posted on 2020-07-13, 22:03authored byZhonghua Li, Qing Zhang, David Ashline, Yuyang Zhu, Yi Lasanajak, Tatiana Chernova, Vernon Reinhold, Richard D. Cummings, Peng G. Wang, Tongzhong Ju, David F. Smith, Xuezheng Song
Mucin-type
O-glycans play key roles in many cellular processes,
and they are often altered in human diseases. A major challenge in
studying the role of O-glycans through functional O-glycomics is the
absence of a complete repertoire of the glycans that comprise the
human O-glycome. Here we describe a cellular O-glycome preparation
strategy, Preparative Cellular O-Glycome Reporter/Amplification (pCORA),
that introduces 4-N3-Bn-GalNAc(Ac)3 as a novel
precursor in large-scale cell cultures to generate usable amounts
of O-glycans as a potential O-glycome factory. Cultured human non-small
cell lung cancer (NSCLC) A549 cells take up the precursor, which is
extended by cellular glycosyltransferases to produce 4-N3-Bn-α-O-glycans that are secreted into the culture medium.
The O-glycan derivatives can be clicked with a fluorescent bifunctional
tag that allows multidimensional HPLC purification and production
of a tagged glycan library, representing the O-glycome of the corresponding
cells. We obtained ∼5% conversion of precursor to O-glycans
and purified a tagged O-glycan library of over 100 O-glycan derivatives,
many of which were present in >100 nmol amounts and were sequenced
by sequential MS fragmentation (MSn). These O-glycans were
successfully printed onto epoxy glass slides as an O-glycome shotgun
microarray. We used this novel array to explore binding activity of
serum IgM in healthy persons and NSCLC patients at different cancer
stages. This novel strategy provides access to complex O-glycans in
significant quantities and may offer a new route to discovery of potential
diagnostic disease biomarkers.