Fluorescent CMP-sialic acids as a tool to study the specificity of the CMP-sialic acid carrier and the glycoconjugate sialylation in permeabilized cells

• Verfasst von: Groß, Hans Jürgen [VerfasserIn]
• Titel: Fluorescent CMP-sialic acids as a tool to study the specificity of the CMP-sialic acid carrier and the glycoconjugate sialylation in permeabilized cells
• Verf.angabe: Hans Jürgen Gross
• E-Jahr: 1992
• Jahr: January 1992
• Umfang: 7 S.
• Fussnoten: Elektronische Reproduktion der Druck-Ausgabe 3. März 2005 ; Gesehen am 06.05.2025
• Titel Quelle: Enthalten in: EJB
• Ort Quelle: Oxford [u.a.] : Wiley-Blackwell, 1967
• Jahr Quelle: 1992
• Band/Heft Quelle: 203(1992), 1-2, Seite 269-275
• ISSN Quelle: 1432-1033
• DOI: doi:10.1111/j.1432-1033.1992.tb19856.x
• Datenträger: Online-Ressource
• Sprache: eng
• K10plus-PPN: 1924808188

Abstract

The specificity of the Golgi carrier for CMP-sialic-acids and the lumenal sialylation of glycoconjugates in mechanically permeabilized cells (semi-intact CHO 15B cells) was studied with CMP-activated fluorescent sialic acids as sensitive markers. Semi-intact cells represent a well-established cellular model for studies on the constitutive secretion pathway because the perforated plasma membrane allows membrane-impermeable CMP-sialic-acids to gain access to cellular organelles. The subcellular structures of semi-intact cells remain morphologically intact and hence synthetic CMP-sialic-acids can be assayed as substrates for the corresponding Golgi sugar-nucleotide transporter. The results prove that the CMP-sialic-acid carrier is able to translocate fluorescent CMP-glycosides, despite the bulky fluoresceinyl residue located at position C5 or C9 of the sialic-acid moiety; the data suggest a slightly higher affinity of the carrier for the C9-substituted CMP-glycoside, whereas the affinity of cellular sialyltransferases is fourfold higher for CMP-5-N-fluoresceinyl-aminoacetylneuraminic acid (5-FTIUNeuAc; 5-N-fluoresceinylaminoneuraminic acid). Using CMP-9-fluoresceinylthioureido-N-acetylneuraminic acid (CMP-9-FTIUNeuAc), an easy and sensitive fluorometric assay was established for the lumenal sialylation in semi-intact cells. Cellular proteins and gangliosides are both labelled by covalent incorporation of the fluorescent N-acetylneuraminic acid analogue. The assay allows rapid screening for small biomolecules or proteins that influence cellular sialyl transport and sialyl transfer; the lumenal fluorescence incorporation does not require ATP or cytosolic compounds. The suitability of fluorescent CMP-glycosides as markers for intracellular sialylation, proven in this paper, introduces the use of synthetic sialic acids for visualisation of cellular sialic acid pathways by fluorescence microscopy. Based on the data presented here, specific CMP-N-acetylneuraminic-acid analogues can be produced and used for the characterization of the Golgi CMP-sialic-acid carrier.