Related Experiment Video
Updated: Aug 24, 2026
Isolation, Fixation and Characterization of Juvenile Gilthead Seabream Head Kidney Leukocytes by Flow Cytometry
Published on: May 9, 2025
Isolation and characterization of complement component C3 from Atlantic cod (Gadus morhua L.) and Atlantic halibut
Sigrun Lange1, Alister W Dodds, Bergljót Magnadóttir
1Institute for Experimental Pathology, University of Iceland, Keldur, IS-112 Reykjavík, Iceland. sigrun1@hi.is
Insights
Researchers isolated and characterized complement component C3 from cod and halibut plasma. They found structural similarities to higher vertebrates but identified species-specific differences in glycosylation.
Area of Science:
- Immunology
- Marine Biology
- Biochemistry
Background:
- Complement component C3 is crucial for innate immunity in vertebrates.
- Understanding fish C3 provides insights into immune evolution and disease resistance in aquaculture.
Purpose of the Study:
- To isolate and characterize complement component C3 from cod (Gadus morhua L.) and halibut (Hippoglossus hippoglossus L.).
- To compare the structural and functional properties of fish C3 with those of higher vertebrates.
- To investigate potential species-specific differences in C3 glycosylation.
Main Methods:
- Purification of C3 using Fast Protein Liquid Chromatography (FPLC) with ion exchange and gel filtration.
- Analysis of purified C3 by SDS-PAGE and N-terminal amino acid sequencing.
- Preparation of specific antibodies for Western blotting and testing proteolytic fragmentation with various enzymes and bacterial products.
Main Results:
- Cod and halibut C3 exhibited a two-chain alpha- and beta-chain structure, similar to higher vertebrates.
- Intra-chain thioesters were identified in the alpha-chains of both fish C3 proteins.
- Trypsin and papain cleaved C3, while pepsin and Aeromonas salmonicida extracellular product did not.
- Differences in C3 glycosylation were observed between cod and halibut after PNGase treatment.
Conclusions:
- Cod and halibut C3 share conserved structural features with vertebrate C3.
- Proteolytic susceptibility of fish C3 varies, with trypsin and papain being effective cleaving agents.
- Species-specific glycosylation patterns in fish C3 suggest evolutionary adaptations in immune function.
Abstract:
Complement component C3 was isolated from the plasma of cod (Gadus morhua L.) and halibut (Hippoglossus hippoglossus L.). Fast protein liquid chromatography (FPLC) techniques, involving ion exchange and gel filtration columns, were used. The purified proteins were analysed by SDS-PAGE which showed a two-chain structure, alpha- and beta-chains, as seen in higher vertebrates. Both proteins had intra-chain thioesters located within their alpha-chains and N-terminal amino acid sequencing confirmed their identity with reference to known C3 amino acid sequences from other species. Specific antibodies were prepared against cod and halibut C3 and tested in Western blotting on sera and purified C3. The proteolytic fragmentation of C3 was tested with trypsin, pepsin, papain and the extracellular product (ECP) from the bacterium Aeromonas salmonicida ssp. achromogenes (Asa). Both trypsin and papain were successful in cleaving C3 whereas pepsin and ECP had no effect. Carbohydrate moieties were detected in the alpha- and beta-chains of cod and halibut C3 and N-linked oligosaccharides were removed from the C3 with PNGase treatment, revealing a difference in C3 glycosylation between the two species.
