Moleculur
und Cellulur
c) 1992 Elsevier
MOLCEL
Endocrinology,
Scientific
Publishers
02776
Polyclonal
antibodies against the polypeptide and carbohydrate of recombinant human choriogonadotropin P-subunit Wenyong
Depurtmmt
Antibody,
polyclonal;
Polypeptide
5 February
epitope;
epitopes
Chen and Om P. Bahl
of’ Biologicul Sciences, Siutc Utliwxity (Received
I&J, words:
57
86 (1992) 51-66 Ireland. Ltd. 0303-7207/92/$05.00
of New
York ut Buffulo,
19Y2; accepted
Carbohydrate
epitope;
18 March
Buj$h,
NY 14260, USA
1992)
Recombinant
human
choriogonadotropin
/3-subunit
Summary In our previous paper (Chen et al. (1991) J. Biol. Chem. 266, 4081-4087) we reported the preparation and characterization of recombinant human choriogonadotropin p subunit (hCG/3) using the baculovirus-insect cell expression system. The rhCGP was found to contain high mannose type N-linked carbohydrates and 3-4 serine-linked disaccharide chains. Despite the carbohydrate structural variation, the rhCGP was similar to hCGP in in vitro immunological and biological properties. In order to evaluate its in vivo immunological properties, rabbit antiserum against rhCG@ was produced. The antiserum was found to be almost identical to anti-hCGP in binding to hCG@ as well as in its crossreactivity with human lutropin (hLH), hCG and human follitropin (hFSH) as indicated by radioimmunoassays using ‘2sI-hCGP as a tracer. Further characterization of the anti-rhCGp antiserum revealed that there are three types of antibodies in terms of antigenic specificity present in the anti-rhCGp antisera pool as shown by dot blot and radioimmunoassays. The carbohydrate-specific antibodies were separated by affinity chromatography using an ovalbumin-glycopeptide-Sepharose column. The antibodies held on the ovalbumin affinity adsorbent were specific for the high mannose type carbohydrates such as those present in rhCG/3, rhCG and thyroglobulin and failed to react with transferrin, cu,-acid glycoprotein and hCGa, all containing complex type carbohydrates. This was further supported by the fact that the recombinant unglycosylated hCG or periodate oxidized rhCGP also did not show any reactivity with the carbohydrate specific antibodies. Two types of peptide epitopes seemed to be present in rhCGP since when the flowthrough fraction from the ovalbumin-glycopeptide-affinity column was passed through the hCG@-Sepharose column, the antibodies in the flowthrough from the latter column were specific to the unique antigenic determinants present only in the rhCGP and not in hCGP. The eluate from the hCGP-Sepharose column contained the third type of antibodies, being the predominant ones, directed to the common epitopes between rhCGP and hCGj3. The high mannose type specific antibodies are potentially useful in differentiating between the high mannose and complex type of N-linked carbohydrates present in a glycoprotein. Also, the antibody could provide an effective reagent in studying the
Correspondence to: Om P. Bahl, Department of Biological Sciences, State University of New York at Buffalo, 347 Cooke Hall, Buffalo, NY 14260, USA. Supported by U.S.P.H.S. Grant HDOX766. Abbreviations: hCG, human choriogonadotropin; rhCGfl. recombinant hCGfi (should not be confused with rhesus monkey C.G.); SelhCG, selenomethionyl recombinant hCG; hFSH, human follitropin: hLH, human lutropin; HPLC, high performance liquid chromatography; RER, rough endoplasmic reticulum; SDS, sodium dodecyl sulfate: PAGE, polyacrylamide gel electrophoreSIS.
intracellular processing of the N-linked oligosaccharides. The antibodies unique to rhCG polypeptide probably were against those epitopes which were either masked or were present in a different conformation in the native hCGp indicating that the high mannose type carbohydrates might have a different steric or subtle conformational effect than the complex type carbohydrate on the rhCGB polypeptide chain.
Introduction Human choriogonadotropin (hCG), one of the four structurally related and well characterized human glycoprotein hormones, is produced by placental trophoblast cells. Its primary function in pregnancy is the stimulation of progesterone synthesis and thereby preparation of the corpus luteum for the implantation and maintenance of the fertilized ovum (Pierce and Parsons, 1981). hCG is a hcterodimer composed of N and p subunits (Bahl, 1969). The cy subunit is 92 amino acid residues long with two N-linked carbohydrates at 52 and 78 positions, while the 145 amino acid-residue p subunit has, in addition to the two N-linked carbohydrates at position 13 and 30, four O-linked oligosaccharides at serine residues 121, 127, 132 and 138, on its carboxyl terminal extension (Kessler et al., 197Ya, b). All the Nlinked carbohydrates in hCG exist predominantly as complex type while the O-linked oligosaccharides in the p subunit are simple structures such as those present in mucins (Endo et al., 1979). Due to the physiological importance of hCG, this hormone and its antibodies have been widely used clinically. Recently we have reported the feasibility of using the recombinant DNA methodology to obtain large amounts of homogencous recombinant hCG and its hCGP subunit from insect cells (Chen et al., 1991; Chen and Bahl, 1991a). Although the recombinant hCGP was found to contain, in addition to 3-4 simple disaccharides, two N-linked high mannose type carbohydrates instead of the complex type present in the native hCG@, it was similar to the native hCGP in its ability to bind with antibodies against native hCGP and to recombine with native hCGLv (Chen et al., 1991). The reconstituted heterodimer (hCGarp) and the recombinant hCG were able to stimulate in vitro CAMP accumulation and steroidogenesis comparable to that of
native hCG (Chen et al., 1991; Chen and Bahl, 1991a). These in vitro studies suggested the potential of recombinant hCG and hCGp in clinical usage. Therefore, the present work was undertaken to further study the in vivo immunological properties of rhCGP. In this communication, we report the production of polyclonal antibodies against the recombinant hCG/3 subunit and comparison of their properties with similarly raised antibodies against the native hCG/3. The recombinant hCGp was found to be highly immunogenic and the antiserum produced, as indicated by radioimmunoassay, was almost identical to the anti-native hCGP antiserum in its specificity and sensitivity. However, because of the differences in the type of carbohydrates present in rhCGP from those of the native hCGP, a small population of the antibodies against rhCGP were found unique to its polypeptide and carbohydrate epitopes. These unique antibodies imply that the high mannose type and complex type carbohydrates differ in their conformation and thus have different effect on the polypeptide epitopes. Materials
and methods
The hormones hCGa, hCG/3, SelhCG, rhCG and hCG were prepared in this laboratory as reported (Bahl, 1969; Chen and Bahl, 1991a, b). hFSH and hLH were obtained from the National Pituitary Agency. The rabbit anti-hCGP antisera, prepared as described (Bahl et al., 1976), had a titer of 1: 50,000 for 30% binding of the tracer under the radioimmunoassay (RIA) conditions. Ovalbumin glycopeptides were prepared by exhaustive digestion of ovalbumin with Pronase followed by gel filtration on Sephadex G-50 (Kamiyama et al., 1962). AffiGel 10 was obtained from BioRad. The Freund’s complete and incomplete adjuvants were purchased from Gibco. The alkaline phosphatase conjugated purified goat
anti-rabbit IgG was from Cooper Biomedical. Human transferrin, human (Y,-acid glycoprotein and bovine thyroglobulin were from Sigma. SDS-PAGE, Western blotting and dot blotting The discontinuous gel system of Laemmli with 4%’ stacking gel at pH 6.8 and 12% separation gel at pH 8.8 was used. The sample buffer containing 125 mM Tris-HCl, pH 6.8, 10% glycerol, 2% bromophenol blue was supplemented with 5% P-mercaptoethanol when desired. The gels were silver stained as described (Sambrook et al., 1989). For the Western blotting, the proteins were transferred to the nitrocellulose filter and subsequently incubated with the appropriate antibodies in phosphate buffered saline containing 2% periodate treated bovine serum albumin. The periodate oxidation of bovine serum albumin was carried out as described (Glass et al., 1981). Dot blot assay was carried out in a BioRad Bio-Dot apparatus using a nitrocellulose filter. Blotting was performed in phosphate buffered saline containing 2% periodate treated bovine serum albumin. Preparation of rhCGP, unglycosylated rhCG and periodate-oxidized rhCG/3 The rhCG@ was prepared by recombinant DNA methodology in the baculovirus expression system and was purified from cell culture medium by immunoaffinity chromatography using highly specific monoclonal antibodies against hCG/3 as described earlier (Chen et al., 1991). The homogeneity of the resulting material was ascertained by reverse phase HPLC and SDS-PAGE. The biosynthetically unglycosylated rhCG was obtained by coinfecting the insect SF9 cells with recombinant baculoviruses containing hCGa cDNA and hCGP cDNA in the presence of 0.8 pg/ml of tunicamycin (Chen et al., in preparation). The unglycosylated rhCG was purified by one step immunoaffinity chromatography with anti-hCG monoclonal antibody B17 (Chen and Bahl, 1991b), and the traces of glycosylated material if any in the preparation were removed by concanavalin A affinity chromatography. The unglycosylated rhCG was characterized by carbohydrate compositional analysis and radioimmunoassay (Chen .et al., in preparation).
