Structure determination, by cryo-EM, of a mollusk hemocyanin with substantial biomedical impact

• Grant number: 21/03998-9
• Support Opportunities: Scholarships in Brazil - Master
• Start date: September 01, 2021
• End date: August 31, 2023
• Field of knowledge: Biological Sciences - Biochemistry - Chemistry of Macromolecules
• Principal Investigator: Andre Luis Berteli Ambrosio
• Grantee: Matías Nahuel Ellena
• Host Institution: Instituto de Física de São Carlos (IFSC). Universidade de São Paulo (USP). São Carlos , SP, Brazil

Abstract

This proposal for a Master's scholarship seeks to enable the research activities expected in the FAPESP-Conicyt grant 2019/13318-5. The outstanding progress in structural biology made possible by single-particle cryo-electron microscopy (cryo-EM) originated the so-called "resolution revolution" for its ability to provide high-resolution structural information of large and dynamic protein complexes. The state-of-the-art Titan Krios microscope in operation at the Nanotechnology National Laboratory (LNNano) established Brazil as the cryo-EM leader in Latin America; thus, it becomes urgent to capacitate the structural biology community across the region to optimize the use of such a facility. Through collaborative efforts between research groups from Chile and Brazil, a wide-spread access to this methodology represents an invaluable opportunity to promote further regional scientific development employing a cutting-edge method. Here, we propose a synergistic approach to determine the cryo-EM structure of the hemocyanin from Concholepas concholepas (CCH), a marine organism endemic of the southern Pacific coasts. Hemocyanins are megadalton-sized oligomers that participate in oxygen transport in multiple invertebrate organisms. Currently, hemocyanins are natural, non-toxic, and nonspecific immunostimulants with critical biomedical and clinical applications; therefore, understanding the precise molecular mechanisms of hemocyanins is critical for future targeted improvement. So far, the most well-studied hemocyanin from a structural and immunological standpoint is KLH (Keyhole Limpet Hemocyanin) from Megathura crenulate. Through a combination of cryo-EM and X-ray crystallography, the atomic details of the functional units that compose KLH have been revealed, displaying highly symmetric homodecameric rings featuring extensively glycosylated regions. However, homologous hemocyanins from other marine organisms such as CCH have demonstrated a significant degree of structural diversity, especially concerning glycosylation patterns, which might explain their unequal efficiency in generating appropriate adjuvant outcomes on immunologically oriented therapies. Studies conducted by Chilean researchers, who are also associated with this proposal, recently showed that CCH is one of the most promising hemocyanins for biomedical applications. CCH has superior stability and solubility compared to homologs, and it is the most effective in inducing immunological responses, as observed in preclinical studies of cancer vaccines and immunocastration. Nevertheless, despite the unique biomedical potential of CCH, its structure and even its amino acid sequence remain unknown. Preliminary biochemical characterization of CCH has demonstrated that two distinct polypeptide chains forming heterodimeric rings, as opposed to the homodimeric rings of KLH. Each CCH subunit appears to have eight globular oxygen-binding domains, termed functional units (FU), which are differentially glycosylated. However, any confirmed structural information remains elusive until now. In this context, we propose the primary goal to determine the structure of CCH by single-particle cryo-EM. We seek to combine the access to Concholepas concholepas specimens and the expert knowledge regarding the molecular biology and biochemistry of this organism by the Chilean researchers with our experience in structure determination using cryo-EM. An additional advantage on the Brazilian side is the considerably easier geographical access to sophisticated multiuser instrumentation dedicated to structural biology, such as the Titan Krios (at LNNano/CNPEM). Substantial preliminary analysis by negative staining electron microscopy of CHH increases the chances of success in this proposal. The student's expertise and experience will be transferred to the Chilean group by online and local training of students and researchers. Simultaneously, the Chilean group is working on the primary sequence identificatio... (AU)

Academic Publications

(References retrieved automatically from State of São Paulo Research Institutions)

ELLENA, Matías Nahuel. Structural determination, by cryo-EM, of the hemocyanin from the mollusk Concholepas concholepas with substantial biomedical impact. 2023. Master's Dissertation - Universidade de São Paulo (USP). Instituto de Física de São Carlos (IFSC/BT) São Carlos.