Solid-phase synthesis of a pentavalent GalNAc-containing glycopeptide (Tn antigen) representing the nephropathy-associated IgA hinge region.
Incomplete or aberrant glycosylation leading to Tn antigen (GalNAcalpha1-Ser/Thr) expression on human glycoproteins is strongly associated with human pathological conditions, including tumors, certain autoimmune diseases, such as the idiopathic IgA nephropathy, and may modulate immune homeostasis. In addition, the Tn antigen is highly expressed by certain pathogens and plays a role in host-pathogen interactions. To enable experimental approaches to study interactions of the Tn antigen with the immune system and analyze anti-Tn antibody responses in infection or disorders, we generated a Tn-expressing resource that can be used for high-throughput screening. In consideration of IgA nephropathy in which the hinge region is incompletely glycosylated, we used this hinge sequence that encodes five potential glycosylation sites as the ideal template for the synthesis of a Tn antigen-expressing glycopeptide. Inclusion of an N-terminal biotin in the peptide enabled binding to streptavidin-coated ELISA plates as monitored using Helix pomatia agglutinin or anti-Tn monoclonal antibody. We also found that the biotinylated IgA-Tn peptide is a functional acceptor for beta1-3-galactosylation using recombinant T-synthase (beta1-3-galactosyltransferase). Besides its immunochemical functionality as a possible diagnostic tool for IgA nephropathy, the peptide is an excellent substrate for glycan elongation and represents a novel template applicable for glycan-antigen-associated diseases.
Bolscher
Jan G M
JG
Department of Oral Biochemistry, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University Amsterdam, Amsterdam, Van der Boechorststraat 7, NL-1081 BT, The Netherlands. jgm.bolscher@vumc.nl
Brevoord
Judith
J
Nazmi
Kamran
K
Ju
Tongzhong
T
Veerman
Enno C I
EC
van Wijk
Joanna A E
JA
Cummings
Richard D
RD
van Die
Irma
I
D015295:Antigens, Tumor-Associated, Carbohydrate / Q000737:chemistry* / Q000276:immunology; D019921:Biotinylation; D005922:Glomerulonephritis, IGA / Q000276:immunology; D006020:Glycopeptides / Q000138:chemical synthesis*; D006801:Humans; D007070:Immunoglobulin A / Q000737:chemistry; D037102:Lectins / Q000276:immunology
D015295:Antigens, Tumor-Associated, Carbohydrate; D006020:Glycopeptides; C038095:Helix lectin; D007070:Immunoglobulin A; D037102:Lectins; C040286:Tn antigen