Identification Of The Spectrin And F-actin-binding Sites In Amphioxus Protein4.1

The unique membrane structure is needed to maitain the morphology of theerythrocyte and the protein skeleton network is needed to maintain the erythrocytemembrane structre. This membrane cytoskeleton is mainly composed of spectrin,actin, protein4.1, ankyrin and so on. Protein4.1and ankyrin provide two links thatfunction independently to tether the network to transmembran proteins. Vertebrateshave four highly conserved genes encoding protein4.1; their protein products areknown as4.1R (red cell type),4.1N (neuron type),4.1G (general type), and4.1B(brain type). Protein4.1R is the first member of protein4.1identified from red bloodcells, and is thus regarded as the prototypical member of protein4.1family. All fourproteins in vertebrate have a common organizaton of domains, including three highlyconserved domains: N-terminal domain FERM (four point oneprotein-ezrin-radixin-moesin), spectrin-actin-binding domain (SAB), and C-terminaldomain (CTD). Adjacent to the FERM domain is a FERM-adjacent (FA) domain. Allknown invertebrate genomes have a single copy of protein4.1genes and it lacks theSAB domain. The SAB domain of the4.1R in vertebrate could bind to spectrin andactin to provide the cytoskeletal linkage. We want to know whether protein4.1ininvertebrate could bind to spectrin and actin, and how the SAB domain of protein4.1in vertebrate evolved.Amphioxus belongs to the cephalochordate and is between invertebrate andvertebrate. Therefore, it is a model animal for the study of evolution and origin ofvertebrate. We have cloned a cDNA encoding an amphioxus protein4.1(BjP4.1) fromamphioxus (Branchiostoma japonicum), and expressed the BjP4.1recombinantprotein. ELISA analysis showed that it could bind to spectrin and actin. The affinity ofrBjP4.1to spectrin and actin is closed to that of human4.1R. To determine thestructure-activity relationship and explore the active sites of the BjP4.1, varioustruncated BjP4.1peptides (FUC, UC, U2/3, and U17/68), with the specific domains deleted, were expressed in E. coli and subjected to binding analysis. ELISA assaysshowed that the truncated peptide FUC has an affinity to spectrin and actin anddecreased by1/3, comparable to the full rBjP4.1. Similarly, the truncated peptides UC,U2/3, and U17/68retained strong affinities to spectrin and actin, although theirbinding activities were reduced by1/2. However, the truncated peptide FA has noaffinity to spectrin and actin.As U17/68retained considerable capacity to bind to spectrin and actin, thedifferent peptides derived from U2/3, i.e. U1/16, U17/33, U34/50, and U51/68, weresynthesized by Sangon Biotech Co. Ltd (Shanghai, China), and subjected to thebinding assay. The experimental results showed that U1/16and U34/50are incapableof binding to spectrin and actin, U17/33has a weak affinity, whereas U51/68has astrong capacity of binding to spectrin and actin. The point mutation approach resultsshowed that the acidic amino acids D387mutation in U2/3make the affinity decrease.In summary, the U2/3region in amphioxus protein4.1has the binding activity tospectrin and actin. We predict that the U2/3region is most likely the prototype of theSAB domain of protein4.1in vertebrate. Our results also showed that the C-terminal18amino acids of the U2/3region may be the key residues to the BjP4.1binding tospectrin and actin.