Ceruloplasmin is a positive acute-phase protein, belongs to family of multinuclear copper oxidase. It has a potent immune defence role in microbial infection or tissue injury

1. Ceruloplasmin: Its Role in the
Physiological & Pathological Processes in
fish
Rajive Kumar Brahmchari

2. Introduction
• hepatic protein, belongs to family of multinuclear copper
oxidase
• glycoprotein of α2 globulin fraction of the serum
• contains 95% of the copper in the plasma
• multitude of binding sites – for Cu, Fe2+, Fe3+, Na+ and Ca2+
• regulates E0
o- E0
r, transport & utilization of iron
• positive acute-phase protein (APP)
[An APP whose plasma conc. ↑ 25% during inflammatory conditions
or cell injury are called  positive APP
or when they decrease in similar proportion  negative APP]

3. • 1st described human caruloplasmin was isolated in 1944
– consists of a single chain of 1046 amino acids forming six
structural domains
– Mol. Wt. – 132 kDa
• copper atoms bound to ceruloplasmin - half in Cu2+ state
& half in Cu+ state
• Cp- larger than albumin, and is of same size as antibodies

4. Structure & Evolution
Ceruloplasmin protein
Cupredoxin
• homologous to cupredoxin
• 3-D structure -- reveals six domains
arranged in a ternary symmetry
– domains 1 and 2, 3 and 4, and 5 and 6 interact
with each other.
– domains 2, 4, and 6 bind type I blue copper sites
• possess multiple copper sites
classified as type I, type II, and type
III sites
– Type I cupredoxin - with strong absorption
maximum at 610 nm
– Type 2 with no specific absorption, and
– Type 3 with a maximum absorption at 330 nm
• type II and type III copper ions forms
the trinuclear copper cluster --- play
role in oxidation of Fe2+ into Fe3+

5. • found to be conserved throughout vertebrate evolution
• evolutionary precursor → Cupredoxin
• Presence in green algae, some archaea & bacterial sp. →
prokaryotic origin
• There are 20 copper-binding residues in Cp gene -- 16 are
conserved in fish and mammals
• At AA position 1011 – an Ile in most fish sp. such as zebra fish,
rohu, nile tilapia
– Leu in channel catfish & mammals
• But, channel catfish protein differs from human & rest fishes
at AA position 721 with His to Asp transition

6. Predicted 3D model of L. rohita
ceruloplasmin protein in I-TASSER. C-
score=1.02, Estimated TM-score=
0.85±0.08, Estimated RMSD=6.8 ±
4.0 Å.
Predicted 3D structure of Cp protein
showing ligand binding sites for (a)
Monooxygen, (b) Doubleoxygen, (c)
Copper and (d) Calcium with C-scores
0.21, 0.09, 0.17 and 0.11

7. • Rohu Cp -- binding sites for only copper and calcium
• human Cp -- iron, cobalt, and sodium binding sites
• Gene ontology analysis
… rohu Cp categorize into copper and chaperon binding
protein having ferroxidase and oxidoreductase activity
… human Cp – many other biological activities

8. Phylogenetic analysis of rohu Cp constructed using neighbor-joining method in bootstrap test
based on a ClustalW multiple sequence alignment of aa sequences

9. Nucleotide sequence
• characterized in many fish species
– zebrafish (Danio rerio),
– rohu (L. rohita),
– Indian medaka (Oryzias melastigama),
– catfish (Ictalurus punctatus),
– icefish (Chionodraco rastrospinosus),
– goldfish (Carassius auratus) and
– yellow perch (Perca flaviscens)
• fish Cp gene → complete coding sequence >3.2 kb &
codes for 1000 - 1100 AA residues.
• Human Cp gene → codes for 1046 amino acid residues

10. • total Cp gene sequence in fish ≈13 kb (20 exons, 19 introns)
• 19 exons are similar to mammalian species
– except exon 9 which is not found in higher vertebrates
• In human, in different tissue types --different isoforms of
Cp gene described arisen from
– use of different polyadenylation site within the 3′UTR region or
– by alternative splicing of exons 18, 19 and 20
• no such isotypic variants detected in fish
Schematic diagram of ceruloplasmin gene structure
Exons - black boxes, non-coding regions - white boxes. Values above boxes - length of the region in base pairs. Values in parentheses - length of introns. Double slashes - non-proportional representation of the introns.

11. Functions
In mammals
• expression in human -- all major lymphocytes especially
NKcells
• Membrane bound forms -- anchored with glycosyl-
phosphatidylinositol (GPI-CP)
• --found in surface of * mammalian astrocytes,
* rat leptomeningeal cells and
* Sertoli cells
• carries 70% of the total copper in human plasma
• half-life – 5.5 days

12. Multifunctional activity of ceruloplasmin gene (cp) in mammalian tissues
 Copper IoI signalling molecule NO
 helps in utilization of copper
- biosynthesis of cytochrome C
oxidase
- transfers copper to superoxide
dismutase
 potent inhibitor of MPO enzyme
 acts as a serum ferroxidase -- IoI toxic
plasma Fe2+ to Fe3+ transported by
transferrin

