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Aplicación de la biología molecular en alergia a alimentos

Aplicación de la biología molecular en alergia a alimentos

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S. B. Lehrer, et al<br />

of cow’s milk proteins, caseins and ß-<strong>la</strong>ctoglobulin, have<br />

be<strong>en</strong> i<strong>de</strong>ntified as major allerg<strong>en</strong>s. The caseins are a family<br />

of chemically re<strong>la</strong>ted proteins. The frequ<strong>en</strong>cy of reactivity<br />

to differ<strong>en</strong>t casein variants has not be<strong>en</strong> systematically studied.<br />

ß-<strong>la</strong>ctoglobulin is a whey protein that composes approximately<br />

20% of total milk proteins. It has a molecu<strong>la</strong>r<br />

weight of 18 kD, and at least six g<strong>en</strong>etic variants have be<strong>en</strong><br />

i<strong>de</strong>ntified. The whey proteins α-<strong>la</strong>ctalbumin and bovine<br />

serum albumin (BSA) have be<strong>en</strong> i<strong>de</strong>ntified as minor cow’s<br />

milk allerg<strong>en</strong>s 9 .<br />

Egg Allerg<strong>en</strong>s<br />

Food allergy to proteins from egg of the domestic chick<strong>en</strong><br />

(Gallus domesticus) is one of the most frequ<strong>en</strong>tly implicated<br />

causes of immediate food allergic reactions of childr<strong>en</strong><br />

in the United States and Europe 12 . Ovomucoid has be<strong>en</strong> i<strong>de</strong>ntified<br />

as the major egg white allerg<strong>en</strong> Gal d 1. It is a glycoprotein<br />

with a molecu<strong>la</strong>r weight of 28 kD and an isoelectric<br />

point of 4.1. Ovalbumin has be<strong>en</strong> i<strong>de</strong>ntified as the major egg<br />

white allerg<strong>en</strong> Gal d 2. It is a monomeric phosphoglycoprotein<br />

with a molecu<strong>la</strong>r weight betwe<strong>en</strong> 43 and 45 kD and an isoelectric<br />

point of 4.5. Ovotransferrin or conalbumin has be<strong>en</strong><br />

i<strong>de</strong>ntified as the major allerg<strong>en</strong> from egg white Gal d 3 with a<br />

molecu<strong>la</strong>r weight of 77 kD and an isoelectric point of 6.0.<br />

Lysozyme (Gal d 4) from egg has a molecu<strong>la</strong>r weight of 14.3<br />

kD and an isoelectric point of 10.7. In addition, a variety of<br />

other egg proteins have be<strong>en</strong> <strong>de</strong>scribed as minor allerg<strong>en</strong>s.<br />

These inclu<strong>de</strong> Ovomucin, Ovoinhibitor, Ovaf<strong>la</strong>voprotein<br />

(ribof<strong>la</strong>vin-binding protein), Apovitell<strong>en</strong>in I, and Apovitell<strong>en</strong>in<br />

VI.<br />

Fish Allerg<strong>en</strong>s<br />

The consumption of fish 13 is a frequ<strong>en</strong>t cause of IgEmediated<br />

reactions. Fish is one among the most commonly<br />

implicated allerg<strong>en</strong>ic foods and has be<strong>en</strong> incriminated in<br />

fatal anaphy<strong>la</strong>ctic reactions. Species-specific analysis of<br />

IgE reactivities has not be<strong>en</strong> performed and most studies<br />

refer only to cod or g<strong>en</strong>erally to fish. One of the first and<br />

most compreh<strong>en</strong>sive analysis of a food allerg<strong>en</strong> was the<br />

purification and characterization of the major codfish<br />

allerg<strong>en</strong>, Gad c 1. Gad c 1, originally <strong>de</strong>signated allerg<strong>en</strong><br />

M, from Baltic cod, Gadus cal<strong>la</strong>rias, has be<strong>en</strong> docum<strong>en</strong>ted<br />

to be the major codfish allerg<strong>en</strong>. It belongs to a group<br />

of muscle proteins called parvalbumins 7 and constitutes<br />

approximately 0.05% to 0.1% of the white cod muscle tissue.<br />

Minor cod fish allerg<strong>en</strong>s distinct from Gad c 1 were<br />

i<strong>de</strong>ntified by CRIE but have not yet be<strong>en</strong> further characterized.<br />

