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Heterogeneity of human α-fetoprotein as revealed by isoelectric focusing in urea-containing gels
165
Biochimica et Biophysica Acta, 536 ( 1 9 7 8 ) 1 6 5 - - 1 7 1 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
BBA 37996
HETEROGENEITY OF HUMAN ~-FETOPROTEIN AS REVEALED BY ISOELECTRIC FOCUSING IN UREA-CONTAINING GELS
E R I C P. L E S T E R , J. B R U C E M I L L E R a n d S T A N L E Y Y A C H N I N *
Department of Medicine, The University of Chicago, and The Franhlin McLean Memorial Research Institute, Chicago, Ill. 60637 (U.S.A.) (Received March 23rd, 1978)
Summary The heterogeneity of human ~-fetoprotein has been studied by analytical isoelectric focusing in polyacrylamide gel slabs in the presence of 8 M urea. Six major isoelectric variants could be identified over a pH range of 6.0--6.2. Verification of their identity was achieved by crossed immunoelectrophoresis into agarose gel containing monospecific antiserum to human ~-fetoprotein. Complete desialylation of the protein did not abolish the heterogeneity; a complex pattern of major ~-fetoprotein bands persisted over a more alkaline pH range. We have been able to correlate the pattern of ~-fetoprotein heterogeneity seen following extended agarose gel electrophoresis with that obtained during isoelectric focusing in the presence of urea. The quantity of certain a-fetoprotein charge isomers in various ~-fetoprotein isolates may be important in considering certain biological functions of this protein.
Human a-fetoprotein has been shown to be heterogeneous by a variety of techniques including ion exchange chromatography [1,2], isoelectric focusing [3,4] and agarose gel electrophoresis, with and without crossed immunoelectrophoresis [2,5,6]. Since human ~-fetoprotein is capable of self-aggregation and may subsequently form disulfide-linked dimers and trimers [3], we undertook to study its molecular heterogeneity by means of isoelectric focusing in the presence of 8 M urea. We now present evidence that the microgeterogeneity of monomeric human ~-fetoprotein is more complex than suspected, since at least six major charge isomers of the protein may be identified by this technique.
* To whom reprint requests
should be addressed.
166 Materials and Methods Human a-fetoprotein was isolated from the serum and/or ascitic fluid of patients with hepatoma and gastric carcinoma. In addition, human a-fetoprotein was isolated from a saline homogenate of fetal livers of 15- to 20-week-old stillborn human abortuses, and from a saline homogenate of adult hepatoma tissue obtained at postmortem. Purification was accomplished by passage over an anti-human a-fetoprotein immunoadsorbent column, and trace impurities and polymeric human a-fetoprotein were eliminated by subsequent passage over an anti-whole human serum immunoadsorbent column and Sephadex G-150 [3,6]. Most human a-fetoprotein preparations were pure as judged by immunoelectrophoresis and polyacrylamide gel electrophoresis, although one preparation (Lu. I) contained trace amounts of human serum albumin. Extended electrophoresis at pH 8.6 in agarose gels was performed as previously described [6]. For analytic isoelectric focusing, 5% polyacrylamide gel slabs were used with the LKB Multiphor system according to the method of Vesterberg [7]. 15% cross-linking was achieved with the use of N,N-diallyl tartardiamide (Polyscience, Inc., Warrington, Pa.) and most gels contained 8 M urea (ultra-pure grade, Schwarz-Mann, Orangeburg, N.Y.). Ampholytes with a pH range of 5--7 or 4--6 (LKB) were used; the pH gradient was verified with a surface pH electrode (Model No. 47310, Ingold Electrode, Inc., Lexington, Mass.). 10-pg samples of purified human a-fetoprotein in a 5-pl volume were applied to the surface of the gel. Focusing was performed at 5 ° with an initial voltage of 300 V; the voltage was increased in 100 V increments every 15 min. The final period of electrofocusing was 2 h at 1000 V. Gels were fixed overnight in the following solution: 150 ml methanol/350 ml water/17.25 g sulfosalicyclic acid/57.5 g trichloracetic acid. They were then washed three times over a 48 h period in acetic acid/ethanol/water (8 : 25 : 67, v/v) and stained with a solution of Coomassie Blue R-250 (230 mg/200 ml acetic acid/ethanol/water) at 50 ° C. Gels were destained by repeated washing in acetic acid/ethanol/water until the background was clear. Gels were scanned at 540 nm with a linear transport chromatogram spectrophotometer (Zeiss PMQ II) for quantitation of the human a-fetoprotein isoelectric variants. In certain experiments, after completion of isoelectric focusing, the gel segment containing electrofocused protein was cut out, embedded in agarose gel containing monospeciflc rabbit anti-human a-fetoprotein, and subjected to electrophoresis at right angles to the isoelectric pH gradient. In other experiments after human a-fetoprotein was subjected to extended agarose gel electrophoresis [6], the gel segment containing the a-fetoprotein electrophoretic variants was removed, laid on the surface of a urea-acrylamide gel and subjected to isoelectric focusing at right angles to the direction of electrophoretic separation. Complete desialylation of human a-fetoprotein was achieved by digestion with Clostridium perfringens neuraminidase (Sigma, Type VI, St. Louis, Mo.) with monitoring of the amount of free sialic acid released so as to assure 100% liberation. Free and protein-bound sialic acid concentrations were determined by a modification of the method of Warren [6].
