Camel Proteins Research Articles

Corrigendum: Camel proteins and enzymes: a growing resource for functional evolution and environmental adaptation

Corrigendum: Camel proteins and enzymes: a growing resource for functional evolution and environmental adaptation

Cloning and Molecular Characterization of HSL and Its Expression Pattern in HPG Axis and Testis during Different Stages in Bactrian Camel.

Hormone-sensitive lipase (HSL) is a key enzyme in animal fat metabolism and is involved in the rate-limiting step of catalyzing the decomposition of fat and cholesterol. It also plays an important regulatory role in maintaining seminiferous epithelial structure, androgen synthesis and primordial germ cell differentiation. We previously reported that HSL is involved the synthesis of steroids in Bactrian camels, although it is unclear what role it plays in testicular development. The present study was conducted to characterize the biological function and expression pattern of the HSL gene in the hypothalamic pituitary gonadal (HPG) axis and the development of testis in Bactrian camels. We analyzed cloning of the cDNA sequence of the HSL gene of Bactrian camels by RT-PCR, as well as the structural features of HSL proteins, using bioinformatics software, such as ProtParam, TMHMM, Signal P 4.1, SOPMA and MEGA 7.0. We used qRT-PCR, Western blotting and immunofluorescence staining to clarify the expression pattern of HSL in the HPG axis and testis of two-week-old (2W), two-year-old (2Y), four-year-old (4Y) and six-year-old (6Y) Bactrian camels. According to sequence analysis, the coding sequence (CDS) region of the HSL gene is 648 bp in length and encodes 204 amino acids. According to bioinformatics analysis, the nucleotide and amino acid sequence of Bactrian camel HSL are most similar to those of Camelus pacos and Camelus dromedarius, with the lowest sequence similarity with Mus musculus. In adult Bactrian camel HPG axis tissues, both HSL mRNA and protein expression were significantly higher in the testis than in other tissues (hypothalamus, pituitary and pineal tissues) (p < 0.05). The expression of mRNA in the testis increased with age and was the highest in six-year-old testis (p < 0.01). The protein expression levels of HSL in 2Y and 6Y testis were clearly higher than in 2W and 4Y testis tissues (p < 0.01). Immunofluorescence results indicate that the HSL protein was mainly localized in the germ cells, Sertoli cells and Leydig cells from Bactrian camel testis, and strong positive signals were detected in epididymal epithelial cells, basal cells, spermatocytes and smooth muscle cells, with partially expression in hypothalamic glial cells, pituitary suspensory cells and pineal cells. According to the results of gene ontology (GO) analysis enrichment, HSL indirectly regulates the anabolism of steroid hormones through interactions with various targets. Therefore, we conclude that the HSL gene may be associated with the development and reproduction of Bactrian camels in different stages of maturity, and these results will contribute to further understanding of the regulatory mechanisms of HSL in Bactrian camel reproduction.

Camel Proteins and Enzymes: A Growing Resource for Functional Evolution and Environmental Adaptation.

In less agroecological parts of the Asian, Arabian, and African deserts, Camelus dromedarius play an important role in human survival. For many years, camels have been employed as a source of food, a tool of transportation, and a means of defense. They are becoming increasingly important as viable livestock animals in many desert climates. With the help of camel genetics, genomics and proteomics known so far, this review article will summarize camel enzymes and proteins, which allow them to thrive under varied harsh environmental situations. An in-depth study of the dromedary genome revealed the existence of protein-coding and fast-developing genes that govern a variety of metabolic responses including lipid and protein metabolism, glucoamylase, flavin-containing monooxygenase and guanidinoacetate methyltransferase are other metabolic enzymes found in the small intestine, liver, pancreas, and spleen. In addition, we will discuss the handling of common medications by camel liver cytochrome p 450, which are different from human enzymes. Moreover, camels developed several paths to get optimum levels of trace elements like copper, zinc, selenium, etc., which have key importance in their body for normal regulation of metabolic events. Insulin tolerance, carbohydrate and energy metabolism, xenobiotics metabolizing enzymes, vimentin functions, behavior during the rutting season, resistance to starvation and changes in blood composition and resistance to water loss were among the attractive aspects of camel enzymes and proteins peculiarities in the camels. Resolving the enigma of the method of adaptation and the molecular processes linked with camel life is still a developing repository full of mysteries that need additional exploration.

