Metabolic Glycoengineering of Sialic Acid Using N-acyl-modified Mannosamines

Summary

Sialic acid is a typical monosaccharide-unit found in glycoconjugates. It is involved in a plethora of molecular and cellular interactions. Here we present a method to modify cell surface sialic acid expression using metabolic glycoengineering with N-acetylmannosamine derivatives.

Abstract

Sialic acid (Sia) is a highly important constituent of glycoconjugates, such as N- and O-glycans or glycolipids. Due to its position at the non-reducing termini of oligo- and polysaccharides, as well as its unique chemical characteristics, sialic acid is involved in a multitude of different receptor-ligand interactions. By modifying the expression of sialic acid on the cell surface, sialic acid-dependent interactions will consequently be influenced. This can be helpful to investigate sialic acid-dependent interactions and has the potential to influence certain diseases in a beneficial way. Via metabolic glycoengineering (MGE), the expression of sialic acid on the cell surface can be modulated. Herein, cells, tissues, or even entire animals are treated with C2-modified derivatives of N-acetylmannosamine (ManNAc). These amino sugars act as sialic acid precursor molecules and therefore are metabolized to the corresponding sialic acid species and expressed on glycoconjugates. Applying this method produces intriguing effects on various biological processes. For example, it can drastically reduce the expression of polysialic acid (polySia) in treated neuronal cells and thus affects neuronal growth and differentiation. Here, we show the chemical synthesis of two of the most common C2-modified N-acylmannosamine derivatives, N-propionylmannosamine (ManNProp) as well as N-butanoylmannosamine (ManNBut), and further show how these non-natural amino sugars can be applied in cell culture experiments. The expression of modified sialic acid species is quantified by high performance liquid chromatography (HPLC) and further analyzed via mass spectrometry. The effects on polysialic acid expression are elucidated via Western blot using a commercially available polysialic acid antibody.

Introduction

Sialic acid is a monosaccharide that can typically be found at the non-reducing termini of glycoconjugates, such as N- and O-glycans or glycolipids. Among all monosaccharides, sialic acid has some unique chemical characteristics. It has a 9 C-atom backbone, a carboxylic group in the C-1 position, that is deprotonated and thereby negatively charged under physiological conditions, and an amino function in the C-5 position. Although over 50 naturally occurring variants of sialic acid have been characterized to date¹, the predominant form of sialic acid found in humans is N-acetylneuraminic acid (Neu5Ac). Other mammals al....

Protocol

1. Preparation of Buffers and Reagents

1. Preparation of 3 mM sodium methoxide solution
   1. Dissolve 8.1 mg sodium methoxide in 50 mL methanol (3 mM) in a 100 mL glass bottle with a stir bar. Store at room temperature (RT) for several weeks.
2. Preparation of Tris-HCl buffer
   1. Combine 8.766 g NaCl, 157 mg Tris-HCl, and 146 mg EDTA in a 100 mL glass bottle with a stir bar and dissolve in 80 mL water.
   2. Add sodium hydroxide (1 M, in water) or HCl (20%, in water) to the stirring solution, while observing the pH with a pH-meter, and adjust the pH to 8.0.
   3. Add the volume of....

Results

HPLC chromatograms of the fluorescent labeled Neu5Ac and the Neu5Gc standards are depicted in Figure 2. Using the herein described method, DMB-labeled Neu5Gc typically elutes between 7 - 9 min elution time, and DMB-Neu5Ac between 10 - 12 min. Several smaller peaks in the chromatogram usually appear between 2 - 6 min. These peaks represent unreacted DMB and reaction intermediates²⁵.

