the comparable o l i g o s a c c h a r i d e u n i t s contain 9, 7, 4, 8, and 6 monosaccharide residues each. Considering the cumulative e r r o r s and the expected i n h e r ent v a r i a t i o n , the data are i n reasonably good accord. The p i c ture that emerges i s many small o l i g o s a c c h a r i d e u n i t s attached to about 1 out o f 10 of the amino a c i d residues u t i l i z i n g about 2/3 of the a s p a r t i c acid/asparagine r e s i d u e s . The high carbohydrate content of CEA suggested the p o s s i b i l i t y that the a n t i g e n i c s i t e as detected i n the radioimmunoassay might r e s i d e there. Although the p r e c i s e nature of t h i s s i t e i s s t i l l undefined and may vary depending on the a n t i s e r a used to define i t , the cumulative weight of evidence i n d i c a t e s that the p r o t e i n p o r t i o n of the molecule plays the major, i f not e x c l u s i v e , r o l e i n determining the s i t e . This does not exclude the p o s s i b i l i t y that many CEA a n t i s e r a contain a n t i b o d i e s to carbohydrate. Under the c o n d i t i o n s of the usual radioimmunoassay only a r a t h e r narrow p o p u l a t i o n of high a f f i n i t y a n t i b o d i e s p l a y s a s i g n i f i cant r o l e (4). A consequence of t h i s s e l e c t i o n i s that the antiserum used may contain l a r g e populations of a n t i b o d i e s p l a y i n g an i n s i g n i f i c a n t r o l e i n the immunoassay t i t r a t i o n , but quite capable of r e a c t i n g with the antigen under the more concentrated c o n d i t i o n s p r e v a i l i n g i n immunodiffusion t e s t s . For t h i s reason i t i s dangerous to assume that phenomena observed by various immunodiffusion techniques are n e c e s s a r i l y involved i n immunoassay t i t r a t i o n s ( 4 _ ) .



residues

Terminal 4 3,4

Terminal 2 6 2,4 2,6 3,6

103 43 61 5 11 5 125 9 9 Trace 20 16 23 77 Trace 92 63 155

Terminal Terminal 3 2 6 3,4 and 2,3

a

x

CEA

23

linkage

Terminal

Glyoosidio

103 72 22 18 2 2 116 14 7 Trace 18 18 25 83 16 94 76 186

11

2

CEA

of h

95 63 18 9 4 4 98 13 7 Trace 13 18 22 73 20 110 83 213

13

CEA

CEA

96 4 14 Trace 18 23 18 77 5 83 41 129

110 36 11 36 13

18

h

CEA-14

A l l tumors were l i v e r

104 58 45 18 11 2 134 9 9 Trace 22 20 11 71 32 88 58 178

34

Z

CEA

5

8-10 g

T h e s u b s c r i p t s 1 to 4 i n d i c a t e CEA p u r i f i e d from d i f f e r e n t tumors. metastases that had o r i g i n a t e d i n the colon. ^Data from Hammarstrom et al. (33).

a

Total

Total N-Acetylglucosamine

Total Mannose

N-Acetylneuraminic acid Fucose Galactose

Carbohydrate

Moles/1.