Effect of pH on the physicochemical characteristics and the surface chemical composition of camel and bovine whey protein’s powders

This study investigated the effect of pH on the denaturation extent, the surface chemical composition, the water sorption isotherm and the glass transition temperature of camel and bovine whey protein’s powders. The LC-MS analysis indicated that the β-Lactoglobulin was the most denatured protein in bovine whey powders regardless the pH value, while this protein was totally absent in camel whey. The α-Lactalbumin was relatively heat stable after drying and predominated the powder surface (X-ray photoelectron spectroscopy results) in both camel and bovine whey powders regardless the pH (neutral (6.7) or acidic (4.3 and 4.6)). Analysis of the water sorption isotherms indicated that decreasing the pH induced the increase of the water activity of lactose crystallization for camel and bovine whey powders. Finally, decreasing the pH led to the decrease of the glass transition temperature of camel and bovine whey powder (at 0.13, 0.23, and 0.33 of water activity).

Comparative study on emulsifying and physico-chemical properties of bovine and camel acid and sweet wheys

Main goal of this research was to examine the emulsifying properties of acid and sweet wheys extracted from bovine and camel fresh milks after heating at 70 and 90 °C for 30 min at laboratory scale. Specifically, emulsifying properties (emulsification stability (ESI) and activity (EAI) indexes) and the physico-chemical characteristics (surface hydrophobicity, ζ-potential, interfacial tension and denaturation rate) of wheys were assessed. Maximum EAI and ESI were found for sweet wheys (EAI~ 2 m2/g; ESI~ 65%), with higher EAI values for the camel whey. This behavior was explained by the strongest electrostatic-repulsive forces between oil droplets under conditions away from the isoelectric-point of proteins in agreement with the ζ-potential measurements.Findings indicate that heating affected the physico-chemical properties of camel and bovine whey proteins in acidic conditions by increasing surface hydrophobicity and the ability to reduce the interfacial tension. These results confirmed the protein-protein aggregation of heated acid wheys as proved by electrophoreses.

Purification of Lactoferrin from Camel colostrum and Protein Profiles of Camel and Bovine Milk

Human and bovine lactoferrin have been studied extensively, but very few reports exist on camel (Camelus dromedarius) lactoferrin. This iron-binding glycoprotein is present primarily in milk. It has been shown to be involved in various physiological and protective functions, including homeostasis and cell proliferation, and it has antibacterial, antifungal, antiviral, antioxidant, immunomodulatory and anticancer activities. This study aimed to compare the protein profiles of camel milk and bovine milk by gel electrophoresis and to isolate camel lactoferrin from colostrum. Camel milk proteins profile lacked β-lactoglobulin. Lactoferrin was isolated from colostrum by cation exchange chromatography and identified by its molecular weight after gel electrophoresis as a single band of about 78 kDa, demonstrating the purity of the isolated protein. The study demonstrates a simple one-step method to purify lactoferrin from camel colostrum.

Stability of antibodies and proteins in camel whey powder treated by Gamma-irradiation during radurisation process

Camel milk and milk derived products have a growing potential in pharmacy, food and biotechnology industry. Its therapeutic value has been investigated and documented in many studies before. The contamination of camel whey powder during manufacturing and transportation may impact economy; re-pasteurisation could cause dramatic effect on antibodies and proteins of camel whey powder. Radurisation (0.4 to 10 kGy) process could be a choice in sterilising camel whey powder. This study aimed to investigate the stability of camel antibodies and proteins in whey powder before and after Gamma-irradiation. Efficacy of radiation dose on microbial content was also studied. Four batches of hyper immune camel whey powder containing antibodies against Propionibacterium acnes were subjected to six Gamma-radiation doses (1.5, 3, 4.5, 6, 7.5 and 9 kGy). Results revealed marked decline in the total microbial count (3-5 log cycles) after escalating the doses of irradiation. Minor effect on antibody activity (7.4 % reduction) was observed after treating the whey by 9 kGy. Moreover, the concentration of total camel IgG was reduced by 13% using the same irradiation dose. In addition, no effect on major whey proteins (Lactoferrin, Casein, and α-lactoalbumin) was revealed in protein identification using SDS-PAGE. In conclusion, using 9 kGy dose of Gamma-irradiation for sterilising camel whey powder is effectively reducing bacterial contamination with limited effects on antibodies activity and protein stability.