167
Results
Fig. l a shows the isoelectric focusing pattern of nine different human a-fetoprotein isolates from a variety of sources. Six major isoelectric variants in varying proportions can be discerned over a pH range of 6.0--6.2; these are termed human a-fetoprotein la, having the highest isoelectric point, 2a, 2b, 3a, 3b, and human a-fetoprotein 3c, having the lowest isoelectric point. In certain human -fetoprotein isolates, bands 2a, 3b, and 3c are split, and a suggestion of minor variants on the acid side of human a-fetoprotein 3c is seen. These patterns are highly reproducible over a period of months, even on samples which have been frozen (--80°C) and thawed on repeated occasions. Total desialylation of human a-fetoprotein alters, but does not abolish heterogeneity by isoelectric focusing (Fig. lb). The complex pattern of major human a-fetoprotein bands is moved to a more alkaline pH range. In order to verify that each protein band seen in isoelectric focusing is a human a-fetoprotein variant, such separations were analyzed by crossed immunoelectrophoresis into an anti-human a-fetoprotein-containing agarose gel. Reproducible patterns of human a-fetoprotein precipitation were obtained which closely resembled the spectrophotometric scans of the Coomassie Bluestained isoelectric focusing patterns (Fig. 2a). Similar heterogeneity was demonstrable when fresh-frozen human a-fetoprotein-containing sera were subjected to sequential isoelectric focusing and crossed immunoelectrophoresis (Fig. 2b), although the low concentrations of the fetoprotein in the sera prevented the visualization of some minor isoelectric variants.
HAFP hi 202630 3b 3c O ~ Liver
"-
~'.,
~9
b) Desialylated HAFP O d . X - Native o~z-o
i t~
Od.X-D Od. L i v e r - D Fet. Liver - O Ho-D
t~
Cr- D Lu Z - D Lu .n,.O
DH Fig. 1. M i c r o h e t e r o g e n e i t y o f h u m a n ~ - f e t o P r o t e i n ( H A F P ) as d i s p l a y e d b y i s o e l e c t r i c f o c u s i n g in p o l y a c r y l a m i d e gel c o n t a i n i n g 8 M u r e a . 10-/~g s a m p l e s o f h u m a n ~ - f e t o p r o t e i n i s o l a t e s f r o m h u m a n h e p a t o m a s e r u m ( O d . If, L u . I, L u . If, a n d M e F ) , ascitie f l u i d ( O d . I, H o . ) , t u m o r h o m o g e n a t e ( O d . liver), f e t a l liver h o m o g e n a t e and gastric c a r c i n o m a s e r u m (Cr.) w e r e s u b j e c t e d t o i s o e l e c t r i e f o c u s i n g i n p o l y a e r y l a m i d e gel p l a t e s c o n t a i n i n g 8 M u r e a a n d 2% a m p h o l y t e s , p H 5 - - 7 , a t 5 ° C f o r 4 h , w i t h a f i n a l p o w e r o f 1 0 0 0 V a n d 1 5 m A , u s i n g a n L K B M u l t i p h o r a p p a r a t u s . T h e p H r a n g e is i n d i c a t e d b e n e a t h e a c h f o c u s e d s a m p l e . N a t i v e s a m p l e s are d i s p l a y e d o n t h e l e f t (a) a n d s a m p l e s s u b j e c t e d t o c o m p l e t e , e n z y m a t i c d e s i a l y l a t i o n a r e d i s p l a y e d o n t h e r i g h t (b). T h e c e n t e r t o p d i s p l a y s a n e n l a r g e m e n t o f t h e n a t i v e O d . liver h u m a n ~ - f e t o p r o t e i n p a t t e r n w i t h l a b e l i n g o f e a c h ~ - f e t o p r o t e i n s p e c i e s ( l a , 2a, 2 b , 3a, 3 b , a n d 3 c ) . B a n d s 2 a a n d 3 b are split.
168 o) L u ] l : - Purified HAFP
b) Lu 11" Serum
/ I I~\\ HAFP I
I
6.3
6.1 pH
I I 5.9 6.3
i 6.1 pH
t 5.9
Fig. 2. Crossed i m m u n o e l e c t r o p h o r e s i s p a t t e r n s of h u m a n a - f e t o p r o t e i n ( H A F P ) isoelectric f o c u s i n g in 8 M u r e a - a c r y l a m i d e gels. S a m p l e s of (a) p u r i f i e d s e r u m a - f e t o p r o t e i n f r o m p a t i e n t Lu., or (b) n a t i v e s e r u m f r o m p a t i e n t Lu. w e r e e l e c t r o f o c u s e d in p o l y a c r y l a m i d e gel, p H 5--7, c o n t a i n i n g 8 M u r e a , c u t o u t , a n d s u b j e c t e d to crossed i m m u n o e l e c t r o p h o r e s i s i n t o a n a g a r o s e gel c o n t a i n i n g a 1 : 8 d i l u t i o n o f m o n o s p e cific r a b b i t a n t i - h u m a n a - f e t o p r o t e i n a n t i s e r u m . T h e p a t t e r n o f m i c r o h e t e r o g e n e i t y of p u r i f i e d L u . II h u m a n a - f e t o p r o t e i n is d i s p l a y e d b e n e a t h the c o r r e s p o n d i n g i m m u n o p r e c i p i t a t e on t h e left. T h e p H r a n g e is i n d i c a t e d b e n e a t h each s p e c i m e n . E a c h p r o t e i n b a n d is seen t o consist o f i m m u n o r e a c t i v e h u m a n a - f e t o p r o t e i n a n d t h e e x i s t e n c e o f e x t e n s i v e m i c r o h e t e r o g e n e i t y o f a - f e t o p r o t e i n in n a t i v e h e p a t o m a s e r u m is c o n f i r m e d .
