Functional and structural characterization and immune response evaluation of a recombinant serine protease from Crotalus durissus collilineatus modified by PEGylation

Author(s)

Ernesto Lopes Pinheiro Júnior

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC)
• Defense date: 2021-02-09

Examining board members

Eliane Candiani Arantes Braga; Veridiana de Melo Rodrigues Ávila; Flávio Henrique da Silva; Karina Furlani Zoccal

• Advisor: Eliane Candiani Arantes Braga; Johara Boldrini França

Abstract

Snake venom serinoproteases (SVSPs) are enzymes capable of affecting the human hemostatic system, given their similarity with enzymes present in its different pathways. Thus, they are considered promising therapeutic agents, in addition to potential biotechnological tools. PEGylation, a process in which polyethylene glycol (PEG) is conjugated to proteins, aims to reduce the immunogenicity and to increase the drugability of these biomolecules in vivo. In this context, this work aimed the purification of an SVSP (collinein-1) from Crotalus durissus collilineatus venom, as well as the expression of its recombinant form (rCollinein-1) in Pichia pastoris system. Both proteins were PEGylated (PEG-collinein-1 and PEG-rCollinein-1) and the structural and functional characterization, in addition to the evaluation of the immune response, were compared between the four forms of the enzyme. Collinein-1 was isolated from the crude venom by two reversed-phase chromatographic steps, while rCollinein-1 was purified in three steps, in columns of affinity to immobilized metal ions (IMAC) and cation exchange. Then, a mono-PEGylated population of both proteins was obtained. The modelling of collinein1 was obtained using the SWISS-MODEL tool, revealing that the protein has a structure like other SVSPs. The thermal stability analysis indicated that all forms of the protein have similar unfolding temperatures. PH 5.0 was the condition with the lowest unfolding temperature, while pH 7.0 was considered as the best condition for the stability of all forms of the enzyme. A significant increase in protein size was observed after PEGylation (collinein-1: 4.0±0.4 nm, PEG-collinein-1: 9,9±1,1 nm, rCollinein-1: 4.9±1.3 nm; PEG-rCollinein-1: 10,7±1,3 nm). On the other hand, no significant changes were seen in the content of secondary enzyme structures. The content of alpha-helixes (7%, 5%, 2%, 15%), beta sheets (31%, 31%, 31%, 22%), turns (21%, 19%, 16%, 17%) and random coil elements (41%, 45%, 51%, 46%) for collinein-1, PEGcollinein-1, rCollinein-1 and PEG-rCollinein-1, respectively, showed that PEGylation did not interfere in the protein folding. The determination of kinetic parameters showed similar values of Km between its different forms (collinein-1: 0.920 ± 0.079 mM; rCollinein-1: 1.243 ± 0.113 mM; PEG-collinein-1: 1.317 ± 0.117 mM and PEG-rCollinein-1: 1.4 ± 0.119 mM). However, the catalytic constant (kcat) and the specificity constant (kcat/Km) were slightly different. rCollinein-1 showed higher values of these parameters (kcat = 2.934 ± 0.118 s-1 and kcat/Km = 2.4 ± 0.115 mM.s-1 ) when compared to collinein-1 (kcat = 0.497 ± 0.017 s-1 and kcat/Km = 0.5 ± 0.048 mM.s-1 ), PEG-collinein-1 (kcat = 0.502 ± 0.020 s-1 and kcat / Km = 0.4 ± 0.068 mM.s-1 ) and PEG-rCollinein-1 (kcat = 1.286 ± 0.050 s-1 and kcat/Km = 0.9 ± 0.084 mM.s-1 ). However, these results indicate that PEGylation was not harmful to the catalytic activity of native and recombinant forms, corroborating the results of fibrinogen degradation, which showed that all forms of the enzyme have catalytic activity on this substrate. However, it was observed that the inhibitory activity of PEGylated proteins was significantly lower in the hEAG1 channel (45.5 ± 13.4% to 8.2 ± 4.7% and 50.3 ± 6.0% to 5.7 ± 3.7%, at 5 µM, for the native and recombinant proteins, respectively), while there was an increase in activity in hERG1, with IC50 values of 22.4 ± 1.1 µM and 10.0 ± 0.4 µM for PEG-collinein-1 and PEG-rCollinein-1, respectively. Other serinoproteases were tested on hEAG1 and the essential amino acid residues for the activity of these enzymes in this channel have been proposed. The absence of cytotoxicity in PBMC cells and the lack of in vivo immunogenicity evaluated in mice of PEGylated proteins are important results in the quest for therapeutic applicability. Overall, the PEGylation of these proteins directed their activity towards hemostasis control, broadening their possibilities to be used as defibrinogenant agents in pathological conditions such as stroke, thrombosis, and pulmonary embolism. (AU)

• FAPESP's process: 16/04761-4 - Functional and structural characterization and immune response evaluation of a recombinant serine protease from Crotalus durissus collilineatus modified by PEGylation
• Grantee: Ernesto Lopes Pinheiro Junior
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)