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2, 1992 December 15, 1992

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GENERATION ALTERATION

OF

SPECIFICITY-VARIANT IN LIGHT ANTIBODIES MONOCLONAL

ANTIBODIES CHAIN

BY

OF CARBOHYDRATE

OF HUMAN

Hirofumi Tachibana*, Sanetaka Shirahata and Hiroki Murakami Graduate School of Genetic Resources Technology, Kyushu University, Hakozaki, Higashi-ku, Fukuoka, 812, Japan
Received October 13, 1992

SUMMARY: A hybridoma line, CSTN, produced human monoclonal antibody of which light chain had N-linked carbohydrate chain within the variable region. Some molecular-weight variants of light chain of the antibody were produced by CSTN variants resistant to cytotoxic effect of concanavalin A. The variant antibodies significantly altered the original cross-reactivity with antigens or lost the ability of antigen binding. The variants variously trimmed their carbohydrate chains by glycosidases,showed the changed reactivity or acquired the ability to bind for antigens. The carbohydrate-deficient antibodies from tunicamycin-treated CSTN and the variant clones behaved in a similar manner on antigen-binding reactivity. Furthermore, comparison of antibodies of which light chains have carbohydrate chains sensitive and resistant to some glycosidases showed that carbohydrate chain in variable region of light chain can influence their reactivity with antigen. 0 1992Academic Press,Inc.

Immunoglobulin

(Ig) molecules are glycoproteins of which carbohydrates are usually

located in their constant regions of heavy chain (1). Such carbohydrates have been indicated to be of significant importance in complement fixation, secretion of Ig molecules (2,3). Studies on a large number of human myeloma protein have revealed that the light chain of some Ig molecules (15% of human myeloma proteins examined) contains additional asparagine-linked carbohydrates attached to the variable region of the chain (4,5). Such carbohydrates have been found in variable regions in both )c and K chains. However, the role of the carbohydrate for antigen recognition has not been established yet (6). It has been well known that variations in amino acid sequence of variable region contribute to many different binding specificities, and immunochemical studies of the antibodies have given insights into the diversification in structure of Ig variable regions in Ig.

* To whom correspondence should be addressed. Abbreviations used: Con A, concanavalin A; Ig, immunoglobulin; bovine carboxypeptidase A, Cpase; Candida cytocrome C, Cyt C; double-stranded DNA, ds DNA; FCS, fetal calf serum; End H, end-P-N-acetylglucosaminidase H.
0006-291X/92 $4.00

625

Copyright 0 1992 by Academic Press, inc. All rights of reproduction in any form reserved.

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In the present study, by altering the configuration of antigen-binding site by changing the structure of carbohydrate located in the variable region, we attempted to clarify the role of carbohydrate chain in the variable region. For this purpose, we utilized human hybridoma CSTN which produce IgM(h) reactive to bovine carboxypeptidase A, Candida cytocrome C and double-stranded (ds)DNA, and the h chain was N-glycosylated (7). Variant cell clones that were producing Igs with altered molecular weights and reactivities with antigens were isolated for resistance to cytotoxic effect of concanavalin A (Con A), which has been used to select variants possessed structurally altered carbohydrates (8,9). Furthermore, antibodies obtained from mutants were treated with some glycosidases.