The basis for the nomenclature we have utilized for human a-fetoprotein isoelectric variants is clarified when human a-fetoprotein isolates previously separated by agarose gel electrophoresis are analyzed by isoelectric focusing (Fig. 3). Human a-fetoprotein 1, the most positively charged native electrophoretic variant, appears as human a-fetoprotein la, while human a-fetoprotein 2 consists of 2a and 2b. Human a-fetoprotein 3 is even more complex and resolves into human a-fetoproteins 3a, 3b, and 3c by isoelectric focusing. In certain experiments the resolution is not entirely absolute (i.e., 2b may correspond in part to 3 as well as predominantly to 2) but whether this is a technical problem of diffusion within the agarose and isoelectric focusing gels cannot yet be stated with certainty. Discussion Our initial attempts to detail the microheterogeneity of human a-fetoprotein using isoelectric focusing in polyacrylamide gels revealed a specific and reproducible pattern of microheterogeneity for each human a-fetoprotein isolate
169
" 6.3
pH
Ha.
6.3 6.1 pH 5.9
0
AcJoroseElectrophoresi~ (~
Fig. 3. T w o d i m e n s i o n a l analysis o f h u m a n ~ - f e t o p r o t e i n ( H A F P ) . T h e r e l a t i o n s h i p b e t w e e n m i c r o h e t e r o g e n e i t y as d i s p l a y e d in e x t e n d e d agarose gel e l c c t r o p h o r e s i s f o l l o w e d b y isoelectric f o c u s i n g in 8 M u r e a a c r y l a m i d e gel. Purified h u m a n ~ - f e t o p r o t e i n f r o m p a t i e n t Od. ( t o p p a n e l ) or H o . ( b o t t o m p a n e l ) w a s subj e c t e d to e x t e n d e d agarose gel e l e c t r o p h o r e s i s , r e s u l t i n g in t h r e e b a n d s , h u m a n ~ - f e t o p r o t e i n s 1, 2, a n d 3, as d i s p l a y e d in t h e l o w e r left insets in e a c h p a n e l . R e p l i c a t e agarose gel strips c o n t a i n i n g t h e i s o m e r s a f t e r e l e c t r o p h o r e s i s w e r e c u t o u t , p l a c e d o n t h e s u r f a c e of an a e r y l a m i d e gel c o n t a i n i n g 8 M u r e a a n d 2% L K B a m p h o l y t e s , p H 5 - - 7 , a n d s u b j e c t e d to isoelectrie focusing. T h e l o w e r l e f t i n s e t in e a c h p a n e l is p o s i t i o n e d to i n d i c a t e the l o c a t i o n of the a g a r o s e gel strips relative t o t h e p o s i t i o n of t h e ~ - f e t o p r o t e i n i s o m e r s a f t e r e l e c t r o f o c u s i n g . T o t h e r i g h t in each p a n e l is a s a m p l e o f 10 p g o f h u m a n ~ - f e t o p r o t e i n w h i c h was e l e c t r o f o c u s e d s i m u l t a n e o u s l y . T h e d i r e c t i o n of a g a r o s e gel e l e c t r o p h o r e s i s is i n d i c a t e d on t h e b o t t o m a n d the p H r a n g e a f t e r e l e c t r o f o c u s i n g is i n d i c a t e d on t h e right. H u m a n ~ - f e t o p r o t e i n I is seen t o consist o f h u m a n ~ - f e t o p r o t e i n l a , 2 t o consist o f 2a a n d 2b, a n d 3 to consist o f h u m a n a - f e t o p r o t e i n s 3a, 3b, a n d 3c.
