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

Two heavily O-glycosylated proteins and albumin co-purified with anti-α-galactoside (anti-Gal), the chief xenograft-rejecting antibody and anti-ß-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.

Human plasma anti-α-galactoside antibody forms immune complex with autologous lipoprotein(a).

Anti-α-galactoside antibody (anti-Gal) from human plasma that bound to α-galactoside-bearing guar galactomannan gel and was eluted with specific sugar (affinity-purified anti-Gal ; APAG) invariably contained apo(a) and apo B subunits in a proportion close to that in plasma lipoprotein(a) [Lp(a)]. Since LDL does not contain apo(a), result suggested Lp(a) as a component of APAG. Lp(a) in APAG was complexed with anti-Gal since plate-coated anti-apo(a) captured Lp(a) along with the antibody. Association of Lp(a) with anti-Gal in APAG was considerably lower in presence of anti-Gal-specific sugar, suggesting that Lp(a) occupied the sugar-binding site of anti-Gal. Content of Lp(a)-bound anti-Gal in APAG, though a minor fraction of total antibody, increased steadily with total Lp(a) content of plasma. Further, Lp(a) released from immune complex-rich fraction of plasma by anti-Gal- specific sugar was proportional to total plasma Lp(a). Anti-Gal titre decreased with increasing Lp(a) concentration among 114 plasma samples. Results indicate the potential of anti-Gal molecules with its binding site partially occupied by Lp(a) molecule(s) to a) use the remaining binding site(s) to recognize other macromolecules or cells and b) transport Lp(a) across Fc receptor-bearing cells.

apoB-independent enzyme immunoassay for lipoprotein(a) by capture on immobilized lectin (jacalin).

Enzyme immunoassay for lipoprotein(a) [Lp(a)] using antibodies to both apoB and apo(a) subunits (a-B assay) is shown to be affected by differential masking of apoB by apo(a) and the presence of LDL-Lp(a) adducts. An apoB-independent immunoassay by capturing Lp(a) through its O-glycans on microplate-coated lectin jacalin and quantitation using peroxidase-labeled anti-apo(a) (J-a assay) is described. J-a assay response is linear, more than twice as sensitive as a-B assay, and is suppressed only 18 ± 5% by non-Lp(a) O-glycan-containing proteins of serum. Wide variations in IgA did not significantly affect Lp(a) binding to jacalin (CV = 6.4%).