MATERIALS

AND METHODS

Cells and Cell Culture. The CSTN is a subclone of human hybridoma HB4C5 (lo), and produces antibody (IgM, A) cross-reactive to bovine carboxypeptidase A (Cpase), Candidu cytocrome C (Cyt C) and double-stranded DNA (dsDNA) (7, 11). The light chain of HB4C5 antibody has a N-glycosylation site actually glycosylated in the complementarity-determining region 1. The cells were maintained in ERDF medium supplemented with 5% fetal calf serum (FCS) at 37C in a humidified atmosphere of 5% CO2/95% air. Selection of Con A Resistant Clones. The serum-free medium was used to select Con A-resistant clones because cytotoxic complex was formed by interaction of Con A with FCS components (9). Cells were suspended with ERDF medium (Kyokuto Seiyaku, Tokyo, Japan) supplemented with 10 &ml of insulin, 20 &ml of human transferrin, 20 uM of ethanolamine, 25 nM of selenite (12) and 1 ug/ml of Con A. The cells were innoculated into 96 well culture plates, and maintained in the same medium for 2 weeks. The resistant clones obtained were maintained in serum-free ERDF medium without Con A for 3 weeks. To remove Con A-sensitive revertants the cells were again selected with the serum-free medium with Con A for 2 weeks. The resistant clones were cultured in 5% FCS-ERDF medium for about 4 weeks, and Ig collected from the medium was used throughout the experiments. Inhibition of Glycosylation. Cells were cultured in the 5% FCS-ERDF medium containing 10 ugJmlof tunicamycin for 6 h, and then the supernatants were discarded. The cells were cultured in the fresh medium containing tunicamycin for further 24 h. The cultured medium obtained were passed through a Con A-agarose column in order to remove incompletely-deglycosylated Ig. Unbound fraction was used carbohydratedepleted Ig solution. Glycosidase Digestion. Five micrograms of Igs were treated in O.lM sodium citrate buffer (pH4.5) containing 1 mM phenyl methyl sulfonyl fluoride at 37C for 18 h with the following glycosidase(s): (a), a-mannosidase (0.1 unit); (b), neuraminidase (O.lunit); (c), a mixture of neuraminidase (0.1 unit) and /3-galactosidase (0.1 unit); (d), a mixture of neuraminidase (0.1 unit), p-galactosidase (0.1 unit) and p-N-acetylhexosaminidase (0.05 unit); (e), End H (6 m unit). The antibodies treated with varying glycosidase preparations were assayed for their reactivities with antigens. Antibody Binding Assay. Reactivity of antibodies were assessed by the method of Kato et. al (13). For anti Cpase and Cyt C assay, Cpase or Cyt C was dissolved in 50 mM sodium bicarbonate at the concentration of 10 ug/ml and 96 well immunoplates (NUNC, Denmark) were coated with them. After washing the wells with phosphate buffered saline (PBS) containing 0.05% Tween 20 (TPBS), the wells were blocked with a blocking solution (0.5% bovine serum albumin+0.2% gelatin/PBS). Antibodies were reacted with antigens for 1 h at 37C, then washed 3 times with TPBS. Antibody bound for antigens were detected by peroxidase-conjugated anti human u chain antibody (TAGO, CA, USA) at 37C for 1 h. The enzymic color reaction was visualized by addition of a substrate solution containing 0.33 mg/ml of 2,2-azino-di-3-ethyle benzthiazolin sulfonic acid, 0.03% Hz02 in O.lM citrate buffer, (pH 4.0). Absorbance of the color reaction was measured at 405 nm by a plate reader. Anti dsDNA assay was performed by the same way as of anti Cpase assay described above, using dsDNA 626

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immobilized with 0.001% protamine sulfate having been trapped to immunoplate. The assay of concentration of antibodies were measured by use of anti human B chain antibody. Western Blotting. The antibodies from original and Con A-resistant clones were applied to SDS-polyacrylamide gel electrophoresis (lO%T) followed by Western blotting for assay of +e molecular weight (13). The blotted nitrocellulose filter was blocked with Block Ace (blocking reagent, Dainippon Pharmaceuticals, Japan) overnight at 4C. After washing with TPBS, the blotted filter was incubated with peroxidase-conjugated anti human @and h chain antibodies (TACO, CA, USA). The color reaction was developed using 4-chloro-1-naphthol as substrate.

RESULTS

AND

DISCUSSION

We have analyzed the role of carbohydrate chains bound for variable region in light chains of antibodies with regard to their reactivity to antigens. To our knowledge, it is the first case to show that antibody-antigen reactivity is affected by the carbohydrate chain on variable region in the light chain. The CSTN is a subclone of HB4C5 producing antibody specific to human lung cancer, and produces monoclonal antibody crossreactive to Cpase, Cyt C and dsDNA. N-linked carbohydrate chain has been found at position 25 in CDR 1 of the light chain of HB4CS antibody (unpublished data). To obtain antibodies altered in their carbohydrate chains, carbohydrate-variants were isolated by screening of Con A-resistance of CSTN. Somatic mutants with defects in the ability to glycosylate proteins have been isolated from the resistants to cytotoxic plant lectins such as Con A (8,9). The CSTN cells were cultured with serum-free ERDF medium containing Con A in 96 well plates, and survived cells were further cultured in the medium. Of 60 wells selected at first screening, two clones, C5TN-C4 (C4) and CSTN9T (9T) were obtained. The molecular sizes of both heavy and light chains of Ig derived from C4 (C4Ig) and that derived from 9T (9TIg) were examined by SDS-PAGE followed by Western blotting. Differences in migration pattern of their heavy and light chains were seen on the SDS-PAGE. Both heavy and light chains of C4Ig migrated slightly lower than those of C5TNIg. On the other hand, the heavy chain of 9TIg migrated identically with that of C41g, and the molecular size of 9T light chain was smaller than that of CSTN but bigger than the light chain derived from CSTN having been treated with tunicamycin (Figure 1). However, heavy and light chains of C4Ig produced by treating C4 with tunicamycin migrated at the same rate as those of CSTNIg. These results indicate that the difference in molecular sizes result from alterations of carbohydrate structure on the Ig molecules. Although tunicamycin-treated 9TIg heavy chain migrated faster than that of normal one, light chain of 9TIg produced by treating with or without even 20 pg/ml tunicamycin was indistinguishable. The light chain of 9TIg was detected with peroxidase-conjugated Con A, a mannose binding lectin, indicating that the chain was glycosylated. To obtain these variants, we did not use any mutagenesis in this experiment, and the variants were not isolated for the alterations of the reactivity to antigen but only for resistance to cytotoxic effect of Con A. Therefore, this sample suggests that some variants with regard to alteration of glycosylation could have existed at a lower frequencies and that the different molecular sizes of the antibodies including 9T 627

