NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health.

Nishihara S, Angata K, Aoki-Kinoshita KF, et al., editors. Glycoscience Protocols (GlycoPODv2) [Internet]. Saitama (JP): Japan Consortium for Glycobiology and Glycotechnology; 2021-.

Enzyme assay of sialate-pyruvate lyase (SPL)

, Doctor of Agriculture, Ph.D.
iGCORE, Nagoya University
, Master of Agriculture
iGCORE Nagoya University
, Doctor of Science, Ph.D.
iGCORE, Nagoya University
Corresponding author.

Created: ; Last Revision: March 30, 2022.

Introduction

Sialate-pyruvate lyase (SPL), whose NCBI gene name is NPL, is also denoted as N-acylneuraminate pyruvate-lyase or sialic acid aldolase. Sialic acids (Sias) comprise N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc), and deaminoneuraminic acid (Kdn). Neu5Ac and Neu5Gc are collectively denoted as N-acylneuraminic acid (Neu5Acyl). SPL is the terminal enzyme of Sia catabolisms (Figure 1) because it catalyzes the cleavage of Sias into pyruvate and a counterpart 6-carbon sugar, such as N-acetylmannosamine (ManNAc), N-glycolylmannosamine (ManNGc), and mannose (Man). ManNAc and ManNGc are collectively denoted as ManNAcyl. Sialic acid 9-phosphate synthase (SPS) catalyzes the following reactions:

Neu5Acyl → ManNAcyl + pyruvate;

Kdn → Man + pyruvate.

It also catalyzes the reverse reaction or the synthesis of Sia (ManNAcyl or Man + pyruvate → Neu5Acyl or Kdn) in Escherichia coli (1, 2).

In vertebrates, SPL is located in the cytosol and degrades excess amounts of free Sia to prevent the overexpression of Sia-glycoconjugates on the cell surface and to provide pyruvate as an energy source. SPL occurs not only in higher animals on the deuterostome lineage but also in microbial species, such as E. coli (3, 4) and Clostridium perfringens (5). Biallelic mutations in SPL lead to sialic aciduria in humans with a clinical phenotype of progressive cardiac myopathy and mid-skeletal myopathy. The myopathy phenotype is also observed in the SPL knockdown zebrafish (6). It is indicated that the Sia catabolism is important for myogenesis in vertebrates.

SPLs from E. coli, C. perfringens, and the pig kidney are purified, sequenced, and characterized. The optimal pH of SPL is in the neutral range, which is matched with its cytosolic localization. The thermostability of SPLs is different depending on organism species. For example, the optimal temperature of C. perfringens SPL is 65°C, while that of E. coli SPL is 80°C. The crystal structure and gel filtration results showed that E. coli SPL protein forms a tetramer with an active site at the C-terminal end of eight-stranded β-barrel (7).

Protocol

There are several methods for measuring SPL activity, which are divided into two groups. One is to monitor an increase in the amount of ManNAcyl (Morgan–Elson Assay; modified from (8)) and pyruvate. The other method is to monitor a decrease in the amount of Sia (9). In this chapter, an assay for measuring a decrease in Sia amount is described.

Materials

1.

E. coli strain BL21(DE3)pLysS.

2.

pET32b(+) vector (Novagen, Madison, WI)

3.

VIVASPIN® 20 (10K) ultrafiltration spin column

4.

Ni2+-NTA agarose (QIAGEN, Valencia, CA)

5.

Neu5Ac, bovine serum albumin (BSA), and isopropyl-1-thio-β-D-galactopyranoside (IPTG) (Sigma, St. Louis, MO)

6.

Kdn; prepared as described previously (10)

7.

0.025 M ortho-periodic acid in 0.125 N H2SO4

8.

1.6% sodium arsenite in 0.4 N HCl

9.

0.1 M thiobarbituric acid (TBA) in 0.1 N NaOH

10.

Anti-6His antibody (Cosmo Bio Co., LTD., Japan)

Instruments

1.

Incubator or heat block

2.

Spectrophotometer U5100 (HITACHI, Tokyo, Japan)

3.

An electrophoresis and blotting system for western blotting

4.

Detection system for western blotting

Methods

1.

Protocol for the preparation of recombinant SPL protein using the E. coli expression system.

a.

