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Topic:Proteomics
Proteomics is the large-scale study of proteins, particularly their structures and functions, in horses. This field encompasses the analysis of the complete set of proteins expressed by the equine genome, known as the proteome. Proteomics research in horses aims to understand the diverse roles of proteins in various biological processes, including growth, development, and response to environmental stimuli. Techniques such as mass spectrometry and protein microarrays are commonly employed to identify and quantify proteins, assess post-translational modifications, and investigate protein-protein interactions. This page compiles peer-reviewed research studies and scholarly articles that explore the methodologies, findings, and implications of proteomics in equine health and disease.
Stallion epididymal fluid proteome: qualitative and quantitative characterization; secretion and dynamic changes of major proteins. Proteins present in and secreted into the lumen of various regions of the stallion epididymis were characterized qualitatively and quantitatively by two-dimensional electrophoresis. Using this proteomic approach, 201 proteins were found in the lumen and 117 were found that were secreted by the epithelium in various parts of the organ. Eighteen proteins made up 92.6% of the total epididymal secretory activity, lactoferrin (41.2%) and clusterin (24.8%) being the most abundant. Procathepsin D, HE1/CTP (cholesterol transfer protein), GPX (glutathione peroxidase), beta-N-acetyl-hexosaminidase, and ...
Identification of proteins in the equine embryonic capsule. An acellular embryonic capsule envelops equine conceptuses between day 6 and day 23 after ovulation. As all of the factors mediating embryo-mother signalling must pass through the capsule, it acts like a 'mailbox'. Therefore, we have started to map the proteins in this special extracellular matrix at the interface between mother and embryo. In the present study, one- and two-dimensional gel electrophoresis were used to examine a range of proteins. Use of western blotting identified three specific proteins in the capsules of equine conceptuses recovered on day 16 after ovulation: insulin-like g...
Mass accuracy and sequence requirements for protein database searching. To elucidate the role of high mass accuracy in mass spectrometric peptide mapping and database searching, selected proteins were subjected to tryptic digestion and the resulting mixtures were analyzed by electrospray ionization on a 7 Tesla Fourier transform mass spectrometer with a mass accuracy of 1 ppm. Two extreme cases were examined in detail: equine apomyoglobin, which digested easily and gave very few spurious masses, and bovine alpha-lactalbumin, which under the conditions used, gave many spurious masses. The effectiveness of accurate mass measurements in minimizing false protein match...
Equine placental cup cells show glycan expression distinct from that of both chorionic girdle progenitor cells and early allantochorionic trophoblast of the placenta. Using lectin histochemistry on plastic-embedded material, the glycosylation patterns of equine girdle and cup cells, and associated endometrial glands, have been investigated from 37 to 67 days gestation. Results were compared with the glycosylation of the 50-day allantochorionic trophoblast of the established equine placenta that will later form the microcotyledons. The differentiated cup cells, which secrete equine chorionic gonadotropin (eCG), showed a pattern of glycosylation that was distinct both from the progenitor girdle cells and the allantochorionic trophoblast, with granules that bo...
Presence and comparison of angiotensin converting enzyme in commercial cell culture sera. This study was conducted to determine the presence of the angiotensin converting enzyme in commercial sera used in cell culture medium. The aim of the research was to bring the presence of proteinases (angiotensin converting enzyme) to cell culture users' knowledge and to give some data for solving problems about the development of peptides as useful drugs. The enzymes, purified from foetal bovine, adult bovine, foetal equine, adult equine, and human sera, showed molecular weights of about 170 kDa. Captopril and lisinopril inhibited enzyme activities at nanomolar concentrations. The enzymes we...
A 105- to 94-kilodalton protein in the epididymal fluids of domestic mammals is angiotensin I-converting enzyme (ACE); evidence that sperm are the source of this ACE. SDS-PAGE analysis of luminal fluid from the ram testis and epididymis revealed a protein of about 105 kDa in the fluid in the caput epididymal region. The molecular mass of this fluid protein shifted from 105 kDa to 94 kDa in the distal caput epididymidis and remained at 94 kDa in the lower regions of the epididymis. The possible sperm origin of this protein was suggested by the decrease in intensity of a 105-kDa compound on the sperm plasma membrane extract and by its total disappearance from the fluid of animals with impaired sperm production caused by scrotal heating. The 94-kDa protein was...
