Abstract
5-N-acetylneuraminic acid, commonly known as sialic acid (Sia), constitutes a family of N- and O-substituted 9-carbon monosaccharides. Frequent modification of O-acetylations at positions C-7, C-8, or C-9 of Sias generates a family of O-acetylated sialic acid (O-AcSia) and plays crucial roles in many cellular events like cell–cell adhesion, proliferation, migration, etc. Therefore, identification and analysis of O-acetylated sialoglycoproteins (O-AcSGPs) are important. In this chapter, we describe several approaches for successful identification of O-AcSGPs. We broadly divide them into two categories, i.e., invasive and noninvasive methods. Several O-AcSias-binding probes are used for this purpose. Detailed methodologies for step-by-step identification using these probes have been discussed. We have also included a few invasive analytical methods for identification and quantitation of O-AcSias. Several indirect methods are also elaborated for such purpose, in which O-acetyl group from sialic acids is initially removed followed by detection of Sias by several approaches. For molecular identification, we have described methods for affinity purification of O-AcSGPs using an O-AcSias-binding lectin as an affinity matrix followed by sequencing using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF-TOF) mass spectroscopy (MS). In spite of special attention, loss of O-acetyl groups due to its sensitivity towards alkaline pH and high temperature along with migration of labile O-acetyl groups from C7–C8–C9 during sample preparation is difficult to avoid. Therefore there is always a risk for underestimation of O-AcSias.
Key words
- Achatinin-H
- Acetylacetone method
- Crab lectin
- Fluorimetric high-performance liquid chromatography (HPLC)
- Influenza C hemagglutinin-esterase
- Mild periodic acid Schiff (mPAS) staining
- O-acetylated sialic acid, O-acetylated sialic acid-binding lectin
- O-acetylated sialoglycoprotein (O-AcSGP)
- 9-O-acetylated sialoglycoprotein (Neu5,9Ac2GP)
- Sialic acid-binding lectin, sequencing, Siglecs
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Acknowledgement
This work received financial support from the Council of Scientific and Industrial Research (CSIR, TLP-006, IAP-001, NWP-004, HCP-004), Department of Biotechnology (GAP-235), Indian Council of Medical Research (GAP-266), New Delhi, India. CM acknowledges support from Sir J.C. Bose National Fellowship, Department of Science and Technology, Govt. of India.
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Mandal, C., Mandal, C. (2013). Identification and Analysis of O-Acetylated Sialoglycoproteins. In: Hake, S., Janzen, C. (eds) Protein Acetylation. Methods in Molecular Biology, vol 981. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-305-3_6
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