Subclone the coding DNA sequence of SPL gene into a prokaryotic expression plasmid, such as pET32b(+) vector (Novagen, Madison, WI) (see Notes 1 and 2).

b.

Transform the E. coli cells with the expression plasmid.

c.

Isolate a single colony of transformed cells and culture it in 1 mL of Luria broth supplemented with 50 mg/μL of ampicillin (LA) medium at 37°C overnight (an overnight culture).

d.

Grow an overnight culture in 500 mL of LA medium at 37°C until the OD600 value is between 0.4 and 0.8.

e.

Add IPTG to the culture medium at the final concentration of 0.4 mM and incubate at 15°C for 4 h.

f.

Harvest the E. coli cells using centrifugation (8,000 ×g, 4°C, 5 min).

g.

Disrupt the cell pellet sonically in 50 mL of 50 mM of Tris-HCl (pH 8.0) containing 2 mM of ethylenediaminetetraacetic acid, 1 mM of phenylmethylsulfonyl fluoride, and 1 μg/mL each of aprotinin, leupeptin, and pepstatin A.

h.

Collect the supernatant using centrifugation (16,000 ×g, 4°C, 10 min).

i.

For the purification of the recombinant SPL protein, mix the supernatant with an equal volume of 10 mM of imidazole, 1.0 M NaCl, and 40 mM of Tris-HCl (pH 8.0).

j.

Apply the mixture to a Ni2+-NTA agarose column (1.4 × 1.3 cm), which has been equilibrated with 5 mM of imidazole, 0.5 M NaCl, and 20 mM of Tris-HCl (pH 8.0).

k.

Wash the column with 10 mL of 30 mM of imidazole, 0.5 M NaCl, and 20 mM of Tris-HCl (pH 8.0).

l.

Elute recombinant SPL proteins with 5 mL of 100 mM of imidazole, 0.5 M NaCl, and 20 mM of Tris-HCl (pH 8.0).

m.

Exchange the medium of the eluted fraction to 10 mM of sodium phosphate buffer (pH 7.2) and 0.15 M NaCl using VIVASPIN® 20 (10K) ultrafiltration spin column.

n.

Remove N-terminal tags (S-tag, Thioredoxin tag, and His tag) by the enterokinase treatment, and obtain the purified SPL as a pass-through fraction of a Ni2+-NTA agarose column.

o.

Quantify the purified SPL protein amount either using bicinchoninic acid protein assay, Coomassie Brilliant Blue staining, or western blotting using anti-His tag antibody.

2.

Protocol for in vitro SPL activity assay

a.

Prepare 40 μL of the reaction mixture containing an appropriate amount of purified SPL in 50 mM of HEPES-NaOH (pH 7.0), 1.0 mM of Sia (Neu5Ac, Neu5Gc, or Kdn), and 1 ng/μL of BSA (Note 3).

b.

Incubate the reaction mixture at 37°C for 1 h.

c.

Stop the reaction by heating at 100°C for 10 min.

d.

Measure the remaining Sias in the reaction mixture using the TBA method (see step 3 of Methods).

3.

Protocol for quantification of Sia amount in the reaction mixture using TBA assay

a.

Add 20 μL of 0.025 M periodate in 0.125 N H2SO4 to 40 μL of the reaction mixture (Note 4).

b.

Incubate the mixture at 37°C for 30 min.

c.

To kill the excess periodate, add 20 μL of 1.6% sodium arsenite in 0.4 N HCl to the mixture and mix well until the color disappeared.

d.

Add 200 μL of 0.1 M TBA in 0.1 N NaOH to the mixture.

e.

Incubate at 100°C for 10 min.

f.

After the sample is cooled to room temperature, add 400 μL of 2-methoxyethanol.

g.

Measure the absorbance at 549 nm of the mixture (f) using a spectrophotometer U5100 (see Notes 5 and 6).

Notes

1.

The eukaryote expression plasmid can be used for recombinant SPL expression.

2.

SPL is a soluble protein without glycan modifications and can easily be prepared by the E. coli system.

3.

To avoid nonspecific enzyme absorption to the sample tube, the low binding tubes should be used.

4.

In this step, ortho-periodic acid is used.

5.

Samples should be diluted with deionized water, when the absorbance at 549 nm of the sample exceeds 1.0. The Sia concentration should be more than 50–70 ng/μL.