Discrimination of mammalian growth hormones by peptide-mass mapping. Recognition by the legal authorities that growth hormones (GHs) may be abused to improve sporting performance and/or physique has led to the implementation of controls that make it an offence to produce, supply, possess or import and export GHs, with intent to supply, without the authority to do so. A method is described for the discriminatory analysis of human, equine, porcine and bovine GHs for forensic purposes. Peptide-mass mapping by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry following tryptic digestion gave sequence coverages of 97.4%, 93.7...
Structural characterisation and comparison of the native and A-states of equine lysozyme. Native state 1H NMR resonance assignments for 125 of the 129 residues of equine lysozyme have enabled measurement of the hydrogen exchange kinetics for over 60 backbone amide and three tryptophan indole hydrogen atoms in the native state. Native holo equine lysozyme hydrogen exchange protection factors are as large as 10(6), the most protected residues being located in elements of secondary structure. High exchange protection in the domain interface correlates with the binding of Ca2+ in this region. Equine lysozyme differs from most non-Ca2+ binding lysozymes in forming a highly populated par...
Isolation and characterization of heparin- and phosphorylcholine-binding proteins of boar and stallion seminal plasma. Primary structure of porcine pB1. In the bovine, seminal plasma heparin-binding proteins bind to sperm lipids containing the phosphorylcholine group and mediate the capacitating effects of heparin-like glycosaminoglycans during sperm residence in the female genital tract. We report the characterization of heparin- and phosphorylcholine-binding proteins of stallion and boar seminal plasma. Horse seminal plasma proteins HSP-1 and HSP-2, and boar protein pB1, belong to the same family as the bull heparin- and phosphorylcholine-binding proteins BSP-A1/2, BSP-A3, and BSP-30K. We have determined the amino acid sequence and posttrans...
Characterization of polypeptides synthesized and secreted by oviductal epithelial cell explants obtained from young, fertile and aged, subfertile mares. To compare the electrophoretic patterns of proteins synthesized and secreted by oviductal epithelial cell (OEC) explants obtained from young, fertile and aged, subfertile mares. Methods: Young, fertile (n = 5; 2 to 7 years old) and aged, subfertile (n = 5; 17 to 24 years old) mares. Methods: 2-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and computerized densitometry. Results: Variation in the synthesis and secretion of polypeptides from young, fertile mare OEC (YOEC) and aged, subfertile mare OEC (AOEC) was evidenced by differences in the intensity of radiolabeled pol...
Characterisation of proteins in the seminal plasma of stallions, geldings and supplemented with testosterone. The major proteins in stallion seminal plasma were characterised by two-dimensional polyacrylamide gel electrophoresis, and compared with the patterns of proteins in normal geldings (castrated males) and geldings supplemented with testosterone. The major proteins or groups of proteins identified according to their approximate relative molecular weight in kilodaltons (kDa) and apparent isoelectric point (pl) were: 1) 60 kDa. pl 7; 2) 23 kDa, pl 4-5; 3) 25-30 kDa, pl 5.5-6; 4) 23 kDa, pl 7-8; and 5) 15-20 kDa, pl 6-7.5. Protein groups 1 and 2 were more prominent in the seminal plasma from the st...
Cross-antigenicity of horse serum albumin with dog and cat albumins: study of three short peptides with significant inhibitory activity towards specific human IgE and IgG antibodies. Horse serum albumin is present in the near vicinity of the animal, while dog and cat serum albumins are very common allergens present in house dust. Human patients clinically defined as allergic to horse could react with horse serum albumin by means of IgE or IgG antibodies. Studies regarding the specificities of these antibodies by inhibition enzyme-linked immunosorbent assay (ELISA) and depletion experiments have demonstrated that they are directed against dog serum albumin and cross-react not only with horse serum albumin but with other serum albumins from different origins. To investigate ...
Inhibition of the protease activity in tracheobronchial aspirates of horses with chronic obstructive pulmonary disease. To clarify the role of proteolytic enzymes in the pathogenesis of chronic obstructive pulmonary disease (COPD) in horses, and to investigate new possibilities for treatment of this disease by interfering in the proteolytic process. Methods: Effect of antiproteolytic activity of selected protease inhibitors on tracheal aspirates was studied in vitro, and the inhibition profiles were compared with those of purified proteases. Methods: Respiratory tract secretions with antiproteolytic activity from 9 horses with COPD. Methods: Caseinolytic agar-diffusion assay. Results: The protease-inhibition pr...
Proteolytic cleavage of VP2, an outer capsid protein of African horse sickness virus, by species-specific serum proteases enhances infectivity in Culicoides. Purified African horse sickness virus (AHSV) was fed, as part of a blood meal, to adult females from a susceptible colony of Culicoides variipennis, established in the insectories at the Institute for Animal Health, Pirbright Laboratory, UK. The meal consisted of heparinized blood obtained from ovine, bovine, equine (horse and donkey) or canine sources spiked with AHSV serotype 9 (AHSV9). The infectivity levels observed for C. variipennis varied significantly, according to the source of the blood sample. Comparison of the protein profiles obtained from AHSV9 incubated with the individual serum...
