Structure of hepatitis B surface antigen. Correlation of subtype with

THE JOURNAL OF BIOLOGICAL CHEMISTRY

Printed in U.S.A. Vol. 257, No. 17, Issue of September 10, pp. 10414-10420. 1982

Structure of Hepatitis B Surface Antigen CORRELATION OF SUBTYPE WITH AMINO ACID SEQUENCE AND LOCATION OF THE CARBOHYDRATE MOIETY*

(Received for publication, December 8, 1981)

Darrell L. Peterson$& Narapendra Nathy, and Francisco GavilanesS From the +Department of Biochemistry, Medical College of Virginia, Richmond, Virginia 23298 and the YAmerican Red Cross, Blood Research Center, Bethesda, Maryland 20014

Hepatitis B surface antigens (HBsAg) of both the adw and ayw subtypes have been purified from four different sources. These antigens have been compared by comparison of the products of tryptic hydrolysis performed under conditions which do not disrupt the overall particle morphology of HBsAg. The resultant peptides were compared by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and high performance liquid chromatograpby followed by amino acid analysis and Edman degradation of the isolated peptides. The same techniques were also applied to HBsAg which had been labeled with tritium in the carbohydrate moiety of the glycoprotein gp-30. These studies demonstrate that residues 122-150 of the protein p-25 and glycoprotein gp-30 occupy an exposed region of the HBsAg lipoprotein particle and contain the major attachment site for carbohydrate in the case of gp-30. The two subtypes were found to differ at two specific positions in this region, suggesting that this is an antigenically important area of the protein.

HBsAg’ is a complex macromolecular aggregate (Mr = 2-4 X lo6) composed of a major protein (p-25), a major glycoprotein (gp-30), and lipid (1-4). We have previously shown that p-25 and gp-30 have identical amino acid sequences and differ only by the presence of carbohydrate in gp-30 (5). Other minor protein components which are observed upon SDS-polyacrylamide gel electrophoresis have been shown to be aggregates of the major components (5, 6). Also, some contaminating normal human plasma proteins may be present in trace amounts. HBsAg is also antigenically complex, containing at least three distinct determinants (4). All HBsAg contain the group specific “a” determinant. In addition, HBsAg contain either the “d” or “y” determinant, and either the “w” or “r” determinant. Thus, there are four major antigenic types of HBsAg, adw, ayw, adr, and ayr (7). It is generally accepted that these antigenic activities are largely a function of the protein moiety of HBsAg, since the isolated major protein, p- 25, has been shown to possess both group-specific and subtype-specific antigenic activities (8-12). However, the structural basis for these various antigenic activities has not been determined.

There is considerable interest in identifying the structural features which specify the antigenic activities of HBsAg, since

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

8 Recipient of Research Grant AI 15955 from the National Insti- tutes of Health.

’ The abbreviations used are: HBsAg, hepatitis B surface antigen; SDS, sodium dodecyl sulfate; PTH, phenylthiohydantoin.

this might allow the chemical synthesis of peptides containing these determinants for use in a synthetic vaccine against hepatitis B virus. Indeed, some success in this regard has been recently reported (13, 14). Although the complete antigenic structures of a few proteins have been reported (see Ref. 15 for review), the structural complexities of proteins in general, and the difficulty in working with the extremely hydrophobic proteins of HBsAg in particular presents a formidable prob- lem.

One approach toward identifying experimentally the antigenic determinants of HBsAg is to locate external features of the protein, as it exists in the intact antigen particle and then to identify the structural differences in these regions of the molecule which are correlated with differences in the subtypes of HBsAg. Since the subtype of the antigen is under the genetic control of the virus, it is presumed that these antigenic differences will reflect differences in the amino acid sequences of the surface antigen protein p-25.

The complete amino acid sequence of the p-25 protein of HBsAg has been deduced from partial amino acid sequence data and from the nucleotide sequence of the viral gene which codes for this protein (11, 16-18). The complete nucleotide sequence of this gene has been determined by three different groups (16-18). Although the amino acid sequences of the protein predicted from these three studies are nearly identical, they do differ at 20 positions. These differences could reflect differences in the subtype of the antigen used or be due to heterogeneity in the viral proteins unrelated to the subtype. However, from the reported data, this cannot be decided, since only Charnay et al. (18) reported the subtype of their antigen as one of the usual subtypes, ayw. Pasek et al. (17) reported that their antigen is of an unusual subtype (adyw) and Valenzuela et al. (16) did not determine the subtype of their antigen.