13. Abnormal Cp Level/ Clinical applications
• Decreased levels
– Deficiency in Cp level -- mutation in Cp gene →
aceruloplasminemia
 characterized by iron overload in the brain, liver, pancreas and retina
leading to iron deficient anemia
 Wilson's disease and Menkes disease
– ↓ level also observed -- hepatic disease, nephrotic syndrome,
malabsorption and malnutrition (trace metal deficiency)
• Elevated level
– During pregnancy
– In acute & chronic infection (late acute phase protein)
• treat few neurodegenerative diseases → Alzheimer's,
Parkinson's disease → binding of increased free copper
and iron causing production of neurotoxins H2O2

14. In fish
❶ Ontogenic & tissue level expression
• not a parentally blessed protein
• Fish synthesize with the development of liver
• In rohu → 9 h post-fertilization and increased there after
• In zebrafish →16 h post-fertilization and later in early
hepatic cells in the yolk sac indicate liver development
• In Japanese medaka →3 day post-fertilization, exact hour
not being looked into
• Liver -- main tissue in fish
• Mild expression -- catfish spleen, brain, gill, intestine,
muscle, skin and stomach tissues
• In rohu -- also in kidney, eye and heart

15. ❷ immune response to pathogens
Pangasiodon hypophthalmus Thaparocleidus sp. Blood dependent on the
parasite load
• ↓ during massive
infection
• ↑ infection intensity
abated
*
*
Epizootic hematopoietic necrosis

16. ❸indirect marker for disease resistance
• Serum Cp →parameter to monitor/ select resistant fish
stocks
– established in rohu against A. hydrophila infection
– Serum Cp → positive correlation with survival (%) of different
full sib families of rohu challenged with A. hydrophila

17. ❹ During inflammatory response & toxicity
• ↑ Cp level in serum -- used as a biomarker for inflammation
• during inflammatory response in fish -- estimated as ferroxidase
activity, prominent after pathogen infection in the trout and
common carp
• anti-inflammatory role of Cp during hormonal, drug and heavy
metal mediated toxicity
• ↑ plasma Cp level noticed
–administered with prolactin and growth hormones
–fed in a diet with different doses of sulfamerazine
• ↑ expression to waterborne arsenic exposure
• dose-dependent reduction in Cp level in Triclosan
exposure

18. ❺Copper & ceruloplasmin
• Metals induces oxidative stress in aquatic organisms
– by producing ROS
– by impairing antioxidant defences
• Metal-binding proteins → ferritin, ceruloplasmin,
metallothioneins play major role in oxidative defence
• Cp acts as an antioxidant in plasma through its
ferroxidase activity
• Ceruloplasmin expression - highly modulated by copper
exposure
• Pre-exposure to copper -- ↑ Cp activity in fish serum

19. Estimation
• checked at → transcript level & protein level
• specific primers available → rohu, icefish and catfish
• At transcript level,
– by relative quantification
– by absolute quantification
• At protein level using fish serum
– measured as p-phenylenediamine (PPD) oxidase activity
following a standard protocol

20. Modulation of Cp expression in fish for health benefits
• Dietary Cu can modulate the expression → dose-dependent
• Hedayati et al. (2016) reported a lower copper absorption by
fish in the nano-copper suspension compared to ionic
copper
– Copper in ionic form - adverse effects
• ionic copper absorbed via gill, nano-copper absorbed via gut
• nano-copper in fish feed -- much less dose ↑ expression

21. Thoughts going forward
• potent immune defence role in microbial infection or
tissue injury
• multitude of binding sites provides multifunctional
activity,
– Inflammatory response
– hormonal,
– drug and heavy metal mediated toxicity
• potent antioxidant
• Indirect marker for selection for disease resistance
fish or biomarker of inflammation

22. References
• P. Bielli & C. Lilia, Structure to function relationships in ceruloplasmin: a 'moonlighting‘
protein. Cellular and Molecular Life Sciences CMLS 59.9 (2002): 1413-1427.
• S. Das, & P.K, Sahoo, Ceruloplasmin, a moonlighting protein in fish. Fish & shellfish
immunol. 82 (2018) 460-468.
• H. Liu, E. Peatman, W. Wang, J. Abernathy, S. Liu, H. Kucuktas, J. Terhune, D.H. Xu, P. Klesius,
Z. Liu, Molecular responses of ceruloplasmin to Edwardsiella ictaluri infection and iron
overload in channel catfish (Ictalurus punctatus), Fish Shellfish Immunol. 30 (2011) 992–997.
• P.K. Sahoo, S. Das, K.D. Mahapatra, J.N. Saha, M. Baranski, J. Ødegård, N. Robinson,
Characterization of the ceruloplasmin gene and its potential role as an indirect marker for
selection to Aeromonas hydrophila resistance in rohu, Labeo rohita, Fish Shellfish Immunol.
34 (2013) 1325–1334.
• Zena Leah Harris, Ceruloplasmin. Clinical and Translational Perspectives on Wilson Disease.
Academic Press (2019) 77-84.
• Bera, K. K., Kumar, S., Paul, T., Prasad, K. P., Shukla, S. P., & Kumar, K.. Triclosan induces
immunosuppression and reduces survivability of striped catfish Pangasianodon
hypophthalmus during the challenge to a fish pathogenic bacterium Edwardsiella
tarda. Environmental research (2020), 186, 109575.