Divergent Anticancer Activity of Free and Formulated Camel Milk α-Lactalbumin

ABSTRACTAlpha-lactalbumin (α-LA), a small milk calcium-binding globular protein, is known to possess noticeable anticancer activity, which is determined by the ability of this protein to form complexes with oleic acid (OA). To date, in addition to human and bovine α-LA, the ability to form such anti-tumor complexes with OA was described for goat and camel α-LA. Although the mechanisms of the anticancer activity of human and bovine α-LA are already well-studied, little is currently known about the anticancer action of this camel protein. The goal of this study was to fill this gap and to analyze the anticancer and pro-apoptotic activities of camel α-LA in its free form (α-cLA) and as an OA-containing complex (OA-α-cLA) using four human cancer cell lines, including Caco-2 colon cancer cells, PC-3 prostate cancer cells, HepG-2 hepatoma cells, and MCF-7 breast cancer cells as targets. The anti-tumor activities of OA-α-cLA and α-cLA were analyzed using MTT test, annexin/PI staining, cell cycle analysis, nuclear staining, and tyrosine kinase (TK) inhibition methods. We show here that the OA-α-cLA complex does not affect normal cells but has noticeable anti-cancer activity, especially against MCF-7 cells, thus boosting the anticancer activity of α-cLA and improving the selectivity of OA. The OA-α-cLA complex mediated cancer cell death via selective induction of apoptosis and cell-cycle arrest at lower IC50 than that of free α-cLA by more than two folds. However, OA induced apoptosis at higher extent than OA-α-cLA and α-cLA. OA also caused unselective apoptosis-dependent cell death in both normal and cancer cells to a similar degree. The apoptosis and cell-cycle arresting effect of OA-α-cLA may be attributed to the TK inhibition activity of OA. Therefore, OA-α-cLA serves as efficient anticancer complex with two functional components, α-cLA and OA, possessing different activities. This study declared the effectiveness of OA-α-cLA complex as a promising entity with anticancer activity, and these formulated OA-camel protein complexes constitute an auspicious approach for cancer remedy, particularly for breast cancer.

Foaming and adsorption behavior of bovine and camel proteins mixed layers at the air/water interface

The aim of this work was to examine foaming and interfacial behavior of three milk protein mixtures, bovine α-lactalbumin-β-casein (M1), camel α-lactalbumin-β-casein (M2) and β-lactoglobulin-β-casein (M3), alone and in binary mixtures, at the air/water interface in order to better understand the foaming properties of bovine and camel milks. Different mixture ratios (100:0; 75:25; 50:50; 25:75; 0:100) were used during foaming tests and interfacial protein interactions were studied with a pendant drop tensiometer. Experimental results evidenced that the greatest foam was obtained with a higher β-casein amount in all camel and bovine mixtures. Good correlation was observed with the adsorption and the interfacial rheological properties of camel and bovine protein mixtures. The proteins adsorbed layers are mainly affected by the presence of β-casein molecules, which are probably the most abundant protein at interface and the most efficient in reducing the interfacial properties. In contrast of, the globular proteins, α-lactalbumin and β-lactoglobulin that are involved in the protein layer composition, but could not compact well at the interface to ensure foams creation and stabilization because of their rigid molecular structure.

α-Lactalbumin: Of Camels and Cows.

Since camel milk has been attributed with various medicinal properties not found in bovine milk, we are systematically examining the differences between different proteins in bovine and camel milk. The purpose of this study is to investigate the structural differences between the bovine and camel α- lactalbumins. α-Lactalbumin is a highly abundant protein present in the milk of all mammalian species. Here we found several structural differences between bovine and camel α-lactalbumins: camel protein is more stable towards thermal and pHmediated denaturation but less stable towards guanidine hydrochloride-mediated unfolding, aggregates faster and is predicted to be more disordered than bovine α- lactalbumin.

