Expeditious chemoenzymatic synthesis of CD52 glycopeptide antigens.
CD52 is a glycosylphosphatidylinositol (GPI)-anchored glycopeptide antigen found on sperm cells and human lymphocytes. Recent structural studies indicate that sperm-associated CD52 antigen carries both a complex type N-glycan and an O-glycan on the polypeptide backbone. To facilitate functional and immunological studies of distinct CD52 glycoforms, we report in this paper the first chemoenzymatic synthesis of homogeneous CD52 glycoforms carrying both N- and O-glycans. The synthetic strategy consists of two key steps: monosaccharide primers GlcNAc and GalNAc were first installed at the pre-determined N- and O-glycosylation sites by a facile solid-phase peptide synthesis, and then the N- and O-glycans were extended by respective enzymatic glycosylations. It was found that the endoglycosidase-catalyzed transglycosylation allowed efficient attachment of an intact N-glycan in a single step at the N-glycosylation site, while the recombinant human T-synthase could independently extend the O-linked GalNAc to form the core 1 O-glycan. This chemoenzymatic approach is highly convergent and permits easy construction of various homogeneous CD52 glycoforms from a common polypeptide precursor. In addition, the introduction of a latent thiol group in the form of protected cysteamine at the C-terminus of the CD52 glycoforms will enable site-specific conjugation to a carrier protein to provide immunogens for generating CD52 glycoform-specific antibodies for functional studies.
Huang
Wei
W
Institute of Human Virology and Department of Biochemistry & Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
Zhang
Xinyu
X
Ju
Tongzhong
T
Cummings
Richard D
RD
Wang
Lai-Xi
LX
D000117:Acetylglucosamine / Q000138:chemical synthesis / Q000737:chemistry; D000595:Amino Acid Sequence; D015703:Antigens, CD / Q000737:chemistry / Q000378:metabolism*; D000951:Antigens, Neoplasm / Q000737:chemistry / Q000378:metabolism*; D055162:Biocatalysis; D000074301:CD52 Antigen; D002236:Carbohydrate Conformation; D002851:Chromatography, High Pressure Liquid; D006020:Glycopeptides / Q000737:chemistry / Q000378:metabolism*; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D015703:Antigens, CD; D000951:Antigens, Neoplasm; D000074301:CD52 Antigen; C000613862:CD52 protein, human; D006020:Glycopeptides; D006023:Glycoproteins; D011134:Polysaccharides; D006026:Glycoside Hydrolases; D000117:Acetylglucosamine