Two approaches have been used to identify exposed regions of the protein in HBsAg: susceptibility to proteolytic cleavage and identification of the attachment site for carbohydrate. In this paper, we report data which support the conclusions that residues 122-150 of protein p-25 and glycoprotein gp-30 OC- cupy an exposed position on the HBsAg lipoprotein particle and contain the major (perhaps only) attachment site for carbohydrate. Furthermore, the adw and ayw subtypes of HBsAg were found to differ at two specific positions in this region of the molecule. Thus, it is likely that this region of the molecule is antigenically important.

EXPERIMENTAL PROCEDURES

Materials Electrophoresis reagents were obtained from Bio-Rad, and sequen-

ator reagents were from Beckman. Trypsin (dicyclohexylcarbodi- imide-treated) and carboxypeptidase A were from Sigma. AU solvents for high performance liquid chromatography were spectrophotomet-

104 14

Structural Comparisons of HBsAg Proteins of Different Subtypes 10415

ric grade from Baker, Phillipsburg, NJ. Amino acid analyzer reagents and buffers were obtained from Dunurn, Sunnyvale, CA.

Methods Asssay and Purification of HBsAg-HBsAg was assayed and

purified from the plasma of high titer chronic carriers of HBsAg of either the adw or ayw subtype by the methods which we have previously described (5). No changes in the procedure were needed regardless of the subtype being isolated. The subtype of the HBsAg was determined with the starting plasma by the method of Prince et al. (19).

Polyacrylamide Gel Electrophoresis-Analytical polyacrylamide gel electrophoresis was performed with a Bio-Rad slab gel apparatus with 12 to 23% linear gradient polyacrylamide gels. A 4% stacking gel was used. The gel and buffer formulations were those of OFarrell (20).

Reduction and Aminoethylation-Aminoethylation of HBsAg or its component proteins was performed as previously described (5) except that no SDS was used.

Amino Acid Analysis-Aliquots of peptide (0.1-0.5 nmol) were hydrolyzed in sealed, evacuated tubes with constantly boiling HCl for 24 h at 110 "C. Amino acid analysis was performed with a Durmm MBF amino acid analyzer,

Sequence Analysis-Edman degradations were performed either manually or with a Beckman 890C Sequencer equipped with a cold trap. For automatic sequencing, approximately 20 nmol of protein were applied with the standard sample application subroutine. Edman degradation was then performed with the 0.1 M Quadrol program (Beckman Program No. 121078). The released thiazolinone derivatives were converted to phenylthiohydantoins by treatment with 1 N HC1 in methanol at 50 "C for 10 min. The PTH derivatives were then identified by high performance liquid chromatography on a Beckman column. The PTH derivatives were eluted with a linear gradient between solvent A (5% tetrahydrofuran, 95% 5.25 nm sodium acetate, pH 5.02) and solvent B (10% tetrahydrofuran, 90% acetonitrile). A gradient from 0 to 40% solvent B in 20 min at 1.3 ml/min was used. The elution was monitored at 269 nm. Alternatively, the PTH derivatives were hydrolyzed with HI and identified by amino acid analysis (5). Manual Edman degradations were performed by the method of Tarr (21). Generally, 5-10 nmol of peptide were used. AU solvents contained 100 $/liter of ethanethiol. The released PTH derivatives were identified as described above.

Carboxypeptidase Digestion-Carboxypeptidase digestion were performed as previously described (5).

Tryptic Digestion of HBsAg-Tryptic digestion of HBsAg was performed in 0.05 M ammonium bicarbonate, a t an enzyme-to-sub- strate ratio of 1:50 to 1:14. Mercaptoethanol, if used, was present at a concentration of 1%. Generally, the protein concentration was 1 m g / d and the digestion was allowed to proceed for 2-6 h at 37 "C.

High PerfoFance Liquid Chromatography of Peptides-The separation of the soluble tryptic peptides of reduced and aminoethylated HBsAg was performed with a Varian Model 5000 liquid chromatograph on a Varian MCH-10 reverse phase (C-18) column. The column was equilibrated with 54.4 nm sodium phosphate, pH 2.8. The sample was applied in 100 pl of this buffer and then eluted with a gradient of this buffer and acetonitrile as second solvent. The percentage of acetonitrile, initially a t zero, was increased to 12% during the first 13 min, then increased to 28% during the next 65 min, and finally taken to 62% during the final 30 min.

