Lectin Affinity Chromatography for the Discovery of Novel Cancer Glycobiomarkers: A Case Study with PSA Glycoforms...

Many clinical biomarkers in cancer are glycoproteins, but the majority of them only consider the protein levels. Indeed, only alfa-fetoprotein (AFP) in hepatocarcinoma and CA15-3 in breast cancer are clinically monitored for their glycoforms. Aberrant glycosylation occurs frequently in many of the glycoproteins synthesized by tumor cells and often produce changes in protein glycoforms that could be exploited as potential biomarkers for improving diagnosis, prognosis or to study the response to treatment. Ideally, the screening of potential biomarkers should be performed from noninvasive samples like serum or plasma, therefore these glycoproteins with tumor associated-glycoforms should be shed from the tumor cell membrane or secreted into the blood to be detectable. Glycosylation changes that are commonly associated with cancer transformation include fucosylation, sialylation, branching, and polylactosaminylation.Lectins are glycan-binding proteins that bind with great specificity to different glycan moieties. Lectin-based strategies to enrich or fractionate glycoproteins are being extensively used and hold promise in targeted analysis for cancer biomarker discovery. Here we describe the use of lectin chromatography to separate prostate specific antigen (PSA) glycoforms based on their sialic acid linkage from sera of patients with prostate cancer (with PSA levels in the range of 2-20 ng/mL). In particular, agarose-bound Sambucus nigra agglutinin (SNA) lectin which has affinity for terminal α2,6-sialic acids on glycoproteins was used. The protocol included first a previous immunoaffinity step to enrich PSA and to avoid interferences of the most abundant serum glycoproteins. Then, the immunopurified PSA was loaded on the SNA chromatography and two fractions were obtained, the first one (unbound fraction) containing the PSA glycoforms without α2,6-sialic acid (basically α2,3-sialylated PSA glycoforms) and the second one (bound fraction) the α2,6-sialylated PSA glycoforms. The quantification of the PSA eluted in the two fractions allows for the determination of the relative content of both groups of PSA glycoforms. The percentage of the α2,6-sialylated PSA glycoforms is significantly decreased in aggressive prostate cancer compared to indolent prostate cancer and benign prostate hyperplasia, being a promising new glycobiomarker for prostate cancer risk stratification.