Structural and functional characterization of glycosylation in an immunoglobulin G1 to Cryptococcus neoformans glucuronoxylomannan.
Analysis of the N-linked oligosaccharides of the murine IgG1 monoclonal antibody (mAb) to Cryptococcus neoformans by LC/MS revealed five different core fucosylated, biantennary complex-type oligosaccharides at Asn-293, with the major species being a mono-galactosylated oligosaccharide with the glycosyl composition of Hex4HexNAc4Fuc (39% of the total glycan pool). The primary sequence predicted from nucleic acid sequencing differed from that measured by mass spectrometry at position 33 (ASN to ASP), a finding that may represent post-translational modification caused by spontaneous ASP deamination. Analysis of mAb 18B7 from three hybridoma clones revealed the same heterogenous N-glycan pattern, indicating that diversity in oligosaccharide structures originated from individual cells. The binding of native and de-glycosylated mAb 18B7 to cryptococcal Ag was comparable but the de-glycosylated 18B7 had shorter serum half-life and did not activate complement (C). De-glycosylated mAb 18B7 was opsonic for C. neoformans with murine macrophages through a mechanism that involved C-independent ingestion through the C receptor. Passive administration of de-glycosylated mAb 18B7 mediated comparable protective efficacy to the native mAb in mice with lethal infection. The results imply that the contribution of N-glycan structure to immunoglobulin function varies depending on the Ag-Ab system.
Wang
Fang
F
Laboratory for Macromolecular Analysis and Proteomics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
Nakouzi
Antonio
A
Alvarez
Mauricio
M
Zaragoza
Oscar
O
Angeletti
Ruth Hogue
RH
Casadevall
Arturo
A
D000595:Amino Acid Sequence; D000818:Animals; D000908:Antibodies, Fungal / Q000737:chemistry* / Q000276:immunology* / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology* / Q000494:pharmacology; D002942:Circular Dichroism; D003455:Cryptococcus neoformans / Q000276:immunology*; D006031:Glycosylation; D007074:Immunoglobulin G / Q000737:chemistry* / Q000276:immunology* / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects; D051379:Mice; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry; D009895:Opsonin Proteins / Q000737:chemistry / Q000276:immunology / Q000494:pharmacology; D010587:Phagocytosis / Q000187:drug effects; D011499:Protein Processing, Post-Translational
D000908:Antibodies, Fungal; D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D009844:Oligosaccharides; D009895:Opsonin Proteins