Exploring C4 and C5 modifications of sialic acids at increasing levels of complexity: Chemistry, protein interactions, and evolution

Sialic acids (Sias) are a family of over 50 monosaccharides characterized by a nine-carbon backbone and a negatively-charged carboxylate at C1. Sias are found on termini of vertebrate cell-surface glycans. N-acetylneuraminic acid (Neu5Ac) is the predominant Sia found in nature and is the precursor of many modified forms. Chemical substituents can modify Neu5Ac and modifications often show specific expression patterns. Sia modification results in a range of effects: from affecting chemical behavior to evolutionary consequences. Here, an examination of multi-faceted effects of C4 and C5 modified forms of Sia is presented. De-N-acetylation of Neu5Ac results in neuraminic acid (Neu), a rare modification suggested to exist in the melanoma and lymphoma gangliosides (de-N-acetyl-gangliosides). In chapter 2, we show that N-acyl group loss results in resistance of the normally labile glycosidic linkage to acid hydrolysis. We devised a purification scheme that utilized this novel chemical feature to provide the first chemical proof of de-N-acetylated-gangliosides' natural occurrence. In chapter 3 we examine the effect of (5)-N-glycolyl formation (resulting from hydroxylation of the N-acetyl group) on protein binding interactions between Sia and some Siglec Sia-binding receptors. Comparison of Neu5Ac- or N-glycolylneuraminic acid (Neu5Gc)-binding of chimpanzee and human Siglec-7 and -9, four very closely related innate immune system-expressed Siglecs, demonstrates that slight Sia-modification can greatly influence Sia-binding events. The strong preference of chimpanzee and gorilla innate immune cell-associated Siglecs for Neu5Gc contrasts to the behavior human Siglecs which bind both Neu5Ac and Neu5Gc. Sequence analysis of the human and chimpanzee Siglec-7 and -9 indicates directional positive selection specifically in Siglec Sia-binding domains. Previous studies have established that while Neu5Gc is abundant on the cell surfaces of all other mammals studied, humans have a unique deficiency in the Neu5Gc biosynthetic machinery resulting in human cell surfaces lacking Neu5Gc. Together these data{09}indicate that hominid loss of Neu5Gc expression resulted in rapid evolution of Siglecs to accommodate Neu5Ac, the dominant human Sia. Chapter 4 presents work in progress including the differential regulation of Sia 4-O-acetylation in horse blood and a strategy to compare the metabolic fate of Neu5Gc between humans and other vertebrate species.