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. 2009 Oct;69(4):319-32.
doi: 10.1007/s00239-009-9273-y. Epub 2009 Sep 17.

Phylogenetic analysis of the NEEP21/calcyon/P19 family of endocytic proteins: evidence for functional evolution in the vertebrate CNS

Nagendran Muthusamy  1 , Sanaa A Ahmed, Brinda K Rana, Sammy Navarre, David J Kozlowski, David A Liberles, Clare Bergson
Affiliations

Phylogenetic analysis of the NEEP21/calcyon/P19 family of endocytic proteins: evidence for functional evolution in the vertebrate CNS

Nagendran Muthusamy et al. J Mol Evol. 2009 Oct.

Abstract

Endocytosis and vesicle trafficking are required for optimal neural transmission. Yet, little is currently known about the evolution of neuronal proteins regulating these processes. Here, we report the first phylogenetic study of NEEP21, calcyon, and P19, a family of neuronal proteins implicated in synaptic receptor endocytosis and recycling, as well as in membrane protein trafficking in the somatodendritic and axonal compartments of differentiated neurons. Database searches identified orthologs for P19 and NEEP21 in bony fish, but not urochordate or invertebrate phyla. Calcyon orthologs were only retrieved from mammalian databases and distant relatives from teleost fish. In situ localization of the P19 zebrafish ortholog, and extant progenitor of the gene family, revealed a CNS specific expression pattern. Based on non-synonymous nucleotide substitution rates, the calcyon genes appear to be under less intense negative selective pressure. Indeed, a functional group II WW domain binding motif was detected in primate and human calcyon, but not in non-primate orthologs. Sequencing of the calcyon gene from 80 human subjects revealed a non-synonymous single nucleotide polymorphism that abrogated group II WW domain protein binding. Altogether, our data indicate the NEEP21/calcyon/P19 gene family emerged, and underwent two rounds of gene duplication relatively late in metazoan evolution (but early in vertebrate evolution at the latest). As functional studies suggest NEEP21 and calcyon play related, but distinct roles in regulating vesicle trafficking at synapses, and in neurons in general, we propose the family arose in chordates to support a more diverse range of synaptic and behavioral responses.

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Figures

Figure 1
Figure 1
Expression of LOC324971 mRNA during zebrafish (Danio rerio) embryogenesis. (A) Temporal expression of LOC324971 mRNA by RT-PCR analysis (open arrow). Stages are as indicated; hpf, hours post-fertilization. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression is shown for comparison (solid arrow). (B–D) Detection of LOC324971 mRNA by in situ hybridization. (B) 18 hpf. mRNA is expressed in the developing forebrain (open arrow), midbrain (open triangles), and hindbrain (solid arrows). The otic vesicle is marked by an asterisk. (C) 24 hpf. Expression in the telencephalon (open arrow), diencephalon (line arrow), tegmentum (open triangle), hindbrain (solid arrows), and spinal cord (bracket) is marked as indicated. (D) 48 hpf. Symbols the same as in (C) except the midbrain is marked by a bracket. Panels B and D are dorsal views of flat mounted embryos and panel C is a lateral view of whole-mount embryo. Anterior is to the left in panels B–D. Scale bar in panel B is 50μm and in panels C, D is 100μm.
Figure 2
Figure 2
Multiple Sequence Alignment. Clustal W alignment of P19, NEEP21, and calcyon proteins identified either with CDART or with the EST database searches (refer to Table 1). Also shown in parentheses in Table 1 are orthologous proteins that were not identified in CDART searches, but were retrieved in TBLASTN queries of chicken, medaka, and zebrafish EST databases. Consensus strength among orthologs in the alignment is indicated by height and color, with red being the greatest.
Figure 3
Figure 3
Rooted Phylogenetic Tree. Predicted evolutionary relationship of the P19, NEEP21, and calcyon orthologs (shown in Fig. 2) constructed using RAXML (Stamatakis, 2006) with the GTR+Λ+I model. A. The tree branch detected as undergoing positive selection using the Ka/Ks ratio from a non-statistical approach plus from the free ratios model and confirmed using branch and branch-site tests according to PAML (Yang, 2007) is shown in blue, while that from the free ratios model and confirmed by both the branch-sites and branches model is shown in brown. The branch showing positive selection by the free ratios model, but not by the other two models is labeled in pink. B. The same tree is depicted with branches represented proportional to their lengths from RAXML. formula image Positive selection detected with the null model, branches-sites model and ASR+PBL formula image Positive selection detected with the null model and the branchessites model formula image Positive selection detected only with the null model
Figure 3
Figure 3
Rooted Phylogenetic Tree. Predicted evolutionary relationship of the P19, NEEP21, and calcyon orthologs (shown in Fig. 2) constructed using RAXML (Stamatakis, 2006) with the GTR+Λ+I model. A. The tree branch detected as undergoing positive selection using the Ka/Ks ratio from a non-statistical approach plus from the free ratios model and confirmed using branch and branch-site tests according to PAML (Yang, 2007) is shown in blue, while that from the free ratios model and confirmed by both the branch-sites and branches model is shown in brown. The branch showing positive selection by the free ratios model, but not by the other two models is labeled in pink. B. The same tree is depicted with branches represented proportional to their lengths from RAXML. formula image Positive selection detected with the null model, branches-sites model and ASR+PBL formula image Positive selection detected with the null model and the branchessites model formula image Positive selection detected only with the null model
Figure 4
Figure 4
The PPLP motif as a primate innovation. Sequence alignment of mammalian calcyon from diverse species in which identical amino acids are boxed. The most parsimonious ancestral sequence reconstruction scenarios are shown on the tree.
Figure 5
Figure 5
The primate PPLP motif mediates interaction of calcyon with WW domain proteins. A. Diagram of the transmembrane (TM) domain structure of calcyon. The N-terminal 80 residues of murine calcyon and both the P40 and L40 variants of hCalcyon were fused to the STag for ‘overlay’ assays shown in B and C. B. Binding specificity of the calcyon WW domain. Bacteria were transformed with pGEX expressing GST fusion proteins of the indicated WW domains (FBP11, FEB65, and YAP65), or the SH3 domain of Abl. Fusion proteins were induced with isopropyl β-D-thiogalactoside (IPTG). Uninduced (U) and induced (I) bacterial extracts were separated on an SDS-PAGE gel, stained with coomassie blue (lower panel) or transferred to PVDF membrane (upper panel). After blocking, blots were incubated with 1 mg purified S-hWT (1-80) in blocking buffer overnight. Binding of S-Tag-hWT (1-80) was detected with anti-S protein conjugated to HRP, followed by ECL and exposure to autoradiography film. C. The FBP11 WW domain binds the calcyon hWT(P40), but not to either the murine or hP40L S-Tag fusion proteins. Overlay assay performed by incubating purified GST-FPB11 with a blot of uninduced and induced lysates of bacteria transformed with the indicated calcyon S-Tag fusion proteins. Bound GST-FBP11 was detected with HRP conjugated anti-GST antibodies as in B. For both B and C, similar results were obtained in at least two independent experiments.
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