Ceruloplasmin in fish

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, Fe
2+
, Fe
3+,
Na
+
and Ca
2+
•regulates E
0
o
- E
0
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. •1
st
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 Cu
2+
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 Fe
2+
into Fe
3+

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 Fe
2+
to Fe
3+
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 H
2O
2

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.