MGC Products: Application Notes

 

User Report

Professor Tadanobu Takahashi Shizuoka Prefectural University Faculty of Pharmaceutical Sciences Biochemistry

Detection of sialo sugar chain terminal structure using lectin

Products used

• Lectin from Sambucus sieboldiana / SSA-Biotin (Cat No .: MGC-J218)
• Lectin from Maackia amurensis / MAM-Biotin (Cat No .: MGC-J210)

Manufacturer: MGC Woodchem Inc.

Lectins that recognize sialic acid MAM, SSA, WGA

Lectin that recognizes sialic acid
Lectin	Main sugar chains to recognize
MAM	Sia α2-3Gal β 1-4GlcNAc β 1-
SSA	Sia α2-6Gal β 1-4GlcNAc β 1-
	Sia α2-6GalNAc
WGA	Examples of sugar chains in which several sialic acids are present in clusters : birds, tetrasialo N-type complex sugar chains, cluster-like mucin-type sugar chains

Experiment contents

Lectins recognize and bind to sugar chain structures. Therefore, if there is a lectin corresponding to the sugar chain structure to be detected, the expression distribution of the sugar chain can be easily stained. However, it should be noted that it may recognize structures other than the sugar chain structure to be detected. In my research field, sugar chain virology, lectins that bind to sialic acid at the ends of sugar chains are often used. Sugar chains with sialic acid (sialo sugar chains) are used as receptors when influenza virus infects host cells. Influenza A virus can distinguish the binding mode of sialic acid (SA) and galactose (Gal) immediately below it. In this commentary, SA refers to N -acetylneuraminic acid, which is the major molecular species of SA. At the end of the sialo-glycan structure to which influenza A virus binds, the bond between the carbon position number 2 of SA and the carbon position number 3 of Gal (SAα2,3Gal), or the carbon position number 6 of Gal. There is a bond with the position (SAα2,6Gal) (Fig. 1). Avian influenza A virus binds strongly to SAα2,3Gal distributed in the trachea of ​​chickens. On the other hand, human influenza A virus binds strongly to SAα2,6Gal expressed in the upper respiratory tract of humans. Such a difference in the binding property of the virus is regarded as a host-specific barrier that the avian influenza A virus cannot be easily transmitted to humans. The change in receptor binding of avian influenza A virus from SAα2,3Gal to SAα2,6Gal is considered to be one of the factors causing transhost transmission from birds to humans. Maackia amurensis lectin (MAM) for detection of SAα2,3Gal at the terminal of sialo sugar chain to which virus binds , Japanese elderberry Sambucus sieboldiana for detection of SAα2,6GalLectin (SSA) is used (Fig. 1).

As a method for measuring the receptor binding of influenza A virus, there is a method of immobilizing a sugar chain having SAα2,3Gal or SAα2,6Gal as a terminal structure on an ELISA plate. After reacting the virus, the receptor binding property is compared by measuring the amount of virus bound to the sugar chain. A sugar chain polymer obtained by adding a sugar chain structure of SAα2,3 or α2,6Galβ1,4GlcNAc (GlcNAc is N -acetylglucosamine) to a γ-polyglutamic acid polymer shows strong binding to influenza A virus. By immobilizing this sugar chain polymer on an ELISA plate, the sensitivity of the virus receptor binding measurement method can be increased.

In order to confirm that the sugar chain polymer was immobilized on the plate, an attempt was made to detect the sugar chain terminal structure with a lectin. SAα2,3Gal, SAα2,6Gal, and SA-free sugar chain polymer (50 ng / well) diluted in ethanol were added to a 96-well microplate (Costar Universal-BIND) and evaporated at 4 ° C for 2 hours under reduced pressure. It was dried and solidified by UV irradiation (256 nm) for 1 minute. After washing 5 times with PBS, blocking was performed overnight at 4 ° C with a 1% degreased BSA-PBS suspension (300 μL / well). After washing 5 times with PBS, biotin-labeled SSA (J-Oil Mills product number: J218) or biotin-labeled MAM (J-Oil Mills product number: J210) at 100 μL / well (5 μg / mL) at room temperature for 2 hours. It was reacted. After washing 5 times with PBS, the reaction was carried out with HRP-labeled streptavidin (130 μL / well) at room temperature for 1 hour. It was washed 5 times with PBS and colored with 100 mM citric acid-phosphate buffer (100 μL / well) supplemented with o-phenylenediamine hydrochloride and H ₂ O _{2 at room temperature for 10 minutes.} The color reaction was stopped with 1N sulfuric acid (100 μL / well), and the absorbance at 492/630 nm was measured. It was confirmed that SSA was strongly bound to the SAα2,6Gal sugar chain polymer and MAM was strongly bound to the SAα2,3Gal sugar chain polymer, and each sugar chain polymer was immobilized on the plate (Fig. 2).

Since this experiment used a homogenized sugar chain structure, the difference in sugar chain structure could be clearly detected by the lectin. However, it is dangerous to try to clearly interpret the expression of sugar chains of various structures in actual tissues and cells only by lectin staining. MAM does not react with O-type sugar chains, SSA reacts with O-type sugar chains, and also reacts with SAα2,6GalNAc (GalNAc is N -acetylgalactosamine), or lectin reactivity due to differences in SA molecular species. It seems that it is necessary to understand the sugar chain binding characteristics of lectins in the literature, etc. before using them.