The periodate oxidation of rhCG/3 was carried out by incubating it at 1 pg/pl in 0.1 M sodium acetate (pH 4.5) with 10 mM sodium periodate for 10 h at room temperature. The excess periodate was removed by the addition of glycerol to a final concentration of 10 mM (Glass et al., 1981). Preparation of polyclonal antibodies against rhCG/3 and radioimmunoassay Two New Zealand female rabbits, weighing 3-4 lbs each, for immunization were purchased from Becken Farms. A sample of 100 pg of rhCGP in 0.5 ml saline was well mixed with an equal volume of Freund’s complete adjuvant and the resulting emulsion was injected intradermally at multiple sites on the back. Four weeks after the primary immunization, a booster of 50 pg of rhCG@ in the Freund’s incomplete adjuvant was administered again at multiple sites intradermally, and the immunization was repeated every other week. The animals were bled from an ear vein between the immunizations, and the sera were evaluated by radioimmunoassay using ‘*‘IhCG (Bahl et al., 1976). The chloramine T method was used for radioiodination of the hormones (Madnick et al., 1981). Specific activity of the labeled hormones was approximately 1 pCi/pg. The RIA was performed using “‘I-hCGP or 12’I-hCG and antirhCGP or anti-hCGp antibodies as described previously (Chen et al., 1991). All solutions were prepared in 50 mM sodium borate buffer, pH 8.0 containing 0.5% bovine serum albumin (BSA). The total volume of the reaction mixture was 300 ~1 containing 30,000 cpm of ‘251-hCG/3 or “‘IhCG and anti-rhCGP or anti-hCGp antibodies and varying concentrations of the hormones. The incubations were performed at 37°C for 1 h after which the amount of tracer antibodies complex was determined by precipitation with 10% polyethylene glycol-8000. Separation of the carbohydrate and polypeptide specific antibodies To purify the carbohydrate specific antibodies, the high mannose type carbohydrate affinity adsorbent was prepared by coupling ovalbumin glycopeptides with AffiGel 10. The coupling procedure was essentially the same as previously used
60
(Chen et al., 1991). A prewashed 2 ml sample of AffiGel 10 with 100 ml of cold water was mixed with 10 mg of ovalbumin glycopeptides in 3 ml of 0.1 M morpholinosulfonic acid buffer (MOPS), pH 7.5 containing 0.3 M NaCI. The mixture was kept stirring at 4°C for 12 h. The active sites of the gel remaining were blocked by incubation with 0.5 ml of 1 M glycine ethyl ester, pH 8.0 for 1 h. The carbohydrate specific antibodies were purified by passing 5 ml of rabbit anti-rhCGp antisera through a 2 ml affinity absorbent in a disposable pipet in 0.01 M Tris-HCI buffer, pH 8.0 containing 0.14 M NaCl. After washing with about ten bed volumes of the above buffer, the column was eluted with 0.05 M glycine-HC1 buffer, pH 2.3 and the eluate was immediately neutralized with 0.5 M Tris-HCI buffer, pH 8.5 and concentrated with a Centriconmembrane microconcentrator (Amicon). The antibodies against the polypeptide were purified by passing the above flowthrough successively from 2 ml columns of Protein A-Sepharose (Pharmacia) and hCG&Sepharose in disposable pipets pre-equilibrated with 0.01 M Tris-HCI, pH 8.0 containing 0.14 M NaCI. After washing with about ten bed volumes of the buffer, the columns were eluted with 8 ml of 0.05 M Tris-HCl buffer, pH 2.3. After neutralization with 0.5 M Tris-HCI buffer, pH 8.5, the flowthroughs and the eluates from both columns were concentrated in a Centricon-10 membrane microconcentrator. After centrifugation the samples of the purified antibodies were subjected to HPLC using a Dionex HPLC system equipped with a Synchropak GPC-300 (250 X 4.6 mm). The column was eluted with 50 mM ammonium bicarbonate at a flow rate of 500 pl/min for 15 min and the eluate was monitored at 210 nm. Results Preparation of unglycosylated oxidized rhCG/3
rhCG and periodate-
The high expression of rhCG in the Baculovirus insect cell expression system was exploited to prepare unglycosylated rhCG biosynthetically using the glycosylation inhibitor, tunicamycin (Materials and methods). The unglyco-
-24 -16 Fig.
I. SDS-PAGE
of rhCG and unglycosylated rhCG. 300-500
ng of each sample nonreducing
was used. Lanes
and reducing
under nonreducing
conditions;
I
and 3: hCG
under
lanes 2 and 4: rhCG
and reducing conditions:
lane 5: unglyco-
sylated rhCG and partially dissociated (r and p subunits.
sylated rhCG, purified by immunoand lectin affinity chromatography using monoclonal antibody B17 (Chen et al., 1991) and concanavalin A, respectively, was examined for homogeneity by SDS-PAGE (Fig. 1). As shown in lane 5, the intact unglycosylated rhCG gave sharp protein bands as compared with those of rhCG indicating that the carbohydrate heterogeneity was probably responsible for the broad bands of rhCG (lanes 2 and 4). Under the condition used, the reduced rhCGa and p subunits were not resolved by SDS-PAGE (lane 4). However, they did separate on reverse phase HPLC (Chen and Bahl, 1991a). The unglycosylated rhCG had a molecular weight of 35.5 kDa while the reduced unglycosylated rhCGa and /? subunits had molecular weights of 16 kDa and 19 kDa respectively. The carbohydrate analysis of the unglycosylated rhCG by Dionex carbohydrate analyzer showed the absence of N-linked carbohydrate chains while the antibody and receptor binding activities as determined by radioimmunoand radioreceptor assays (Chen et al., in preparation) were comparable to those of rhCG (data not shown). The rhCGP containing high mannose type Nlinked carbohydrate chains, approximately six
TABLE
1
CARBOHYDRATE COMPOSITIONAL PERIODATE OXIDIZED rhCGP Number
Protein
rhCG/3 Periodate
oxidized
rhCGP
ANALYSIS
OF
of sugar residues/m01
Fuc
GalN
GlcN
Gal
Man
2.5 0
2.4 0
4.0 i1 4.0”
3.7 0
11.3 4.x
“ Calculated based on four residues drate chains.
”
of GlcN per two carbohy-
mannose residues on the average per chain and l-2 fucose residues (Chen et al., 1991), was subjected to periodate oxidation. Under the experimental conditions used, the treatment resulted in the oxidation of almost four of the six mannose residues and all fucose residues in a single chain as determined by its carbohydrate analysis (Table 1) by Dionex carbohydrate analyzer using a pulse amperometric detector (Chen et al., 1991). Consequently, the two surviving mannose residues must be linked at the C, position. Based on the oxidation data and the analogy with other high mannose type carbohydrate chains, a tentative structure can be assigned to the carbohydrate in rhCGp as shown in Fig. 2. The positions of the four mannose residues marked by an asterisk are consistent with the periodate oxidation data while the positions of the other two mannose residues need to be determined. The second L-fucose residue is probably located at C, of the innermost N-acetylglucosamine (Weber et al., 1987; Prenner et al., 1991; personal communication from L. Marz).
Characterization of anti-rhCG/3 In order to study the immunological properties of the recombinant hCGp expressed in the insect
Fig. 2. Tentative structure of high mannose type carbohydrate chains in rhCGP. The presence of four mannose residues marked by an asterisk is established by the periodate oxidation data.