In vitro Catabolism of very low density lipoproteins from horse (Equus caballus) by the action of autologous lipoprotein lipase. Incubation of equine very low density lipoproteins with lipoprotein lipase isolated from horse postheparin plasma resulted in the formation of lipoproteins of a higher density. Lipoproteins isolated after incubation and plasma lipoproteins had a different chemical composition and triacylglycerol fatty acid pattern. In vitro-obtained low density lipoproteins contained substantially more phospholipids and triacylglycerols but significantly less cholesteryl esters than native low density lipoproteins. Comparing the triacylglycerol fatty acid pattern of plasma very low density lipoproteins and in ...
Characterisation of a membrane receptor on ruminants and equine platelets and peripheral blood leukocytes similar to the human integrin receptor glycoprotein IIb/IIIa (CD41/61). This paper describes two anti-glycoprotein IIb/IIIa or CD41/61 murine monoclonal antibodies (Co.35E4 and Co.2oA1). The cellular distribution and apparent molecular weight of the antigen detected by these antibodies is consistent with their reaction with ruminant and equine glycoprotein IIb/IIIa. Biochemical analysis of the equine molecule using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) revealed bands of 24, 100 and 110 kDa under reducing conditions and 115 and 80 kDa under nonreducing conditions. Biochemical analysis of ruminant antigen revealed that the 24 kDa band...
Recent developments in cryopreservation of stallion semen with special emphasis on thawing procedure using thermal hysteresis proteins. This research study explores the process of cryopreservation of stallion semen, focusing on improving the thawing procedures using thermal hysteresis proteins (THPs) from Antarctic and Arctic fish in order to […]
Further characterisation of forms of haemosiderin in iron-overloaded tissues. The biochemical and biophysical properties of isolated haemosiderins have been compared to that of another iron-containing protein, termed prehaemosiderin, which sediments through chaotropic potassium iodide only after 20 h of ultracentrifugation, in contrast to that of haemosiderin which is recovered after 2 h of ultracentrifugation. The iron/protein ratio and iron/phosphate ratio were less that that of the corresponding haemosiderin, while the elemental composition was also reduced in many of the prehaemosiderin samples. Mossbauer spectroscopy and electron diffraction identified the predomin...
Proteins induced by recombinant equine interferon-beta 1 within equine peripheral blood mononuclear cells and polymorphonuclear neutrophilic granulocytes. Peripheral blood mononuclear cells (PBMC) and polymorphonuclear neutrophilic granulocytes (PMN) as well as embryonic equine dermal fibroblasts and the equine fibroblast line E. Derm which were used as controls, were treated with recombinant equine interferon-beta 1 (rEqIFN-beta 1) in vitro which induced the expression of different proteins in these cells. A 74 kDa protein was induced in PBMC and an 82 kDa protein was additionally found in the equine fibroblast E. Derm cell line following treatment with rEqFN-beta 1. Both proteins reacted with anti-mouse and anti-human Mx protein antisera in im...
Proteolytic processing of the replicase ORF1a protein of equine arteritis virus. To study the proteolytic processing of the equine arteritis virus (EAV) replicase open reading frame 1a (ORF1a) protein, specific antisera were raised in rabbits, with six synthetic peptides and a bacterial fusion protein as antigens. The processing of the EAV ORF1a product in infected cells was analyzed with Western blot (immunoblot) and immunoprecipitation techniques. Additional information was obtained from transient expression of ORF1a cDNA constructs. The 187-kDa ORF1a protein was found to be subject to at least five proteolytic cleavages. The processing scheme, which covers the entire OR...
Isolation, primary structures and metal binding properties of neuronal growth inhibitory factor (GIF) from bovine and equine brain. Human neuronal growth inhibitory factor (GIF) impairs the survival of cultured neurons and is deficient in the brains of Alzheimer's disease victims. We have isolated and sequenced analogous proteins from bovine and equine brain. By comparing their primary structures with those of human, mouse and rat GIFs, a consensus GIF sequence was obtained. Although this exhibits ca. 65% similarity with primary structures of mammalian metallothioneins (MTs), some significant differences are expected in the content of helix and turn secondary structures. In contrast to MTs, which usually bind 7 Zn(II) ions...
