FRET analysis using sperm-activating peptides tagged with fluorescent proteins reveals that ligand-binding sites exist as clusters

Abstract

Long-range cellular communication between the sperm and egg is critical for external fertilization. Sperm-activating peptides (SAPs) are diffusible components of the outer layer of eggs in echinoderms, and function as chemoattractants for spermatozoa. The decapeptide named speract is the best-characterized sea urchin SAP. Biochemical and physiological actions of speract have been studied with purified or chemically synthesized peptides. In this work, we prepared recombinant speract fused to a fluorescent protein (FP; FP-speract) using three color variants: a cyan (eCFP), a yellow (mVenus) and a large Stokes shift yellow (mAmetrine) FP. Although these fluorescence tags are 20 times larger than speract, competitive binding experiments using mAmetrine-speract revealed that this FPsperact has binding affinity to the receptor that is comparable (7.6-fold less) to that of non-labeled speract. Indeed, 10 nmol l −1 eCFPsperact induces physiological sperm responses such as membrane potential changes and increases in intracellular pH and Ca 2+ concentrations similar to those triggered by 10 nmol l −1 speract. Furthermore, FP-speract maintains its fluorescence upon binding to its receptor. Using this property, we performed fluorescence resonance energy transfer (FRET) measurements with eCFP-speract and mVenus-speract as probes and obtained a positive FRET signal upon binding to the receptor, which suggests that the speract receptor exists as an oligomer, at least as a dimer, or alternatively that a single speract receptor protein possesses multiple binding sites. This property could partially account for the positive and/or negative cooperative binding of speract to the receptor.

Quantitative Proteome Reveals Variation in the Condition Factor of Sea Urchin Strongylocentrotus nudus during the Fishing Season Using an iTRAQ-based Approach

“…Thus, RL36 in S. nudus gonads may participate in ribosomal subunit formation and may be affected by the temperature during individual developments during the fishing season. Sperm activated by SAPs was observed to have higher intracellular pH and Ca 2+ concentrations [50]. It is speculated that the increased content of SAPs could be related to the condition factor of S. nudus gonads during the fishing season.…”