.-P -E s
80
60
,L.d
10
100
“g
Fig. 3. Radioimmunoassay of hCGP (3) and rhCG/3 (01 with rabbit anti-hCGP serum at 1 :65,000 dilution (A) and antirhCG/3 serum at 1:55,000 dilution (Bl using “‘I-hCGP tracer.
cells, antibodies against rhCG@ were raised in two rabbits as described in Materials and methods. Both antisera displayed high titer and showed under the radioimmunoassay conditions a binding of more than 30% with ‘2sI-hCG at 1 :65,000 dilution. The specificity of the antiserum against rhCGP was evaluated by radioimmunoassay as shown in Fig. 3A. For comparison the assays of hCGP and rhCGP using antiserum against native hCG/3 were also performed (Fig. 3B) under identical conditions. The data indicated that both antisera could bind with hCGP and rhCG/3 equally competitively and there was no significant difference in their immuno specificity, further supporting the conformational and antigenic similarity of hCG@ and rhCGP.
62
\
I
1MI
got
x
B
-
60 5
70.
f
60.
E
50-
E 40n
301
201 lotI 10 1W “9 Fig. 4. Radioimmunoassay of hCG CO), hLH (0) and hFSH (x) with (A) rabbit anti-hCGp serum and (B) anti-rhCGp serum using ‘251-hCG tracer. 1
The similarity of the antisera against rhCG/3 and hCG@ was also evident from their interactions with hCG, hLH and hFSH as indicated by radioimmunoassay (Fig. 4). Using 12’I-hCG as a tracer, the displacement curves of hCG, hLH and hFSH with antisera against rhCGP and hCGP were quite comparable suggesting no significant specificity differences between the two antisera. Carbohydrate and polypeptide specific antibodies Since rhCGP is different from the native hCGP in that it possesses two N-linked high mannose type carbohydrates instead of the complex type, an attempt was made to determine if the antisera contained any antibodies directed against the carbohydrate. The antiserum was passed through an affinity column using the high mannose type carhohydrate containing glycopeptides from ovalbumin. The column was eluted to yield the carbohydrate specific antibodies. The antibodies against rhCGP polypeptide in the flowthrough were fur-
ther purified by protein A-Sepharose affinity column. The purified antibodies were examined and quantitated by HPLC equipped with a Synchropak-300 GPC column. The single peak of both antibodies in HPLC suggested the homogeneity of the purified materials (data not shown). Further quantification of the antibodies indicated that about 0.07% of antibodies in the anti-rhCGp antiserum were against the high mannose type carbohydrate based on their relative peak size on HPLC. The specificity of the purified antibodies was assessed using different glycoproteins by a dot blot assay. As shown in Fig. 5 (lanes A and B), the protein A purified antibodies were specific to hCGP polypeptide backbone and could bind with hCGj3, rhCG@, SelhCG and rhCG but not other glycoproteins or glycopeptides. The carbohydrate specific antibodies eluted from the ovalbumin glycopeptide column could bind with the glycoproteins containing high mannose type carbohydrates only, such as those present in thyroglobulin, rhCGP, rhCG and SelhCG and failed to bind with the complex type carbohydrate containing glycoproteins such as a,-acid glycoprotein and transferrin. To further confirm that the antibodies eluated from the ovalbumin glycopeptide column were carbohydrate specific, biosynthetically prepared unglycosylated rhCG and periodate treated rhCGP were used in the dot blot assay as shown in Fig. 5 (lanes C and D). The ovalbumin affinity purified antibodies failed to react with the unglycosylated rhCG or periodate treated rhCGP. Thus, it is clear that the antibodies against the carbohydrate moiety of rhCGP were specific for the high mannose type rather than complex type carbohydrates. rhCGP polypeptide specific antibodies The substitution of the complex type carbohydrates with high mannose type chains raises the possibility of change in the peptide epitopes due to differences in the masking effects of different carbohydrate structures probably because of their conformational differences. To investigate this possibility, the protein A purified antibodies from antisera against rhCG/3, after absorbing with the ovalbumin glycopeptide column, were passed through the hCGP coupled Sepharose column.
63
A
B
C
D
6 7
SDS-PAGE under reducing conditions using silver staining for the visualization of protein bands (Fig. 5). As stated previously (Chen et al., 1991), rhCGB and native hCGB behave differently under reducing and nonreducing conditions. This differential behavior is attributed to the differences in their carbohydrate structures, in particular, due to the lack of sialic acid in the rhCGB. As shown in Fig. 6B and Fig. 7, the eluate could bind with rhCG and hCGB equally well within experimental error while the flowthrough displayed preferential binding to rhCGB suggesting the emergence of some new epitopes in rhCGB due to change in the carbohydrate structure from the complex type present in hCGB to the high mannose type in the recombinant hCGB. Immunological characterization of three populations of antibodies in rabbit anti-rhCG/3 by radioimmunoassays In order to quantify the immunological activity of the above three populations of the antibodies fractionated from the anti-rhCGB, radioim-
6 9
64.0 47.0
10 Fig. 5. Dot blot analysis of various glycoproteins with rhCG/3 polypeptide epitope specific antibody (lanes A and D) and rhCGP carbohydrate epitope specific antibody (lanes B and 0. 500 ng of each sample was used. Lanes A and B: (1) hCG@, (2) hCGn, (3) SelhCG, (4) rhCG, (5) rhCGp, (6) cu,-acid glycoprotein, (7) transferrin, (8) thyroglobulin, (9) ovalbumin-glycopeptides, and (10) buffer blank as control. Lanes C and D: (1) rhCGP, (2) rhCG, (3) periodate treated rhCGP, and (4) unglycosylated rhCG.
The flowthrough as well as the eluate were subjected to the dot blot assay and Western blotting with rhCGB and hCGB. To rule out the possibility of any trace contaminant, the homogeneity of rhCGB used in the assay was ascertained by
33.0 24.0 '16.0
Fig. 6. SDS-PAGE and Western blot analyses of rhCGP and hCGP. A: About 200 ng samples of rhCG@ (lane 1) and hCGP (lane 2) were subjected to SDS-PAGE in 12% gel under reducing conditions and subsequently stained with silver nitrate. B: About 100 ng samples of rhCGP (lane 1) and hCG/3 (lane 2) were applied to 12% SDS-PAGE under nonreducing conditions. After transferring to nitrocellulose filter, the proteins were detected by polypeptide specific antibodies (see the text for details).
munoassays using ‘2’I-hCGb and each antibody were performed. As shown in Fig. 8A, the predominant component of anti-rhCG/? antibody at 1 : 50,000 dilution had similar reactivity toward hCG and rhCG. On the other hand, the carbohydrate specific antibody at 1: 100 dilution showed reactivity with rhCGP with insignificant crossreactivity with native hCGp indicating that the antibody was specific to the high mannose type carbohydrate present in rhCGP rather than the complex type that was present in the native hCGP (Fig. 8 B 1. Similarly, the antibody crossreacted with thyroglobulin, a high mannose type carbohydrate containing glycoprotein. A 50% inhibition of binding of ‘2sl-rhCGP to the carbohydrate specific antibody by thyroglobulin was achieved at the 24.2 ng level as compared to the 10.5 ng level with rhCGP (Fig. 8B). Finally, the polypeptide specific antibody hCGP at 1 : 250 dilution showed
100
go- A 80 % 70g
60-
E
50-
E 40=
3020 10 -
I
I
1
10 v
I
I
I
1
10 ng
M
10
1
ng Fig. 8. Radioimmunoassays using “iI-rhCGp and (A) antirhCGP polypeptide antibody at 1:SO.OOO dilution, (B) the carbohydrate specific antibody at 1: 100 dilution, CL‘) the rhCGP polypeptide specific antibody at 1 :200 dilution. A: rhCG (01, hCG (0) and hFSH (X ).B; rhCGP (0). hCGp (0) and thyroglobulin (X );C: rhCGP (01 and hCGp (3).
no significant (Fig. 8C).
