| Sažetak (engleski) | X
INTRODUCTION
Red deer (Cervus elaphus) is economically and culturally one of the most important
wildlife and game species, not only in Croatia, but also in the world context. From a scientific
point of view, red deer is particularly interesting in terms of phylogenetic analyses, especially
in case of decreasing populations, the settlement of other individuals with different genetic
characteristics, but also through the anthropogenic impact on the original populations. In
addition, the deer occupies a special place in terms of the production of high-valuable meat and
velvet antlers, both due to their medicinal potential and as a potential model for the study of
tumors. Finally, scientists are also interested in deer from the point of infectious and parasitic
diseases, and of other diseases that potentially can be transmitted to humans and domestic
animals. With the advancement of molecular methods, it is understandable that proteomic
research becomes increasingly used in the previously mentioned research on animals. The
largest part of these studies has been done on the proteome of growing antlers, followed by
research on diseases and meat. Knowledge of proteomes of a healthy population is a
prerequisite for all these studies. This is especially important if known that the implementation
of classic research on wild animals with the creation of control and experimental (case) groups
is extremely difficult, especially when it comes to a large animal model. Precisely for the stated
reason, it is expected that this research will offer a basis for further studies in the field of deer
proteomics, where initial data of serum and liver proteomes are necessary.
REVIEW OF THE CURRENT KNOWLEDGE
According to scientific classification, red deer (Cervus elaphus), belongs to the order
Artiodactyla (ungulates), suborder Ruminantia (ruminants), family Cervidae (deer, red deer),
genus Cervus (deer) and species Cervus elaphus. From a molecular point of view, the
classification is somewhat different, so deer are included in the superorder Laurasiatheria, the
order Cetartiodactyla and the family Cervidae. In addition, deer are divided into true and false
deer, and can be classified into four subfamilies: Hydropotinae (monotypic species),
Capreolinae (roe deer, white-tailed deer, reindeer and elk), Muntinacinae (mullet), Cervinae
(axis deer, red deer, sika...). Historically speaking, red deer appeared in Europe most likely in
the Middle Pleistocene, that is, some 800,000 years ago. Those early forms are classified in the
paleosubspecies Cervus elaphus acoronatus. In addition to this, some other species are known XI
from the Middle Pleistocene period, so Cervus elaphus rianensis lived in today's Italy, and
Cervus elaphus siciliae lived in Sicily in the late Pleistocene. More recent studies, which are
based on genetic features, classify European deer into two lineages, western deer (elaphoid)
and eastern (maraloid). Geographically, we find the western line in the area of most of Europe,
North Africa and the western part of Asia. The eastern line lives in North America and the
eastern part of Asia. Studies on mitochondrial DNA revealed the existence of three clusters
made up of haplogroups A, B and C. Of these, haplogroup C inhabits mostly the Balkans.
Although there are reports of the presence of the so-called Pannonian deer (C. e. pannoniensis)
in the area of Slavonia and Baranja, recent studies show that the differences between the western
and eastern parts of Croatia are minimal. Red deer is one of the largest deer species that can
grow 170 to 250 cm in length, and reaches 120 to 150 cm in height. Body mass is between 140
and 300 kg. Females are smaller than males. Dental formula is I 0/3, C 1(0)/1, P 3/3, M 3/3,
comprising 34 permanent teeth in total (preceded by 22 deciduous teeth). Upper canines are
present only in males. The summer coat is reddish in colour, while ventral part of the abdomen
is whitish. The winter coat is thicker and darker (usually dark brown) in colour. The main
characteristics of this family are antlers, which are built entirely like bone. Antlers, with the
exception of reindeer/caribou, are worn exclusively by males, and only exceptionally by
females. Under the influence of hormones, the antlers grow and are shed every year, and this
cycle is strictly periodic in deer of temperate areas. Deer antlers are true, solid bone, made of
spongiosa and compacta. Antlers are growing from bony outgrowths on the forehead, which we
call pedicles. While growing, the antlers are covered with a specific skin (velvet). Following
the mineralization of the antlers, this skin is removed. As the age of the deer increases, the
antlers become larger, richer in tines, more massive and heavier, until the age of full maturity,
which according to older literature is around 12-14 years. Hinds are reaching sexual maturity at
the age of two. Pregnancy lasts 240-260 days. Before calving, the hinds separates from the herd
and usually gives birth to one calf with an average weight of around 15 kg, and returns to the
herd in about two weeks. Calves are born with spots that disappear by the end of summer. A
deer lives over 20 years in captivity, and about 10 to 12 years in nature. In habitats with fewer
predators, they can live more than 15 years.
