Recombinant ArtinM activates mast cells

Abstract

Background: Mast cells are hematopoietically derived cells that play a role in inflammatory processes such as allergy, as well as in the immune response against pathogens by the selective and rapid release of preformed and lipid mediators, and the delayed release of cytokines. The native homotetrameric lectin ArtinM, a D-mannose binding lectin purified from Artocarpus heterophyllus seeds, is one of several lectins that are able to activate mast cells. Besides activating mast cells, ArtinM has been shown to affect several biological responses, including immunomodulation and acceleration of wound healing. Because of the potential pharmacological application of ArtinM, a recombinant ArtinM (rArtinM) was produced in Escherichia coli. The current study evaluated the ability of rArtinM to induce mast cell degranulation and activation.

Results: The glycan binding specificity of rArtinM was similar to that of jArtinM. rArtinM, via its CRD, was able to degranulate, releasing β-hexosaminidase and TNF-α, and to promote morphological changes on the mast cell surface. Moreover, rArtinM induced the release of the newly-synthesized mediator, IL-4. rArtinM does not have a co-stimulatory effect on the FcεRI degranulation via. The IgE-dependent mast cell activation triggered by rArtinM seems to be dependent on NFkB activation.

Conclusions: The lectin rArtinM has the ability to activate and degranulate mast cells via their CRDs. The present study indicates that rArtinM is a suitable substitute for the native form, jArtinM, and that rArtinM may serve as an important and reliable pharmacological agent.

Keywords: ArtinM; Degranulation; Lectin; Mast cells; rArtinM.

Fig. 1

Analysis of rArtinM and jArtinM and analytical ultracentrifugation assay. a Lane 1: undenatured rArtinM. Lane 2: rArtinM after thermal dissociation. Lane 3: undenatured jArtinM. Lane 4: jArtinM after thermal dissociation. 3 μg of protein were loaded to each lane. 12.5 % SDS-PAGE stained with Coomassie blue G-250. b Size distribution obtained from the sedimentation velocity profiles of rArtinM at 20 °C. Fit and residuals after fitting to a c(S) were calculated in SEDFIT. Plot of the distribution of sedimentation coefficients (C(s)) versus (S), where S is plotted in Svedberg units. The shape of the major peak shows oligomeric structures of rArtinM. By non-linear fitting, the average molecular weight (Mw) for rArtinM was determined to be 50 kDa (displayed inside the plot)

Fig. 2

Carbohydrate microarray analyses of jArtinM and rArtinM. Binding of jArtinM (a) and rArtinM (b) to a glycan array of 255 lipid-linked oligosaccharide probes. Biotinylated ArtinM (50 μg/mL) was overlaid, and binding was detected after adding streptavidin conjugated to Alexa Fluor 647. Glycoarray data analysis was performed with dedicated software. Numerical scores of the binding signals are means of duplicate spots at 5 fmol/spot (with error bars). The various types of oligosaccharide linkage are indicated by the colored panels

Fig. 3

High doses of rArtinM are required for mast cell mediator release. a In the absence of IgE only 20 and 40 μg/ml of rArtinM induced β-hexosaminidase release, and in the presence of IgE all doses induced β-hexosaminidase release. b In the absence of IgE only 40 μg/ml of rArtinM stimulated release of TNF-α, whereas in IgE sensitized cells 10 and 40 μg/ml of rArtinM induced TNF-α release. c The release of LTC4 was seen only in IgE sensitized cells. d rArtinM stimulation of IL-4 release did not require IgE. Data are expressed as mean ± SEM and are representative of three separate experiments. *p < 0.05 or ** p < 0.01 or *** p < 0.001 between samples and the controls

Fig. 4

rArtinM induces degranulation via its CRDs. When rArtinM (40 μg/ml) was preincubated with D-mannose for 1 h, β-hexosaminidase release was abolished (black arrows). Data are expressed as mean ± SEM and are representative of three independent experiments. *** p < 0.001 between samples and the controls

Fig. 5

Activation induced by rArtinM occurs through a CRD-dependent manner. By scanning electron microscopy (SEM) deep ruffles are observed on the surface of IgE sensitized mast cells after activation with DNP₄₈-HSA and on the surface of rArtinM treated mast cells both in the absence or presence of IgE. When rArtinM was preincubated with D-mannose, the cells are in spindle-shape and covered by fine microvilli on surface, similar to unstimulated cells (Control). Bars = 10 μm

Fig. 6

rArtinM does not have co-stimulatory effect on mast cell degranulation via FcεRI. Cells sensitized with IgE and stimulated with the combination of DNP₄₈-HSA + rArtinM (40 μg/ml) had a decrease in β-hexosaminidase release. Data are expressed as mean ± SEM and are representative of three separate experiments. ** p < 0.01 between samples and the controls

Fig. 7

rArtinM activates NFkB, but not NFAT. rArtinM (40 μg/ml) induced NFkB activation only in the presence of IgE (a), but did not activate the transcription factor NFAT either in the absence or in the presence of IgE (b). Data are expressed as mean ± SEM and are representative of three separate experiments. *** p < 0.001 between samples and the controls