Fig. 7. Dot blot analysis of rhCGP and hCG@ with (A) hCGp polypeptide antibody and (B) rhCGP specific antibody. The amounts of hCGp and rhCGP applied were: (1) 12.5 ng; (2) 25 ng; (3) 50 ng; (4) 100 ng; (5) 200 ng; (6) buffer control.
crossreactivity
with
native
hCGp
Discussion The data presented here indicate that rhCGP from insect cells was quite immunogenic and pro-
65
duced a high titer and high affinity antisera in rabbits. In this study, we have characterized the anti-rhCGP antiserum in detail and have further fractionated the antiserum into three different immuno specificities. The data indicate that there are three types of antibodies, those which are directed against the peptide epitopes common to both hCG/3 and rhCG/3, these being the predominant ones, antibodies to the peptide epitopes unique to rhCGP, and the antibodies directed against the carbohydrate epitopes. The antiserum displayed close similarity to the antiserum against native hCG/? in both specificity and sensitivity as evident by radioimmunoassays using ‘2”I-hCGP and anti-rhCG@ or anti-hCG/3. Furthermore, the anti-rhCG/3 showed similar crossreactivity with hCG, hLH and hFSH as the antibody to the native hCG/?, again indicating similarity between the two antibodies. Therefore, for immunodiagnostic applications it should be possible to use rhCGP in place of native hCG@ both as a standard or as an antigen for raising antibodies. Carbohydrates in glycoproteins, by virtue of their hydrophilic nature, are usually present on the surface of proteins and can significantly influence the physicochemical properties as well as the immunological and biological characteristics of glycoproteins (Green et al., 1986; Feizi et al., 1987). The removal of the carbohydrate moiety of hCG has been found to alter its antigenic structure (Rebois and Liss, 1987; Ryan et al., 1987; Sairam et al., 1988). Because of the carbohydrate structural differences between the rhCG/3 and native hCG/?, in this study we have been able to show that a significant though small percentage of the polyclonal antibodies were specific to the high mannose type carbohydrates present in rhCG. Similarly, a small percentage of the antibodies were directed against peptide epitopes unique to rhCGP. In our studies, we have separated the antibodies against the carbohydrate and rhCGj3 specific peptide epitopes. The carbohydrate specificity of the antibodies was confirmed by their ability to crossreact with rhCG/3 and rhCG and their inability to recognize the biosynthetically prepared unglycosylated hCG, devoid of carbohydrates, or periodate treated rhCG/I in which the neutral sugars were oxidized. The antibodies against carbohydrate epitopes were spe-
cific to the high mannose type carbohydrates as shown by their binding with the high mannose type carbohydrate containing glycoproteins including thyroglobulin, insect cell derived rhCG and rhCGP, but not to the glycoproteins containing the hybrid or complex type carbohydrates such as those present in hCGa, hCGP, a,-acid glycoprotein and transferrin. Thus, the anticarbohydrate antibodies provide a valuable reagent for various basic studies. They can be used in determining the type of carbohydrate whether complex or high mannose type present in a glycoprotein. The antibodies may be useful in investigating the intracellular processing of the N-linked carbohydrates in glycoproteins and intracellular trafficking. Subsequent to the transfer of the oligosaccharides Glc,Man,GlcNAc,from the oligosaccharide lipid intermediate to the growing polypeptide chain in the rough endoplasmic reticulum (RER), the carbohydrate undergoes a series of processing reactions. These involve the cleavage of all glucose residues and one mannose residue in the lumen of RER before the glycoprotein enters the Golgi. This is followed either by phosphorylation of Man,GlcNAc,and sequestration of the protein in lysosomes or further removal of mannose and incorporation of peripheral sugars, GlcNAc, Gal and sialic acid in mid and trans Golgi for secretion (Paulson, 1989). It is conceivable that the high mannose type carbohydrate containing proteins may be enrouted from the endoplasmic reticulum directly to another subcellular compartment without further modification in the Golgi apparatus. This antiserum could be helpful in investigating this possibility. The carbohydrate structure in glycoproteins undergoes alterations in various developmental as well as pathological states (Kornfeld et al., 1985). The carbohydrate specific antibodies may provide an effective means of studying the carbohydrate changes during various pathologic and developmental states. The N-linked carbohydrates of glycoproteins are generally regarded as non-antigenic in mammalian species. It is probably because of the exposure of the immune system to a diverse array of heterogeneous carbohydrate structures during the development of the organism and thus resulting in the immunosuppression or tolerance to the
66
carbohydrates The present work represents one of the few examples of rabbit antibodies directed specifically against N-linked carbohydrates of a glycoprotein. Since the carbohydrate specific antibodies were isolated from the pool of antibodies that were generated against insect cell derived rhCG& rhCGp probably had a unique carbohydrate structure(s) that was antigenic. Recent reports have suggested that antigenicity of carbohydrates of insect cell glycoproteins is probably due to differences in the mode of fucosylation (Weber et al., 1987; Prenner et al., 1991). It is interesting to note that anti-rhCGP contained, though a small fraction of the total antibodies, antibodies unique to the polypeptide chain. Thus, it appears that the high mannose type carbohydrates present in rhCG@ unmask certain peptide epitopes which are not exposed in the native hCGp containing complex type carbohydrates. Presumably, the conformation of the two types of carbohydrates is different and they may have a different steric effect on surface peptide epitopes or the two types of carbohydrates may have a subtle but different effect on the conformation of the polypeptide chain and thereby on their epitopes. In conclusion, we have obtained a rabbit antirhCG/3 antiserum with high titer, which is similar in specificity and sensitivity to the antiserum against native hCGP. The antibodies against the N-linked carbohydrates and the polypeptide backbone have been purified by affinity chromatography using ovalbumin glycopeptide and protein A and native hCG/3, respectively. The antibodies against carbohydrate epitopes were found to be highly specific for high mannose type carbohydrates and they may be potentially useful in the characterization and in the study of in vivo assembly of carbohydrates of glycoproteins. Further, such antibodies may be useful in studying carbohydrate variation during various developmental and pathologic states. The presence of antibodies unique to the peptide epitope(s) of rhCG/3 in the antiserum may reflect conformational differences between the high mannose and the complex type carbohydrates and thus different steric or conformational effects on the polypeptide chain.
Acknowledgements We wish to thank Dr. Rana Samuel for performing dot blot assay experiments, Juchun Fu and Huawei Zeng for technical support, Diane McMaster for help in the preparation of the manuscript, and James Stamos for photo-illustration work. References Bahl, O.P. (1969) J. Biol. Chem. 244, 567-574. Bahl, O.P., Pandian, M.R. and Ghai, R.D. (1976) Biochem. Biophys. Res. Commun. 70, 525-530. Chew W. and Bahl, O.P. (1991a) J. Biol. Chem. 266, 81928197. Chen, W. and Bahl, O.P. (1991b) J. Biol. Chem. 266, 93559358. Chen, W. and Bahl, O.P., manuscript in preparation. Chen, W., Shen, Q.-X. and Bahl, O.P. (1991) J. Biol. Chem. 266, 4081-4087. Endo, Y., Yamoshita, K., Tachibana, Y., Tojo, S. and Kobata, A. (1979) J. Biol. Chem. 254, 669-670. Feizi, T. and Childs, R.A. (1987) Biochem. J. 245, I-I 1. Glass, II, W.F., Briggs, R.C. and Hrilica, L.S. (1981) Anal. Biochem. 115, 219-224. Green, E.D., Boime, I. and Baenziger, J.V. (1986) Mol. Cell. Biochem. 72, 81-100. Kamiyama, S. and Schmid, K. (1962) Biochim. Biophys. Acta 58, X0-85. Kessler, M.J., Reddy, M.S., Shah, R.H. and Bahl, O.P. (1979a) J. Biol. Chem. 254, 7901-7908. Kessler, M.J., Mise, T., Ghai. R.D. and Bahl, O.P. (197Yb) J. Biol. Chem. 254, 7909-7914. Kornfeld, R. and Kornfeld, S. (1985) Ann. Rev. Biochem. 54, 63 l-664. Madnick, H.M., Kalyan, N.K., Segal, H.L. and Bahl, O.P. (1981) Arch. Biochim. Biophys. 212, 432-442. Paulson, J.C. (1989) Trends Biochem. Sci. 14, 272-276. Pierce, J.G. and Parson, T.F. (1981) Annu. Rev. Biochem. 50, 465-495. Prenner, C., Mach, L., Glossoal, J. and Marz. L. (1991) Glycoconj. J. 8, 241. Rebois, R.V. and Liss, M.T. (1987) J. Biol. Chem. 262, 38Yl3896. Ryan, R.J., Keutman, H.T., Charlesworth, M.C., McCormick, D.J., Millius, R.P., Calve, F.O. and Vityavanich, T. (1987) Recent Prog. Horm. Res. 43, 383-429. Sairam, M.R., Linggen, J. and Bhargavi, G.N. (1988) Biosci. Rep. 8, 271-278. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Vol. 3, p. 1856, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Weber, A., Schroder, H., Thalberg, K. and Marz, L. (1987) Allergy 42, 464-470.