The term "proteomics" appears officially for the first time in 1995, and is defined as a
comprehensive characterization and analysis of gene expression and the entire set of proteins
found in cells, tissue or organism called proteome. The growth of proteomics is based on the
development of DNA and protein sequence databases, advances in protein separation methods,
field of mass spectrometry, and the development of computer algorithms for searching
databases. Proteomics is based on the global analysis of all proteins within the system at once,
as well as on the possibility of studying the complex interactions of proteins. The proteome is
a dynamic and complex system subject to qualitative and quantitative changes. Therefore, the
study of the proteome becomes necessary for the understanding of biological systems and their
functioning. Proteomics combines knowledge on molecular biology, biochemistry and
bioinformatics. Mass spectrometry (MS) is an analytical method that provides information
about the mass of peptides or their fragments based on the ratio of mass and charge, and
provides the base for protein identification. Qualitative protein analysis determines the primary
amino acid sequence of a protein, while the quantitative analysis determines the representation
of a particular protein in the sample. Finally, proteomics provides a good insight into the biology
of an organism, given that the representation of proteins, for example, does not necessarily
correspond to the transcription and representation of genes. Also, the dynamics of proteins
during the life are not the same, nor are their turnover, interactions, placement, etc. All of the
above indicates that the various combinations and interactions of proteins on which the
phenotype depends are not always noticeable through the observation of the individual's
genome.
So far, a relatively small number of proteomic studies on deer tissues is available in the
scientific literature, mainly dealing with meat quality and antler growth cycle. In the case of
antlers, the aim of studies was mainly to determine their regeneration potential, the role of stem
cells in that process, and the role of environmental factors in their growth and development. A
smaller proportion of studies was oriented on the reproductive cycle and diseases. A recent
study analysed changes in the liver proteome of red deer after infection with large American
fluke (Fascioloides magna) on a sample of 25 livers. In that research, 234 proteins were
identified with different representation between the control and experimental groups.
According to the authors, the research results point to the fact this infection is associated with
changes in fatty acid metabolism, oxidative stress and fibrosis.
HYPOTHESIS AND AIMS OF THE RESEARCH
The hypothesis of the research is that the application of the proteomic approach with
the help of mass spectrometry of high precision and sensitivity will enable the generation anhypothesis is that proteins participating in the immune response will predominate in the serum
due to the specificity of the species and living conditions; while proteins with a metabolic role
will predominate in the liver.
The main aims of the research are:
a) to perform a qualitative and semi-quantitative analysis of serum and liver proteins of red deer
(Cervus elaphus)
b) to characterise serum and liver proteins using bioinformatics methods (functional analysis
and classification of identified proteins with the help of a proteomic/genomic databases)
c) to compare serum and liver proteomes.