Labeling of the Sialic Acid Moiety of HBsAg Glycoprotein-The method of Van Lenten and Ashwell (22) was modifled and applied to HBsAg. Hepatitis B surface antigen was delipidated by extraction with chloroform/methanol. The insoluble protein was collected by centrifugation and freeze dried. The freeze-dried protein (7 mg) was suspended in 700 pl of 0.1 M sodium acetate, pH 5.6, and then 300 p1 of 0.024 M sodium periodate was added. The reaction was allowed to proceed for 15 min at 0 "C and then terminated by dilution with 5 d of cold buffer and centrifugation of the insoluble protein. The protein precipitate was washed five times with 5 ml of 0.05 M sodium phosphate, pH 7.5, and then suspended in 500 /.tl of this buffer. Sodium borotritiide (1 mCi) was then added and allowed to react for 30 min. Excess borohydride was then added. The labeled protein was washed by repeated suspension and centrifugation until all free tritium was removed. The specific activity of the protein was determined by counting an aliquot after dissolving in 1% SDS. [3H]Toluene was used as an internal standard for calculating counting efficiency.

2 9 40 U E N I T S G F L G P L L V L Q A G F F L L T R I L T I P Q S L D S W W T S L N

S P I T T ; 60 T 89

P L G G T T V C L C Q N S Q S P T S N H S P T S C P P T C P G Y R W U C L R R f

109 T P S

I I F L f I L L L C L I P L L V L L D Y Q G M L P V C P L I P G S T T T S T G P

K T P N f A R T P I Y 149 :

C R T C U T T A Q G T S H Y P S C C C T K P S D G N C T C I P I P S S W A F G K G '

-,'I P N F 4 -

1 2 T 3

Y V T A P A 189 I V 209 ~ L ~ E ~ ~ ~ A ~ F ~ ~ L ~ L L ~ P F ~ Q ~ F ~ G L ~ P T ~ ~ L ~ ~ I ~ ~ ~ ~ ~

V L ; 229 ::

W G P S L K S I L S P F L P L L P I F F C L W V K I T

FIG. 1. Amino acid sequence of p-25 and gp-30. The sequence data shown are a summary of all the available data, taken from the DNA sequences (16-18) and from our protein sequence data. The complete sequence shown is that of Charnay et al. (18). Directly above this sequence are those amino acids which are different from this sequence in the antigen described by Pasek et al. (17), and above this are those amino acids which are different from this sequence in the antigen of Valenzuela et al. (16). Below the sequence are those amino acids which we have found to differ from the sequence shown, in the case of the adw antigen. In addition, residues 125 and 127 of our ayw antigens differed from that shown by Charnay and were identical with the adw antigen.

RESULTS

Automatic Edman Degradation of HBsAg-We have previously shown that the adw and ayw antigens have identical NH2-terminal sequences for 30 residues (5). This was, however, based on only one example of each subtype. We have repeated these studies with three additional sources of each subtype obtained from the Washington, D. C. area, and have identified the NHz-terminal 20-25 amino acids from each additional sources of each subtype. No variations were observed, and the sequences obtained were identical with that of Fig. 1.

Carboxyl-terminal Sequence Determinations-We have also previously shown that the adw and ayw antigens have the same carboxyl-terminal sequence (5). Examination of these additional sources of antigen by the same carboxypeptidase methods has shown no variation in the carboxyl-ter- mind sequence (Fig. 1).

Tvptic Digestion of HBsAg-When HBsAg (either adw or ayw, four examples each) is incubated with trypsin (trypsin- to-protein ratios of 1:50 to 1:14 by weight) in the absence of reducing agents, no digestion occm. However, when the digestion is performed in the presence of 1% mercaptoethanol, the proteins are rapidly cleaved (Fig. 2) . Lane B shows an adw antigen sample taken at 0 time. Even in the short time required for sample preparation, a small amount of digestion occurs. Identical results were also obtained for the ayw antigen at 0 time (not shown). Lanes A and C show the results of digestion of the adw and ayw subtypes of HBsAg with 2% trypsin for 2 h, respectively. No differences are observed between the cleavage products obtained under these conditions. Also, identical results are observed after 18 h of digestion, even with 7% trypsin (data not shown). When stained for carbohydrate by the periodate-Schiff reaction, only gp-30 stains at 0 time (lane E ) . After digestion, residual undigested gp-30 stains, but is not readily observed in Fig. 2 due to its low concentration. The only tryptic fragment which stains for carbohydrate in either adw or ayw HBsAg, after digestion, is

