(1) (2) (3)(4) Coeliac symposium 1998 facta. Prolamins in cereals

The Finnish Coeliac Society. Tampere. 10-12 July 1998.

Changing Features of Coeliac Diseases.

Edited by Susanna Lohiniemi, Pekka Collin, Markku Mäki

Herbert Wieser. Prolamins in Cereals. Deutsche Forschungsanstalt für Lebensmittelchemie, Germany.

1. Proteins in cereals

Cereals constitute one of the most important basic components in human nutrition and are cultivated almost all over the world. They are species of grasses (family Pooideae) with highly developed seeds characterised by high starch (32-73 %) and low water content (11-14 %). Wheat, rice and maize account for more than 80 80 % of total cereal production (1996: 2050 Mt), but the other common cereals - barley, sorghum, oats, millet and rye- are also of great importance for specific regions. Cereals which evidently activate coeliac disease (wheat, rye, abrley) belong to the tribe Triticeae, and share a close tqaxonomic relationship. Oats, controversial with respect to coeliac disease, are related more distantly to the Triticeae. The non-toxic cerelas maize, sorghum, millet and rice, however, show separated evolutionary lines within the grass family.

Usually cereal grains contains 7-16 % protein, which, in terms of function, can be classified into three types: 1: structural proteins like membrane proteins located in the outer parts of the kernels; 2: metabolic proteins, e.g. enzymes and enzyme inhibitors, present mainly in the aleurone layer and embryo, and 3: storage proteins occurring exclusively in the starchy endosperm. The proteins of the latter type make up about 70- 80 % of the total grain protein ; they are unique to cereals, and their main function is to provide the developing embryo with nitrogen and amino acids.

Traditionally, storage protein have been grouped into two fractions, the prolamines, soluble in aqueous alcohols and the glutelins insoluble in aqueous alcohols. In this article, the term, ”prolamines” is not used for both alcohol-soluble and insoluble storage proteins as proposed by Shewry et al., but in the classical definition: prolamins are cereal endosperm proteins soluble in aqueous alcohols without reduction of disulphide bonds. While the prolamins occur in grains and flour predominantly as monomers, the glutelins are linked by disulphide bonds and are present in an aggregated state with molecular weights up to millions. 

2.  Characterization of prolamin fractions

Herbert Wieser. Prolamins in Cereals. Deutsche Forschungsanstalt für Lebensmittelchemie, Germany.

The prolamin fractions of the various cereals have been given trivial names; gliadin (wheat), secalin (rye), hordein (barley), avenin (oats), zein ( maize), kafirin (sorghum, millet) and oryzin ( rice. The prolamin content on cereal flours varies considerably and depends on species, variety and growing conditions. On an average, wheat has the highest content of prolamin ( 3-6 g/100 g flour) followed by maize, whereas rice is almost free of prolamins.

The name ”prolamins” reflects the characteristics of amino acid compositions: high contents of proline (Pro, P) and glutamine (Gln, Q), which is particularly true for coeliac toxic gliadin, secalin and hordein (Table 1).

 Prolamins of rice, millet, sorghum and maize are lower in glutamine (Q) and proline (P), but rich in leucine (L) and alanine (A); avenin of oats is in medium position.

Altogether, the proportions of the major amino acids, in particular of glutamine (Q) and proline (P), in prolamins reflect very well their coeliac toxicity. 

Features common to all prolamins are low contents of the essential amino acids methionine (M), lysine (K) and tryptophan (W). For this reason, the biological value of prolamins is rather low, and from nutritional point of view, a gluten-free diet is not disadvantageous for consumers. 

The range of molecualr masses shown in table 1 indicates that the prolamins of wheat, rye and barley differ from other prolamins by significantly higher values. 