MATERIAL AND METHODS
The study was funded by the Croatian Science Foundation project IP 8963 "Hostparasite interaction: the relationship between three different hosts and Fascioloides magna
infection". The Committee for Ethics in Veterinary Medicine approIn this cross-sectional study, the sample size was calculated based on the meta-analysis
of literature data using online available statistical calculators. The database for cattle (Bos
taurus) was searched, as a ruminant model most closely related to deer, with well-researched
and edited gene databases. The health status of the deer was assessed based on the external
appearance, fattening condition, the appearance of the hair cover and behaviour of the
individual before the actual shooting, and after the shooting based on the presence of any visible
wounds, discharge from natural openings and after opening the individual based on the
macroscopic appearance of the organs. Only individuals that were declared healthy according
to the mentioned criteria were included in the further research. Blood samples (from large veins
of the neck or heart) and liver of red deer were collected immediately after the hunt, or, in the
shortest possible time. Blood was centrifuged directly in the field using a portable centrifuge,
and serum was separated and stored at -80 oC until analysis. Liver samples were collected
during evisceration of the animals. For further analysis, tissues were sampled only from those
livers in which no signs of invasion were observed. Tissue samples were labelled and stored at
-80 oC until further analysis.ved the research (Class:
640-01118-17/60, Reg. number: 251-61-44-18-02)
In this cross-sectional study, the sample size was calculated based on the meta-analysis
of literature data using online available statistical calculators. The database for cattle (Bos
taurus) was searched, as a ruminant model most closely related to deer, with well-researched
and edited gene databases. The health status of the deer was assessed based on the external
appearance, fattening condition, the appearance of the hair cover and behaviour of the
individual before the actual shooting, and after the shooting based on the presence of any visible
wounds, discharge from natural openings and after opening the individual based on the
macroscopic appearance of the organs. Only individuals that were declared healthy according
to the mentioned criteria were included in the further research. Blood samples (from large veins
of the neck or heart) and liver of red deer were collected immediately after the hunt, or, in the
shortest possible time. Blood was centrifuged directly in the field using a portable centrifuge,
and serum was separated and stored at -80 oC until analysis. Liver samples were collected
during evisceration of the animals. For further analysis, tissues were sampled only from those
livers in which no signs of invasion were observed. Tissue samples were labelled and stored at
-80 oC until further analysis.
Proteomic analysis of serum and liver tissues was performed using a semi-quantitative
proteomic approach with isobaric tags that enable multiplexing - simultaneous analysis of six
samples according to the previously described protocol. Samples labelled with tandem mass
tags (TMT) were analysed using Dionex UltiMate 3000 RSLCnano system coupled to a Q
Exactive Plus mass spectrometer with a hybrid quadrupole and Orbitrap mass analyser by the
data dependent acquisition (DDA) procedure, in accordance with previous descriptions. The
SEQUEST algorithm within the software Proteome Discoverer was used for protein
identification and relative quantification. The Cervus elaphus database downloaded from
UniprotKB, which contained 19,262 entries, was searched according to the following
parameters: two missed trypsin cleavage sites allowed, an error tolerance of the measured mass
of precursors and fragments of 10 ppm and 0.05 Da, carbomidomethyl as a fixed peptide
modification, oxidation and TMT sixplex as dynamic peptide modifications. The percentage of
false discovery rate (FDR) during peptide identification was calculated using the Percolator
algorithm within the Proteome Discoverer. Proteins with at least two unique peptides and 1%
FDR were considered reliably identified and were used in further analyses. Proteins that were
present in at least 50% of the samples were included in statistical analysis. Since the majority
of identified proteins did not follow a normal distribution (as determined by the Shapiro-Wilk
test), the relative protein abundance is presented as median and interquartile range (1st and 3rd
quartiles). For bioinformatic analysis, the unique protein ID was first converted to the official
gene symbol using the UniProtKB ID mapping tool. Proteins for which there was no red deer
gene annotation or were listed as “uncharacterized protein” were replaced using the UniProt
BLAST tool with the closest matching ortholog from the Bos taurus database (at least 70%
match). Gene Ontology (GO) functional analysis and Kyoto Encyclopedia of Genes and
Genomes (KEGG) biological pathway analysis were performed using the ShinyGO tool. All
obtained terms had FDR < 0.05, where the nominal P value was obtained based on the
hypergeometric test. Analysis of biological pathways associated with the liver and serum
proteome was performed using the Reactome tool with the human genome as background and
pathways with FDR < 0.05 were selected as significant. The fold enrichment factor was defined
as the ratio of identified genes that belong to a certain GO term and the total number of genes
in the background. Western blot was used for the validation of selected proteins.
