Enzymatically mediated, glycosidic conjugation of immunoglobulins with viral epitopes.
We developed a novel enzymatic procedure to couple a peptide to the sugar moieties of immunoglobulins (Igs). The synthesis of the conjugates consists in galactose (Gal) oxidation of desialylated Igs followed by covalent attachment of the peptides with concurrent stabilization of the Schiff bases upon mild reduction. The peptide used in this study, corresponds to the amino acid residues 110-120 of hemagglutinin (HA) of PR8 A virus and is recognized by CD4 T helper cells in association with I-Ed class II major histocompatibility complex (MHC). The degree of coupling as determined by competitive inhibition of radioimmunoassay (IRIA) using FPLC purified conjugates was estimated at 11.4 peptides per IgG molecule. Coupling of HA110-120 peptide to the sugar moiety of various mouse and human Igs was confirmed by Western blot analysis developed with anti-HA110-120 antibodies. Complete detachment of the peptide from the conjugates by N-deglycosylation with PGNase F indicated a defined specificity of coupling HA peptide to the N-linked oligosaccharides of Igs. To facilitate quick release of the peptides from the conjugates into the lysosomal compartment of the antigen processing cells (APC) we introduced at the alpha amino terminus of the peptide (HAc110-120), a cleavage site for cathepsins (AAAL). The immunoglobulin-galactose-HAc110-120 conjugates (IGP) were able to activate HA110-120 specific T hybridoma cells as efficient as influenza PR8 A virus and 40-100-fold higher than the synthetic peptide itself.
Brumeanu
T D
TD
Department of Microbiology, Mount Sinai School of Medicine, New York, NY 10029, USA.
Dehazya
P
P
Wolf
I
I
Bona
C A
CA
D000595:Amino Acid Sequence; D000818:Animals; D000939:Epitopes; D005690:Galactose / Q000276:immunology*; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral / Q000276:immunology*; D006801:Humans; D007136:Immunoglobulins / Q000276:immunology*; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology*
D000939:Epitopes; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral; D007136:Immunoglobulins; D010446:Peptide Fragments; D005690:Galactose