Proteomic profiling of camel and cow milk proteins under heat treatment

Cow and camel milk proteins before and after heat treatment at 80°C for 60min were identified using LC/MS and LC–MS/MS following monodimensional electrophoresis. The database used for the identification of camel and cow proteins was set from http://www.uniprot.org/. The obtained results showed that, after heating, camel milk at 80°C for 60min, camel α-lactalbumin (α-la) and peptidoglycan recognition protein (PGRP) were not detected while camel serum albumin (CSA) was significantly diminished. When heating cow milk at 80°C for 60min, α-lactalbumin (α-la) and β-lactoglobulin (β-lg) were not significantly detected. Moreover, 19 protein bands from SDS-PAGE were analyzed and a total of 45 different proteins were identified by LC–MS/MS. Casein fractions were kept intact under a heat treatment of 80°C during 60min of both camel and cow milks. Camel and bovine whey proteins were affected by a heat treatment of 80°C for 60min.

Camel milk: alternative milk for human consumption and its health benefits

Purpose – Camel as a livestock plays an important role in desert ecosystem and its milk has potential contribution in human nutrition in the hot and arid regions of the world. This milk contains all the essential nutrients as found in other milk. Fresh and fermented camel milk has been used in different regions in the world including India, Russia and Sudan for human consumption as well as for treatment of a series of diseases such as dropsy, jaundice, tuberculosis, asthma and leishmaniasis or kala-azar. The present paper aims to explore the possibility of camel milk as an alternative milk for human consumption. Design/methodology/approach – Recently, camel milk and its components were also reported to have other potential therapeutic properties, such as anti-carcinogenic, anti-diabetic, anti-hypertensive and renoprotective potential; and for autism, and has been recommended to be consumed by children who are allergic to bovine milk. Findings – It has also been reported to alleviate oxidative stress and lipid peroxidation in rats. Camel milk differs from bovine milk in composition. It contains low total solids and fat; however, proteins and lactose are in equal amount but of higher quality than cow milk. Because of the high percentage of β-casein, low percentage of α-casein, deficiency of β-lactoglobulin and similarity of the immunoglobulins, it become safer for persons who are allergic to bovine milk. It contains protective proteins in higher amount which contributes to its functionality. The fermentation and enzymatic hydrolysis of camel protein produce different types of bioactive peptides which exerts different activity in in vitro and in vivo conditions. Originality/value – Because of its unique quality and functionality, this milk has potential application in management of different diseases and application in food industries.

Fouling Behavior of Camel and Cow Milks Under Different Heat Treatments

Using a developed laboratory-scale device, different heat treatment conditions were applied on camel and cow milks. After each fouling experiment, photos of stainless steel plates were taken and dry deposit weights were determined. The thermal behavior of camel and cow proteins was studied by electrophoresis (sodium dodecyl sulphate polyacrylamide gel electrophoresis, SDS-PAGE), differential scanning calorimetry (DSC), and free thiol group concentration evolution. The obtained results have shown that heating both camel and cow milks at 70 °C for 2 h generates deposit formation. The fouling rate was more important when heating camel milk than after heating cow milk for all heat conditions except at 90 °C for 2 h. Electrophoresis patterns indicated that after heating camel milk at 90 °C, α-lactalbumin (α-la), camel serum albumin (CSA), and κ-casein bands decreased. Bovine serum albumin (BSA) disappeared from the electrophoresis patterns after heating cow milk at 70 °C while β-lactoglobulin (β-lg) and α-la bands disappeared only at 90 °C. DSC thermograms of camel milk showed that the denaturation temperature of camel proteins is 77.8 °C, lower than that of cow proteins which is 81.7 °C. The results of free thiol group evolution versus temperature and heating time showed that camel protein denaturation starts between 70 and 80 °C. However, for cow milk, the whole denaturation phenomenon happens after heat treatment at 70 °C for 30 min.

Identification by FT-ICR-MS of Camelus dromedarius α-lactalbumin variants as the result of nonenzymatic deamidation of Asn-16 and Asn-45

Nonenzymatic deamidation of asparaginyl residues can occur spontaneously under physiological conditions principally when a glycyl residue is at the carboxyl side of Asn and leads to formation of aspartyl and isoaspartyl residues. This modification can change the biological activity of proteins or peptides and trigger an auto-immune response. The α-lactalbumins of members of the Camelidae family are the only of described α-lactalbumins that carry two AsnGly sequences. In the present study, high-resolution mass spectrometry, which enables accurate mass measurement has shown that Asn16 and Asn45 underwent a nonenzymatic deamidation, the sequence Asn45–Gly46 being deamidated spontaneously at near-neutral and basic pH and Asn16–Gly17 rather at basic pH. The 16–17 sequence was probably stabilized at near-neutral pH by hydrogen bonds according to the molecular modelisation performed with the camel protein.