6.

When lipids are contaminated, the absorbance at 530 nm becomes higher than the absorbance at 549 nm.

References

1.
Rodríguez-Aparicio LB, Ferrero MA, Reglero A. N-acetyl-D-neuraminic acid synthesis in Escherichia coli K1 occurs through condensation of N-acetyl-D-mannosamine and pyruvate. Biochem J. 1995;308(Pt2):501–5. [PMC free article: PMC1136953] [PubMed: 7772033] [CrossRef]
2.
Ferrero MA, Reglero A, Fernandez-Lopez M, Ordas R, Rodriguez-Aparicio LB. N-Acetyl-D-neuraminic acid lyase generates the sialic acid for colominic acid biosynthesis in Escherichia coli K1. Biochem J. 1996;317(Pt1):157–65. [PMC free article: PMC1217457] [PubMed: 8694758] [CrossRef]
3.
Vimr ER, Troy FA. Identification of an inducible catabolic system for sialic acids (nan) in Escherichia coli. J Bacteriol. 1985;164:845–53. [PMC free article: PMC214328] [PubMed: 3902799] [CrossRef]
4.
Vimr ER, Troy FA. Regulation of sialic acid metabolism in Escherichia coli: role of N-acylneuraminate pyruvate-lyase. J Bacteriol. 1985;164:854–60. [PMC free article: PMC214329] [PubMed: 3902800] [CrossRef]
5.
Schauer R, Sommer U, Krüger D, van Unen H, Traving C. The terminal enzymes of sialic acid metabolism: acylneuraminate pyruvate-lyases. Biosci Rep. 1999;19:373–83. [PubMed: 10763805] [CrossRef]
6.
Wen XY, Tarailo-Graovac M, Brand-Arzamendi K, Willems A, Rakic B, Huijben K, Da Silva A, Pan X, El-Rass S, Ng R, Selby K, Philip AM, Yun J, Ye XC, Ross CJ, Lehman AM, Zijlstra F, Abu Bakar N, Drögemöller B, Moreland J, Wasserman WW, Vallance H, van Scherpenzeel M, Karbassi F, Hoskings M, Engelke U, de Brouwer A, Wevers RA, Pshezhetsky AV, van Karnebeek CD, Lefeber DJ. Sialic acid catabolism by N-acetylneuraminate pyruvate lyase is essential for muscle function. JCI Insight. 2018;3 [PMC free article: PMC6338320] [PubMed: 30568043] [CrossRef]
7.
Izard T, Lawrence MC, Malby RL, Lilley GG, Colman PM. The three-dimensional structure of N-acetylneuraminate lyase from Escherichia coli. Structure. 1994;2:361–9. [PubMed: 8081752] [CrossRef]
8.
Reissig JL, Storminger JL, Leloir LF. A modified colorimetric method for the estimation of N-acetylamino sugars. J Biol Chem. 1995;217:959–66. [PubMed: 13271455]
9.
Hara S, Yamaguchi M, Takemori Y, Furuhata K, Ogura H, Nakamura M. Determination of mono-O-acetylated N-acetylneuraminic acids in human and rat sera by fluorometric high-performance liquid chromatography. Anal Biochem. 1989;179:162–6. [PubMed: 2757191] [CrossRef]
10.
Angata T, Matsuda T, Kitajima K. Synthesis of neoglycoconjugates containing deaminated neuraminic acid (KDN) using rat liver alpha2,6-sialyltransferase. Glycobiology. 1998;8:277–84. [PubMed: 9451037] [CrossRef]

Footnotes

The authors declare no competing or financial interests.

Figures

Figure 1: . Schematic diagram of the biosynthetic pathway of Sia-glycoconjugates in vertebrates.

Figure 1:

Schematic diagram of the biosynthetic pathway of Sia-glycoconjugates in vertebrates. Sialate-pyruvate lyase (SPL), which is located in the cytosol, catalyzes the cleavage of Sias into pyruvate and N-acylmannosamine or Man. SPL catalyzes the reverse reaction or a synthetic reaction of Sia in E. coli (1, 2).

Copyright Notice

Licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 Unported license. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

Bookshelf ID: NBK593990PMID: 37590719
Contents