when it was focused in gels containing ampholytes in a pH range of 4--6 and 12.5% sucrose without urea. While the isoelectric points (pI) of the human alpha-fetoprotein variants ranged between 4.8 and 5.2, and thus agreed well with the pI of human ~-fetoprotein [2,3,4,8], the resolution of the charge isomers present was inadequate for quantitative purposes. The addition of a variety of non-ionic detergents did n o t alter the pI of human ~-fetoprotein b u t did improve resolution of the molecular variants to some ext~nt. Only 8 M urea provided excellent resolution of the microheterogeneity present [8]. The
170 pI of the human ~-fetoprotein variants was altered by 8 M urea and the possible exposure of charged groups on the molecule which are normally hidden in the absence of 8 M urea. Furthermore, the presence of 8 M urea decreases the activity coefficient of hydrogen ions, to give apparently higher pK and pI values of the carrier ampholytes and proteins [9]. In the present paper, we detail only the number and relative proportions of molecular charge variants of human ~-fetoprotein, recognizing that the isoelectric points determined in 8 M urea differ from those obtained when human ~-fetoprotein is in its native configuration. The detection of these variants in 8 M urea suggests that the charge differences between them depend on variations in molecular structure as opposed to conformational heterogeneity. It is unlikely that the use of urea has produced artifactual microgeterogeneity by carbamylation of human ~-fetoprotein because: {1) the pattern of microgeterogeneity was constant and reproducible for each individual human ~-fetoprotein preparation rather than random, as one would expect if it were produced solely by carbamylation; (2) the presence of large amounts of ampholytes {2%} serves to protect human ~-fetoprotein from carbamylation {the -NH~ groups of the ampholyte molecules acting as scavengers of any cyanate formed from urea) [10,12]; ( 3 ) t h e use of ultrapure urea, a low gel temperature (5°C}, and short focusing times {4 h) during which the samples exposed to urea reduce the possibility of carbamylation; and (4) the correlation observed between the pattern of heterogeneity seen in agarose gel electrophoresis and that found with isoelectric focusing in 8 M urea argues that the patterns seen in 8 M urea are not the product of artifactual alteration of human ~-fetoprotein; the most electronegative forms of human ~-fetoprotein seen with agarose gel electrophoresis display the lowest isoelectric points, as expected. In fact, the use of 8 M urea is now an accepted technique in isoelectric focusing of many proteins [9--13]. We have demonstrated that human ~-fetoprotein displays extensive microheterogeneity, as revealed by crossed immunoelectrophoresis following extended electrophoresis in agarose gel [6], and that this pattern of heterogeneity can be correlated with the charge isomer patterns displayed by the same human ~-fetoprotein during isoelectric focusing in the presence of 8 M urea. Thus, those human ~-fetoprotein isolates which consist predominantly of human ~-fetoprotein 3 by the former technique contain a high proportion of 3a, 3b, and 3c, while those which have a high proportion of human ~-fetoprotein 1 by electrophoresis display a large proportion of l a by isoelectric focusing. The appearance of a new electrophoretic species, human ~-fetoprotein 0, following enzymatic desialylation of human ~-fetoprotein [6] is also correlated with the appearance of a new, more positively charged human ~-fetoprotein isoelectric variant (Fig. lb}; indeed, close examination of the isoelectric focusing patterns obtained following desialylation suggests fine differences between most of the desialylated species and the native human ~-fetoprotein isoelectric forms from which they are derived. We, as well as others, have suggested that variation in sialic acid content of native human ~-fetoprorein could account only partially for the heterogeneity seen by electrophoresis [1,4,6]. It appears from our isoelectric focusing patterns of desialylated human ~-fetoprotein that in addition to variation in sialic acid content, heterogeneity is based upon multiple other charge differences in the human ~-fetoprotein
171 molecule, the nature of which remain to be determined. While the possibility exists that these charge differences are due to a genetically determined difference in amino acid composition, we believe that these charge differences are at least in part a post-synthetic modification of the human a-fetoprotein molecule which results in a shift from negatively to positively charged isomers. This conclusion is based upon two observations: (1)The human a-fetoprotein isolates obtained from fetal liver and hepatoma tissue are the most negatively charged preparations we have encountered [6,14]. (2) We have isolated human a-fetoprotein from the serum, ascitic fluid, and tumor of a single hepatoma patient and have found that tumor human a-fetoprotein is relatively rich in human a-fetoprotein 3 (particularly 3a), and that the proportion of 3a diminishes in the serum isolate and is almost absent in ascitic fluid human a-fetoprotein; conversely the proportion of l a increases in a mirror image pattern [14] (see Fig. la, specimens Od. I, Od. II and Od. liver). As the amount of 3a diminishes there is a dramatic parallel decrease in the immunosuppressive potency of these three a-fetoprotein isolates which cannot be attributed to a change in sialic acid content, suggesting, along with other evidence, that 3a is at least one of the negatively charged human a-fetoprotein isomers responsible for the inhibition of lymphocyte transformation in vitro [6,14]. Elucidation of the structural basis for the charge differences amongst human a-fetoprotein isomers may clarify the biochemical mechanism for modulation of the biological potency of human a-fetoprotein charge isomers.