Partial aminoacid composition (mol-%)

Amino acid name	Gliadin	Secalin	Hordein	Avenin	Oryzin	Kafirin	Zein
Gln (Q)	37	35	35	34	20	22	19
Pro (P)	17	18	23	10	5	8	10
Leu (L)	7	6	6	11	12	13	19
Ala (A)	3	3	2	6	9	14	14
Met (M)	1	1	1	2	1	2	1
Lys (K)	1	1	1	1	1	0	0
Trp (W)	0	0	1	0	1	2	0

Range of molecular masses

Gliadin 32 000- 74 000

Secalin 40 000- 53 000

Hordein 35 000 -72 000

Avenin 20 000 - 30 000

Oryzin 11 000 - 23 000

Kafirin 20 000 - 24 000

Zein 19 000 - 26 000.

3. Classification of prolamin components

Herbert Wieser. Prolamins in Cereals. Deutsche Forschungsanstalt für Lebensmittelchemie, Germany.

The prolamin fraction consist of numerous, in part closely related proteins. According to their amino acid sequences, amino acid compositions and molecular masses, they can be classified into different types.

Gliadin, secalin and hordein have two common types: the omega-type ( kreikk. ω): ω -gliadin, ω-secalin and ω- hordein) and the gamma-type (kreikk. γ) (γ-gliadins, γ-secalins and γ-hordein); gliadin additionally contains alfa-type (kreikk. α) ( α-gliadins).

Omega (ω) -Type prolamins (omega-type) have high contents of glutamine ( Q, Gln) 48 mol-%), prolamine (P, Pro) about 25 mol-% and phenylalanine (F, Phe) ( about 8 mol-%), which together accounts for about 80 % of the total composition Their molecular masses are in a range from 53 000- 64 000. The complete amino acid sequences of these proteins have not yet (1998) been described, with the exception of a C-hordein, the corresponding gene of which has been proposed to be silent, but some information on partial sequencess does exists.

Omega (ω)-prolamins consist almost entirely of repetitive sequences with only short, non-repetitive N- and C-terminal sequences. The most dominant repeat appears to be QQPQQPFP with numerous modifications of single residues.

Gamma (γ)- type prolamins of wheat, rye and barley have molecular masses of about 30 000 and are homologous to a high degree. Their primary structures are divided into two completely different domains. Their primary structures are divided into two completely different domains.

The N-terminal domain ( Division I about 140 amino acid residues) has short N-terminal sequences which are non-repetitive. Then, repetitive sequences follow; they have motifs similar to those of omega(ω)-gliadins and are rich in glutamine (Q), proline(P) and phenylalanine (F). ( Table 2).

The C-terminal domain (divisions III-V, about 160 amino acid residues) possesses a more usual amino acid composition with less glutamine (Q) and proline(P), but more charged residues: glutamic acid ( Glu, E), lysine (Lys, K), arginine (Arg, R) and hydrophobic residues: leucine (Leu, L), isoleucine (Ile, I), valine ( Val, V). In this domain, cysteine (Cys, C) residues are also present, forming intramolecular disulphide bonds (-s-s-). Partially, the sequences show homology with seed proteins of other plants, e.g. rape. castor bean.

Alfa( α) -type gliadins are unique to the wheat.

Typically these proteins have an N-terminal domain with two different sequence divisions: the N-terminal division I is partially non-repetitive (about 32 residues), partially repetitive, consisting of motifs like QPQPFPPQQPYP, which are mostly repeated five times. Furthermore, one or two poly-Gln-sequences (Poly-Q-sequences) (division II with maximal 18 residues of Gln, (Q) are present. The amino acid composition (Table 2) is similar to that of the gamma (γ)-type, but with lower proline (P) and higher tyrosine (Y) contents.

The C-terminal domain of the alfa(α)-type is homologous in divisions III and V with the (γ)-type, and contain three intramolecular disulphide bonds (-s-s-).

Avenins, the prolamins of oats, are homologous with the alfa (α)- and gamma(γ)-type prolamins within division III and V of the C-terminal domain. (Figure1). Eight cysteine residues (Cys) are present in these divisions, all forming intramolecular disulphide bonds. Unique to avenins are the repetitive sequences in divisions IV with repetitive motifs like QQQVFQPL or QQQFFQPQM, which are repeated four times and frequently modified.

The N-terminal domain is characterised by non-repetitive sequences ( about 16 residues) and by short repetitive sequences consisting of three repeats of the motif QQQQPFV or QQQQMLL.

