Improved methods to characterize the length and quantity of highly unstable PolySialic acids subject category: (Carbohydrates, chromatographic techniques)

Abstract

Polysialic acid (polySia) is a linear homopolymer of α2-8-linked sialic acids that is highly expressed during early stages of mammalian brain development and modulates a multitude of cellular functions. While degree of polymerization (DP) can affect such functions, currently available methods do not accurately characterize this parameter, because of the instability of the polymer. We developed two improved methods to characterize the DP and total polySia content in biological samples. PolySia chains with exposed reducing termini can be derivatized with DMB for subsequent HPLC analysis. However, application to biological samples of polySia-glycoproteins requires release of polySia chains from the underlying glycan, which is difficult to achieve without concurrent partial hydrolysis of the α2-8-linkages of the polySia chain, affecting its accurate characterization. We report an approach to protect internal α2-8sia linkages of long polySia chains, using previously known esterification conditions that generate stable polylactone structures. Such polylactonized molecules are more stable during acid hydrolysis release and acidic DMB derivatization. Additionally, we used the highly specific Endoneuraminidase-NF enzyme to discriminate polysialic acid and other sialic acid and developed an approach to precisely measure the total content of polySia in a biological sample. These two methods provide improved quantification and characterization of polySia.

Keywords: DMB derivatization; Degree of polymerization; Lactonization; Polysialic acid.

Fig. 1.

Overview of polysialic acid lactonization, and Lactonization-Protection-DMB procedure A) Structure of α2-8 linked Neu5Ac (above), and the same following acidic lactonization. B) Overview of Lactonization-Protection-DMB procedure for DP analysis of glycoprotein polysialic acid moieties: (1) polysialic acid moeties are lactonized, (2) mild acid hydrolysis releases sialic acid structures from underlying glycans, (3) sialic acid structures are labeled with DMB fluorophore, (4) lactonization is reversed, (5) labeled structures are quantitated via anion exchange HPLC.

Fig. 2.

DMB derivatization of colominic acid also hydrolyzes 2–8 linkages A) DMB Derivatization of Colominic acid at 50 °C. B) DMB Derivatization of Colominic acid at 4 °C. C) Same as B, following mild acid hydrolysis. D) Same as C, following lactonization. E) Close-up view of all chromatograms from 60 to 90 min.

Fig. 3.

Lactonization protects high DP polysialic acids during release from mouse brain glycopeptides A) Overview of brain glycopeptide preparation: (1) brains are homogenized and delipidated, (2) using proteinase digestion, brain proteins are digested into glycopeptides. B) DMB-HPLC analysis of neonate mouse brain glycopeptides after lactonization step. C) DMB-HPLC analysis of neonate mouse brain glycopeptides with no lactonization.

Fig. 4.

Lactonization-protection-DMB HPLC long-chain polysia analysis and EndoN-sensitive Neu5Ac analysis during analysis of early postnatal mouse brains. Brains subjected to Lactonization-protection-DMB HPLC analysis: A) Infant mouse (P1). B) Juvenile mouse (P14). C) Adult mouse (10 weeks).

Fig. 5.

DMB Analysis of EndoN-sensitive Neu5Ac A) Overview of EndoN-sensitive Neu5Ac purification: (1) polysialic acid moieties are digested into oligosialic fragments, which are then (2) separated from glycopeptides and terminal sialic acid structures using a centrifugal filtration device. B) HPLC analysis of spin filter flow-through following EndoN digestion of colominic acid. C) HPLC analysis of spin filter flow-through of untreated colominic acid.