Acknowledgments E.P.L. was the recipient of a USPHS-NCI Postdoctoral Research Fellowship Award (No. 1 F 32 CA05337-02). This work was supported by a grant from the Leukemia Research Foundation, by Grant No. 1-PO CA19266-02 awarded by the National Cancer Institute, DHEW and by training grant 5 T32 AM0713403 (NIAMD). The F.M.I. is operated by the University of Chicago for the U.S. Energy Research and Development Administration under Contract No. EY-76C-02-0069. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Lester, E.P., Miller, J.B., Baron, J.M. and Yaehnin, S. (1978) I m m u n o l o g y 34, 189--198 Alpert, E., Drysdale, J.W., Isselbacher, K.J. and Schur, P.H. (1972) J. Biol. Chem. 247, 3792--3798 Yachnin, S., Hsu, R., Heinrikson, R.L. and Miller, J.B. (1977) Biochim. Biophys. Acta 4 9 3 , 4 1 8 - - 4 2 8 Alpert, E. and Perencevich, R.C. (1975) Ann. N.Y. Acad. Sci. 259, 131--135 Purves, L.R., Van der Merwe, E. and Bersohn, T. (1970) S. Aft. Med. J. 44, 1264--1268 Lester, E.P.0 Miller, J.B. and Yachnin, S. (1976) Proc. Natl. Acad. Sci. U.S. 73, 4645--4648 Vesterberg, O. (1972) Biochim. Biophys. Acta 257, 11--19 Lester, E.P., Miller, J.B. and Yachnin, S. (1978) [mmunol. Comm. 7 , 1 3 7 - - 1 6 1 Righetti, P.B. and Drysdale, J.W. (1974) J. Chromatogr. 9 8 , 2 7 1 - - 3 2 1 Bhakdi, S., Knuferrnann, H. and Wallaeh, D.F.H. (1975) Biochim. Biophys. Acta 394, 550--557 Miller, D.W. and Elgin, S.C.R. (1974) Anal. Biochem. 60, 142--148 Catsimpoolas, N. and Wang, J. (1971) Anal. Biochem. 44, 436--444 Ui, N. (1971) Biochim. Biophys. Acta 2 2 9 , 5 6 7 ~ 5 8 1 Lester, E.P., Miller, J.B. and Yachnin, S. (1977) Proc. Natl. Acad. Sci. U.S. 74, 3988--3992
Biochimica et Biophysica Acta, 536 ( 1 9 7 8 ) 1 6 5 - - 1 7 1 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
BBA 37996
HETEROGENEITY OF HUMAN ~-FETOPROTEIN AS REVEALED BY ISOELECTRIC FOCUSING IN UREA-CONTAINING GELS
E R I C P. L E S T E R , J. B R U C E M I L L E R a n d S T A N L E Y Y A C H N I N *
Department of Medicine, The University of Chicago, and The Franhlin McLean Memorial Research Institute, Chicago, Ill. 60637 (U.S.A.) (Received March 23rd, 1978)
Summary The heterogeneity of human ~-fetoprotein has been studied by analytical isoelectric focusing in polyacrylamide gel slabs in the presence of 8 M urea. Six major isoelectric variants could be identified over a pH range of 6.0--6.2. Verification of their identity was achieved by crossed immunoelectrophoresis into agarose gel containing monospecific antiserum to human ~-fetoprotein. Complete desialylation of the protein did not abolish the heterogeneity; a complex pattern of major ~-fetoprotein bands persisted over a more alkaline pH range. We have been able to correlate the pattern of ~-fetoprotein heterogeneity seen following extended agarose gel electrophoresis with that obtained during isoelectric focusing in the presence of urea. The quantity of certain a-fetoprotein charge isomers in various ~-fetoprotein isolates may be important in considering certain biological functions of this protein.
Human a-fetoprotein has been shown to be heterogeneous by a variety of techniques including ion exchange chromatography [1,2], isoelectric focusing [3,4] and agarose gel electrophoresis, with and without crossed immunoelectrophoresis [2,5,6]. Since human ~-fetoprotein is capable of self-aggregation and may subsequently form disulfide-linked dimers and trimers [3], we undertook to study its molecular heterogeneity by means of isoelectric focusing in the presence of 8 M urea. We now present evidence that the microgeterogeneity of monomeric human ~-fetoprotein is more complex than suspected, since at least six major charge isomers of the protein may be identified by this technique.
* To whom reprint requests
should be addressed.
166 Materials and Methods Human a-fetoprotein was isolated from the serum and/or ascitic fluid of patients with hepatoma and gastric carcinoma. In addition, human a-fetoprotein was isolated from a saline homogenate of fetal livers of 15- to 20-week-old stillborn human abortuses, and from a saline homogenate of adult hepatoma tissue obtained at postmortem. Purification was accomplished by passage over an anti-human a-fetoprotein immunoadsorbent column, and trace impurities and polymeric human a-fetoprotein were eliminated by subsequent passage over an anti-whole human serum immunoadsorbent column and Sephadex G-150 [3,6]. Most human a-fetoprotein preparations were pure as judged by immunoelectrophoresis and polyacrylamide gel electrophoresis, although one preparation (Lu. I) contained trace amounts of human serum albumin. Extended electrophoresis at pH 8.6 in agarose gels was performed as previously described [6]. For analytic isoelectric focusing, 5% polyacrylamide gel slabs were used with the LKB Multiphor system according to the method of Vesterberg [7]. 15% cross-linking was achieved with the use of N,N-diallyl tartardiamide (Polyscience, Inc., Warrington, Pa.) and most gels contained 8 M urea (ultra-pure grade, Schwarz-Mann, Orangeburg, N.Y.). Ampholytes with a pH range of 5--7 or 4--6 (LKB) were used; the pH gradient was verified with a surface pH electrode (Model No. 47310, Ingold Electrode, Inc., Lexington, Mass.). 10-pg samples of purified human a-fetoprotein in a 5-pl volume were applied to the surface of the gel. Focusing was performed at 5 ° with an initial voltage of 300 V; the voltage was increased in 100 V increments every 15 min. The final period of electrofocusing was 2 h at 1000 V. Gels were fixed overnight in the following solution: 150 ml methanol/350 ml water/17.25 g sulfosalicyclic acid/57.5 g trichloracetic acid. They were then washed three times over a 48 h period in acetic acid/ethanol/water (8 : 25 : 67, v/v) and stained with a solution of Coomassie Blue R-250 (230 mg/200 ml acetic acid/ethanol/water) at 50 ° C. Gels were destained by repeated washing in acetic acid/ethanol/water until the background was clear. Gels were scanned at 540 nm with a linear transport chromatogram spectrophotometer (Zeiss PMQ II) for quantitation of the human a-fetoprotein isoelectric variants. In certain experiments, after completion of isoelectric focusing, the gel segment containing electrofocused protein was cut out, embedded in agarose gel containing monospeciflc rabbit anti-human a-fetoprotein, and subjected to electrophoresis at right angles to the isoelectric pH gradient. In other experiments after human a-fetoprotein was subjected to extended agarose gel electrophoresis [6], the gel segment containing the a-fetoprotein electrophoretic variants was removed, laid on the surface of a urea-acrylamide gel and subjected to isoelectric focusing at right angles to the direction of electrophoretic separation. Complete desialylation of human a-fetoprotein was achieved by digestion with Clostridium perfringens neuraminidase (Sigma, Type VI, St. Louis, Mo.) with monitoring of the amount of free sialic acid released so as to assure 100% liberation. Free and protein-bound sialic acid concentrations were determined by a modification of the method of Warren [6].