RESULTS
In total, 234 proteins were identified in deer serum using at least two unique peptides
and with 1% FDR. Only proteins that were present in at least 50% of the samples, i.e. 87 of
them, were included in the further statistical and bioinformatic processing. According to the
gene ontology for biological processes, the largest number of genes (N = 31) is included in the
process of "response to external stimulus", followed by (N = 29) "immunological response" and
"proteolysis". Most biological processes are closely related to the immune system. Gene
ontology for cellular localization showed that the vast majority of proteins belong to the
extracellular region (N = 56) or the extracellular space (N = 44). Individual proteins attached
to the cell membrane, respective parts of lipoprotein particles, complexes with fibrinogen and
hemoglobin, and the content of alpha granules of platelets secreted into the circulation were
also identified. The largest number of proteins belongs to the regulators of molecular functions
(N = 23), that is, to the regulation of enzymatic activities (N = 22), namely peptidases (N = 18)
and inhibitors of (endo)peptidases (N = 17). In addition to enzymatic regulation, serum proteins
participate in binding to signal receptors (N = 13), lipids (N = 9), and other molecules
(glycosaminoglycans, heparin, phospholipids and others). The Reactome analysis showed that
53 biological pathways are statistically significant (FDR < 0.05). After determining the
additional criterion that at least 10 genes are present in each biological pathway, this number
was reduced to 14. Most of the proteins identified and quantified in the serum proteome are
involved in the immune system (N = 68), i.e. the innate immune system (N = 43). Identified
proteins were further included in complement cascade (N = 24), degranulation of platelets (N
= 21) and neutrophils (N = 16), regulation of insulin-like growth factor (IGF) transport and
uptake (N = 18), and others. Some of the proteins are related to haemostasis (N = 34), such as
platelet adhesion, activation and aggregation (N = 21) and coagulation cascade (N = 12). Eleven
proteins are involved in the processes of binding and taking up ligands using scavenger
receptors. Fibrinolysis, complement activation, humoral immune response and humoral
immune response mediated by circulating immunoglobulins, regulation of coagulation and
hemostasis are distinguished as significantly enriched biological processes (based on the fold
enrichment) in the serum. Most of the enriched biological processes are related to the immune
system and the immune system response. KEGG database analysis determined that the majority
of proteins are related to the complement and coagulation cascade (N = 29).
Using a proteomic approach based on TMT labelling, 22 liver samples from healthy
deer were analysed. Analysis showed that a total of 493 proteins were identified using at least two unique peptides and with 1% FDR. Only proteins present in at least 50% of the samples,
i.e. 387 of them, were included in further statistical and bioinformatic processing. Gene
ontology for biological processes determined that the largest number of genes is involved in the
metabolism of small molecules (N = 110), metabolism of organic acids (N = 82), catabolic
processes (N = 74) and lipid metabolism (N = 53). It should also be noted the processes of
transport of organic substances (N = 41), and the creation of precursors of metabolites and
energy (N = 35). The results of the gene ontology analysis for cellular localization showed that
most proteins originate from mitochondria (N = 73) and endoplasmic reticulum (N = 54). The
Reactome analysis showed that 66 biological pathways were statistically significant (FDR <
0.05). With the criterion that there are at least 30 genes in each pathway, the number of
significant biological pathways was reduced to 19. The largest number of liver proteins is
involved in metabolic processes (N = 234), metabolism of amino acids and derivatives (N =
66), biological oxidation (N = 48), neutrophil degranulation (N = 45) and translation (N = 41).
Based on the fold enrichment, the significantly enriched biological processes in the liver are the
metabolic/catabolic processes of amino acids, small molecules and fatty acids, as well as
nucleotides, and compounds with purine nucleobases. Finally, seventeen proteins were found
to be common to serum and liver.