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Figure 1. SDS-PAGE analysis of lg molecules by Con A-resistant lines. Culture superna~~nts from the cells treated with or without tunicamvcin were analyzed on reducing gel and by Western blotting. Ig heavy and light chains were detected with peroxidase-conjugated anti human m and I chain antibodies. Lane I: CSTN Ig, Lane 2: C4 Ig, Lanes 3 and 7: 9T Ig, Lanes 4,s and 6: CSTN, C4 and 9T lgs after tunicamycin treatment, respectively.

light chain result from alteration of carbohydrate chain, but not from the alteration of primary structure. The antibodies produced
by

the variants were assessed for the reactivities to Cpase,

Cyt C and dsDNA. C4 and 9TIgs showed dramatical alteration in cross-reactivities to antigens (Figure 2). C4 Ig reacted to Cyt C with very lower strength and completely lost the ability to bind Cpase and dsDNA. The 9TIg showed an elevated reactivity for Cyt C. The reactivity for Cpase was lower than that of CSTNIg, and could not bind to dsDNA. If the alterations of reactivity result from differences in the amino acid sequence of mutant antibodies, the alteration of the reactivities could have been observed when carbohydratedeficient antibodies were compared. In order to elucidate whether there are alterations in primary structure or carbohydrate chain, deglycosylated Igs were obtained by treating CSTN and C4 cells with tunicamycin. The deglycosylated C5TNIg reacted to Cpase and Cyt C with much lower strength, and did not bind to dsDNA (Figure 3). On the other hand, the C4Ig without carbohydrate gained the ability to bind to Cpase and Cyt C, and the patterns of reactivities were similar with
that of

CSTN

deglycosylated-Ig.

These

results indicate that the antigen binding alterations are due to the alterations on the carbohydrate structure. This was recently substantiated by assessing that nucleotide sequences in the variable regions in these Igs were completely identical (data not shown). Accordingly, correlation between the antigen binding activity and the structural changes of carbohydrate chain in the antibody molecule was examined in detail. A preliminary experiment using glycosidase-digestion followed by Western blot analysis revealed that carbohydrate chain on light chain of CSTNIg was sensitive to neuraminidase and End H. Based on the finding, we selected several glycosidases to modify the carbohydrate moieties. The CSTN, C4 and 9TIgs were treated with different 628

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1.5

ds DNA
5 $ -2 8 5 -f 4 . .l 1 11 0 0.0 1 10 100 1.0

0.5

11 00

Ig ConcernZion

(n$l)

Ig Concentration (@ml)

Ig Concentration (@ml) Figure 2. ELISA analysis comparing the reactivities of Igs from C4 and 9T with CSTN lg. The Igs were assayed for the reactivity to Cpase, Cyt C and ds DNA Assays were done as described in MATERIALS AND METHODS. CSTNlg (0); C4lg (A); OTlg (m)

1.5 2 1.5

Cpase

5
3 LO-

ds DNA

3 9
I l.O-

z $ e

0.5 -

10

100

1000

00-A

10

100

Ig Concentration

(rig/ml)

Ig Concentration

(q/ml)

Ig Concentration (@ml) Fi ure 3 ELBA analysis of N-linked carbohydrate .-+-y+ c am e lclent Igs produced by tunicamycin-treated cells. Assays were done as descrived in MATERIALS AND METHODS. C5TNIg from tunucamycin treated cells(O); C4Ig from tunicamycin treated cells(A). 629

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Table 1.

