Substrate Specificity of the Sialic Acid Biosynthetic Pathway

Abstract

Unnatural analogues of sialic acid can be delivered to mammalian cell surfaces through the metabolic transformation of unnatural N-acetylmannosamine (ManNAc) derivatives. In previous studies, mannosamine analogues bearing simple N-acyl groups up to five carbon atoms in length were recognized as substrates by the biosynthetic machinery and transformed into cell surface sialoglycoconjugates [Keppler, O. T., et al. (2001) Glycobiology 11, 11R-18R]. Such structural alterations to cell surface glycans can be used to probe carbohydrate-dependent phenomena. This report describes our investigation into the extent of tolerance of the pathway toward additional structural alterations of the N-acyl substituent of ManNAc. A panel of analogues with ketone-containing N-acyl groups that varied in the length or steric bulk was chemically synthesized and tested for metabolic conversion to cell surface glycans. We found that extension of the N-acyl chain to six, seven, or eight carbon atoms dramatically reduced utilization by the biosynthetic machinery. Likewise, branching from the linear chain reduced metabolic conversion. Quantitation of metabolic intermediates suggested that cellular metabolism is limited by the phosphorylation of the N-acylmannosamines by ManNAc 6-kinase in the first step of the pathway. This was confirmed by enzymatic assay of the partially purified enzyme with unnatural substrates. Identification of ManNAc 6-kinase as a bottleneck for unnatural sialic acid biosynthesis provides a target for expanding the metabolic promiscuity of mammalian cells.

Preparation and immunological studies of protein conjugates of N-acylneuraminic acids

“…Our preliminary results [40] have also shown that the phenylacetylated derivative of mannosamine is a very good substrate for Neu5Ac adolase that is involved in the bioengineering of sialic acid and sialoglycoconjugates on cells [41]. These studies indicate great promise for the application of derivatized forms of sialooligosaccharides as immunological targets and guide the choice of derivatizations for further studies of glycoengineering of tumor cells and development of novel and efficient cancer immunotherapies.…”

Peracetylated N-Acetylmannosamine, a Synthetic Sugar Molecule, Efficiently Rescues Muscle Phenotype and Biochemical Defects in Mouse Model of Sialic Acid-deficient Myopathy

“…The use of O-acyl-modified (peracetylated) ManNAc analogs has been shown to increase metabolic efficiency up to 900-fold (35), and among these, Ac 4 ManNAc was the most efficient in increasing total sialic acid levels (34). This distinct efficiency exhibited by Ac 4 ManNAc may be explained by two factors.…”

Distribution ofN-acetylneuraminic acid and sialylglycan in eggs of the Silky fowl

“…Sialylglycoconjugates are also known to modulate cell-cell interactions and viscosity, and determine negative charge on the surface of cells (Schauer, 1982). While sialic acid can be taken up by eukaryotic cells (Oetke et al, 2001), its primary origin in animals is through biosynthesis ( Jacobs et al, 2001). Although sialic acid and sialylglycoconjugates have been prepared from animal tissues (Wang et al, 2001), hen's eggs and by synthesis (Maru et al, 1998), there are no reports of sialic acid from the egg of the Silky fowl.…”