Lectin-binding and dissociation/reconstitution studies on the trichocysts of the dinoflagellate Oxyrrhis marina

Abstract

Trichocyst-enriched fractions were isolated from the dinoflagellate Oxyrrhis marina and subjected to protein staining and lectin-binding studies, to dissociation experiments using heat, and to solubilization/reconstitution experiments using 6 M guanidine hydrochloride. The trichocysts could be stained by Alcian Blue and became labeled by the lectin Concanavalin A, but not by wheat germ agglutinin. The trichocysts did not dissociate when the fractions were heated for 5 min at 40 or 50 °C. Heating at 60 °C resulted in the dissociation of trichocysts into irregular filamentous structures. These filaments were still present when the fractions were incubated for 5 min at temperatures of 70 and 80 °C. Reassembly was not achieved by subsequent cooling steps. The disintegration of trichocysts was also achieved in 6 M guanidine hydrochloride, and reassembly into filamentous structures, similar to those obtained by heat, occurred after dialysis against distilled water. Electron microscopy revealed that the filaments created either by heat or using guanidine hydrochloride by far did not resemble native trichocysts. They were much thinner (5–7 nm in width), missed the characteristic striation of electron-dense and -transparent lines along the longitudinal axes, and showed much more bending. Furthermore, they tend to merge to thicker shapeless structures and blob-like aggregates. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that fractions enriched either in trichocysts or in reconstituted filamentous structures obtained in the guanidine hydrochloride solubilization experiments were dominated by proteins with relative molecular weights in the range of approximately 15 to 29 kDa. Minor amounts of larger proteins were also detected. Tryptic in gel digestion followed by mass spectrometry confirmed the presence of almost the same set of proteins within the both, the trichocyst-enriched fractions, and the fractions of filaments reconstituted thereafter. These proteins were previously proposed to represent the matrix polypeptides of the trichocysts of Oxyrrhis marina (Rhiel et al., Protoplasma 255: 217–230, 2018).