STRUCTURAL UNITS OF THE POLYSACCHARIDE PORTION OF CEA

TABLE 2


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The evidence supporting the r o l e of the p r o t e i n p o r t i o n i n determining the a n t i g e n i c s i t e may be summarized as f o l l o w s . S e r i a l p e r i o d a t e o x i d a t i o n , Smith degradation (36), of CEA demonstrated p e r s i s t e n c e of a n t i g e n i c a c t i v i t y even a f t e r removal o f 90? of the carbohydrate ( 2 3 , 3 3 , 3 7 ) . A n t i s e r a to CEA can detect as l i t t l e as l O ' * M o f CEA. Such high b i n d i n g a f f i n i t i e s are c h a r a c t e r i s t i c a l l y achieved a g a i n s t p r o t e i n a n t i gens but not a g a i n s t carbohydrates (38). These high b i n d i n g a f f i n i t i e s can not be explained by b i v a l e n t antibody b i n d i n g to adjacent s i t e s on the CEA molecule, s i n c e e q u i v a l e n t concentrat i o n s of b i n d i n g s i t e s of monovalent Fab' fragments of CEA a n t i bodies were e q u a l l y e f f e c t i v e as the i n t a c t antibody i n the CEA radioimmunoassay (39_). D i l u t e a l k a l i treatment of CEA destroys i t s a n t i g e n i c a c t i v i t y , but does not a f f e c t i t s l e c t i n b i n d i n g ability (33). I t i s w e l l known that p r o t e i n s t r u c t u r e i s r a p i d l y degraded by a l k a l i , whereas the carbohydrate l i n k a g e s o f asparagine l i n k e d g l y c o p r o t e i n s are r e l a t i v e l y i n s e n s i t i v e to base. Westwood et al. (40) have shown that c o n t r o l l e d a c i d h y d r o l y s i s o f CEA destroys i t s a n t i g e n i c a c t i v i t y , producing N-terminal p r o l i n e peptides with i n t a c t carbohydrate chains except f o r the l o s s of s i a l i c a c i d and fucose. I t had p r e v i o u s l y been shown that d e s t r u c t i o n of s i a l i c a c i d and fucose by periodate o x i d a t i o n does not a l t e r the a n t i g e n i c a c t i v i t y (23). Reduction and a l k y l a t i o n o f the c y s t i n e d i s u l f i d e bonds i n CEA destroys i t s a n t i g e n i c a c t i v i t y (22,33,41,42). C l e a r l y the secondary and t e r t i a r y s t r u c t u r e s o f CEA are c r i t i c a l i n maintaining i t s a n t i genic nature.


1

The sequence o f the f i r s t 24 amino a c i d s at the N-terminus of CEA was r e a d i l y determined by standard automated Edman techniques. T h i s sequence i s presented i n Figure 2 (43). I t has been confirmed s e v e r a l times (44) and extended an a d d i t i o n a l 6 r e s i d u e s (45), although the choice between glutamic a c i d and glutamine at c e r t a i n r e s i d u e s has not been uniform (46). A d d i t i o n a l sequencing encountered t e c h n i c a l problems a r i s i n g from the high degree of g l y c o s y l a t i o n . These d i f f i c u l t i e s began with the r e s i s t a n c e to s p e c i f i c cleavage, presumably due to s t e r i c hinderance by the carbohydrate groups. Since the carbohydrate s u b s t i t u e n t s are h i g h l y heterogeneous i n degree of s u b s t i t u t i o n among a p o p u l a t i o n o f CEA molecules, s p e c i f i c cleavage methods d i d not g i v e uniform or s t o i c h i o m e t r i c y i e l d s of expected p e p t i d e s . Once peptides were obtained, t h e i r p u r i f i c a t i o n was rendered d i f f i c u l t by these heterogeneous carbohydrate substituents. Glycopeptides have broad, i l l d e f i n e d peaks i n separations based on c o n v e n t i o n a l s i z i n g techniques. Edman degradations o f g l y c o p e p t i d e s f r e q u e n t l y come to an abrupt h a l t when an amino a c i d to which carbohydrate i s attached i s encountered. The f i r s t of these d i f f i c u l t i e s was solved by the use of the detergent T r i t o n X100 (0.25?), which rendered CEA suscept i b l e to t r y p s i n cleavage (47). Although CEA c o n t a i n s s u f f i c i e n t a r g i n i n e and l y s i n e r e s i d u e s to produce about 40 t r y p t i c p e p t i d e s ,


TODD AND SHIVELY

Carcinoembryonic

Antigen

,0H

v

0-R

R-0

R-0

OCH,

HO

0-R

OCH,

OCH. 0

CH OH

CH OCCH

2


I

HCOCH3

HCOCH,

© (D (D

CH OCH

CH 0CH

3

3

I 0 HCOCCH,

HCOH

I

lo HCOCCH,

HCOH

I CH OCH 2

CH OCH 2

3

3

Cancer Research Figure 1. Methylation analysis. Numbers indicate preferred order of bond rupture under electron bombardment in the mass spectrometer.

