rEqu c 1 Horse

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Code: e227
Latin name: Equus caballus
Source material: An E. coli strain carrying a cloned cDNA encoding Equus caballus allergen Equ c 1
Common names: Lipocalin, Ag6

Horse

Recombinant allergens

rEqu c 1


Equ c 1

Allergen

rEqu c 1. (1 , 2, 3, 4, 5)

Biological function

A lipocalin.

Mw

Approximately 22 - 25 kDa.

Other allergens isolated

At least 16 allergens have been isolated from the Horse. (6, 7, 8, 9, 10, 11) Several allergens have been shown to be glycoproteins, including a 27 and a 31 kDa protein. (12)

Allergens characterised to date include:

  • Equ c 1, a 25 kDa protein and a lipocalin, from skin. (1, 2, 3, 4, 5, 13, 14, 15, 16, 17)
  • Equ c 2, a 16 kDa protein and a lipocalin, from skin. (13, 14, 15, 18, 19)
  • Equ c 3, a 67 kDa protein and an albumin (Ag3), from skin, serum, milk, and muscle. (15, 19, 20, 21)
  • Equ c 4, an 18.7 kDa protein, latherin, from skin, sweat and salivary glands. (13, 15, 22)
  • Equ c 5, a 16.7 kDa protein, a lipid-binding serum glycoprotein, from skin. (13, 15)

Allergen description

Equ c 1, a protein of approximately 22 to 25 kDa in size, belongs to the lipocalin protein family. Equ c 1 is produced in both skin and saliva. (17) However, lipocalins are highly concentrated in saliva. (23) Urine contains little of the allergen. (24) The expression of mRNA Equ c 1 has been demonstrated in horse liver and in sublingual and submaxillary salivary glands. (1) Equ c 1 mRNA expression is about 100-fold higher in sublingual salivary glands than in submaxillary salivary glands or the liver. (1) The protein content of horse dander is more than double that of horse hair and skin scrapings, while the carbohydrate content is of the same order. (25)

Although other horse allergens are found in horse dander, the most important allergen appears to be the lipocalin, Equ c 1. IgE against Equ c 1 is found in the sera of 76% of horse-allergic subjects. (16) Equ c 1 is reported to account for 55% of skin-prick test reactivity of horsehair and dandruff extract. (25)

Equ c 1 exhibits a considerable amino acid identity with cat Fel d 4 (67%). Its amino acid identity with a pig salivary lipocalin is 61%, and with the major urinary proteins of rodents about 50%. (1, 25) The amino acid identity with human epididymal-specific lipocalin-9 is 37%. There are several isoforms of the allergen. It has been proposed to bind histamine. Unlike some other allergens, such as Equ c 2 (also a lipocalin), Equ c 1 has a surfactant-like property. (13, 25)

An analysis of the IgE-binding epitopes of Equ c 1 suggests that the dominant epitopes are localised in a restricted region of the molecule. (2 )Carbohydrates may have some impact on IgE binding. (2, 13)

The lipocalins are a large, diverse group of at least 50 proteins. Lipocalins are a large protein group comprising small, extracellular proteins present in vertebrate and invertebrate animals, plants, and bacteria. These proteins are present in humans, and it is notable that almost all important respiratory allergens from mammals belong to this protein family. (26)

Lipocalins are typically small, acidic glycoproteins present in body fluids and secretions produced by the liver or secretory glands. They share common biological functions, predominantly related to the transport of small hydrophobic molecules such as odorants, steroids, vitamins and pheromones; i.e. their most prominent feature is their ability to bind small hydrophobic molecules, such as steroids. Although they were originally characterised as transport proteins for diverse molecules (such as odorants, steroids, and pheromones), they are involved in a wide range of other biological functions. (24) Some lipocalins show immunomodulatory activity. ß-lactoglobulin (Bos d 5) and tear lipocalin have been reported to have nonspecific endonuclease activity. (24)