DISCUSSION
The majority of the proteins in the deer serum are involved in the immune response,
especially in the part of non-specific (innate) immunity. Among the serum proteins, acute phase
proteins were also found, representing a nonspecific and complex reaction of the organism to
infections, injuries, inflammatory processes, etc. Despite the uniform nature of the acute phase
response, there are numerous different features between various animal species. Diagnostically,
the most important acute phase proteins in ruminants are haptoglobin (Hp) and serum amyloid
A, followed by fibrinogen, ceruloplasmin, alpha-1-acid glycoprotein, alpha-1 antitrypsin,
lactoferrin, and as acute phase negative proteins - albumin, transferrin and transthyretin. Studies
on the acute-phase response in reindeer are relatively rare, and include reports on elevated
concentrations of fibrinogen in diseased reindeer, which is in line with studies on red deer,
where its role as an acute-phase reactant was also observed. In addition, the serum Hp of red
deer showed a high predictive value in the diagnosis of proliferative tuberculosis. In the case of
other deer species, the determination of the physiological values of red deer Hp and fallow deer
fibrinogen should be highlighted. Some of the identified acute phase proteins in the serum proteome inhibit proteases released by phagocytes or pathogens in order to reduce damage to
healthy tissue (α1-anti-trypsin, α2- macroglobulin). Others, such as haptoglobin, have the
ability to scavange and bind metabolites that are released by cellular decomposition, while for
example alpha-1-acid glycoproteins has antibacterial activity.
The second most abundant protein system in deer serum is related to haemostasis. Thus,
coagulation factors, anticoagulants, components of the fibrinolytic system and vascular
endothelium are present in the serum proteome. Processes such as fibrinolysis, complement
activation, humoral immune response and response mediated by circulating immunoglobulins,
regulation of coagulation and haemostasis were shown to be significantly enriched biological
processes in red deer serum. Finally, changes in the composition of the blood can also indicate
environmental pollution. Biochemical analysis of doe serum showed exposure to pesticides and
artificial fertilizers used in agriculture. Therefore, the living conditions and habitat of red deer
determine the dominance of immune system-related proteins in the serum proteome.
The results of gene ontology analysis for liver proteins showed that most of the proteins
originate from mitochondria and endoplasmic reticulum. The most represented are
oxidoreductases, while largest proportion of proteins is involved in metabolic processes,
translation and elongation, and biological oxidation. Metabolism is the most dominant category
present in the red deer liver proteome. Thus, proteins participating in the metabolism were
identified. A large part of the red deer liver proteome consists also of ribosomal proteins (10
proteins of the small 40S ribosomal subunit and 11 proteins of the large 60S ribosomal subunit),
nucleosome components (histones H2B and H3), ribonucleoproteins (HNRNPA3, HNRNPL,
HNRNPH1), proteasome subunits (PSMB4, PSMB6), elongation factors, proteins associated
with ubiquitination (USP5, UBA1). In the red deer liver proteome, several members of the heat
shock protein family that play a role in enhancing the host's immune response have been
identified, which was also shown in the response to infection with Fascioloides magna fluke.
Among the proteins significantly represented in the liver proteome, members of the cytochrome
P450 system with other proteins are responsible for the detoxification of xenobiotics. Enzymes
involved in the metabolism of xenobiotics were shown to be significantly altered in deer
infected with F. magna. Additionally, in the liver proteome, a multitude of proteins related to
the immune response were identified, and 45 of them are included in the prominent biological
pathway "neutrophil degranulation". Among them are acute phase proteins (haptoglobin, alpha-
1-acid glycoprotein, A1BG, transferrin, ceruloplasmin), components of the antioxidant system
(peroxiredoxins, glutathione-S-transferase, glutathione reductase, superoxide dismutase, cytochrome P450, paraoxonase), and other proteins that act on the modulation of the immune
response.
In conclusion, proteomic analysis of liver and serum can contribute to a better
characterization of the red deer proteome, identification of biomarkers and molecular
description of organ specialization. While the serum proteome is the most informative from a
medical point of view, because almost all cells directly or indirectly communicate with the
blood, and many after damage or death release their contents into the bloodstream; the liver
proteome, as the main chemical factory of the organism, provides important information about
metabolic changes in animals. |