CARCINOEMBRYONIC ANTIGEN A m i n o - T e r m i n a l Residues 5 10 Leu Thr He Glu Ser Thr Pro Phe A s n 15 Vol Ala Glu Gly Lys Glu Vol L e u Leu Leu vol His Asn L e u Lys

f at the N-terminus of CEA 2

S

e

q

u

e

n

c

e

0

a

m

i

n

0

a c i d s

—[AAAAAAAA)— ORIGINAL GENE

I —[AAAAAAAA]

|AAAAAAAA|—

DUPLICATED GENES ^ —fvvwvw] [wwwv^j— DIVERGENT GENES

Figure 3. Proposed genetic evolui f th protein chains of CEA and related antigens

t

on

or

e


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only 10 were i s o l a t e d . These were f r a c t i o n a t e d by a combinat i o n o f anion and c a t i o n exchange r e s i n s using high pressure l i q u i d chromatography. In some cases these peptides could be sequenced through 20 Edman degradation c y c l e s . D e t a i l s are presented elsewhere (47). C u r r e n t l y methods of d e g l y c o s y l a t i n g CEA are being evaluated with the o b j e c t i v e o f s i m p l i f y i n g the sequencing problem. I t has long been recognized that radioimmunoassay o f serum from normal i n d i v i d u a l s f o r CEA always showed some v a r i a b l e low l e v e l o f CEA r e a c t i v i t y . The demonstration i n normal t i s s u e s o f m a t e r i a l c r o s s r e a c t i v e with but d i s t i n g u i s h a b l e from CEA appeared to e x p l a i n the observed normal l e v e l s . These molecules included NCA (48), NGP (49), CCEA-2 (50), CEX (51), and 6 p r o t e i n (52). These molecules a l l have lower molecular weights than does CEA. Subsequently other groups (53-55) reported the presence i n normal serum and normal t i s s u e e x t r a c t s o f m a t e r i a l with CEA-like a c t i v i t y and s i m i l a r to CEA i n e l u t i o n from g e l f i l t r a t i o n c o l umns. Unfortunately the concentration o f t h i s substance i n normal t i s s u e was so low that s u f f i c i e n t m a t e r i a l could not be obtained f o r chemical c h a r a c t e r i z a t i o n . In 1974 Go et al. (56) measured the s e c r e t i o n i n various p o r t i o n s o f the g a s t r o i n t e s t i n a l t r a c t o f m a t e r i a l that i n h i b i t e d i n the assay f o r CEA. The colon secreted the l a r g e s t q u a n t i t y o f the m a t e r i a l . I t e l u t e d from a Sephadex G-200 column i n the same p o s i t i o n as CEA. The presence o f these q u a n t i t i e s of m a t e r i a l with CEA-like a c t i v i t y i n the colon lavages o f healthy i n d i v i d u a l s made i t p o s s i b l e to i s o l a t e s u f f i c i e n t m a t e r i a l f o r chemical characteri z a t i o n and d i r e c t comparison with CEA (57). The m a t e r i a l with CEA-like a c t i v i t y from c o l o n i c lavages was p u r i f i e d by g e l f i l t r a t i o n on Sepharose 6B and Sephadex G200 followed by a f f i n i t y chromatography on concanavalin A l i n k e d to Sepharose. The p u r i f i e d m a t e r i a l migrated i n polyacrylamide-sodium dodecyl s u l f a t e e l e c t r o p h o r e s i s as a s i n g l e d i f f u s e band with m o b i l i t y i d e n t i c a l to that o f tumor CEA. I t possessed the same s p e c i f i c a c t i v i t y as CEA i n radioimmunoassay. The carbohydrate and amino a c i d compositions were s i m i l a r to those o f CEA. Moreover, methylat i o n a n a l y s i s demonstrated that the monosaccharide linkages were s i m i l a r t o those i n CEA (57), and i t s N-terminal amino a c i d sequence was homologous to CEA (j>8). Thus by a l l c r i t e r i a a p p l i e d i t was i n d i s t i n g u i s h a b l e from CEA. I t thus seems l i k e l y that CEA i s a normal t i s s u e product. This r a i s e s the question of whether the elevated serum l e v e l s observed i n malignancy r e s u l t from increased c e l l u l a r s y n t h e s i s or by r e d i r e c t i o n to the blood stream o f m a t e r i a l normally e l i m i n a t e d i n the g a s t r o i n t e s tinal tract. During the i s o l a t i o n o f CEA from l i v e r metastases o f c o l o n i c adenocarcinoma, i t was observed that a lower molecular weight component possessing CEA a c t i v i t y i n the radioimmunoassay was present i n the eluate from the Sepharose 4B column. This materi a l , which we have designated TEX, has now been i s o l a t e d and puri f i e d (59). Like CEA, TEX i s a g l y c o p r o t e i n . I t binds to E