Through the eye of an electrospray needle: mass spectrometric identification of the major peptides and proteins in the milk of the one‐humped camel (Camelus dromedarius)

The milk of the one-humped camel (Camelus dromedarius) reportedly offers medicinal benefits, perhaps because of its unique bioactive components. Milk proteins were determined by (1) two-dimensional gel electrophoresis and peptide mass mapping and (2) liquid chromatography-tandem mass spectrometry (LC-MS/MS) following one-dimensional polyacrylamide gel electrophoresis. Over 200 proteins were identified: some known camel proteins including heavy-chain immunoglobulins and others exhibiting regions of exact homology with proteins from other species. Indigenous peptides were also identified following isolation and concentration by two strategies: (1) gel-eluted liquid fraction entrapment electrophoresis and (2) small-scale electrophoretic separation. Extracts were analyzed by LC-MS/MS and peptides identified by matching strategies, by de novo sequencing and by applying a sequence tag tool requiring similarity to the proposed sequence, but not an exact match. A plethora of protein cleavage products including some novel peptides were characterized. These studies demonstrate that camel milk is a rich source of peptides, some of which may serve as nutraceuticals.

Improvement of the Antimicrobial and Antioxidant Activities of Camel and Bovine Whey Proteins by Limited Proteolysis.

The compositions and structures of bovine and camel milk proteins are different, which define their functional and biological properties. The aim of this study was to investigate the effects of enzymatic hydrolysis of camel and bovine whey proteins (WPs) on their antioxidant and antimicrobial properties. After enzymatic treatment, both the antioxidant and the antimicrobial activities of bovine and camel WPs were improved. The significantly higher antioxidant activity of camel WPs and their hydrolysates as compared with that of bovine WPs and their hydrolysates may result from the differences in amounts and/or in accessibilities of antioxidant amino acid residues present in their primary structures and from the prevalence of alpha-lactalbumin and beta-lactoglobulin as proteolytic substrates in camel and bovine whey, respectively. The results of this study reveal differences in antimicrobial and antioxidant activities between WP hydrolysates of bovine and camel milk and the effects of limited proteolysis on these activities.

Identification of Multiple HLA-DR-Restricted Epitopes of the Tumor-Associated Antigen CAMEL by CD4+ Th1/Th2 Lymphocytes

CD4(+) Th cells play an important role in the induction and maintenance of adequate CD8(+) T cell-mediated antitumor responses. Therefore, identification of MHC class II-restricted tumor antigenic epitopes is of major importance for the development of effective immunotherapies with synthetic peptides. CAMEL and NY-ESO-ORF2 are tumor Ags translated in an alternative open reading frame from the highly homologous LAGE-1 and NY-ESO-1 genes, respectively. In this study, we investigated whether CD4(+) T cell responses could be induced in vitro by autologous, mature dendritic cells pulsed with recombinant CAMEL protein. The data show efficient induction of CAMEL-specific CD4(+) T cells with mixed Th1/Th2 phenotype in two healthy donors. Isolation of CD4(+) T cell clones from the T cell cultures of both donors led to the identification of four naturally processed HLA-DR-binding CAMEL epitopes: CAMEL(1-20), CAMEL(14-33), CAMEL(46-65), and CAMEL(81-102). Two peptides (CAMEL(1-20) and CAMEL(14-33)) also contain previously identified HLA class I-binding CD8(+) T cell epitopes shared by CAMEL and NY-ESO-ORF2 and are therefore interesting tools to explore for immunotherapy. Furthermore, two CD4(+) T cell clones that recognized the CAMEL(14-33) peptide with similar affinities were shown to differ in recognition of tumor cells. These CD4(+) T cell clones recognized the same minimal epitope and expressed similar levels of adhesion, costimulatory, and inhibitory molecules. TCR analysis demonstrated that these clones expressed identical TCR beta-chains, but different complementarity-determining region 3 loops of the TCR alpha-chains. Introduction of the TCRs into proper recipient cells should reveal whether the different complementarity-determining region 3 alpha loops are important for tumor cell recognition.