10416 Structural Comparisons of HBsAg Proteins of Different Subtypes

A B C

GP-30- P-25

P-25-I;GP-30-I t 2 - L - P-25 -2" llb

the higher molecular weight fragment, as shown in lanes D and F.

These results are entirely consistent with the results which we have previously obtained for the digestion of purified p-25 and gp-30 obtained from HBsAg (ayw), which was shown to be cleaved at residue 122 (5).

From these results, we can conclude that, in the intact HBsAg lipoprotein particle, both adw and ayw, residue 122 occupies an exposed position and is a site for tryptic cleavage. It should be noted that the conditions used for the digestions have been shown not to destroy the overall particle structure of HBsAg (24).

Tryptic Digestion of Reduced and Aminoethylated HBsAg-HBsAg (four examples of both adw and ayw) was reduced under relatively mild conditions (1% mercaptoethanol, 0.2 M Tris-HC1, pH 8.2, HBsAg, 1.0 mg/ml) for 4 h, and then aminoethylated by the addition of a 10-fold excess of ethylenimine over total thiol. Following dialysis against 0.05 M ammonium bicarbonate, the aminoethylated HBsAg was digested with trypsin for 6 h (trypsin-to-protein ratio of 1:50). The resultant digest was dried. The peptides which were insoluble in 54.4 m~ sodium phosphate, pH 2.8, were then examined by SDS-polyacrylamide gel electrophoresis, while those that were soluble were examined by high performance liquid chromatography (Figs. 3 and 4).

Fig. 3 shows that the cleavage patterns obtained upon SDS- polyacrylamide gel electrophoresis of the insoluble tryptic peptides of the adw and ayw antigens appear identical, both with respect to protein staining (lanes A and B) and to carbohydrate staining (lanes C and D).

Fig. 4 shows the separation of the soluble tryptic peptides and aminoethylated HBsAg, both adw and ayw, by high performance liquid chromatography. The profdes are very similar, although differences in the absolute amount of some peaks are observed. However, we observe similar differences between different digests of the same HBsAg and have con- cluded that such differences must represent minor differences in reaction or digestion conditions. We have previously identified these peptides for the ayw antigen as arising from residues 123-150 of both p-25 and gp-30 and have shown that these peptides elute as doublets and triplets, apparently be- cause of incomplete aminoethylation and cleavage of a Cys- Cys-Cys sequence (residues 137-139, Fig. 1) (5).

Amino acid analysis was performed on an aliquot of each

D E F FIG. 2. SDS-polyacrylamide gel

electrophoresis of HBsAg after digestion with trypsin. The samples were dissolved in 25 pl of 0.0625 M Tris- HC1, pH 6.8, containing 10% mercaptoethanol and 2.3% sodium dodecyl sulfate, and then heated in boiling water for 1 min. The samples were then made 10% in glycerol containing bromphenol blue as a tracking dye and electrophoresed on a 0.75-mm thick, 12-23% linear gradient, polyacrylamide gel at 15 rnA until the dye eluted. Lane A shows digested HBsAg (adw), lane B shows the 0 time control HBsAg (adw), and lane C shows the digested HBsAg (ayw). AU samples contained 150 pg of HBsAg. Lanes D, E, and F are identical with A, B, and C except that instead of being stained with Coomassie blue tbe gel was stained by periodate-Schiff.