Several lipocalins are able to bind and transport small hydrophobic ligands such as retinol. (3, 27, 28, 29) Major respiratory allergens of dogs, mice, rats, horses and cows belong to the lipocalin group of proteins. (30) In addition to mammalian respiratory allergens, the milk allergen (Bos d 5) (ß-lactoglobulin), the cockroach allergen (Bla g 4), a ‘kissing bug’ (Triatoma protracta) allergen (Tria p 1), (31) and a pigeon tick (Argas reflexus) allergen (Arg r 1) (32) belong to the lipocalin group. (24)

Several lipocalins have been described as allergenic proteins, including: (2)

Mouse

Mus m 1

18-21 kDa

mouse major urinary protein mMUP

Rat

Rat n 1

17-21 kDa

rat urinary a2-globulin

Cow

Bos d 2

Bos d 5

 

bovine dander lipocalin

bovine milk ß-lactoglobulin

Cockroach

Bla g 4

 

cockroach lipocalin

Dog

Can f 1

Can f 2

 

dog lipocalin

dog lipocalin

Horse

Equ c 1

Equ c 2

 

horse lipocalin

horse lipocalin

Guinea pig

Cav p 1

Cav p 2

 

guinea pig lipocalin

guinea pig lipocalin

Cat

Fel d 4

 

cat lipocalin

Rabbit

Ory c 1

Ory c 2

 

rabbit lipocalin

rabbit lipocalin

 

In addition to Equ c 1, another horse lipocalin allergen, Equ c 2, is expressed. (23) Lipocalin allergens are usually present in hair dandruff.

Lipocalins are members of the calycin superfamily. Despite the diversity at the primary sequence level and a sequence identity often less than 20%, all lipocalins share a conserved folding pattern, a similar three-dimensional structure and one to three conserved regions: an 8-stranded ß-barrel flanked by an a-helix at the C-terminal end of the polypeptide chain. (3, 26) The central cavity of the lipocalin ß-barrel serves for the binding and transport of small hydrophobic molecules such as retinol (retinol-binding protein), odorant molecules (bovine odorant-binding protein), and pheromones (as in mouse and rat: mouse major urinary protein, mMUP1, and rat urinary a2-globulin). (2)

Although the overall amino acid identity between lipocalins is usually below 20%, in some cases the sequential identity over animal species can be well above 20%. For example, dog Can f 1 exhibits a 57% identity with human tear lipocalin (von Ebner's gland protein), and human epididymal-specific lipocalin-9 exhibits a 40% to 50% identity with rodent lipocalins. (24) Lipocalins exist as both monomers and dimers, and they can be either glycosylated or nonglycosylated. (24)

Immune reactivity to lipocalin allergens is not completely understood. Although the cellular immune response to lipocalin allergens has not yet been fully characterised, in general it appears to be weak. Researchers have noted that this is surprising, as humans mount a strong IgE response against these allergens; and therefore it was proposed that the presence of endogenous lipocalins might be a factor contributing to the T helper type 2 (Th2) deviation of the immune response against exogenous lipocalin allergens. (25)

Other researchers have argued that in Bos d 5, the IgE-binding epitopes are spread along the molecule, whereas in Bos d 2 the C terminus appears to contain the human B cell epitopes, and that Bos d 5 contains several murine T cell epitopes. They therefore propose that the allergenicity of lipocalins may be a consequence of molecular mimicry between lipocalin allergens and endogenous lipocalins at the T cell level. (33) And although Fel d 1 and the animal lipocalins appear to bind ligands, these proteins have quite different structures: Fel d 1 is a complex heterodimeric protein with 8 alpha-helices, whereas the lipocalins usually have 8 beta-sheets with a short alpha-helical C-terminus. Unlike mite proteolytic allergens, ligand-binding function per se does not appear to have direct effects on IgE production or inflammation. (34)

