Limited Proteolysis of β‐Conglycinin and Glycinin, the 7S and 11S Storage Globulins from Soybean [Glycine Max (L.) Merr.]

Abstract

The G2 (A2BIa) glycinin subunit from soybean (Clycine mux L. Merr.) was purified and renatured to the homohexameric holoprotein. This protein along with purified P-conglycinin were subjected to limited proteolysis by trypsin. The generated polypeptide fragments were separated via SDS/PAGE and the amino acid sequence of the N-terminals was determined. Four cleavage points were detected in the a-chain A2 of glycinin as well as in the a'-chain of P-conglycinin. From the known three-dimensional structure of 7 s globulin and the hypothetical model of 7 s globulin-like 11s globulin structure, it was possible to draw the conclusion that two distinct types of susceptible sites for proteolytic cleavage are characteristic of the subunits of both globulins. The first includes the sequences linking N-and C-terminal domains of both globulins and the sequence of N-terminal extensions of 70-kDa subunits from the vicilinlike 7 s globulins. The second type includes the loop between P-strands E and F of the N-terminal domain of 11s globulins and of the C-terminal domain of 7 s globulins. A statistically significant similarity was found between the N-terminal extension of the a'-chain of P-conglycinin and the interdomain linker regions of soybean glycinin and pea legumin. It is proposed that the three sequence regions which form the first type of susceptible sites are of similar structural function and might have evolved from the Nterminal segment of a putative single-domain ancestor.Keywords: protein structure ; seed storage globulins ; gene evolution ; limited proteolysis ; Glycine rnax (L.) Merr.Glycinin and P-conglycinin from soybean seeds belong to the legumin-like 11s and vicilin-like 7s storage globulin families, respectively. Secondary structure predictions and multiple alignment of amino acid sequences [I] suggest that structural similarities should exist between 1 IS and 7s seed storage globulins. The trimer which is formed from 11s globulin subunit precursors before their cleavage into a-and /)-chains [2, 31 as well as the trimeric half-molecule from the mature hexameric 11s globulin holoprotein [4] appear to be structurally similar to the trimeric 7 s globulin molecule. X-ray-crystallographic analysis of two 7 s globulins, canavalin from jack bean [5] and phaseolin from garden bean [6, 71, revealed that their subunits are composed of two similar domains. Each domain is composed of a /]-barrel followed by a region of a-helices (Fig. 1, A1 and BI).A canonical model for the domain structure of 7 s globulins, including both 50-kDa and 70-kDa subunits, was postulated 171. However, the structural role of the extended N-terminal sequence upstream from the N-terminal domain of the 70-kDa subunits remains unclear. Different alignment methods that have been employed to compare the domains of the 7 s globulin subunits with corresponding regions from the 11s globulin subunits suggest that similar N-and C-terminal domains should exist in the legumin-like storage globulins [7, 81 (Fig. 1, A2 has been predicted that all four stru...

Identification and analysis of a conserved immunoglobulin E-binding epitope in soybean G1a and G2a and peanut Ara h 3 glycinins

“…Interestingly, this G2a epitope was part of a corresponding region of a G1a epitope identified by our group in an earlier study [17] and was also adjacent to an Ara h 3 epitope (G240-A254) identified by Rabjohn et al [16]. Structurally, this potentially allergenic epitope is located in a domain between two variable regions of legume glycinins, which have been identified as residing on the surface of the protein and susceptible to protease cleavage [12,23]. In the tertiary structure of the glycinin trimer, this domain corresponds to the subunit interface region ( Figure 6A).…”

C-Terminal 23�kDa polypeptide of soybean Gly�m�Bd 28�K is a potential allergen

“…This linker region seems to be a hypervariable site among legume seed storage proteins and contains specific sites for proteolytic cleavage in vivo. For example, the soybean glycinins are proteolytically processed in vivo in the linker region joining the cupin domains to form the acidic and basic chains of the mature proteins (Shutov et al 1995(Shutov et al , 1996(Shutov et al , 1998(Shutov et al , 1999. Therefore, these regions of plant cupin allergens could be points of attack in the mammalian digestive tract, yielding potentially allergenic peptides.…”

Soybean Glycinin G1 Acidic Chain Shares IgE Epitopes with Peanut Allergen Ara h 3

“…Both epitope A and epitope B reside in a domain between two variable regions (variable region 2, residues Q188–E216, and the hypervariable region, residues T269–K306 [24]) that may represent a particularly allergenic domain of legume seed storage proteins. This glycinin acidic chain domain, between variable region 2 and the hypervariable region [24], corresponds to α-helices α1, α2, α3 and β-strand J′ of the proposed model of 11S legumin protein structure [29, 30]. This domain is located at the subunit interface of the 7S trimer that corresponds to half of the 11S hexamer [30].…”

“…The primary structure of glycinin, in comparison with similar legume family proteins, possesses three variable regions [24]. These regions have been identified as residing at the surface of the glycinin subunit since proteolytic cleavage occurs at or near these variable regions [24, 29]. Both epitope A and epitope B reside in a domain between two variable regions (variable region 2, residues Q188–E216, and the hypervariable region, residues T269–K306 [24]) that may represent a particularly allergenic domain of legume seed storage proteins.…”

“…Lower identity scores are obtained for soybean glycinin with the 7S proteins French bean phaseolin, soybean α-subunit of β-conglycinin, and peanut vicilin (17–20%). However, legumin-like 11S and vicilin-like 7S seed storage proteins are thought to have evolved from a common ancestor [28], and limited proteolysis of 7S and 11S proteins from soybean revealed that they share similar structural elements [29]. The determined crystal structure of phaseolin and proposed canonical structure of 7S proteins [30]has already been used to model a structure of the peanut allergen Ara h 1 [10].…”