Induction of CAMEL/NY-ESO-ORF2-specific CD8+ T cells upon stimulation with dendritic cells infected with a modified Ad5 vector expressing a chimeric Ad5/35 fiber.

Delivery of the full-length tumor antigen might be more successful in immunotherapy than single peptides and has the advantage that patients no longer need to be selected for their HLA type. In this study, we tested the in vitro induction of CAMEL/NY-ESO-ORF2-specific T cells by dendritic cells infected with an adenovirus (Ad) type 5 vector containing the fiber shaft and knob of human serotype Ad35 (Ad5F35 vector). Our data show induction of CD8(+) T cells specific for the known HLA-A(*)0201-binding CAMEL/NY-ESO-ORF2(1-11) epitope by DC infected with Ad5F35-CAMEL, but not by DC pulsed with the recombinant CAMEL protein. In one healthy donor, even CD8(+) T cells specific for a new HLA-B7-binding CAMEL/NY-ESO-ORF2(46-54) epitope were raised. In conclusion, the in vitro induction of CAMEL/NY-ESO-ORF2-specific CD8(+) T cells in healthy donors by DC infected with Ad5F35-CAMEL strongly supports further investigation of the Ad5F35 vector as a vehicle for gene transfer into DC for the generation of tumor antigen-specific CD8(+) T cell responses in vivo.

CD4+ Th2 cell recognition of HLA-DR-restricted epitopes derived from CAMEL: a tumor antigen translated in an alternative open reading frame.

Tumor Ag NY-ESO-1 is an attractive target for immunotherapy of cancer, since both CD8(+) CTL and CD4(+) Th cells against NY-ESO-1 have been described. Moreover, NY-ESO-1 as well as the highly homologous tumor Ag LAGE-1 are broadly expressed in various tumor types. Interestingly, the NY-ESO-1 and LAGE-1 genes also encode for proteins translated in an alternative open reading frame. These alternatively translated NY-ESO-ORF2 and CAMEL proteins, derived from the NY-ESO-1 and LAGE-1 genes, respectively, have been demonstrated to be immunogenic, since CTL specific for these proteins have been isolated from melanoma patients. In this study a panel of advanced melanoma patients was screened for the presence of Th cells specific for the alternatively translated tumor Ags NY-ESO-ORF2 and CAMEL. PBMC of melanoma patients were stimulated for 4 days with mixes of overlapping peptides covering the entire NY-ESO-ORF2 and CAMEL protein sequences and were tested for the release of type 1 (IFN-gamma) and type 2 (IL-13) cytokines in ELISPOT assays. In three of 15 patients, T cells specific for two CAMEL peptides (CAMEL(71-92) and CAMEL(81-102)) could be detected. From one of these patients, CD4(+) T cell clones specific for CAMEL(81-102) could be generated. These clones recognized a naturally processed epitope presented in both HLA-DR11 and HLA-DR12 and produced high levels of IL-4, IL-5, and IL-13. In conclusion, this study shows the presence of Th cells specific for the alternatively translated tumor Ag CAMEL in melanoma patients and is the first report that describes the isolation of tumor Ag-specific CD4(+) Th 2 clones.

Purification and partial characterization of camel ( Camelus Dromedarius) ceruloplasmin

Adult and young camel ceruloplasmin (Cp) were isolated and purified using the single-step chromatography on amino ethyl-activated sepharose. There are no differences between the adult and the young camel protein. The molecular mass of the protein, as estimated by SDS-PAGE (denaturant conditions), was approximately 130 000 Da. The electrophoretic mobility of camel Cp is slightly higher as compared to human and sheep protein suggesting that the camel Cp is homogeneous, compact and more acid. The copper content was estimated to be 5.8±0.3 atoms per molecule. The spectrocopic feature includes an absorption maximum at 610 nm, which could be attributed to type 1 copper. The EPR spectrum was completely devoid of any typical signal of the type 2 copper. The kinetic parameters of the adult camel Cp for the specific activity as p-phenylendiamine oxidase were determined as K m=0.42 mM and V max=0.93 μM NADH/mn/mg Cp. The optimum pH for the activity was 5.7.