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SummaryImmunoglobulin G (IgG) molecules are glycosylated in C.2 at Asn297; the N-linked carbohydrates attached there have been shown to contribute to antibody (Ab) stability and various effector functions. The carbohydrate attached to the IgG constant region is a complex biantennary structure. Alterations in the structure of oligosaccharide have been associated with human diseases such as rheumatoid arthritis and osteoarthritis. To study the effects of altered carbohydrate structure on Ab effector function, we have used gene transfection techniques to produce mouse-human chimeric IgG1 Abs in the Chinese hamster ovary (CHO) cell line Lec 1, which is incapable of processing the high-mannose intermediate through the terminal glycosylation steps. We also produced IgG1 Abs in Pro-5, the wild-type CHO cell line that is the parent of Lec 1. The Pro-5-produced Ab (IgGl-Pro-5) was similar to IgG1-My 1, a myeloma-produced IgG1 Ab of the same specificity, in its biologic properties such as serum half-life, ability to effect complementmediated cytolysis, and affinity for FcylLI. Although the Lec 1-produced Ab, IgG1-Lec 1, was properly assembled and retained antigen specificity, it was incapable of complement-mediated hemolysis and was substantially deficient in complement consumption, Clq binding, and C1 activation. IgG1-Lec 1 also showed reduced but significant affinity for Fcq/R.1 receptors. The in vivo half-life of IgG1-Lec 1 was shorter than that of either the myeloma-or Pro-5-produced counterpart, with more being cleared during the or-phase and with more rapid clearance during the E-phase. Clearance of IgG1-Lec 1 could be inhibited by the administration of yeast-derived mannan. Thus the uptake of IgG1-Lec 1 appears to be accelerated by the presence of terminally mannosylated oligosaccharide. Therefore, certain Ab functions as well as the in vivo fate of the protein are dramatically affected by altered carbohydrate structure. Expression of Igs in cell lines with defined glycosylation mutations is shown to be a useful technique for investigating the contribution of carbohydrate structure to Ab function. 1Abs are glycoproteins and are glycosylated at characteristic positions according to their isotype. The IgG molecule has one conserved glycosylation site, at Asn297, within the C.2 domain of each of its two heavy chains. The oligosaccharides attached there are accommodated within the internal space between the two C,2 domains (1) and are thought to stabilize the molecule and to contribute to the tertiary structure of the Fc (2). Aglycosylated IgGs have been shown to be deficient in such effector functions as complement activation, Fc receptor recognition, and Ab-dependent cell-mediated cytotoxicity (ADCC) 1 (3-5).The oligosaccharides associated with the C.2 of IgG are a Abbreviations used in this paper:

The presence of N‐linked carbohydrate at Asn58 in the VH of the antigen binding site of an antibody specific for alpha(1‐‐‐‐6)dextran (TKC3.2.2) increases its affinity for dextran 10‐ to 50‐fold. Site‐directed mutagenesis has now been used to create novel carbohydrate addition sequences in the CDR2 of a non‐glycosylated anti‐dextran at Asn54 (TST2) and Asn60 (TSU7). These antibodies are glycosylated and the carbohydrates are accessible for lectin binding. The amino acid change in TSU7 (Lys62‐‐‐‐Thr62) decreases the affinity for antigen; however, glycosylation of TSU7 increased its affinity for antigen 3‐fold, less than the greater than 10‐fold increase in affinity seen for glycosylated TKC3.2.2. The difference in impact of glycosylation could result either from the position of the carbohydrate or from its structure; unlike the other antibodies, TSU7 attaches a high mannose, rather than complex, carbohydrate in CDR2. In contrast, glycosylation of TST2 at amino acid 54 inhibits dextran binding. Thus slight changes in the position of the N‐linked carbohydrate in the CDR2 of this antibody result in substantially different effects on antigen binding. Unlike what was observed for the anti‐dextrans, a carbohydrate addition site placed in a similar position in an anti‐dansyl is not utilized.

We have now produced mouse-human chimeric IgG1 in wild-type Chinese hamster ovary (CHO) cell lines Pro-5 as well as in the glycosylation mutants Lec 2, Lec 8, and Lec 1. Analysis of the attached carbohydrates shows those present on IgG1-Lec 1 were mannose terminated. Carbohydrate present on IgG1-Lec8 was uniformly biantennary terminating in N-acetylglucosamine. The glycosylation profiles of IgG1-Lec 2 and IgG1-Pro-5 were heterogeneous. Only IgG1-Pro-5 was sialylated with sialic acid present on only a small percentage of the carbohydrate structures. When the in vivo fate of antibodies labeled with (125)I-lactotyramine was determined, it was found that the majority of all of the antibodies, irrespective of the structure of their attached carbohydrate, is catabolized in the skin and muscle. However, the attached carbohydrate structure does influence the amount that is catabolized in the liver and the liver serves as a major site for the catabolism of proteins bearing carbohydrate with the Lec2 (with terminal galactose) or Lec1(with terminal mannose) structure.

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