Cytidine 5'-Monophospho-N-acetylneuraminic Acid or a Related Compound is the Low Mr Factor from Human Red Blood Cells Which Induces Gonococcal Resistance to Killing by Human Serum

1 Department of Microbiology, University of Birmingham, PO Box 363, Birmingham B15 2TT, UK 2 Department of Biochemistry, University of Birmingham, PO Box 363, Birmingham B15 2TT, UK 3 Macromolecular Analysis Service, University of Birmingham, PO Box 363, Birmingham B15 2TT, UK ABSTRACT A low- M r factor which induces gonoccocal resistance to complement-mediated serum killing has been partially purified from lysates of mixed red and buffy coat cells from human blood. The lysates were dialysed against Tris buffer for 24 h at 25 °C with the diffusate being continuously recycled through a column of QAE-Sephadex A25. After elution in an NaCl gradient, the active fractions were both desalted and further purified on Sephadex G10. A second fractionation on QAE-Sephadex A25 and desalting with Sephadex G10 preceded further purification by repeated high-pressure liquid chromatography (HPLC) using a DEAE anion exchange column and desalting with Sephadex G10. Less than 500 µg of material showing one peak in HPLC was obtained from 1 litre of blood. After NMR had indicated the possible presence of pyrimidine nucleotide, carbohydrate and N -acetyl groups, nanogram quantities of a commercial preparation of cytidine 5'-monophospho- N -acetylneuraminic acid (CMP-NANA) were shown to induce gonococci to serum resistance. The synthetic CMP-NANA also co-eluted with the preparation from blood cells in HPLC, and the two materials were indistinguishable in their patterns of acid and heat lability. Furthermore, the resistance-inducing activity of both materials was inhibited by cytidine monophosphate, which is known to inhibit sialylation reactions by CMP-NANA. It appears therefore that the resistance-inducing factor is CMP-NANA or a closely related compound. If the factor is CMP-NANA, biological activities indicated that the cell lysate from 1 litre of blood contained about 40 µg, and the most purified preparation contained only about 1%. With this minute amount in a mixture, the presence of CMP-NANA or a closely related analogue could not be established unequivocally by NMR. Keywords: CMP-NANA, cytidine 5'-monophospho- N -acetylneuraminic acid, NANA, N -acetylneuraminic acid, RBC, red blood cells, RIF, resistance inducing factor