Other researchers have argued that the allergenicity of lipocalin is a consequence of molecular mimicry between endogeneous lipocalins and exogenous lipocalin allergens at the T-cell level. (35)

Despite the low amino acid sequence identity, often being less than 20%, and the diversity at the primary sequence level, due to all lipocalins sharing a conserved folding pattern, cross-reactivity between lipocalins is possible. (3, 26)

However, the clinical significance of IgE cross-reactivity between mammalian non-serum-derived respiratory allergens remains unclear. (25) The cross-reactivity appears to be mostly weak to moderate. Earlier analyses were based on inhibiting IgE binding to an allergen extract by another extract: the inhibition usually found to be individually variable. Further, the extracts often showed an unequal inhibitory capacity, suggesting that only a part of the IgE-binding epitopes were common. (25)

A number of studies have shed some light on possible cross-reactivity between Equ c 1 and other lipocalin allergens.

A study evaluating IgE cross-reactivity of five lipocalin allergens (cow Bos d 2, dog Can f 1 and Can f 2, horse Equ c 1, and mouse Mus m 1) and one human endogenous lipocalin (tear lipocalin (TL)), found that human IgE could exhibit cross-reactivity between lipocalin proteins, including TL. An assessment of sera from 42 atopic patients and control subjects found the prevalence of dog-allergic patients sensitised to Can f 1 and Can f 2 to be 42% and 16%, respectively. Eighty-three percent of cow-allergic patients were sensitised to Bos d 2, whereas 76% of horse-allergic patients were sensitised to Equ c 1. Sixty-six percent of mouse-allergic patients had specific IgE to mouse lipocalin, Mus m 1.

Inhibition studies demonstrated IgE cross-reactivity between Can f 1 and human TL, between Can f 1 and Can f 2, and between Equ c 1 and Mus m 1. Low levels of IgE to human TL were found in the sera of seven dog-allergic patients, of whom six were IgE-positive for Can f 1. Several lipocalins exhibited IgE cross-reactivity, probably due to the sequential identity of the proteins and to similarities in their three-dimensional structures. However, as the authors pointed out, the clinical significance of the findings needs to be elucidated; and low-level IgE cross-reactivity could play a role in regulating immune response to lipocalin allergens. (4)

A study describing the characterisation of Fel d 4, a cat dander lipocalin, reported high sequence identity to boar salivary lipocalin and the horse lipocalin Equ c 1. IgE binding to Fel d 4 could be blocked by an allergen extract from cow, and to a lesser degree by extracts from horse and dog. (36)

Recombinant Equ c 1 behaves similarly to native Equ c 1 in several immunological tests with the IgE antibodies of allergic patients. It has been shown to have an amino acid sequence identity of 49-51% with rodent urinary proteins from mouse and rat. (1)

Horse allergy occurs among people who handle horses regularly, either professionally or for recreational purposes, and results in the induction or exacerbation of asthma, allergic rhinitis, allergic conjunctivitis and occupational asthma. (29, 37, 38, 39, 40, 41, 42, 43, 44) Horse allergy has also been reported to result in angio-oedema, respiratory distress, and poorly-controlled asthma. (43) Horse dander may be carried on clothing and also represents a ‘hidden’ allergen because exposure is either indirect or does not immediately precede the development of symptoms. A detailed clinical history is therefore essential for determining the causal allergen. (43)

See Horse - epithelium / dander e3 for clinical information and further details on horse dander allergy. See also Horse - serum proteins e205 and Horse – meat f321 for horse meat allergy.

