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A biotinylated sialylglycan was prepared enzymatically by endo-M, and binding behavior of an SSA lectin was studied on a different coverage of a sialylglycan-immobilized 27 MHz quartz-crystal microbalance (QCM).
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Glycoconjugates such as glycolipids and glycoproteins on cell membranes play an important role of receptor to lectins, toxins and viruses in living systems, and are significantly related to the formation of organized systems such as cell adhesion, differentiation and morphogenesis.1,2
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In order to elucidate the function of glycoconjugates on the cell surface, it is necessary to design a suitable glyco-microarray.
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However, the immobilization of carbohydrates on a substrate or chip due to complexed structures of glycoconjugates is still unexploited compared with that for nucleic acids and proteins.3,4
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The simple way for this purpose is to immobilize directly naturally-occurring glycoconjugates such as glycoproteins and glycolipids on a substrate; however, this is not suitable for studying the role of the carbohydrate itself such as in molecular recognition.
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It was developed to immobilize only the synthetic sugar chains on a substrate by using chemical methods such as Diels–Alder reactions and activated esterification;5,6 however, it required complicated chemical treatments such as the selective protection and de-protection of certain functional hydroxyl groups.
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It is also common to achieve the immobilization through a Schiff's base linkage using an aldehyde group of a reducing end of a carbohydrate with an amine or hydrazide group;2 however, this method has a disadvantage of collapsing the reducing end.
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In this communication, we have prepared a biotinylated naturally-occurring sialylglycan by enzymatic transglycosylation of endo-β-acetylglucosaminidase from Mucor hiemalis, as shown in a step (a) of Fig. 1.
Type: Experiment |
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The biotinylated sialylglycan was immobilized on an avidin-covered 27 MHz quartz-crystal microbalance (QCM) [step (b)].
Type: Experiment |
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SSA lectin from Sambucus sieboldiana could recognize specifically the sialyl(α2-6)Gal/GalNAc sequence independent of the surface coverage (5–80%) of the carbohydrate [step (c)].
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The 27 MHz QCM is known as a mass measuring device, which has been calibrated as the 1 Hz frequency decrease corresponding to the 0.62 ng cm−2 mass increase on the QCM electrode in the aqueous solution as well as in the air phase.7–9
Type: Experiment |
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ConceptID: Exp2
Type: Experiment |
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In the preparation of the biotinylated sialylglycan, we chose endo-β-N-acetyl glucosaminidase from Mucor hiemalis (EC 3.2.1.96) (endo-M) as an enzyme, which is a unique endoglycosidase for the hydrolysis of N,N′-diacetylchitobiosyl linkages in oligosaccharides bound to asparaginyl residues of various glycoproteins to leave one N-acetylglucosamine residue on the protein moieties10–12.Endo-M was found to hydrolyze not only the high mannose type but the complex type and the hybrid type of asparagine-linked oligosaccharides.12,13
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Furthermore, endo-M also showed transglycosylation activity.12–18
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Sialylglycopeptide (SGP) was separated from hen's egg yolk according to a previous method.18
Type: Experiment |
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SGP (340 µg, 12 mM) was reacted with biotinylated GlcNAc (38 µg, 2.1 mM) in the presence of endo-M (30 mU in 30 µL) in 10 mM phosphate buffer, pH 6.0 at 37 °C [step (a) in Fig. 1].
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The biotinylated sialylglycan could be prepared in 55–60% yield after 5 h of reaction, and it was purified by HPLC (C18 column, eluent: CH3CN containing trifluoroacetic acid) and confirmed by MALDI-TOF MS (matrix: DHB, +Na+: 2619.4, calibrated with insulin).
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The yield was comparable to that of the transglycosylation to glucose and aliphatic alcohols as acceptors (30–50%).10
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Type: Experiment |
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NeutrAvidin was chosen as a biotin-binding protein because carbohydrates on the avidin have been removed to reduce non-specific binding.
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When an aqueous solution of sialylglycan was injected to the NeutrAvidin-covered QCM plate, the frequency decreased (mass increased) depending on the concentration of sialylglycan (10–600 nM), and the immobilized amount could be controlled to be 5–60 ng cm−2 (1.9–23 pmol cm−2) on the QCM through biotin–avidin linkages [step (b) in Fig. 1].
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This corresponds to approximately 3–60% coverage of the NeutrAvidin surface on the QCM.
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Type: Observation |
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The state and orientation of the sialylglycan on the QCM chip was confirmed by binding of SSA lectin from Sambucus sieboldiana, which is known to recognize specifically the NeuAc(α2-6)Gal/GalNAc sequence of carbohydrate chains of glycoconjugates [step (c) in Fig. 1].19–21
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As shown in Fig. 2, when SSA lectin was injected to the sialylglycan surface (5–80% coverage) on the QCM, the frequency decreased (mass increased) with saturation behavior.
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SSA lectin bound weakly to the GlcNAc surface and NeutrAvidin surface due to the non-specific binding to the surface.
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Thus, SSA lectin specifically recognizes the surface of NeuAc(α2-6)Gal/GalNAc of the immobilized sialylglycan.
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From saturation binding measurements, the maximum binding amount (Δmmax) and binding constant (Ka) could be obtained according to eqn. 1,19–21where [SSA]0 is the initial concentration of SSA lectin.
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Values for Δmmax and Ka were obtained at different immobilized amounts of sialylglycan on the QCM, and the results are shown in Fig. 3.
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Both Ka and Δmmax values were independent of the immobilized amount of sialylglycan (5–80% of surface coverage).
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Ka value was obtained as 2.5 ± 0.4 × 107 M−1, which is similar to Ka = 6.7 × 106 M−1 obtained for SSA lectin binding to the NeuAc(α2-6)Gal(β1-4)Glc moiety by a surface plasmon resonance method,19 and Ka = 9.5 × 106 M−1 for SSA lectin binding to glycolipid monolayers having NeuAc(α2-6)Gal headgroups.20
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The slightly low Ka value of 3.4 × 105 M−1 was obtained in the bulk solution.21
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The maximum binding amount of SSA lectin was estimated to be 410 ng cm−2 as a monolayer adsorption of globular proteins of Mw = 160 kDa, which is consistent with the obtained value of Δmmax = 480 ± 40 ng cm−2.
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ConceptID: Res5
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Thus, SSA lectin can recognize the NeuAc(α2-6)Gal(β1-4)GlcNAc sequence of sialylglycan independent of the surface coverage of 5–80%.
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SSA lectin is an A2B2-type tetramer and a sectional area is roughly calculated to be 16 nm2.
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This is very large relative to that of sialylglycan (0.6–0.8 nm2), and ca. 30 sialylglycan molecules are calculated to be covered under one SSA lectin binding.
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This means that the existence of less than 3% of sialylglycan is enough to bind SSA lectin as a monolayer adsorption.
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In conclusion, we can prepare the biotinylated naturally-occurring sialylglycan by enzymatic transglycosylation in a high yield, and the state or orientation of sialylglycan can be assayed by using a QCM technique.
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We believe that the combination of the biotinylation of naturally-occurring carbohydrates by the endo-M enzyme and the QCM chip assay will be very useful for the elucidation of roles of carbohydrates and studies on glycomics.
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ConceptID: Bac5