und Cellulur
c) 1992 Elsevier
MOLCEL
Endocrinology,
Scientific
Publishers
02776
Polyclonal
antibodies against the polypeptide and carbohydrate of recombinant human choriogonadotropin P-subunit Wenyong
Depurtmmt
Antibody,
polyclonal;
Polypeptide
5 February
epitope;
epitopes
Chen and Om P. Bahl
of’ Biologicul Sciences, Siutc Utliwxity (Received
I&J, words:
57
86 (1992) 51-66 Ireland. Ltd. 0303-7207/92/$05.00
of New
York ut Buffulo,
19Y2; accepted
Carbohydrate
epitope;
18 March
Buj$h,
NY 14260, USA
1992)
Recombinant
human
choriogonadotropin
/3-subunit
Summary In our previous paper (Chen et al. (1991) J. Biol. Chem. 266, 4081-4087) we reported the preparation and characterization of recombinant human choriogonadotropin p subunit (hCG/3) using the baculovirus-insect cell expression system. The rhCGP was found to contain high mannose type N-linked carbohydrates and 3-4 serine-linked disaccharide chains. Despite the carbohydrate structural variation, the rhCGP was similar to hCGP in in vitro immunological and biological properties. In order to evaluate its in vivo immunological properties, rabbit antiserum against rhCG@ was produced. The antiserum was found to be almost identical to anti-hCGP in binding to hCG@ as well as in its crossreactivity with human lutropin (hLH), hCG and human follitropin (hFSH) as indicated by radioimmunoassays using ‘2sI-hCGP as a tracer. Further characterization of the anti-rhCGp antiserum revealed that there are three types of antibodies in terms of antigenic specificity present in the anti-rhCGp antisera pool as shown by dot blot and radioimmunoassays. The carbohydrate-specific antibodies were separated by affinity chromatography using an ovalbumin-glycopeptide-Sepharose column. The antibodies held on the ovalbumin affinity adsorbent were specific for the high mannose type carbohydrates such as those present in rhCG/3, rhCG and thyroglobulin and failed to react with transferrin, cu,-acid glycoprotein and hCGa, all containing complex type carbohydrates. This was further supported by the fact that the recombinant unglycosylated hCG or periodate oxidized rhCGP also did not show any reactivity with the carbohydrate specific antibodies. Two types of peptide epitopes seemed to be present in rhCGP since when the flowthrough fraction from the ovalbumin-glycopeptide-affinity column was passed through the hCG@-Sepharose column, the antibodies in the flowthrough from the latter column were specific to the unique antigenic determinants present only in the rhCGP and not in hCGP. The eluate from the hCGP-Sepharose column contained the third type of antibodies, being the predominant ones, directed to the common epitopes between rhCGP and hCGj3. The high mannose type specific antibodies are potentially useful in differentiating between the high mannose and complex type of N-linked carbohydrates present in a glycoprotein. Also, the antibody could provide an effective reagent in studying the
Correspondence to: Om P. Bahl, Department of Biological Sciences, State University of New York at Buffalo, 347 Cooke Hall, Buffalo, NY 14260, USA. Supported by U.S.P.H.S. Grant HDOX766. Abbreviations: hCG, human choriogonadotropin; rhCGfl. recombinant hCGfi (should not be confused with rhesus monkey C.G.); SelhCG, selenomethionyl recombinant hCG; hFSH, human follitropin: hLH, human lutropin; HPLC, high performance liquid chromatography; RER, rough endoplasmic reticulum; SDS, sodium dodecyl sulfate: PAGE, polyacrylamide gel electrophoreSIS.
intracellular processing of the N-linked oligosaccharides. The antibodies unique to rhCG polypeptide probably were against those epitopes which were either masked or were present in a different conformation in the native hCGp indicating that the high mannose type carbohydrates might have a different steric or subtle conformational effect than the complex type carbohydrate on the rhCGB polypeptide chain.
Introduction Human choriogonadotropin (hCG), one of the four structurally related and well characterized human glycoprotein hormones, is produced by placental trophoblast cells. Its primary function in pregnancy is the stimulation of progesterone synthesis and thereby preparation of the corpus luteum for the implantation and maintenance of the fertilized ovum (Pierce and Parsons, 1981). hCG is a hcterodimer composed of N and p subunits (Bahl, 1969). The cy subunit is 92 amino acid residues long with two N-linked carbohydrates at 52 and 78 positions, while the 145 amino acid-residue p subunit has, in addition to the two N-linked carbohydrates at position 13 and 30, four O-linked oligosaccharides at serine residues 121, 127, 132 and 138, on its carboxyl terminal extension (Kessler et al., 197Ya, b). All the Nlinked carbohydrates in hCG exist predominantly as complex type while the O-linked oligosaccharides in the p subunit are simple structures such as those present in mucins (Endo et al., 1979). Due to the physiological importance of hCG, this hormone and its antibodies have been widely used clinically. Recently we have reported the feasibility of using the recombinant DNA methodology to obtain large amounts of homogencous recombinant hCG and its hCGP subunit from insect cells (Chen et al., 1991; Chen and Bahl, 1991a). Although the recombinant hCGP was found to contain, in addition to 3-4 simple disaccharides, two N-linked high mannose type carbohydrates instead of the complex type present in the native hCG@, it was similar to the native hCGP in its ability to bind with antibodies against native hCGP and to recombine with native hCGLv (Chen et al., 1991). The reconstituted heterodimer (hCGarp) and the recombinant hCG were able to stimulate in vitro CAMP accumulation and steroidogenesis comparable to that of
native hCG (Chen et al., 1991; Chen and Bahl, 1991a). These in vitro studies suggested the potential of recombinant hCG and hCGp in clinical usage. Therefore, the present work was undertaken to further study the in vivo immunological properties of rhCGP. In this communication, we report the production of polyclonal antibodies against the recombinant hCG/3 subunit and comparison of their properties with similarly raised antibodies against the native hCG/3. The recombinant hCGp was found to be highly immunogenic and the antiserum produced, as indicated by radioimmunoassay, was almost identical to the anti-native hCGP antiserum in its specificity and sensitivity. However, because of the differences in the type of carbohydrates present in rhCGP from those of the native hCGP, a small population of the antibodies against rhCGP were found unique to its polypeptide and carbohydrate epitopes. These unique antibodies imply that the high mannose type and complex type carbohydrates differ in their conformation and thus have different effect on the polypeptide epitopes. Materials
and methods
The hormones hCGa, hCG/3, SelhCG, rhCG and hCG were prepared in this laboratory as reported (Bahl, 1969; Chen and Bahl, 1991a, b). hFSH and hLH were obtained from the National Pituitary Agency. The rabbit anti-hCGP antisera, prepared as described (Bahl et al., 1976), had a titer of 1: 50,000 for 30% binding of the tracer under the radioimmunoassay (RIA) conditions. Ovalbumin glycopeptides were prepared by exhaustive digestion of ovalbumin with Pronase followed by gel filtration on Sephadex G-50 (Kamiyama et al., 1962). AffiGel 10 was obtained from BioRad. The Freund’s complete and incomplete adjuvants were purchased from Gibco. The alkaline phosphatase conjugated purified goat
anti-rabbit IgG was from Cooper Biomedical. Human transferrin, human (Y,-acid glycoprotein and bovine thyroglobulin were from Sigma. SDS-PAGE, Western blotting and dot blotting The discontinuous gel system of Laemmli with 4%’ stacking gel at pH 6.8 and 12% separation gel at pH 8.8 was used. The sample buffer containing 125 mM Tris-HCl, pH 6.8, 10% glycerol, 2% bromophenol blue was supplemented with 5% P-mercaptoethanol when desired. The gels were silver stained as described (Sambrook et al., 1989). For the Western blotting, the proteins were transferred to the nitrocellulose filter and subsequently incubated with the appropriate antibodies in phosphate buffered saline containing 2% periodate treated bovine serum albumin. The periodate oxidation of bovine serum albumin was carried out as described (Glass et al., 1981). Dot blot assay was carried out in a BioRad Bio-Dot apparatus using a nitrocellulose filter. Blotting was performed in phosphate buffered saline containing 2% periodate treated bovine serum albumin. Preparation of rhCGP, unglycosylated rhCG and periodate-oxidized rhCG/3 The rhCG@ was prepared by recombinant DNA methodology in the baculovirus expression system and was purified from cell culture medium by immunoaffinity chromatography using highly specific monoclonal antibodies against hCG/3 as described earlier (Chen et al., 1991). The homogeneity of the resulting material was ascertained by reverse phase HPLC and SDS-PAGE. The biosynthetically unglycosylated rhCG was obtained by coinfecting the insect SF9 cells with recombinant baculoviruses containing hCGa cDNA and hCGP cDNA in the presence of 0.8 pg/ml of tunicamycin (Chen et al., in preparation). The unglycosylated rhCG was purified by one step immunoaffinity chromatography with anti-hCG monoclonal antibody B17 (Chen and Bahl, 1991b), and the traces of glycosylated material if any in the preparation were removed by concanavalin A affinity chromatography. The unglycosylated rhCG was characterized by carbohydrate compositional analysis and radioimmunoassay (Chen .et al., in preparation).