167
Results
Fig. l a shows the isoelectric focusing pattern of nine different human a-fetoprotein isolates from a variety of sources. Six major isoelectric variants in varying proportions can be discerned over a pH range of 6.0--6.2; these are termed human a-fetoprotein la, having the highest isoelectric point, 2a, 2b, 3a, 3b, and human a-fetoprotein 3c, having the lowest isoelectric point. In certain human -fetoprotein isolates, bands 2a, 3b, and 3c are split, and a suggestion of minor variants on the acid side of human a-fetoprotein 3c is seen. These patterns are highly reproducible over a period of months, even on samples which have been frozen (--80°C) and thawed on repeated occasions. Total desialylation of human a-fetoprotein alters, but does not abolish heterogeneity by isoelectric focusing (Fig. lb). The complex pattern of major human a-fetoprotein bands is moved to a more alkaline pH range. In order to verify that each protein band seen in isoelectric focusing is a human a-fetoprotein variant, such separations were analyzed by crossed immunoelectrophoresis into an anti-human a-fetoprotein-containing agarose gel. Reproducible patterns of human a-fetoprotein precipitation were obtained which closely resembled the spectrophotometric scans of the Coomassie Bluestained isoelectric focusing patterns (Fig. 2a). Similar heterogeneity was demonstrable when fresh-frozen human a-fetoprotein-containing sera were subjected to sequential isoelectric focusing and crossed immunoelectrophoresis (Fig. 2b), although the low concentrations of the fetoprotein in the sera prevented the visualization of some minor isoelectric variants.
HAFP hi 202630 3b 3c O ~ Liver
"-
~'.,
~9
b) Desialylated HAFP O d . X - Native o~z-o
i t~
Od.X-D Od. L i v e r - D Fet. Liver - O Ho-D
t~
Cr- D Lu Z - D Lu .n,.O
DH Fig. 1. M i c r o h e t e r o g e n e i t y o f h u m a n ~ - f e t o P r o t e i n ( H A F P ) as d i s p l a y e d b y i s o e l e c t r i c f o c u s i n g in p o l y a c r y l a m i d e gel c o n t a i n i n g 8 M u r e a . 10-/~g s a m p l e s o f h u m a n ~ - f e t o p r o t e i n i s o l a t e s f r o m h u m a n h e p a t o m a s e r u m ( O d . If, L u . I, L u . If, a n d M e F ) , ascitie f l u i d ( O d . I, H o . ) , t u m o r h o m o g e n a t e ( O d . liver), f e t a l liver h o m o g e n a t e and gastric c a r c i n o m a s e r u m (Cr.) w e r e s u b j e c t e d t o i s o e l e c t r i e f o c u s i n g i n p o l y a e r y l a m i d e gel p l a t e s c o n t a i n i n g 8 M u r e a a n d 2% a m p h o l y t e s , p H 5 - - 7 , a t 5 ° C f o r 4 h , w i t h a f i n a l p o w e r o f 1 0 0 0 V a n d 1 5 m A , u s i n g a n L K B M u l t i p h o r a p p a r a t u s . T h e p H r a n g e is i n d i c a t e d b e n e a t h e a c h f o c u s e d s a m p l e . N a t i v e s a m p l e s are d i s p l a y e d o n t h e l e f t (a) a n d s a m p l e s s u b j e c t e d t o c o m p l e t e , e n z y m a t i c d e s i a l y l a t i o n a r e d i s p l a y e d o n t h e r i g h t (b). T h e c e n t e r t o p d i s p l a y s a n e n l a r g e m e n t o f t h e n a t i v e O d . liver h u m a n ~ - f e t o p r o t e i n p a t t e r n w i t h l a b e l i n g o f e a c h ~ - f e t o p r o t e i n s p e c i e s ( l a , 2a, 2 b , 3a, 3 b , a n d 3 c ) . B a n d s 2 a a n d 3 b are split.
