Structural characterization of the N-glycans of a humanized anti-CD18 murine immunoglobulin G.
This study characterized the N-glycans of a humanized immunoglobulin G4 (IgG4) expressed in NS/O mouse myeloma cells and directed against the CD18 family of adhesion-promoting receptors on leukocytes. The N-glycans were released from the purified recombinant IgG by N-glycanase treatment, purified by Sephadex G50 chromatography, and fractionated by Bio-Gel P-4 chromatography into three oligosaccharide pools. Each pool was analyzed individually by glycosyl composition analysis, high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), 600-MHz 1H-NMR spectroscopy, and electrospray-ionization mass spectrometry. In addition, each of the three pools was subfractionated by HPAEC and the isolated subfractions that contained sufficient material were hydrolyzed and analyzed for glycosyl composition by HPAEC-PAD. The overall results indicate the presence of five oligomannoside-type structures (containing 5 to 8 Man residues) which are not usually found in IgG, and the presence of eight diantennary (mostly truncated) N-acetyllactosamine-type structures which are typical of mouse and human IgGs. The N-acetyllactosamine-type structures were heterogeneous with regard to alpha(1-->6) fucosylation of the linkage GlcNAc, and the presence or absence of GlcNAc and/or Gal beta(1-->4)GlcNAc extending the core pentasaccharide (Man3GlcNAc2). No evidence was found for the presence of sialic acid or bisecting GlcNAc residues on the N-acetyllactosamine-type chains. The latter finding suggests that the N-glycans of this humanized IgG are of the mouse type.
Ip
C C
CC
Department of Cellular and Molecular Biology, Merck Research Laboratories, West Point, Pennsylvania 19486.
Miller
W J
WJ
Silberklang
M
M
Mark
G E
GE
Ellis
R W
RW
Huang
L
L
Glushka
J
J
Van Halbeek
H
H
Zhu
J
J
Alhadeff
J A
JA
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000302:isolation & purification; D015703:Antigens, CD / Q000276:immunology*; D018821:CD18 Antigens; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002850:Chromatography, Gel; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry* / Q000302:isolation & purification; D007962:Leukocytes / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry* / Q000302:isolation & purification; D011134:Polysaccharides / Q000737:chemistry* / Q000302:isolation & purification; D016028:Receptors, Leukocyte-Adhesion / Q000276:immunology; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D018821:CD18 Antigens; D007074:Immunoglobulin G; D009844:Oligosaccharides; D011134:Polysaccharides; D016028:Receptors, Leukocyte-Adhesion; D011994:Recombinant Proteins