A B C D

FIG. 3. SDS-polyacrylamide gel electrophoresis of the insoluble peptides of reduced and aminoethylated HBsAg after digestion with trypsin. The samples were treated and the electrophoresis was performed as described in Fig. 2. Lanes A and C are digested adw antigen. Lanes B and D are digested ayw antigen. Lanes A and B were stained with Coomassie blue. Lanes C and D were stained with periodate-Schiff. GP-C and C-TERM are identified under "Results."

fraction (10% generally) and those fractions which contained peptides whose amino acid composition matched that expected for peptides 1, 2, and 3 were pooled and dried under vacuum. The peptides at this point contained too much salt (sodium phosphate) to be manually sequenced. Therefore, the peptides were dissolved in 100-200 pl of water and desalted by chromatography on a C-18 reverse phase column. In this case, a gradient of (a) 0.1% trifluoroacetic acid in water and (b ) 0.1% trifluoroacetic acid in acetonitrile was used. The gradient was 0-70% b in 30 min at 1.0 ml/min. Under these conditions, peptides 2 and 3 were initially adsorbed on the column while the salt passed through and were then eluted by the gradient in a totally volatile solvent. Amino acid analysis was performed on 10% of the sample, and 90% was saved for sequencing.

The results of amino acid analysis are shown in Table I.


Peptide 1 was found to be identical in both subtypes. However, peptide 2 from the adw antigens was found to differ in composition from that obtained from the ayw antigen by two amino acids. Peptide 3 from three different adw sources was found to differ in amino acid composition from ayw peptide 2 by one amino acid, but a fourth adw antigen peptide 2 was found to have the same amino acid composition as the ayw peptide. All ayw antigens gave peptides 2 with identical amino acid compositions.

These peptides were manually sequenced and the results are shown in Table I1 and Fig. 1. The results can be summarized as follows for the sequence from residue 123-143:

ADW

AY W

10 20 30 40 TIME /rnin.

FIG. 4. High performance liquid chromatograph of the soluble tryptic fragments of aminoethylated HBsAg (adw) and HBsAg (ayw). The peptides labeled 1,2, and 3 correspond to those identified in Fig. 1 and Table I.

adw TCTTPAQGtjSM!PSCCCTKPS(T)

ayw TCTTPAQGrSl.llPSCCCTKPS

The results for the peptides obtained from the ayw antigens were found to be identical with those which we previously reported for this subtype (5). Moreover, in each case, they differed from that reported by Charnay et al. at positions 125 and 127 as indicated in Fig. 1, and were identical with adw at these positions. However, the peptides from the adw antigens were found to differ from the corresponding ayw antigens at the two underlined positions shown above and in Fig. 1. Specifically, ayw antigen always had threonine and tyrosine as residues 131 and 134, respectively, while the adw antigen had asparagine and phenylalanine at these positions. In three cases, the adw antigen also differed from the ayw antigen at residue 143, having threonine instead of serine. However, in

TABLE I1 Sequence of HBsAgpeptides

These peptide correspond to those in Table I and Fig. 1. Peptide 2 Peptide 3

Step adw” aywb adw‘/ayw adwd

Amino acid 2 2 m o l 2 2 m o l %:? nmol

1 Thr 6.3 Thr 7.0 Thr 4.8 Thr 6.1 2 Thr 5.7 Thr 6.6 Lys 4.4 Lys 5.6 3 Pro 5.0 Pro 5.9 Pro 4.1 Pro 5.2 4 Ala 4.9 Ala 5.6 Ser 3.2 Thr 4.3 5 Gln 4.3 Gln 5.0 Asp 3.4 Asp 4.4 6 Gly 4.2 Gly 4.7 Gly 3.2 Gly 4.0 7 Asn 3.5 Thr 4.2 Asn 2.0 Asn 2.6 8 Met 3.5 Met 3.8 9 Phe 3.2 Tyr 3.0

10 Pro 2.4 Pro 3.2

“The amino acids released by manual Edman degradation were identified by high performance liquid chromatography as described under “Methods.” The quantities reported were determined by the ratio of the height of the observed PTH derivative compared to the height of a 3-mol standard and are therefore approximations.

The values reported are for one of four determinations. Essen- tially identical results were obtained in each case.

‘The values reported are those for one of the adw peptides; however, essentially identical results were obtained for all ayw subtypes, as described under “Results.”

dThe values are those for one adw subtype, but identical results were obtained for two additional adw antigens.