168 o) L u ] l : - Purified HAFP
b) Lu 11" Serum
/ I I~\\ HAFP I
I
6.3
6.1 pH
I I 5.9 6.3
i 6.1 pH
t 5.9
Fig. 2. Crossed i m m u n o e l e c t r o p h o r e s i s p a t t e r n s of h u m a n a - f e t o p r o t e i n ( H A F P ) isoelectric f o c u s i n g in 8 M u r e a - a c r y l a m i d e gels. S a m p l e s of (a) p u r i f i e d s e r u m a - f e t o p r o t e i n f r o m p a t i e n t Lu., or (b) n a t i v e s e r u m f r o m p a t i e n t Lu. w e r e e l e c t r o f o c u s e d in p o l y a c r y l a m i d e gel, p H 5--7, c o n t a i n i n g 8 M u r e a , c u t o u t , a n d s u b j e c t e d to crossed i m m u n o e l e c t r o p h o r e s i s i n t o a n a g a r o s e gel c o n t a i n i n g a 1 : 8 d i l u t i o n o f m o n o s p e cific r a b b i t a n t i - h u m a n a - f e t o p r o t e i n a n t i s e r u m . T h e p a t t e r n o f m i c r o h e t e r o g e n e i t y of p u r i f i e d L u . II h u m a n a - f e t o p r o t e i n is d i s p l a y e d b e n e a t h the c o r r e s p o n d i n g i m m u n o p r e c i p i t a t e on t h e left. T h e p H r a n g e is i n d i c a t e d b e n e a t h each s p e c i m e n . E a c h p r o t e i n b a n d is seen t o consist o f i m m u n o r e a c t i v e h u m a n a - f e t o p r o t e i n a n d t h e e x i s t e n c e o f e x t e n s i v e m i c r o h e t e r o g e n e i t y o f a - f e t o p r o t e i n in n a t i v e h e p a t o m a s e r u m is c o n f i r m e d .
The basis for the nomenclature we have utilized for human a-fetoprotein isoelectric variants is clarified when human a-fetoprotein isolates previously separated by agarose gel electrophoresis are analyzed by isoelectric focusing (Fig. 3). Human a-fetoprotein 1, the most positively charged native electrophoretic variant, appears as human a-fetoprotein la, while human a-fetoprotein 2 consists of 2a and 2b. Human a-fetoprotein 3 is even more complex and resolves into human a-fetoproteins 3a, 3b, and 3c by isoelectric focusing. In certain experiments the resolution is not entirely absolute (i.e., 2b may correspond in part to 3 as well as predominantly to 2) but whether this is a technical problem of diffusion within the agarose and isoelectric focusing gels cannot yet be stated with certainty. Discussion Our initial attempts to detail the microheterogeneity of human a-fetoprotein using isoelectric focusing in polyacrylamide gels revealed a specific and reproducible pattern of microheterogeneity for each human a-fetoprotein isolate
169
" 6.3
pH
Ha.
6.3 6.1 pH 5.9
0
AcJoroseElectrophoresi~ (~
Fig. 3. T w o d i m e n s i o n a l analysis o f h u m a n ~ - f e t o p r o t e i n ( H A F P ) . T h e r e l a t i o n s h i p b e t w e e n m i c r o h e t e r o g e n e i t y as d i s p l a y e d in e x t e n d e d agarose gel e l c c t r o p h o r e s i s f o l l o w e d b y isoelectric f o c u s i n g in 8 M u r e a a c r y l a m i d e gel. Purified h u m a n ~ - f e t o p r o t e i n f r o m p a t i e n t Od. ( t o p p a n e l ) or H o . ( b o t t o m p a n e l ) w a s subj e c t e d to e x t e n d e d agarose gel e l e c t r o p h o r e s i s , r e s u l t i n g in t h r e e b a n d s , h u m a n ~ - f e t o p r o t e i n s 1, 2, a n d 3, as d i s p l a y e d in t h e l o w e r left insets in e a c h p a n e l . R e p l i c a t e agarose gel strips c o n t a i n i n g t h e i s o m e r s a f t e r e l e c t r o p h o r e s i s w e r e c u t o u t , p l a c e d o n t h e s u r f a c e of an a e r y l a m i d e gel c o n t a i n i n g 8 M u r e a a n d 2% L K B a m p h o l y t e s , p H 5 - - 7 , a n d s u b j e c t e d to isoelectrie focusing. T h e l o w e r l e f t i n s e t in e a c h p a n e l is p o s i t i o n e d to i n d i c a t e the l o c a t i o n of the a g a r o s e gel strips relative t o t h e p o s i t i o n of t h e ~ - f e t o p r o t e i n i s o m e r s a f t e r e l e c t r o f o c u s i n g . T o t h e r i g h t in each p a n e l is a s a m p l e o f 10 p g o f h u m a n ~ - f e t o p r o t e i n w h i c h was e l e c t r o f o c u s e d s i m u l t a n e o u s l y . T h e d i r e c t i o n of a g a r o s e gel e l e c t r o p h o r e s i s is i n d i c a t e d on t h e b o t t o m a n d the p H r a n g e a f t e r e l e c t r o f o c u s i n g is i n d i c a t e d on t h e right. H u m a n ~ - f e t o p r o t e i n I is seen t o consist o f h u m a n ~ - f e t o p r o t e i n l a , 2 t o consist o f 2a a n d 2b, a n d 3 to consist o f h u m a n a - f e t o p r o t e i n s 3a, 3b, a n d 3c.