The primary structures of the non-toxic prolamins oryzin, kafirin and zein are, as far as is known, completely different from those of toxic prolamins.

Altogether, the N-terminal domains and- in particular, their repetitive sequences are unique to toxic prolamins and mainly characterised by high contents of glutamine (Q), proline(P) and aromatic amino acids ( F,Y,W) (Table 2). Their secondary stricture is characterized by the frequent occurrence of beta-turn conformations. Most in -vivo and in-vitro testing of prolamin (gliadin) peptides demonstrated that regions of the N-terminal domains are involved in activating coeliac disease.

(eräs löytämäni moderni linkki:

http://www.mdpi.com/2072-6643/8/10/644/htm)

N- terminal

IV

V

C- terminal

4. Relation between prolamins and glutelins

Glutelins are the second major protein fractions in cereal endosperm. They comprise aggregated proteins linked by disulphide bonds (-s-s-). After reduction of disulphide bonds, the resulting subunits are as soluble in aqueous alcohol as prolamins.

Glutelin subunits of wheat, rye and barley can be classified into high- molecule-weight (HMW) and low-molecule-weight(LMW) subunits.

The HMW subunits of three cereals are homologous to a high degree. They have molecular masses in a range of 95 000- 136 000 (determined by SDS-PAGE) and contain three different domains: a non-repetitive N-terminal domain with about 100 residues, a central repetitive domain with about 400-700 residues and a non-repetitive C-terminal domain with about 40 residues.

Both N-terminal and C-terminal have relatively well-balanced amino-acid compositions with most or all of the cysteine residues.

The central domain is rich in glutamine (Q), glycine (G) and proline (P) and contains repetitive hexapeptides like QQPGQG as a backbone, which are repeated about 50 to 70 times and frequently modified and interspersed by motifs like YYPTSP an QPG. A remarkable sequence homology to prolamins cannot be detected.

In contrast, LMW subunits of glutelins are partially or highly homologous to the corresponding prolamin.

LMW subunits of glutenin ( glutelin of wheat) ar homologous to alfa-and gamma-type gliadin within division III and V of the C-terminal domain. ( Figure 1). Division IV is less homologous, elongated about 20 residues, and contains one cysteine residue, which forms an intermolecular disulphide bond with other gluten proteins.

The N-terminal domain (division I) differs significantly from those of alfa- and gamma-type prolamins and is characterised by short non-repetitive and long repetitive sequences. The typical repeat motifs contain a series of glutamines (Q) (two to six residues) followed by units such as PPFS. A small portion of omega-, alfa- and gamma-type gliadins is also present in the glutelin fraction. By substitution of a single residue by cysteine, they have an odd number of cysteines and are covalently bound to the glutenin aggregate; consequently, they are not extractable with aqueous alcohols. This could be the reason why glutenin has been described as weakly toxic.

LMW subunits of rye glutelin comprise a group of proteins with a molecular mass of about 75 000 and are named 75 K gamma-secalin, because they are closely related to the corresponding gamam-type prolamins (40 K-gamma (γ)-secalin). It has been proposed that 75 K γ-secalins were formed from 40 K (γ)-secalins by elongation of the glutamine (Q)- and proline (P)-rich repetetive sequences and by insertion of at least one cysteine residue.

LMW subunits of barley glutelin (B-hordeins) have C-terminal domains highly homologous with those of LMW subunits of wheat. The N-terminal domain consists exclusively of repetetive sequences related to those of gamma(γ)-hordeins.

Summarising, one portion of the glutelin subunits , namely the HMW subunits, are totally different from prolamins, whereas the other portion, the LMW subunits, are closely related to corresponding prolamins in the case of rye and abrley. The LMW subunits of wheat, however, have respective sequences distinctly different from those of the gliadins.

(Tabel 1)

Taulukko 2 ei ole kirjoitettu näkyviin.

Kuva 1 ei ole kirjoitettu näkyviin.

Yllä oleva  tekemäni kaava vain selventänee  N- ja C- terminaalin ja  niiden  divisioiden jaon periaatteen.