Anti-α-galactoside and Anti-β-glucoside Antibodies are Partially Occupied by Either of Two Albumin-bound O-glycoproteins and Circulate as Ligand-binding Triplets

ABSTRACT

Two heavily O-glycosylated proteins and albumin co-purified with anti-α-galactoside (anti-Gal), the chief xenograft-rejecting antibody and anti-β-glucan (ABG) antibody isolated from human plasma by affinity chromatography on respective ligand-bearing matrices. Both antibodies and O-glycoproteins co-purified with plasma albumin eluted from albumin-specific matrix. Using components of affinity-purified antibody samples separated by electrophoresis binding of either albumin or antibody to the affinity matrix of the other or binding of O-glycoprotein to either matrix was ruled out. Enzyme-linked immunoassay and ligand-induced fluorescence enhancement of fluorolabeled antibody showed that O-glycoproteins occupied sugar-binding sites of anti-Gal and ABG. Neither antibody recognized albumin. O-Glycoprotein-albumin complexes free in plasma, or released from antibodies by specific sugars, were captured on microwell-coated O-glycan-specific lectin jacalin and detected using labeled anti-albumin. We conclude that circulating anti-Gal and ABG form protein triplets in which either O-glycoprotein bridges between antibody and albumin by binding simultaneously to both. Bound albumin restricted O-glycoprotein occupation on antibodies enabling triplets to bind other ligands using spared binding sites. Free anti-Gal and ABG were undetectable in plasma. Jacalin treatment, but not de-O-glycosylation of O-glycoproteins abolished their recognition by anti-Gal or ABG indicating that antibodies recognized serine- and threonine-rich peptide sequences that underlie the O-glycans and are reported surrogate ligands for anti-Gal. The albumin- and antibody-binding O-glycoproteins AOP1 and AOP2 were single polypeptide proteins of size 107 kDa and 98 kDa, containing 54% and 51% carbohydrate respectively and conformed to no known plasma protein in properties. Prospects of triplet-mediated modulations in autologous tissues expressing antibody ligands are discussed.

KEYWORDS:

• Albumin- and antibody-associated O-glycosylated protein
• anti-carbohydrate antibody-O-glycoprotein-albumin triplet
• anti-α-galactoside antibody
• anti-β-glucoside antibody
• serine- and threonine-rich peptide sequence

Acknowledgments

A Ph.D. fellowship and contingency grant to KS from the host institute is gratefully acknowledged.

Disclosure statement

No potential conflict of interest was reported by the authors.