Discussion

If the chemically synthesized ManNAc derivatives, ManNProp and ManNBut are analyzed via mass spectrometry, only the correct mass peak for both specimens should be identified. Therefore, the products can be assumed to have a purity of over 99%. Small amounts of Neu5Gc, which is normally not found in human cells²⁹, are detected in the membrane fractions of the lysed cells. This most likely occurs through a salvage pathway that recruits Neu5Gc from fetal bovine serum sialoglycoconjugates in the media.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cells	Sigma-Aldrich	92110411
RPMI medium	Sigma-Aldrich	R8758
75 ml tissue culture flasks	Greiner	690175
48-well plates	Corning	3548
FCS	PAA	A15-102
Pen/Strep	Gibco	15140-122
Trypsine	Gibco	15400-054
EDTA	Roth	X986.1
Tris	Serva	37190.03
SDS	Roth	2326.2
SDS-PAGE equipment (tanks, glassware etc., machine	VWR	SDS Gel/Blot
Acrylamide	Roth	3019.1
Protein ladder	Fisher Scientific	267620
Nitrocellulose	GE Healthcare	10600002
Ponceau red	Roth	5938.2
Milk powder	Roth	T145.3
ECL	Millipore	WBLUF 0500
0.5 ml Centrifugal Filter Unit with 3 kDa membrane	Merck-Millipore	UFC500324
15 mL centrifuge tubes	Sigma-Aldrich (Corning)	CLS430791-500EA
2-Mercaptoethanol	Sigma-Aldrich	M6250-10ML
2-Propanol	Sigma-Aldrich	34965-1L	HPLC gradient grade
4,5-Methylenedioxy-1,2-phenylenediamine dihydrochloride	Sigma-Aldrich	D4784-50MG
48 well, flat bottom tissue culture plate	Sigma-Aldrich (Corning)	CLS3548-100EA
50 mL centrifuge tubes	Sigma-Aldrich (Corning)	CLS430829-500EA
Acetonitrile	Sigma-Aldrich	34967-1L	HPLC gradient grade
Aprotinin from bovine lung	Sigma-Aldrich	A1153-10MG	lyophilized powder, 3-8 TIU/mg solid
Butyryl chloride	Sigma-Aldrich	109614-250G
C18 RP column	Phenomenex	00F-4435-E0	110 Å, 3 µm particle size, 4.6 x 150 mm
D-Mannosamine hydrochloride	Sigma-Aldrich	M4670-1G
Dulbecco`s Phosphate Buffered Salt Solution	PAN Biotech	P04-36500
Ethylenediaminetetraacetic acid	Sigma-Aldrich	E9884-100G
Formic acid	Sigma-Aldrich	56302-50ML-GL
Hydrochloric acid solution	Sigma-Aldrich	H1758-100ML	36.5-38.0%, in water
Leupeptin	Sigma-Aldrich	L2884-10MG
Methanol	Carl-Roth	T169.2	HPLC gradient grade
N-Acetyl-D-mannosamine	Sigma-Aldrich	A8176-250MG
N-Acetylneuraminic acid	Sigma-Aldrich	A0812-25MG
N-Glycolylneuraminic acid	Sigma-Aldrich	G9793-10MG
Phenylmethanesulfonyl fluoride	Sigma-Aldrich	P7626-250MG
Propionyl chloride	Sigma-Aldrich	P51559-500G
Safe-Lock Tubes, 1.5 mL, amber (light protection)	Eppendorf	30120191
Safe-Lock Tubes, 1.5 mL, colorless	Eppendorf	30120086
Sodium bisulfite solution	Sigma-Aldrich	13438-1L-R-D	40%, in water
Sodium chloride	Sigma-Aldrich	746398-500G-D
Sodium hydroxide	Sigma-Aldrich	795429-500G-D
Sodium hydroxide solution	Sigma-Aldrich	319511-500ML	1.0 M, in water
Sodium methoxide	Sigma-Aldrich	164992-5G
Trifluoroacetic acid	Sigma-Aldrich	T6508-100ML-D
Tris hydrochloride	Sigma-Aldrich	T5941-100G
Trypsin 0.25 %/EDTA 0.02 % in PBS	PAN Biotech	P10-019100
Water	Carl-Roth	T905.1	HPLC gradient grade
Silica Gel 60	Carl-Roth	9779.1
HPLC	Agilent
ESI-MSD-System	Agilent