The periodate oxidation of rhCG/3 was carried out by incubating it at 1 pg/pl in 0.1 M sodium acetate (pH 4.5) with 10 mM sodium periodate for 10 h at room temperature. The excess periodate was removed by the addition of glycerol to a final concentration of 10 mM (Glass et al., 1981). Preparation of polyclonal antibodies against rhCG/3 and radioimmunoassay Two New Zealand female rabbits, weighing 3-4 lbs each, for immunization were purchased from Becken Farms. A sample of 100 pg of rhCGP in 0.5 ml saline was well mixed with an equal volume of Freund’s complete adjuvant and the resulting emulsion was injected intradermally at multiple sites on the back. Four weeks after the primary immunization, a booster of 50 pg of rhCG@ in the Freund’s incomplete adjuvant was administered again at multiple sites intradermally, and the immunization was repeated every other week. The animals were bled from an ear vein between the immunizations, and the sera were evaluated by radioimmunoassay using ‘*‘IhCG (Bahl et al., 1976). The chloramine T method was used for radioiodination of the hormones (Madnick et al., 1981). Specific activity of the labeled hormones was approximately 1 pCi/pg. The RIA was performed using “‘I-hCGP or 12’I-hCG and antirhCGP or anti-hCGp antibodies as described previously (Chen et al., 1991). All solutions were prepared in 50 mM sodium borate buffer, pH 8.0 containing 0.5% bovine serum albumin (BSA). The total volume of the reaction mixture was 300 ~1 containing 30,000 cpm of ‘251-hCG/3 or “‘IhCG and anti-rhCGP or anti-hCGp antibodies and varying concentrations of the hormones. The incubations were performed at 37°C for 1 h after which the amount of tracer antibodies complex was determined by precipitation with 10% polyethylene glycol-8000. Separation of the carbohydrate and polypeptide specific antibodies To purify the carbohydrate specific antibodies, the high mannose type carbohydrate affinity adsorbent was prepared by coupling ovalbumin glycopeptides with AffiGel 10. The coupling procedure was essentially the same as previously used
60
(Chen et al., 1991). A prewashed 2 ml sample of AffiGel 10 with 100 ml of cold water was mixed with 10 mg of ovalbumin glycopeptides in 3 ml of 0.1 M morpholinosulfonic acid buffer (MOPS), pH 7.5 containing 0.3 M NaCI. The mixture was kept stirring at 4°C for 12 h. The active sites of the gel remaining were blocked by incubation with 0.5 ml of 1 M glycine ethyl ester, pH 8.0 for 1 h. The carbohydrate specific antibodies were purified by passing 5 ml of rabbit anti-rhCGp antisera through a 2 ml affinity absorbent in a disposable pipet in 0.01 M Tris-HCI buffer, pH 8.0 containing 0.14 M NaCl. After washing with about ten bed volumes of the above buffer, the column was eluted with 0.05 M glycine-HC1 buffer, pH 2.3 and the eluate was immediately neutralized with 0.5 M Tris-HCI buffer, pH 8.5 and concentrated with a Centriconmembrane microconcentrator (Amicon). The antibodies against the polypeptide were purified by passing the above flowthrough successively from 2 ml columns of Protein A-Sepharose (Pharmacia) and hCG&Sepharose in disposable pipets pre-equilibrated with 0.01 M Tris-HCI, pH 8.0 containing 0.14 M NaCI. After washing with about ten bed volumes of the buffer, the columns were eluted with 8 ml of 0.05 M Tris-HCl buffer, pH 2.3. After neutralization with 0.5 M Tris-HCI buffer, pH 8.5, the flowthroughs and the eluates from both columns were concentrated in a Centricon-10 membrane microconcentrator. After centrifugation the samples of the purified antibodies were subjected to HPLC using a Dionex HPLC system equipped with a Synchropak GPC-300 (250 X 4.6 mm). The column was eluted with 50 mM ammonium bicarbonate at a flow rate of 500 pl/min for 15 min and the eluate was monitored at 210 nm. Results Preparation of unglycosylated oxidized rhCG/3
rhCG and periodate-
The high expression of rhCG in the Baculovirus insect cell expression system was exploited to prepare unglycosylated rhCG biosynthetically using the glycosylation inhibitor, tunicamycin (Materials and methods). The unglyco-
-24 -16 Fig.
I. SDS-PAGE
of rhCG and unglycosylated rhCG. 300-500
ng of each sample nonreducing
was used. Lanes
and reducing
under nonreducing
conditions;
I
and 3: hCG
under
lanes 2 and 4: rhCG
and reducing conditions:
lane 5: unglyco-
sylated rhCG and partially dissociated (r and p subunits.
sylated rhCG, purified by immunoand lectin affinity chromatography using monoclonal antibody B17 (Chen et al., 1991) and concanavalin A, respectively, was examined for homogeneity by SDS-PAGE (Fig. 1). As shown in lane 5, the intact unglycosylated rhCG gave sharp protein bands as compared with those of rhCG indicating that the carbohydrate heterogeneity was probably responsible for the broad bands of rhCG (lanes 2 and 4). Under the condition used, the reduced rhCGa and p subunits were not resolved by SDS-PAGE (lane 4). However, they did separate on reverse phase HPLC (Chen and Bahl, 1991a). The unglycosylated rhCG had a molecular weight of 35.5 kDa while the reduced unglycosylated rhCGa and /? subunits had molecular weights of 16 kDa and 19 kDa respectively. The carbohydrate analysis of the unglycosylated rhCG by Dionex carbohydrate analyzer showed the absence of N-linked carbohydrate chains while the antibody and receptor binding activities as determined by radioimmunoand radioreceptor assays (Chen et al., in preparation) were comparable to those of rhCG (data not shown). The rhCGP containing high mannose type Nlinked carbohydrate chains, approximately six
TABLE
1
CARBOHYDRATE COMPOSITIONAL PERIODATE OXIDIZED rhCGP Number
Protein
rhCG/3 Periodate
oxidized
rhCGP
ANALYSIS
OF
of sugar residues/m01
Fuc
GalN
GlcN
Gal
Man
2.5 0
2.4 0
4.0 i1 4.0”
3.7 0
11.3 4.x
“ Calculated based on four residues drate chains.
”
of GlcN per two carbohy-
mannose residues on the average per chain and l-2 fucose residues (Chen et al., 1991), was subjected to periodate oxidation. Under the experimental conditions used, the treatment resulted in the oxidation of almost four of the six mannose residues and all fucose residues in a single chain as determined by its carbohydrate analysis (Table 1) by Dionex carbohydrate analyzer using a pulse amperometric detector (Chen et al., 1991). Consequently, the two surviving mannose residues must be linked at the C, position. Based on the oxidation data and the analogy with other high mannose type carbohydrate chains, a tentative structure can be assigned to the carbohydrate in rhCGp as shown in Fig. 2. The positions of the four mannose residues marked by an asterisk are consistent with the periodate oxidation data while the positions of the other two mannose residues need to be determined. The second L-fucose residue is probably located at C, of the innermost N-acetylglucosamine (Weber et al., 1987; Prenner et al., 1991; personal communication from L. Marz).
Characterization of anti-rhCG/3 In order to study the immunological properties of the recombinant hCGp expressed in the insect
Fig. 2. Tentative structure of high mannose type carbohydrate chains in rhCGP. The presence of four mannose residues marked by an asterisk is established by the periodate oxidation data.
