Abstract
Purpose: :
To characterize a peropsin homologue expressed in the basal arthropod, Limulus polyphemus. Peropsins are among the least understood members of the opsin superfamily.
Methods: :
A full-length Limulus peropsin-like sequence (Lp-perops) was cloned from a cDNA library of Limulus ventral eye using standard procedures. The Lp-perops sequence was compared to other peropsins, retinochromes and RGRs in phylogenetic analyses. The distribution of the transcript was determined by PCR using gene specific primers and in situ hybridization using as probe RNA encoding the full-length open reading frame. A specific monoclonal antibody directed against the C-terminus of Lp-perops was applied to Western blots and to fixed frozen sections of Limulus eyes to determine the tissue and cellular distribution of the protein. Immunostained sections were analyzed by confocal microscopy.
Results: :
In phylogenetic analyses, the predicted amino acid sequence of Lp-perops clusters more closely with deuterostome peropsins than it does with the retinochromes of mollusks or the RGRs of vertebrates. The Lp-perops transcript was detected in RNA extracted from all three types of Limulus eyes: lateral compound eyes (LE), median ocelli (ME) and ventral rudimentary eyes (VE). In situ hybridization of VE showed the transcript is located in specialized glia which tightly surround photoreceptors. On Western blots, a single band of Lp-perops immunoreactivity at approximately 38 kDa, close to the protein’s predicted size of 39 kDa, was detected in membrane extracts of LE and VE but so far not in membranes from ME. Using immunocytochemistry, Lp-perops-like immunoreactivity was detected in glia of VE and ME that tightly surround photoreceptors and are closely associated with photosensitive membranes. In LE, it was detected in pigment cells that surround photoreceptors.
Conclusions: :
A homologue of deuterostome peropsin is expressed in the eyes of the basal chelicerate arthropod Limulus polyphemus. Thus, peropsins evolved before the protostome-deuterstome split. The close association between Lp-perops-expressing cells and photoreceptors suggests Lp-perops is involved in vision. The function of this association remains a puzzle because the photopigment in rhabdomeral photoreceptors is bistable.
Keywords: opsins • photoreceptors • immunohistochemistry