Effects of Glycosidase Digestion on Reactivities of Igs Digested with

Contml Ig a. a-hdannosidase

b.
Sialldase

e.
Sialidase + P-Calactosidase

Treated It3 CBTN

Coated substance Cpase

Z%NA

d. Slalidase + b-Galactosidasc+
p-N-tlCQ+

c. EndH

hexosamini dasc

0.490
0.203 0.024 0.003 0.000 0.260 0.330 0.000

0.550 0.030 0.222 0.001 0.000 0.252 0.298 0.000

1.020 0.753 0.056 0.136 0.000 0.046 0.272 0.336 0.000

1.060 0.069 0.874 0.231 0.000 0.076 0.300 0.362 0.000

1.090 0.824 0.071 0.264 0.000 0.081 N. D. N. D. N. D.

0.650 0.280 0.049 0.044 0.000 0.010 N. D. N. D. N. D.

C5TN-C4

Qase i%NNA

C5TN-9T

Cpase WC ds DNA

Reactivitiesweredeterminedby ELISA, and these valuesrepresent absorbance at 405 nm. Details of glycosidase digestions of each preperations were described in MATERIALS AND METHODS. N. D.: Experimentwasnot performed.

combination of glycosidases in a sequential manner, which would decrease the molecular weights gradually. As shown in Table 1, Igs treated with glycosidase(s) were assayed for antigen-binding reactivities. CSTNIg treated with a-mannosidase slightly increased reactivities with Cpase, Cyt C and dsDNA. By removal of sialic acid residues the reactivity for these antigens was significantly increased. Further removal of galactose and N-acetylglucosamine induced more higher reactivity. In contrast, removal of carbohydrate with End H showed lower increment in reactivity than that of the desialylated sample. The results suggest that the enhancement of reactivity is considered to be resulting from not steric hindrance but charge effects from sialic acid. In C4Ig, the sequential removal of glycosides induced the reactivity for Cpase and Cyt C but not for dsDNA. The reactivity was acquired by desialylation and enhanced by treatment with pgalactosidase. The result indicates that the changing of carbohydrate structure affects not only binding strength to antigen but also specificity although the structure of carbohydrate chain on C4Ig is unclear. In addition, 9TIg treated with neuraminidase and End H behaved in an identical manner to normal 9Tlg with regard to antigen-binding reactivity. When 9TIg treated with neuraminidase or End H was assayed by Western blotting, the heavy chain migrated faster than that of normal one but light chain migrated at a same rate with normal one (data not shown). This result indicated that the carbohydrate chain on 9T light chain was resistant to these glycosidases. These findings suggest that the alterations of carbohydrate chain on constant region of heavy chain do not associate with these alteration in antigen-binding reactivities but the changes of carbohydrate chain on light chain is critical in generating altered antibody-antigen reactivity. We generated hybrid hybridomas by fusing HB4C5 with other hybridoma, and the cells produced hybrid antibodies reactive to Cpase and dsDNA and having light chain without carbohydrate chain (15). The antibodies treated with neuraminidase bound to these 630

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antigens with similar reactivity. This finding supports the idea that altered glycosylation on light chain could induce altered antigen binding affinity and specificity. The role of carbohydrate chains on constant region of Ig heavy chains have been established for Ig assembly, transport, secretion and maintenance of Ig cornformation. Some Ig heavy chains contain N-linked carbohydrate chain attached to the variable regions (4,5). Wallick et al. demonstrated that the presence of carbohydrate chain in CDR2 of variable region of heavy chain is critical for the high-affinity binding of anti dextran antibody (16). On the other hand, the role of the carbohydrate chains found in the variable regions of some light chain (15% of human myeloma proteins) has not been established yet. In this study, carbohydrate chain on variable region of light chain of CSTN Ig is shown to play important roles in its antigen binding affinity and specificity. With regard to specificity of antibody, Ohno et al. have proposed that structure of heavy chain is critical for the specificity of the antibody, and light chain is destined to play a subsidiary role for to heavy chain (17). Our findings demonstrate that not only amino acid sequence of variable region of heavy chain but also carbohydrate moiety on variable region of light chain can contribute to determine the specificity of the antibody. Recently, several animal cell lines have been used for the production reports indicate that glycosylation is highly host cell-dependent. Therefore, of it is glycoproteins, and their carbohydrate structure have been reported (18,19,2(l). These important to select host cell which can properly glycosylate each glycoprotein. In the view point, isolation of lectin-resistant cells may be useful in selection of suitable host cells with regard to glycosylation. In addition, it would be an useful technique for generation of specificity-variant antibodies that alteration of carbohydrate moieties by combination the production using lectin-resistant variants of the host cells and digestion of the products using several glycosidases.

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