N-Glycolylneuraminic acid (Neu5Gc) is the second most populous
sialic acid (Sia). The only known biosynthetic pathway of Neu5Gc is the hydroxylation of cytidine-5'-monophosphate-N-acetylneuraminic
acid (CMP-Neu5Ac), catalyzed by
CMP-Neu5Ac hydroxylase (CMAH). Neu5Gc is abundantly found in mammals except for human, in which CMAH is inactivated due to mutation in the CMAH gene. Evidence has accumulated to show occurrence of Neu5Gc-containing
glycoconjugates in sera of
cancer patients, human cancerous tissues and cultured human cell lines. Recently, occurrence of natural
antibodies against Neu5Gc was shown in healthy humans and is a serious problem for clinical
xenotransplantation and stem cell
therapies. Studying human occurrence of Neu5Gc is of importance and interest in a broad area of medical sciences. In this study, using a fluorometric high performance liquid chromatography method, we performed quantitative analyses of Sias both inside and in the external environment of the cell and found that (i) incorporation of Neu5Gc was most prominent in soluble
glycoproteins found both in the extracellular space and inside the cell as the major Sia compounds. (ii) Of the total Neu5Gc in the Sia compounds that the cells synthesized, 90% was found in the secreted
sialoglycoproteins, whereas for Neu5Ac, 70% was found in the secreted
sialoglycoproteins. (iii) The Neu5Gc ratio was higher in the secreted
sialoglycoproteins (as high as 40% of total Sias) than in intracellular
sialoglycoproteins. (iv) The majority of the secreted
sialoglycoproteins was anchored on the culture dishes and solubilized by brief
trypsin treatment. Based on these findings, a new idea on the mechanism of accumulation of Neu5Gc in
cancer cells was proposed.