A novel 65 kDa RNA-binding protein in squid presynaptic terminals

A novel SDS-stable dimer of a heterogeneous nuclear ribonucleoprotein at presynaptic terminals of squid neurons

“…The precise molecular definition of what composes the p65 dimer is still ambiguous. The data support the hypothesis that endogenous p65, a highly basic protein as determined by two-dimensional electrophoresis and ion exchange chromatography (Lico et al, 2010), is a homodimer of hnRNPA/B-like protein 2 (isoelectric point, pI 8.9), but not a homodimer of hnRNPA/B-like protein 1 (pI 7.1). However, we have not excluded the possibility that p65 is a heterodimer containing both hnRNPA/B-like proteins 1 and 2 (combined pI is 8.6).…”

“…Also, comparative staining by immunofluorescence on histological slices of the optic lobe showed similar staining in specific layers within the external plexiform layer by antibodies against synaptic vesicle protein 2 (anti-Sv2), a presynaptic protein, and α-sqRNP2 (FIG 1C). These data and images are similar to those previously obtained with an unrelated antibody that recognized p65 (Lico et al 2010) and substantiate the localization of p65 within the presynaptic terminals. Surprisingly however, in addition to p65 a 37 kDa polypeptide (p37) was also strongly and consistently labeled by α-sqRNP2 on western blots of these fractions (FIG 1A) suggesting that there was a common epitope being recognized or a strong cross reaction.…”

“…Western blotting was done as previously described (Costa et al 1999). Tryptic peptides from appropriate colloidal Coomassie blue stained bands (Imperial Protein Stain, Pierce Rockford, Il) were analyzed by matrix assisted laser desorption, time-of-flight mass spectrometry (MALDI-TOF/TOF) as previously described (Lico et al, 2010). All mass spectra from MALDI-TOF/TOF were submitted to databank searches using MASCOT against nrNCBI, Swiss-pro and squid ESTs.…”

“…In a previous study (Lico et al 2010), we identified and biochemically characterized a highly basic, RNA-binding protein (referred to as p65) that was present in presynaptic terminals of squid optic lobes and stellate ganglia. Based on a sequence deduced from mass spectrometry analysis of tissue-purified p65, we raised a polyclonal antibody in rabbits against the synthetic peptide CKLFIGGLSYDTNEDTIKK , referred to as anti-squid ribonucleoprotein motif 2 (α-sqRNP2) due to its sequence homology to the consensus, RNA-recognition motif, LFIGGL (Maris et al 2005).…”

Identification of molecular motors in the Woods Hole squid, Loligo pealei: An expressed sequence tag approach

“…A lipid binding protein was found to be required for the MgATP stimulation of the Na + /Ca 2+ exchanger [Berberián et al,2009]. Finally, a novel 65 kDa RNA‐binding protein in the presynaptic terminal that is suggested to play a role in the synaptic localization of RNAs [Lico et al,2010].…”