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concanavalin A Sepharose from which i t can be e l u t e d by d i s p l a c e ment with methyl a-D-mannoside. By Sephadex G200 g e l f i l t r a t i o n and by sodium dodecyl s u l f a t e polyacrylamide g e l e l e c t r o p h o r e s i s i t s molecular weight was shown to be about 110,000 d a l t o n s . TEX contained 35% carbohydrate, c o n s i d e r a b l y l e s s than that present i n CEA. Linkage s t u d i e s of the carbohydrate by methylat i o n a n a l y s i s revealed that TEX contained s u b s t a n t i a l l y l e s s t e r minal g a l a c t o s e , as w e l l as l e s s 4-linked i n t r a c h a i n and 3,4branched N-acetylglucosamine than CEA. A s i n g l e treatment with periodate destroyed a l l of the s i a l i c a c i d and fucose and 25% of the N-acetylglucosamine as with CEA, but i n a d d i t i o n twice as much mannose and g a l a c t o s e are destroyed as compared with CEA (59). When the s i z e of the p r o t e i n component i s c a l c u l a t e d from the molecular weight and percent p r o t e i n , i t i s found that t h i s s i z e i s n e a r l y i d e n t i c a l to that of CEA. S i m i l a r l y the amino a c i d composition o f TEX i s n e a r l y i d e n t i c a l to that o f CEA, except f o r the presence of small but r e p r o d u c i b l e amounts of methionine i n TEX, but not i n CEA. The sequence o f the f i r s t 24 N-terminal amino a c i d s of TEX d i f f e r s from CEA only i n the presence o f a l a n i n e , at p o s i t i o n 21 i n l i e u of v a l i n e i n CEA. The combined evidence, both immunological and chemical, i n d i cates that TEX i s c l o s e l y r e l a t e d to CEA, but d i f f e r s i n i t s mode and degree o f g l y c o s y l a t i o n and has at l e a s t one and probably more amino a c i d a l t e r a t i o n s i n i t s polypeptide chain (59). Since TEX, which was i s o l a t e d from a tumor, may be s i m i l a r to NCA and r e l a t e d m a t e r i a l s i s o l a t e d from normal t i s s u e , a preparation o f NCA was obtained from normal spleen t i s s u e by an immunochemical method u t i l i z i n g i n s o l u b i l i z e d a n t i b o d i e s to CEA and NCA (60). The molecular weight of the NCA obtained was found to be about 100,000 d a l t o n s . The carbohydrate composition was 30%. Unfortunately the m a t e r i a l a v a i l a b l e was i n s u f f i c i e n t f o r methylation a n a l y s i s and periodate o x i d a t i o n s t u d i e s . The sample possessed n e a r l y the same amino a c i d a n a l y s i s as CEA, but l i k e TEX contained a small but demonstrable amount of methionine. The sequence o f the f i r s t 26 N-terminal amino a c i d s was i d e n t i c a l to that o f CEA, except a t p o s i t i o n 21, where as i n TEX a l a nine i s present i n s t e a d of the v a l i n e o f CEA (60). The immunological data show that NCA and TEX are i d e n t i c a l i f tested with e i t h e r goat anti-NCA or r a b b i t anti-TEX, but TEX c r o s s r e a c t s with CEA when tested with monkey anti-CEA (59), an antiserum which was p r e v i o u s l y shown not to c r o s s r e a c t with spleen NCA (6l_). An a l t e r n a t i v e i n t e r p r e t a t i o n i s that TEX cont a i n s t r a c e amounts o f CEA]_ , r e c e n t l y described as i d e n t i c a l to CEA i n a l l respects except, perhaps, i n degree of g l y c o s y l a t i o n (62). Whatever may be the i n t e r r e l a t i o n s h i p s o f the v a r i o u s a n t i gens c r o s s r e a c t i v e with CEA (^-52,62), i t appears that from the standpoint o f primary s t r u c t u r e , i.e. sequence, of the p r o t e i n component, CEA, NCA, and TEX are c l o s e l y r e l a t e d . NCA and TEX may or may not d i f f e r from one another i n t h i s respect, but they both d i f f e r from CEA. We b e l i e v e that they a r i s e from d i s t i n c t 0W


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but related genes which have a common evolutionary origin in a single gene. This primoridal gene (Figure Z) has duplicated, or replicated to more than 2 genes, which have subsequently undergone divergent evolution. A more profound understanding of the interaction of these genes and their gene products in benign and malignant disease will hopefully lead to improved clinical utility of this system. ACKNOWLEDGMENTS


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