22<br />

Crustacea Allerg<strong>en</strong>s<br />

The c<strong>la</strong>ss Crustacea belongs to the phylum Arthropoda<br />

and inclu<strong>de</strong>s shrimp, prawns, crabs, lobster, and crawfish.<br />

Crustacea are common causes of hypers<strong>en</strong>sitivity. Like that of<br />

fish allergy, a higher inci<strong>de</strong>nce of crustacea allergy would be<br />

expected in geographic areas where greater amounts of shellfish<br />

are consumed on a regu<strong>la</strong>r basis. A 36 kD allerg<strong>en</strong>, <strong>de</strong>signated<br />

P<strong>en</strong> a 1, was iso<strong>la</strong>ted from boiled brown shrimp, P<strong>en</strong>aeus<br />

aztecus. Sequ<strong>en</strong>cing of a 21 amino acid Lys-C pepti<strong>de</strong> of<br />

P<strong>en</strong> a 1 <strong>de</strong>monstrated significant homology (60%-85%) with<br />

tropomyosin from various species consist<strong>en</strong>t with the conclusion<br />

that P<strong>en</strong> a 1 is tropomyosin 6 . P<strong>en</strong> a 1 constitutes 20% of<br />

the soluble protein and accounts for approximately 80% of<br />

pooled shrimp-allergic sera reactivity to shrimp meat extract.<br />

More than 80% of allergic subjects reacted to this allerg<strong>en</strong> 6 .<br />

P<strong>en</strong> a 1 was cloned and sequ<strong>en</strong>ced; its cDNA sequ<strong>en</strong>ce showed<br />

26 base pair substitutions wh<strong>en</strong> compared with the<br />

sequ<strong>en</strong>ce of Met e 1; these base pair substitutions resulted in<br />

only one amino acid substitution in position 69. Homologous<br />

proteins P<strong>en</strong> i 1, and Met e 1 from Indian shrimp P<strong>en</strong>aeus<br />

indicus and greasyback shrimp Metap<strong>en</strong>aeus <strong>en</strong>sis respectively<br />

have also be<strong>en</strong> studied 14,15 .<br />

EPITOPES IN ALLERGENS OF ANIMAL<br />

ORIGIN: STUDY OF SHRIMP TROPOMYOSIN<br />

Tropomyosin is a major muscle protein pres<strong>en</strong>t in all<br />

living creatures. These molecules appears to be highly conserved<br />

by the substantial amino acid sequ<strong>en</strong>ce i<strong>de</strong>ntity of tropomyosins<br />

from unre<strong>la</strong>ted species. For example, shrimp tropomyosin<br />

amino acid sequ<strong>en</strong>ce shares homology of<br />

approximately 60% with non-allerg<strong>en</strong>ic tropomyosins of vertebrates.<br />

Tropomyosin has a rather unique structure in that it is<br />

composed of two polypepti<strong>de</strong> chains each in alpha helix formation<br />

coiled around one another in the coiled-coil formation<br />

16 . Although the structure of tropomyosin is well known,<br />

little information about its B cell epitopes (IgE-binding epitopes)<br />

and no information about the T cell epitopes was avai<strong>la</strong>ble<br />

until rec<strong>en</strong>tly.<br />

In or<strong>de</strong>r to i<strong>de</strong>ntify areas of the tropomyosin molecule<br />

that contain important IgE binding regions, the following strategy<br />

was used by Ayuso et al 17 . Thirty-six over<strong>la</strong>pping pepti<strong>de</strong>s<br />

(15 amino acids long, offset 6 amino acids) were synthesized<br />

that spanned the <strong>en</strong>tire sequ<strong>en</strong>ce of P<strong>en</strong> a 1, shrimp<br />

tropomyosin allerg<strong>en</strong>. Testing the sera from 18 shrimp-allergic<br />

subjects, reactive to P<strong>en</strong> a 1, these synthetic pepti<strong>de</strong>s were<br />

scre<strong>en</strong>ed for IgE antibody reactivity. Based on the preval<strong>en</strong>ce

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