Clonal analysis of the glycosylation of immunoglobulin G secreted by murine hybridomas.
A panel of 10 hybridomas was assembled to assess the influence of various genetic and biological factors upon glycosylation of secreted monoclonal IgG. After exhaustive Pronase digestion of IgG, glycopeptides were characterized chromatographically by apparent size, charge, and concanavalin A (Con A)-Sepharose and Lens culinaris (LcH)-agarose affinity. Six glycosylation phenotypes were found to be common among all clones studied. Despite this phenotypic heterogeneity in glycosylation of IgG, considerable similarity exists between different clones. In particular, virtually all IgG glycopeptides bear a core fucose residue. Second, the majority of the glycosylation repertoire is comprised of two phenotypes, characterized by glycopeptides which differ in affinity for Con A-Sepharose. Of these, the predominantly expressed phenotype is the same for all clones. The carbohydrate structure derived from this phenotype, elucidated by 500-MHz 1H NMR spectroscopy, is (Formula: see text). Significant variability between different hybridomas exists in the relative expression of the two major phenotypes. Other differences between clones may reflect the expression of an additional site which is glycosylated differently. However, there is no apparent correlation of phenotype with either the hybridoma's parentage or the serologically defined polypeptide structure of the IgG which it secretes. In addition to clonal variability, other sources of variability in phenotypic expression were identified. A generational variability is apparent upon continuous culturing of the same hybridoma. Also, differences in culture medium pH or proliferative state of the cells may have a modest influence upon the glycosylation phenotype.
Rothman
R J
RJ
Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania 19104.
Warren
L
L
Vliegenthart
J F
JF
Härd
K J
KJ
D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002846:Chromatography, Affinity; D002999:Clone Cells; D006020:Glycopeptides / Q000302:isolation & purification; D006031:Glycosylation; D006825:Hybridomas / Q000276:immunology*; D007074:Immunoglobulin G / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D051379:Mice; D008807:Mice, Inbred BALB C; D008809:Mice, Inbred C3H; D008969:Molecular Sequence Data; D011402:Pronase
D006020:Glycopeptides; D007074:Immunoglobulin G; D011402:Pronase