Horse allergens can be carried on clothing and may thus be found in domestic dust samples from urban environments, which may cause angio-oedema, respiratory distress, and poorly-controlled asthma in children. (45)

In a study over a period of 11 years, reviewing children diagnosed with allergy to horses (35 boys and 21 girls, 35 of whom were under 10 years of age), the main clinical signs reported were ocular symptoms (n=36), asthma (n=30) and rhinopharyngitis (n=24). All the children had highly positive skin-specific IgE tests, and 62% had specific IgE (class 3 and 4) and were polysensitised. In several children, the first symptoms occurred at the time of the first known contact with a horse or pony. (46) Horse allergy has been reported to decrease with age. (47)

Horse allergy occurs among people who regularly handle horses, either professionally or for recreational purposes. (37) In a study evaluating occupational allergy to horse in grooms, sensitisation to horse hair was 12.8%, compared to 4.3% in controls. Asthma was found in 14.4% of the grooms and 5.4% of the controls, allergic rhinitis in 42.4% of the grooms and 18.4% of the controls, allergic conjunctivitis in 35.2% of the grooms and 15.2% of the controls, and allergic skin diseases in 32.8% of the grooms and 13% of the controls. (48)

A postal questionnaire sent to a random sample of 2 500 farmers throughout New Zealand reported that asthma prevalence was higher for horse breeders/groomers (16.5%), pig farmers (18.2%), poultry farmers (17.4%), and those working with oats (17.4%). Hay fever was significantly higher in deer and crop farmers, and in farmers working with horses and goats; eczema was higher for goat and deer farmers. (49)

In an Italian study of 1 822 consecutive outpatients, all subjects who had previously experienced an immediate skin reaction to horse dander were studied. Of 1201 skin-prick test-positive patients, 35 (3.43%) were sensitised to horse dander. No patient was mono-sensitised. Six individuals reported having direct contact with a horse, 10 subjects had occasional contact with horse owners (indirect exposure), and 19 denied direct or indirect exposure to horse or horse allergens. Twenty of the 35 horse-sensitised patients reported both nasal and bronchial symptoms, 14 had rhinitis without asthma and one had asthma without rhinitis. The authors suggest that allergic sensitisation to horse allergens is more frequent than is expected in urban-dwelling subjects without direct or occupational exposure to horses. (50)

Skin-specific IgE responses to a variety of allergens, including horse hair, have been reported to be higher in brittle asthma than in non-brittle asthma. (51)

As Equ c 1 is also found in horse saliva, allergic reactions may occur in sensitised individuals following a horse bite, as clinically demonstrated in a report of a 42-year-old woman who experienced anaphylaxis following a horse bite, in which 3 reactive bands were detected: 65 kDa (horse serum albumin Equ c 3), 25 kDa (Ecu c 1) and 18 kDa (Ecu c 2). Ecu c 1 and Ecu c 2 are lipocalins highly concentrated in the saliva. (23)

Recombinant allergens, which are genetically engineered isoforms resembling allergen molecules from known allergen extracts, have immunoglobulin E (IgE) antibody binding comparable to that of natural allergens, and generally show excellent reactivity in in vitro and in vivo diagnostic tests. (52 )To date, many different recombinant allergens of various pollens, moulds, mites, and foods, as well as latex and bee venom, have been cloned, sequenced, and expressed.

Recombinant allergens have a wide variety of uses, from the diagnosis and management of allergic patients, to the development of immunotherapy, to the standardisation of allergenic test products as tools in molecular allergology. Recombinant allergens are particularly useful for investigations of allergies or allergens manifesting wide cross-reactivity.

As the number of important allergens in commercial horse extracts can vary extensively, and as natural preparations may be contaminated with other components, with the potential to cause false-positive skin-specific IgE test results, recombinant Equ c 1 has a role to play in assessing allergy to horse.

Importantly, horse-allergic individuals are sensitised to a heterogenous range of horse allergens. Recombinant allergens enable assessment of sensitisation to specific allergens in the repertoire, and specifically Equ c 1, which has the ability to indicate primary sensitisation to horse. (53) 

Compiled by Dr Harris Steinman, harris@allergyadvisor.com 

References

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As in all diagnostic testing, the diagnosis is made by the physican based on both test results and the patient history.