Life science
Medical care, Drug development

Analysis of endocytosis using synthetic membrane protein
Institute for Protein Research
Professor Hironobu Hojo
https://researchmap.jp/read0014254

Life science

Abstract
Membrane proteins, exist on the cell membrane, exchange information and substances inside and outside the cell. Many of them
are expected to be drug discovery targets, and their rapid functional elucidation is awaited. However, membrane proteins are extremely hydrophobic and difficult to be prepared even with recombinant DNA methods. In addition, they often have post-translational
modifications such as sugar chains, resulting in heterogeneousstructures, making it difficult to obtain highly pure samples required
for functional analysis.
We have been engaged in the chemical synthesis of proteins.
Chemical synthesis can provide proteins with various post-translational modifications at any sites, and their functions can be analyzed accurately. In this study, in order to elucidate the mechanism of
caveolae endocytosis, in which substances are taken up into cells
from cavities on the cell membrane called caveolae, we established a method for chemical synthesis of caveolin, a membraneprotein that constitutes caveolae. As a result, it was proved thatsynthetic caveolin has a three-dimensional structure similar to thatof natural products, showing the success of the synthesis.

▍ Background & Results
When a protein is chemically synthesized, the entire polypeptide
is divided into several peptide segments, and each of them is subjected to machine-assisted synthesis by the solid-phase method.
After purification, they are condensed into full-length polypeptide
chain. A problem in the synthesis of membrane proteins is that thetransmembrane region is highly hydrophobic, so the peptide in thisregion has extremely low solubility, which hinders purification andcondensation with other peptide segments.
In the synthesis of caveolin, for the highly hydrophobic membrane-inserted region, a solubilizing tag is added during thesolid-phase synthesis, purification and the assembly of the entire
polypeptide chain and then, these solubilizing tags are cleaved toobtain the desired caveolin. In addition, caveolin has three palmitoyl groups at the C-terminal region, which makes it much more hydrophobic. Therefore, we decided to introduce palmitic acid after
the peptide chain assembly to avoid an increase in hydrophobicity
during synthesis.
Finally, folding was performed by embedding it in liposomes
and bicelle membranes. The synthetic caveolin showed circular dichroism spectrum similar to that of natural caveolin, indicating thesuccess of the synthesis. In future, we can analyze the function ofcaveolin how it induces endocytosis, and the functions of multiple
phosphorylation at the N-terminus.

▍ Significance of the research and Future perspective
We established a method for the chemical synthesis of a membrane protein, caveolin, by incorporating the solubilizing tag. At theend of the synthesis the tag was easily removed and the caveolin

was successfully embeded in the membrane. Since this method
can be applied to the synthesis of other membrane proteins, webelieve that it will greatly contribute to the elucidation of the functions of membrane proteins.

Figure 1. Structure of caveola. Oligomeric caveolin is important for caveolaformation.

Figure 2. Chemical synthesis of caveolin. P denotes the sites of phosphorylation.

P a t e n t
Tr e a t i s eHojo, Hironobu; Takei, Toshiki; Asahina, Yuya et al. Total Synthesis and Structural Characterization of Caveolin-1 Angew. Chem. Int. Ed. 2021, 60, p.13900–13905, doi: 10.1002/anie.202100826
Asahina, Yuya; Komiya, Shinobu; Ohagi, Ami et al. Chemical Synthesis of O-Glycosylated Human Interleukin-2 by the Reverse Polarity Protection StrategyAngew. Chem. Int. Ed. 2015, 54, p.8226–8230, doi: 10.1002/anie.201501847

URL
K e y w o r d caveolin, membrane protein, chemical synthesis, solubilizing tag, post-translational modification