.-P -E s
80
60
,L.d
10
100
“g
Fig. 3. Radioimmunoassay of hCGP (3) and rhCG/3 (01 with rabbit anti-hCGP serum at 1 :65,000 dilution (A) and antirhCG/3 serum at 1:55,000 dilution (Bl using “‘I-hCGP tracer.
cells, antibodies against rhCG@ were raised in two rabbits as described in Materials and methods. Both antisera displayed high titer and showed under the radioimmunoassay conditions a binding of more than 30% with ‘2sI-hCG at 1 :65,000 dilution. The specificity of the antiserum against rhCGP was evaluated by radioimmunoassay as shown in Fig. 3A. For comparison the assays of hCGP and rhCGP using antiserum against native hCG/3 were also performed (Fig. 3B) under identical conditions. The data indicated that both antisera could bind with hCGP and rhCG/3 equally competitively and there was no significant difference in their immuno specificity, further supporting the conformational and antigenic similarity of hCG@ and rhCGP.
62
\
I
1MI
got
x
B
-
60 5
70.
f
60.
E
50-
E 40n
301
201 lotI 10 1W “9 Fig. 4. Radioimmunoassay of hCG CO), hLH (0) and hFSH (x) with (A) rabbit anti-hCGp serum and (B) anti-rhCGp serum using ‘251-hCG tracer. 1
The similarity of the antisera against rhCG/3 and hCG@ was also evident from their interactions with hCG, hLH and hFSH as indicated by radioimmunoassay (Fig. 4). Using 12’I-hCG as a tracer, the displacement curves of hCG, hLH and hFSH with antisera against rhCGP and hCGP were quite comparable suggesting no significant specificity differences between the two antisera. Carbohydrate and polypeptide specific antibodies Since rhCGP is different from the native hCGP in that it possesses two N-linked high mannose type carbohydrates instead of the complex type, an attempt was made to determine if the antisera contained any antibodies directed against the carbohydrate. The antiserum was passed through an affinity column using the high mannose type carhohydrate containing glycopeptides from ovalbumin. The column was eluted to yield the carbohydrate specific antibodies. The antibodies against rhCGP polypeptide in the flowthrough were fur-
ther purified by protein A-Sepharose affinity column. The purified antibodies were examined and quantitated by HPLC equipped with a Synchropak-300 GPC column. The single peak of both antibodies in HPLC suggested the homogeneity of the purified materials (data not shown). Further quantification of the antibodies indicated that about 0.07% of antibodies in the anti-rhCGp antiserum were against the high mannose type carbohydrate based on their relative peak size on HPLC. The specificity of the purified antibodies was assessed using different glycoproteins by a dot blot assay. As shown in Fig. 5 (lanes A and B), the protein A purified antibodies were specific to hCGP polypeptide backbone and could bind with hCGj3, rhCG@, SelhCG and rhCG but not other glycoproteins or glycopeptides. The carbohydrate specific antibodies eluted from the ovalbumin glycopeptide column could bind with the glycoproteins containing high mannose type carbohydrates only, such as those present in thyroglobulin, rhCGP, rhCG and SelhCG and failed to bind with the complex type carbohydrate containing glycoproteins such as a,-acid glycoprotein and transferrin. To further confirm that the antibodies eluated from the ovalbumin glycopeptide column were carbohydrate specific, biosynthetically prepared unglycosylated rhCG and periodate treated rhCGP were used in the dot blot assay as shown in Fig. 5 (lanes C and D). The ovalbumin affinity purified antibodies failed to react with the unglycosylated rhCG or periodate treated rhCGP. Thus, it is clear that the antibodies against the carbohydrate moiety of rhCGP were specific for the high mannose type rather than complex type carbohydrates. rhCGP polypeptide specific antibodies The substitution of the complex type carbohydrates with high mannose type chains raises the possibility of change in the peptide epitopes due to differences in the masking effects of different carbohydrate structures probably because of their conformational differences. To investigate this possibility, the protein A purified antibodies from antisera against rhCG/3, after absorbing with the ovalbumin glycopeptide column, were passed through the hCGP coupled Sepharose column.
63
A
B
C
D
6 7
SDS-PAGE under reducing conditions using silver staining for the visualization of protein bands (Fig. 5). As stated previously (Chen et al., 1991), rhCGB and native hCGB behave differently under reducing and nonreducing conditions. This differential behavior is attributed to the differences in their carbohydrate structures, in particular, due to the lack of sialic acid in the rhCGB. As shown in Fig. 6B and Fig. 7, the eluate could bind with rhCG and hCGB equally well within experimental error while the flowthrough displayed preferential binding to rhCGB suggesting the emergence of some new epitopes in rhCGB due to change in the carbohydrate structure from the complex type present in hCGB to the high mannose type in the recombinant hCGB. Immunological characterization of three populations of antibodies in rabbit anti-rhCG/3 by radioimmunoassays In order to quantify the immunological activity of the above three populations of the antibodies fractionated from the anti-rhCGB, radioim-
6 9
64.0 47.0
10 Fig. 5. Dot blot analysis of various glycoproteins with rhCG/3 polypeptide epitope specific antibody (lanes A and D) and rhCGP carbohydrate epitope specific antibody (lanes B and 0. 500 ng of each sample was used. Lanes A and B: (1) hCG@, (2) hCGn, (3) SelhCG, (4) rhCG, (5) rhCGp, (6) cu,-acid glycoprotein, (7) transferrin, (8) thyroglobulin, (9) ovalbumin-glycopeptides, and (10) buffer blank as control. Lanes C and D: (1) rhCGP, (2) rhCG, (3) periodate treated rhCGP, and (4) unglycosylated rhCG.
The flowthrough as well as the eluate were subjected to the dot blot assay and Western blotting with rhCGB and hCGB. To rule out the possibility of any trace contaminant, the homogeneity of rhCGB used in the assay was ascertained by
33.0 24.0 '16.0
Fig. 6. SDS-PAGE and Western blot analyses of rhCGP and hCGP. A: About 200 ng samples of rhCG@ (lane 1) and hCGP (lane 2) were subjected to SDS-PAGE in 12% gel under reducing conditions and subsequently stained with silver nitrate. B: About 100 ng samples of rhCGP (lane 1) and hCG/3 (lane 2) were applied to 12% SDS-PAGE under nonreducing conditions. After transferring to nitrocellulose filter, the proteins were detected by polypeptide specific antibodies (see the text for details).
munoassays using ‘2’I-hCGb and each antibody were performed. As shown in Fig. 8A, the predominant component of anti-rhCG/? antibody at 1 : 50,000 dilution had similar reactivity toward hCG and rhCG. On the other hand, the carbohydrate specific antibody at 1: 100 dilution showed reactivity with rhCGP with insignificant crossreactivity with native hCGp indicating that the antibody was specific to the high mannose type carbohydrate present in rhCGP rather than the complex type that was present in the native hCGP (Fig. 8 B 1. Similarly, the antibody crossreacted with thyroglobulin, a high mannose type carbohydrate containing glycoprotein. A 50% inhibition of binding of ‘2sl-rhCGP to the carbohydrate specific antibody by thyroglobulin was achieved at the 24.2 ng level as compared to the 10.5 ng level with rhCGP (Fig. 8B). Finally, the polypeptide specific antibody hCGP at 1 : 250 dilution showed
100
go- A 80 % 70g
60-
E
50-
E 40=
3020 10 -
I
I
1
10 v
I
I
I
1
10 ng
M
10
1
ng Fig. 8. Radioimmunoassays using “iI-rhCGp and (A) antirhCGP polypeptide antibody at 1:SO.OOO dilution, (B) the carbohydrate specific antibody at 1: 100 dilution, CL‘) the rhCGP polypeptide specific antibody at 1 :200 dilution. A: rhCG (01, hCG (0) and hFSH (X ).B; rhCGP (0). hCGp (0) and thyroglobulin (X );C: rhCGP (01 and hCGp (3).
no significant (Fig. 8C).