when it was focused in gels containing ampholytes in a pH range of 4--6 and 12.5% sucrose without urea. While the isoelectric points (pI) of the human alpha-fetoprotein variants ranged between 4.8 and 5.2, and thus agreed well with the pI of human ~-fetoprotein [2,3,4,8], the resolution of the charge isomers present was inadequate for quantitative purposes. The addition of a variety of non-ionic detergents did n o t alter the pI of human ~-fetoprotein b u t did improve resolution of the molecular variants to some ext~nt. Only 8 M urea provided excellent resolution of the microheterogeneity present [8]. The
170 pI of the human ~-fetoprotein variants was altered by 8 M urea and the possible exposure of charged groups on the molecule which are normally hidden in the absence of 8 M urea. Furthermore, the presence of 8 M urea decreases the activity coefficient of hydrogen ions, to give apparently higher pK and pI values of the carrier ampholytes and proteins [9]. In the present paper, we detail only the number and relative proportions of molecular charge variants of human ~-fetoprotein, recognizing that the isoelectric points determined in 8 M urea differ from those obtained when human ~-fetoprotein is in its native configuration. The detection of these variants in 8 M urea suggests that the charge differences between them depend on variations in molecular structure as opposed to conformational heterogeneity. It is unlikely that the use of urea has produced artifactual microgeterogeneity by carbamylation of human ~-fetoprotein because: {1) the pattern of microgeterogeneity was constant and reproducible for each individual human ~-fetoprotein preparation rather than random, as one would expect if it were produced solely by carbamylation; (2) the presence of large amounts of ampholytes {2%} serves to protect human ~-fetoprotein from carbamylation {the -NH~ groups of the ampholyte molecules acting as scavengers of any cyanate formed from urea) [10,12]; ( 3 ) t h e use of ultrapure urea, a low gel temperature (5°C}, and short focusing times {4 h) during which the samples exposed to urea reduce the possibility of carbamylation; and (4) the correlation observed between the pattern of heterogeneity seen in agarose gel electrophoresis and that found with isoelectric focusing in 8 M urea argues that the patterns seen in 8 M urea are not the product of artifactual alteration of human ~-fetoprotein; the most electronegative forms of human ~-fetoprotein seen with agarose gel electrophoresis display the lowest isoelectric points, as expected. In fact, the use of 8 M urea is now an accepted technique in isoelectric focusing of many proteins [9--13]. We have demonstrated that human ~-fetoprotein displays extensive microheterogeneity, as revealed by crossed immunoelectrophoresis following extended electrophoresis in agarose gel [6], and that this pattern of heterogeneity can be correlated with the charge isomer patterns displayed by the same human ~-fetoprotein during isoelectric focusing in the presence of 8 M urea. Thus, those human ~-fetoprotein isolates which consist predominantly of human ~-fetoprotein 3 by the former technique contain a high proportion of 3a, 3b, and 3c, while those which have a high proportion of human ~-fetoprotein 1 by electrophoresis display a large proportion of l a by isoelectric focusing. The appearance of a new electrophoretic species, human ~-fetoprotein 0, following enzymatic desialylation of human ~-fetoprotein [6] is also correlated with the appearance of a new, more positively charged human ~-fetoprotein isoelectric variant (Fig. lb}; indeed, close examination of the isoelectric focusing patterns obtained following desialylation suggests fine differences between most of the desialylated species and the native human ~-fetoprotein isoelectric forms from which they are derived. We, as well as others, have suggested that variation in sialic acid content of native human ~-fetoprorein could account only partially for the heterogeneity seen by electrophoresis [1,4,6]. It appears from our isoelectric focusing patterns of desialylated human ~-fetoprotein that in addition to variation in sialic acid content, heterogeneity is based upon multiple other charge differences in the human ~-fetoprotein
171 molecule, the nature of which remain to be determined. While the possibility exists that these charge differences are due to a genetically determined difference in amino acid composition, we believe that these charge differences are at least in part a post-synthetic modification of the human a-fetoprotein molecule which results in a shift from negatively to positively charged isomers. This conclusion is based upon two observations: (1)The human a-fetoprotein isolates obtained from fetal liver and hepatoma tissue are the most negatively charged preparations we have encountered [6,14]. (2) We have isolated human a-fetoprotein from the serum, ascitic fluid, and tumor of a single hepatoma patient and have found that tumor human a-fetoprotein is relatively rich in human a-fetoprotein 3 (particularly 3a), and that the proportion of 3a diminishes in the serum isolate and is almost absent in ascitic fluid human a-fetoprotein; conversely the proportion of l a increases in a mirror image pattern [14] (see Fig. la, specimens Od. I, Od. II and Od. liver). As the amount of 3a diminishes there is a dramatic parallel decrease in the immunosuppressive potency of these three a-fetoprotein isolates which cannot be attributed to a change in sialic acid content, suggesting, along with other evidence, that 3a is at least one of the negatively charged human a-fetoprotein isomers responsible for the inhibition of lymphocyte transformation in vitro [6,14]. Elucidation of the structural basis for the charge differences amongst human a-fetoprotein isomers may clarify the biochemical mechanism for modulation of the biological potency of human a-fetoprotein charge isomers.
Acknowledgments E.P.L. was the recipient of a USPHS-NCI Postdoctoral Research Fellowship Award (No. 1 F 32 CA05337-02). This work was supported by a grant from the Leukemia Research Foundation, by Grant No. 1-PO CA19266-02 awarded by the National Cancer Institute, DHEW and by training grant 5 T32 AM0713403 (NIAMD). The F.M.I. is operated by the University of Chicago for the U.S. Energy Research and Development Administration under Contract No. EY-76C-02-0069. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14
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