Carbohydrate Sequence of the Prostate Cancer-associated Antigen F77 Assigned by a Mucin O-Glycome Designer Array

Abstract

Background: F77 is a previously uncharacterized prostate cancer-associated antigen.Results: Using a microarray of sequence-defined glycolipids and neoglycolipids, mucin-O-glycan designer arrays, and mass spectrometry, we demonstrate that F77 antibody recognizes blood group H on 6-linked branches of poly-N-acetyllactosamine backbones.Conclusion: F77 antigen is expressed on glycolipids and on glycoprotein O-glycans.Significance: F77 antigen can now be explored rationally as a cancer biomarker.

O-Glycome Beam Search Arrays for Carbohydrate Ligand Discovery

“…Mucins and Mucin-enriched Glycoproteins-Porcine stomach mucin (PSM) was from Sigma (Dorset, UK); other mucins and mucinenriched glycoproteins in supplemental Table S1 have been described previously (22).…”

“…S1. O-Glycans were released from PSM, 1 g dry weight, by reductive alkaline borohydride degradation (22) and fractionated initially by anion-exchange chromatography on an AG1X8 column (Bio-Rad, Hercules, CA, acetate form, 3 ml resin), with sequential elution with 2 mM and 300 mM pyridium acetate, followed by 300 mM Na 2 SO 4 to yield predominantly neutral, sialylated and sulfated fractions, respectively. The neutral fraction was the subject of the present study.…”

“…This is now a state-of-the-art platform with the glycan probes robotically arrayed in a liposomal formulation at low femtomoles per spot (20,21). A "designer" array of NGLs from an epithelial O-glycome, resolved on TLCs enabled the characterization of the elusive prostate cancer-associated antigen F77 (22).…”

“…1) is a significant advance involving, as an early step, microarray analysis of NGL populations resolved by HPLC with mass spectrometry (MS) monitoring rather than TLC plate binding analyses (commonly referred to as chromatogram binding), which necessitates elution of bound components (22). There follows iterative microarray analyses of targeted ligand-positive fractions resolved by appropriate chromatographic separations concomitantly with MS and culminating in micro-immuno-sequencing of a ligand-positive glycan.…”

Binding of CLL Subset 4 B Cell Receptor Immunoglobulins to Viable Human Memory B Lymphocytes Requires a Distinctive IGKV Somatic Mutation

“…Microarray analyses for anti-i-and anti-I-type reactivity Microarray binding analyses were performed at 4°C essentially as described (26). In brief, after blocking with 3% w/v bovine serum albumin (Sigma-Aldrich) in HEPES-buffered saline (5 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM CaCl 2 ), the microarrays were probed with the following CLL mAbs: 141, 282, 183, 240 and 342, all at 10 μg/mL.…”