Fig. 7. Dot blot analysis of rhCGP and hCG@ with (A) hCGp polypeptide antibody and (B) rhCGP specific antibody. The amounts of hCGp and rhCGP applied were: (1) 12.5 ng; (2) 25 ng; (3) 50 ng; (4) 100 ng; (5) 200 ng; (6) buffer control.
crossreactivity
with
native
hCGp
Discussion The data presented here indicate that rhCGP from insect cells was quite immunogenic and pro-
65
duced a high titer and high affinity antisera in rabbits. In this study, we have characterized the anti-rhCGP antiserum in detail and have further fractionated the antiserum into three different immuno specificities. The data indicate that there are three types of antibodies, those which are directed against the peptide epitopes common to both hCG/3 and rhCG/3, these being the predominant ones, antibodies to the peptide epitopes unique to rhCGP, and the antibodies directed against the carbohydrate epitopes. The antiserum displayed close similarity to the antiserum against native hCG/? in both specificity and sensitivity as evident by radioimmunoassays using ‘2”I-hCGP and anti-rhCG@ or anti-hCG/3. Furthermore, the anti-rhCG/3 showed similar crossreactivity with hCG, hLH and hFSH as the antibody to the native hCG/?, again indicating similarity between the two antibodies. Therefore, for immunodiagnostic applications it should be possible to use rhCGP in place of native hCG@ both as a standard or as an antigen for raising antibodies. Carbohydrates in glycoproteins, by virtue of their hydrophilic nature, are usually present on the surface of proteins and can significantly influence the physicochemical properties as well as the immunological and biological characteristics of glycoproteins (Green et al., 1986; Feizi et al., 1987). The removal of the carbohydrate moiety of hCG has been found to alter its antigenic structure (Rebois and Liss, 1987; Ryan et al., 1987; Sairam et al., 1988). Because of the carbohydrate structural differences between the rhCG/3 and native hCG/?, in this study we have been able to show that a significant though small percentage of the polyclonal antibodies were specific to the high mannose type carbohydrates present in rhCG. Similarly, a small percentage of the antibodies were directed against peptide epitopes unique to rhCGP. In our studies, we have separated the antibodies against the carbohydrate and rhCGj3 specific peptide epitopes. The carbohydrate specificity of the antibodies was confirmed by their ability to crossreact with rhCG/3 and rhCG and their inability to recognize the biosynthetically prepared unglycosylated hCG, devoid of carbohydrates, or periodate treated rhCG/I in which the neutral sugars were oxidized. The antibodies against carbohydrate epitopes were spe-
cific to the high mannose type carbohydrates as shown by their binding with the high mannose type carbohydrate containing glycoproteins including thyroglobulin, insect cell derived rhCG and rhCGP, but not to the glycoproteins containing the hybrid or complex type carbohydrates such as those present in hCGa, hCGP, a,-acid glycoprotein and transferrin. Thus, the anticarbohydrate antibodies provide a valuable reagent for various basic studies. They can be used in determining the type of carbohydrate whether complex or high mannose type present in a glycoprotein. The antibodies may be useful in investigating the intracellular processing of the N-linked carbohydrates in glycoproteins and intracellular trafficking. Subsequent to the transfer of the oligosaccharides Glc,Man,GlcNAc,from the oligosaccharide lipid intermediate to the growing polypeptide chain in the rough endoplasmic reticulum (RER), the carbohydrate undergoes a series of processing reactions. These involve the cleavage of all glucose residues and one mannose residue in the lumen of RER before the glycoprotein enters the Golgi. This is followed either by phosphorylation of Man,GlcNAc,and sequestration of the protein in lysosomes or further removal of mannose and incorporation of peripheral sugars, GlcNAc, Gal and sialic acid in mid and trans Golgi for secretion (Paulson, 1989). It is conceivable that the high mannose type carbohydrate containing proteins may be enrouted from the endoplasmic reticulum directly to another subcellular compartment without further modification in the Golgi apparatus. This antiserum could be helpful in investigating this possibility. The carbohydrate structure in glycoproteins undergoes alterations in various developmental as well as pathological states (Kornfeld et al., 1985). The carbohydrate specific antibodies may provide an effective means of studying the carbohydrate changes during various pathologic and developmental states. The N-linked carbohydrates of glycoproteins are generally regarded as non-antigenic in mammalian species. It is probably because of the exposure of the immune system to a diverse array of heterogeneous carbohydrate structures during the development of the organism and thus resulting in the immunosuppression or tolerance to the
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carbohydrates The present work represents one of the few examples of rabbit antibodies directed specifically against N-linked carbohydrates of a glycoprotein. Since the carbohydrate specific antibodies were isolated from the pool of antibodies that were generated against insect cell derived rhCG& rhCGp probably had a unique carbohydrate structure(s) that was antigenic. Recent reports have suggested that antigenicity of carbohydrates of insect cell glycoproteins is probably due to differences in the mode of fucosylation (Weber et al., 1987; Prenner et al., 1991). It is interesting to note that anti-rhCGP contained, though a small fraction of the total antibodies, antibodies unique to the polypeptide chain. Thus, it appears that the high mannose type carbohydrates present in rhCG@ unmask certain peptide epitopes which are not exposed in the native hCGp containing complex type carbohydrates. Presumably, the conformation of the two types of carbohydrates is different and they may have a different steric effect on surface peptide epitopes or the two types of carbohydrates may have a subtle but different effect on the conformation of the polypeptide chain and thereby on their epitopes. In conclusion, we have obtained a rabbit antirhCG/3 antiserum with high titer, which is similar in specificity and sensitivity to the antiserum against native hCGP. The antibodies against the N-linked carbohydrates and the polypeptide backbone have been purified by affinity chromatography using ovalbumin glycopeptide and protein A and native hCG/3, respectively. The antibodies against carbohydrate epitopes were found to be highly specific for high mannose type carbohydrates and they may be potentially useful in the characterization and in the study of in vivo assembly of carbohydrates of glycoproteins. Further, such antibodies may be useful in studying carbohydrate variation during various developmental and pathologic states. The presence of antibodies unique to the peptide epitope(s) of rhCG/3 in the antiserum may reflect conformational differences between the high mannose and the complex type carbohydrates and thus different steric or conformational effects on the polypeptide chain.
Acknowledgements We wish to thank Dr. Rana Samuel for performing dot blot assay experiments, Juchun Fu and Huawei Zeng for technical support, Diane McMaster for help in the preparation of the manuscript, and James Stamos for photo-illustration work. References Bahl, O.P. (1969) J. Biol. Chem. 244, 567-574. Bahl, O.P., Pandian, M.R. and Ghai, R.D. (1976) Biochem. Biophys. Res. Commun. 70, 525-530. Chew W. and Bahl, O.P. (1991a) J. Biol. Chem. 266, 81928197. Chen, W. and Bahl, O.P. (1991b) J. Biol. Chem. 266, 93559358. Chen, W. and Bahl, O.P., manuscript in preparation. Chen, W., Shen, Q.-X. and Bahl, O.P. (1991) J. Biol. Chem. 266, 4081-4087. Endo, Y., Yamoshita, K., Tachibana, Y., Tojo, S. and Kobata, A. (1979) J. Biol. Chem. 254, 669-670. Feizi, T. and Childs, R.A. (1987) Biochem. J. 245, I-I 1. Glass, II, W.F., Briggs, R.C. and Hrilica, L.S. (1981) Anal. Biochem. 115, 219-224. Green, E.D., Boime, I. and Baenziger, J.V. (1986) Mol. Cell. Biochem. 72, 81-100. Kamiyama, S. and Schmid, K. (1962) Biochim. Biophys. Acta 58, X0-85. Kessler, M.J., Reddy, M.S., Shah, R.H. and Bahl, O.P. (1979a) J. Biol. Chem. 254, 7901-7908. Kessler, M.J., Mise, T., Ghai. R.D. and Bahl, O.P. (197Yb) J. Biol. Chem. 254, 7909-7914. Kornfeld, R. and Kornfeld, S. (1985) Ann. Rev. Biochem. 54, 63 l-664. Madnick, H.M., Kalyan, N.K., Segal, H.L. and Bahl, O.P. (1981) Arch. Biochim. Biophys. 212, 432-442. Paulson, J.C. (1989) Trends Biochem. Sci. 14, 272-276. Pierce, J.G. and Parson, T.F. (1981) Annu. Rev. Biochem. 50, 465-495. Prenner, C., Mach, L., Glossoal, J. and Marz. L. (1991) Glycoconj. J. 8, 241. Rebois, R.V. and Liss, M.T. (1987) J. Biol. Chem. 262, 38Yl3896. Ryan, R.J., Keutman, H.T., Charlesworth, M.C., McCormick, D.J., Millius, R.P., Calve, F.O. and Vityavanich, T. (1987) Recent Prog. Horm. Res. 43, 383-429. Sairam, M.R., Linggen, J. and Bhargavi, G.N. (1988) Biosci. Rep. 8, 271-278. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Vol. 3, p. 1856, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Weber, A., Schroder, H., Thalberg, K. and Marz, L. (1987) Allergy 42, 464-470.