TABLE I Amino acid composition of the soluble trypticpeptides of aminoethylated HBsAg

Peptide 1 Peptide 2 Peptide 3

Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cysteine Methionine Tyrosine Phenylalanine

0.21 (0) 0.85 ( l ) a 0.90 (1) 2.57 (3)

1.70 (2) 0.99 (1)

N.D.‘ (2) 1.23 (1) 0.87 (1)

1.0 (1) 1.0 (1) 0.94 (1) 1.00 (1) 0.92 (1)

0.88 (1) 2.10 (2) 2.05 (2) 2.15 (2) 1.75 (2) 1.20 ( 1) 1.85 (2) 1.92 (2)

1.20 (1) 1.25 (1) 0.21 (0)

1.14 (1) 1.25 (1)

N.D. (2) N.D. (1) N.D. (1) N.D. (1) 1.20 (1) 1.15 (1) 1.22 (1) 1.16 (1)

0.92 (1) 0.92 (1) 1.05 (1) 1.10 (1) 1.20 (1) 1.00 (1)

1.04 (1) Lysine 1.00 (1) 1.00 (1) 1.00 (1)

The values reported were determined by amino acid analysis after 24 h of hydrolysis and are the averages of the three or four determinations.

Values in parentheses are those obtained by Edman degradation. e N.D., not determined.


one case, the two subtypes were identical at this position, both containing serine (Fig. 1 and above).

Location of the Carbohydrate in gp-30-When HBsAg was digested after reduction, only gp-30-2 stained for carbohydrate as shown in Fig. 2. This indicates that the carbohydrate must be attached between residues 122 and 226 for both the adw and ayw antigens. When reduced and aminoethylated HBsAg was digested with trypsin, most but not all (about 75%) of the carbohydrate was released into the peptide fraction which was soluble in 54.4 m~ sodium phosphate, pH 2.8 (see below). When the insoluble peptides were examined in order to determine where the remaining carbohydrate was attached, the results shown in Fig. 3 were obtained. Only one carbohydrate- containing fragment was observed, in both subtypes, which migrated slightly faster than gp-30-1 and gp-30-2. This is designated GP-C in Fig. 3, and must have been derived from gp-30-2 by cleavage between residues 122 and 150, since these are the only peptides released into the soluble peptides. A lower molecular weight peptide, designated C-TERM in Fig. 3, which did not stain for carbohydrate (lanes C and D ) , was also observed. Since between residues 170 and 221 (Fig. 1) there are no tryptic cleavage sites, it is expected that C-TERM contains at least these residues. However, this peptide may also contain residues 150-170, since we have not observed peptides containing these residues in the soluble tryptic peptides of aminoethylated HBsAg. Indeed, the apparent molecular weight of C-TERM (10,000) suggests that this peptide derives from residues 150-226 (calculated M, = 9,166). These results are similar to but not identical with the results which we have previously obtained for the digestion of aminoethylated p-25 and gp-30, which were isolated by preparative polyacrylamide gel electrophoresis (5). Under these previous conditions, GP-C was observed; however, instead of a single fragment corresponding to C-TERM, two peptides were observed with slightly lower molecular weights (Mr = 6,700 and 7,900). These would be expected if, under the present conditions of digestion of the intact aminoethylated HBsAg particles, lysine 160 and arginine 169 were not as accessible for tryptic cleavage. The results indicate that the carbohydrate is attached between residues 122 and 150. We have shown above that we can obtain the peptides from region 122-150 by tryptic digestion of reduced and aminoethylated HBsAg, followed by separation by high performance liquid chromatography on a C-18 reverse phase column. We have therefore applied this technique after f i s t incorporating a tritium label into the carbohydrate moiety of gp-30.

HBsAg gp-30 is a sialoglycoprotein (25). A tritium label was introduced into the sialic acid component by the method of Van Lenten and Ashwell (22) as described under “Methods.” HBsAg (ayw) (7.0 mg) was used and a specific activity of 0.027 pCi/mg was obtained. To verify that the label was present in the gp-30 component of the HBsAg, the labeled antigen was subjected to SDS-polyacrylamide gel electrophoresis. The resultant gel was sliced and the gel slices were eluted by soaking in 1% SDS for 24 h at 37 “C with constant agitation. The amount of label was then determined by liquid scintillation counting. The results are shown in Fig. 5. It is readily apparent that gp-30 contains most of the tritium label. P-25 contains only a very small amount of label and probably represents nonspecific incorporation as described by Van Lenten and Ashwell for other proteins (22). Also, it is evident that some label is not associated with the proteins at all, and migrates at the dye front. We have not characterized this material.

