IgG Fc N-Glycosylation Changes in Lambert-Eaton Myasthenic Syndrome and Myasthenia Gravis Maurice H. J. Selman,† Erik H. Niks,‡ Maarten J. Titulaer,‡ Jan J. G. M. Verschuuren,‡ Manfred Wuhrer,*,† and Andre´ M. Deelder† Biomolecular Mass Spectrometry Unit, Department of Parasitology, and Department of Neurology, Leiden University Medical Center, Leiden, The Netherlands Received May 12, 2010
N-glycosylation of the immunoglobulin Fc moiety influences its biological activity by, for example, modulating the interaction with Fc receptors. Changes in IgG glycosylation have been found to be associated with various inflammatory diseases. Here we evaluated for the first time IgG Fc Nglycosylation changes in well-defined antibody-mediated autoimmune diseases, that is, the neurological disorders Lambert-Eaton myasthenic syndrome and myasthenia gravis, with antibodies to muscle nicotinic acetylcholine receptors or muscle-specific kinase. IgGs were purified from serum or plasma by protein A affinity chromatography and digested with trypsin. Glycopeptides were purified and analyzed by MALDI-FTICR-MS. Glycoform distributions of both IgG1 and IgG2 were determined for 229 patients and 56 controls. We observed an overall age and sex dependency of IgG Fc N-glycosylation, which was in accordance with literature. All three disease groups showed lower levels of IgG2 galactosylation compared to controls. In addition, LEMS patients showed lower IgG1 galactosylation. Notably, the galactosylation differences were not paralleled by a difference in IgG sialylation. Moreover, the level of IgG core-fucosylation and bisecting N-acetylglucosamine were evaluated. The control and disease groups revealed similar levels of IgG Fc core-fucosylation. Interestingly, LEMS patients below 50 years showed elevated levels of bisecting N-acetylglucosamine on IgG1 and IgG2, demonstrating for the first time the link of changes in the level of bisecting N-acetylglucosamine with disease. Keywords: antibody • glycopeptides • myasthenia gravis • glycosylation • mass spectrometry • posttranslational modification • LEMS
Introduction Immunoglobulins (Igs) represent an important part of the human immune system and can adapt to many different antigenic challenges. Structural variety of Igs is achieved by the expression of a multitude of hypervariable regions which create the antigen binding site and define the antigen specificity and affinity. Moreover, multiple classes (i.e., IgM, IgD, IgG, IgA, IgE) and subclasses (e.g., IgG1-4) of immunoglobulins are present in humans, differing in temporal and spatial expression patterns as well as in function. Igs are glycosylated, and the attached glycans have been implicated in modulating antibody function.1-5 IgGs represent the most abundant antibody class in the human circulation being present at a concentration of approximately 10 mg/mL.6,7 They consist of two heavy chains and two light chains. Parts of the heavy chains, together with the light chains, form two fragment antigen binding (Fab) moieties, while the remainders of the two heavy chains form the fragment crystallizable (Fc) moiety. A single biantennary * To whom correspondence should be addressed. Manfred Wuhrer, Biomolecular Mass Spectrometry Unit, Department of Parasitology, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands. E-mail:
[email protected]. Fax: +31-71-526-6907. † Biomolecular Mass Spectrometry Unit, Department of Parasitology. ‡ Department of Neurology. 10.1021/pr1004373
2011 American Chemical Society
complex-type N-glycan is attached to the asparagine residue at position 297 in the CH2 domain of each heavy polypeptide chain of the Fc part. As a result of the glycosylation machinery, a variety of biantennary complex-type N-glycans can be present (N-glycan microheterogeneity) that often carry a core-fucose and vary in the number of antenna galactose residues.8-10 Biantennary, core-fucosylated IgG Fc N-glycoforms with no, one, or two galactoses are referred to as G0F, G1F, and G2F, respectively. Moreover, these complex-type N-glycans may contain a bisecting N-acetylglucosamine and a sialic acid residue attached to an antenna galactose. In healthy individuals, IgG glycosylation features are reflected in age, gender and pregnancy.11-13 For both males and females, it has been shown that the percentage of galactosylated complex-type N-glycans on IgG decrease with increasing age. Compared to males of similar age, young females (
