The sperm agglutination antigen-1 (SAGA-1) glycoforms of CD52 are O-glycosylated.
CD52 is composed of a 12 amino acid peptide with N-linked glycans bound to the single potential glycosylation site at position 3, and a glycosylphosphatidylinositol-anchor attached at the C-terminus. Some glycoforms of this molecule expressed in the male reproductive tract are recognized by complement-dependent sperm-immobilizing antibodies in infertile patients making this antigen an important target for immunocontraception and fertility studies. Although the amount of posttranslational modification is already remarkable for such a small polypeptide, O-glycosylation of CD52 has additionally been implicated by several studies, but never rigorously characterized. In this report, we show clear evidence for the presence of O-glycans in CD52 preparations immunopurified using the murine S19 monoclonal antibody generated against sperm agglutination antigen-1 (SAGA-1), a male reproductive tract specific form of CD52. The O-glycans have been characterized by MALDI-TOF and tandem mass spectrometry after reductive elimination and permethylation. The data indicate that the major SAGA-1 O-glycans are core 1 and 2 mucin-type structures, with and without sialic acid (NeuAc(0-2)Hex(1-3)HexNAc(1-2)HexNAcitol). Minor fucosy- lated O-glycans are also present including some struc- tures with putative Le(y) epitopes (NeuAc(0-1)Fuc(1-3)Hex(1-2) HexNAc(0-1)HexNAcitol). Analysis of O-glycopeptides by tandem mass spectrometry provided an additional level of support for the O-glycosylation of SAGA-1. Elucidation of the O-glycosylation of SAGA-1 adds to the complexity of this molecule and may help to explain its biological activity.
Parry
Simon
S
Division of Molecular Biosciences, Imperial College London, London SW7 2AZ, UK.
Wong
Nyet-Kui
NK
Easton
Richard L
RL
Panico
Maria
M
Haslam
Stuart M
SM
Morris
Howard R
HR
Anderson
Peggy
P
Klotz
Kenneth L
KL
Herr
John C
JC
Diekman
Alan B
AB
Dell
Anne
A
D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D015703:Antigens, CD / Q000276:immunology*; D000951:Antigens, Neoplasm / Q000276:immunology*; D000954:Antigens, Surface / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D000074301:CD52 Antigen; D002240:Carbohydrate Sequence; D006023:Glycoproteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007248:Infertility, Male / Q000276:immunology*; D008297:Male; D008969:Molecular Sequence Data; D009077:Mucins / Q000737:chemistry / Q000378:metabolism; D011134:Polysaccharides / Q000276:immunology / Q000378:metabolism*; D012661:Semen / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013094:Spermatozoa / Q000276:immunology*
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D000951:Antigens, Neoplasm; D000954:Antigens, Surface; D000074301:CD52 Antigen; C000613862:CD52 protein, human; D006023:Glycoproteins; D009077:Mucins; D011134:Polysaccharides; C120760:SAGA-1 protein, human