Studies on glycoprotein-derived carbohydrates. This research focuses on the study of glycoproteins, specifically investigating their carbohydrate chains and their various functions in living organisms. The article highlights the challenges in isolating specific carbohydrate chains […]
Characterisation of proteolytic activity of excretory-secretory products from adult Strongylus vulgaris. An excretory-secretory (ES) preparation derived from adult Strongylus vulgaris in vitro was assessed for proteolytic activity using azocasein and synthetic, fluorogenic, peptide substrates. Fractionation was by molecular sieve fast protein liquid chromatography (molecular sieve FPLC) and resolution by gelatin-substrate sodium dodecyl sulphate-polyacrylamide gel electrophoresis (gelatin-substrate SDS-PAGE). The cysteine proteinase activator, dithiothreitol (DTT), enhanced azocaseinolysis and hydrolysis of carbobenzoxy-phenylalanyl-arginine-7-amido-4-methylcoumarin (Z-Phe-Arg-NMec) by the ES pre...
Cellular sources of proteolytic enzymes in equine joints. Isolated equine blood and articular cells were investigated for proteolytic enzyme production by means of gel filtration and analysis on 14C-acetylated collagen and casein substrates. Significant amounts of collagenase and caseinase activity were produced by cultured synoviocytes stimulated with equine interleukin 1, although large amounts of collagenase also originated from neutrophils.
Quantitative analysis of cyanogen bromide-cleaved peptides for the assessment of type I: type II collagen ratios in equine articular repair tissue. Cyanogen bromide was used to solubilise and specifically fragment purified equine Type I and II collagen and equine articular surface repair tissue. The resultant peptides were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and quantified by densitometric scanning. Measurement of the relative amounts of the peptides alpha 2(I) CB3, 5 and alpha 1(II)CB10 provided an accurate method of establishing the ratio of Type I to Type II collagen in mixtures of purified equine collagens. The method was sensitive to 6% Type II collagen when the band areas were corrected for peptid...
Cross-validation of cyanogen bromide-peptide ratios to measure the proportion of type II collagen in pepsin digests of equine articular cartilage, meniscus, and cartilage repair tissue. Collagen type I and type II were purified from equine flexor tendon and articular cartilage, respectively. Equal amounts of these collagens were cleaved with cyanogen bromide, and 11 mixtures containing increasing proportions of type II collagen were separated in seven identical sodium dodecyl sulfate-polyacrylamide gels. The density of bands was measured in wet gels and the peak areas were used to form six ratios of peptide bands that had polynomial relationships with the known proportions of type I and type II collagen in the mixtures. Calibration curves for determining the proportion of typ...
Characterization of equine oviductal proteins synthesized and released at estrus and at day 4 after ovulation in bred and nonbred mares. Proteins synthesized and released in vitro by oviducts collected from horse mares during estrus and at day 4 after ovulation for bred and nonbred mares were examined by two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2-D SDS PAGE) and fluorography. Ampullary and isthmic regions both produced a wide array of nondialyzable proteins in culture. Major proteins or groups of proteins identified according to relative molecular weight (kDa) and apparent isoelectric point (pI) were at 100 kDa, pI 8; 100-200 kDa, pI 6; 150 kDa, pI 4.5; 60-100 kDa, pI 4; and an array of polypep...
Comparative studies of the Spi1 proteins of three equine alpha-1-proteinase inhibitor haplotypes following isolation by affinity chromatography. 1. Antiproteinase deficiency can result in excessive proteinase-induced tissue damage. The major anti-elastase (Spi1) protein of equine alpha 1-proteinase inhibitor (alpha 1-PI) was isolated from the plasma/serum of three common haplotypes (I, L and U). 2. The N-terminal amino acid sequences of the three inhibitors were identical, but were only approx 65-77% homologous with two other published equine Spi1 sequences. 3. All three inhibitors complexed quickly and irreversibly with equine leucocyte proteinase 2A (kass = 2 x 10(7) M-1 sec-1). They were also efficient inhibitors of chymase (rat mas...
Oxidation of methionine residues in equine growth hormone by Chloramine-T. 1. Reactivity of methionine residues towards Chloramine-T was studied in the equine growth hormone. 2. With a 20.0-fold molar excess of reagent over methionine, full oxidation of the four residues of the protein is achieved. 3. Methionine 4 is the most reactive group, followed by methionines 72 and 178--methionine 123 being the less reactive residue. 4. As judged by circular dichroism spectra and binding assays, protein conformation and binding capacity to specific receptors remains unchanged even after full oxidation of all four methionine residues. 5. Results agree with data previously obtai...