To verify that the label was in the sialic acid moiety of the glycoprotein, the labeled proteins were heated for 1 h at 80 “C in 0.1 N HzS04. These conditions readily release sialic acid

I200

IO00

z 8 800

600

400

200

I t I I I I I I I I I

0 GEL SLICE FIG. 5. HBsAg, labeled by the periodate-borotritriide

method of Van Lenten and Ashwell (22). HBsAg (300 pg) labeled as described under “Methods” was prepared and electrophoresis was performed as described in Fig. 2. The gel superimposed over the graph showing the distribution of the radioactivity is the same HBsAg, except that only 20 pg were applied. The radioactivity presented is the observed, uncorrected counts/min.

from sialoglycoproteins (22). Under these conditions, more than 90% of the radioactivity is released into the supernatant from HBsAg proteins. This is consistent with results for other glycoproteins (22).

The labeled proteins were reduced, aminoethylated, and digested with trypsin as described. Approximately 75% of the label was released from the insoluble protein and recovered in the soluble tryptic fraction. These peptides were separated by high performance liquid chromatography as shown in Fig. 6. The fractions were collected, and an aliquot of each was counted by liquid scintillation counting. The results are also shown in Fig. 6. It is apparent that almost all of the label is recovered in a single peak co-eluting with a single peptide peak. Only very small amounts of radioactivity are observed in other regions of the chromatogram.

The radioactive peak material was pooled and subjected to amino acid analysis. By the position of the peak, and the results presented above, we expected this to be the peptide containing residues 140-147 (Figs. 1 and 4). Amino acid analysis confirmed this, giving the composition Asp, 2.08; Thr, 1.18; Ser, 0.97; Gly, 1.20; Lys, 1.00; and aminoethyl-Cys, 1.05. When an aliquot of this peptide was hydrolyzed for 4 h at 100 “C with 4 N HC1 (26), amino acid analysis revealed the presence of approximately 2 m o l of glucosamine/nmol of peptide. It is thus apparent that this peptide contains attached carbohydrate, including sialic acid and glucosamine. These results were obtained with the ayw antigen; however, essentially identical results were obtained with adw antigen, except that the specific activity of the labeled antigen was not identical, and the amino acid composition of the glycopeptide was different by one amino acid as described.


0.2

10 20 30 40 50 FRACTION

1000 2

FIG. 6. High performance liquid chromatograph of the soluble tryptic fragments of aminoethylated HBsAg proteins, labeled in the sialic acid moiety. The solid Line is the absorbance at 215 nm. The dashed 2ine is the counts/min of tritium.

DISCUSSION

Several different preparations of HBsAg of both the adw and ayw subtypes (four each) have been examined in an effort to identify those features of the protein structure which specify the antigenic determinants of the HBsAg particle. It is expected that such determinants would occupy exposed positions on the HBsAg protein, as has been shown for the antigenic determinants of other proteins.

The results presented here indicate that the segment of the protein of both p-25 and gp-30 containing amino acid residues 122-150 occupies such an exposed region, since this region is readily susceptible to cleavage by trypsin under conditions which do not disrupt the overall particle morphology (24). Under conditions similar to o m , Sukeno et al. (24) reported no changes upon electron microscopy. We have also found no difference by sedimentation velocity measurements, buoyant density measurement, electron microscopy, or circular dichro- ism spectroscopy.2 The conclusion that this is an exposed area is not unexpected since it is relatively the most hydrophilic portion of the molecule (see Fig. 1). Also, the demonstration that carbohydrate is attached in this region (probably Asn 146, see below) further supports the conclusion that this is an exterior part of the protein as it exists in the intact HBsAg lipoprotein particle.

The structure of the carbohydrate in gp-30 and its role, if any, in the antigenicity remains undefined. However, it is now clear that the major attachment site for sialic acid-containing carbohydrate is in the peptide containing residues 140-147 of both the adw and ayw subtypes. This peptide also has been shown to contain glucosamine and to contain the only asparagine residue (Am-146) in the proper sequence (Asn-x-Thr/ Ser) for glycosylation. We have not, however, determined the complete carbohydrate composition of this peptide, to determine if indeed this is the only site of carbohydrate attachment in gp-30. Also, since we have not been able to release all of the carbohydrate from gp-30 by tryptic digestion following aminoethylation, we cannot d e out other attachment sites at

D. L. Peterson and F. Gavilanes, unpublished data.

this time. The fact, however, that the carbohydrate is always present only on the largest fragment of gp-30-2 (gp-C) following tryptic digestion after aminoethylation suggests strongly that it is attached only in the general region of the molecule between residues 122 and 150, regardless of the subtype.

Previous studies have revealed no gross differences between the subtypes of HBsAg. The particles are identical in size and density and contain protein and glycoprotein components (p- 25 and gp-30) which are indistinguishable upon SDS-polyacrylamide gel electrophoresis (27). In addition, we were able to demonstrate no differences in the amino acid compositions, NHa-terminal sequences (30 steps), or carboxyl-terminal sequences (3 residues). (5, ll). However, it has been demonstrated that the isolated p-25 are antigenically distinct (8-12), containing the same group-specific and subtype-specific determinants as intact HBsAg.

Since the two subtypes are antigenically distinct, and this difference is retained in the isolated p-25, it might be predicted that these proteins differ in their amino acid sequence in an area which is exposed on the intact HBsAg particle. We have demonstrated that residues 122-150 constitute such a region. Therefore, we compared the two subtypes in this region. Trypsin was shown to cleave both subtypes into identical fragments as judged by SDS-polyacrylamide gel electrophoresis and high performance liquid chromatography. However, the amino acid compositions and the amino acid sequences of the peptides derived from the two subtypes were different at two positions. Specifically, HBsAg (adw) contains asparagine and phenylalanine as residues 131 and 134, respectively, while HBsAg (ayw) contains threonine and tyrosine as these positions.

An examination of all the currently available sequence data on HBsAg, taken from the published DNA sequences of the p-25 gene (17-19), our previous protein data (5, 16, 27), and the data presented here, allows some tentative conclusions to be drawn concerning the subtype antigenic determinants of HBsAg. These data, summarized in Fig. 1, are based on sequence data from 11 different antigens (three based on DNA sequence, eight based on protein sequence data). Of those based on the DNA sequence, only one was of a defined subtype, ayw (18). One was undefined as to subtype (16) and one was of an apparently unusual subtype (17). However, based on the results presented here, we can conclude that these latter two were probably adw and ayw, respectively.

Variation between different antigen sources is observed throughout most of the p-25 sequence. However, no variation has been observed in the NHz-terminal44 amino acids of the protein (DNA and protein data) or between residues 70 and 113 (DNA data only). Since these two regions have identical amino acid sequences in at least one example of an adw and an ayw antigen, it seems unlikely that these residues are of importance as far as subtype specificity is concerned. These regions are also extremely hydrophobic and are likely to be buried in the lipid of HBsAg. No other long stretch of sequence is found to be without variability. However, some of the variations are inconsequential. For example, residue 224 has been predicted to be alanine (ayw) in one case and valine (one ayw and one adw) based on the DNA sequences. We have found this residue always to be valine in om studies of four adw and four ayw antigens. Therefore, we can conclude that, although this residue may vary, it does not affect the subtype. Other residues which vary, but which do not affect the subtype, are residues 57, 114, 120, 125, 127, and 189. When these are taken into account, it seems valid to conclude that residues 1-44 and 47-121 are not likely to be directly involved in the antigenic subtype determinant of HBsAg. In contrast, as shown above, variation at two positions between residues 123


and 150 is correlated with differences in the antigenic subtype, strongly suggesting that this region is at least partially reson- sible for the subtype specificity.

However, we cannot at present conclude that only this area of the protein is involved, since the region containing residues 150-215 is not yet understood in these regards. The only data available are from the DNA sequences, since we have not yet been able to obtain peptides from this portion of the molecule due to expected extreme hydrophobicity. Although the predicted variations do seem to correlate with subtype, these care based on the results from only one example of each subtype, and no conclusions may be drawn until more data are available.

Further support that the sequence between 123 and 150 is antigenically important comes from recently reported data showing that synthetic peptides based on this sequence of the molecule are immunogenic, giving rise to anti-HBsAg antibodies (13, 14). Indeed, we have recently shown that the synthetic peptide residues 122-137 was such an immunogen (28). We have not yet characterized the antibodies produced to determine whether they exhibit subtype specificities. Such studies are currently in progress.

Acknowledgments-We gratefully acknowledge the technical as- sistance of Bonnie Yanchmson. We also thank Dr. Robert Carithers and Edward Galen for supplying the antigen-positive sera necessary for this work.

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