Characterising avenin-like proteins (ALPs) from albumin/globulin fraction of wheat grains by RP-HPLC, SDS-PAGE, and MS/MS peptides sequencing

Abstract

Background: Wheat grain avenin-like proteins (ALPs) belong to a recently discovered class of wheat grain storage protein. ALPs in wheat grains not only have beneficial effects on dough quality but also display antifungal activities, which is a novel observation for wheat storage proteins. Previous studies have shown that ALPs are likely present in the albumin/globulin fractions of total protein extract from wheat flour. However, the accumulation characteristics of these ALPs in the mature wheat grain remains unknown. Results: In the present study, a total of 13 ALPs homologs were isolated and characterized in the albumin/globulin fractions of the wheat protein extract. A combination of multiple techniques including RP-HPLC, SDS-PAGE, MALDI-TOF and peptide sequencing were used for accurate separation and identification of individual ALP homolog. The C-terminal TaALP-by-4AL/7DS, TaALP-by-4AL/7AS/7DS, TaALP-bx/4AL/7AS/7DS, TaALP-ay-7DS, TaALP-ay-4AL, TaALPax-4AL, TaALP-ax-7AS, and TaALP-ax-7DS, were separated as individual protein bands from wheat flour for the first time. These unique ALPs peptides were mapped to the latest wheat genome assembly in the IWGSC database. The characteristic defence related proteins present in albumin and globulin fractions, such as protein disulfide-isomerase (PDI), grain softness protein (GSP), alpha-amylase inhibitors (AAIs) and endogenous alpha-amylase/subtilisin inhibitor were also found to co-segregate with these identified ALPs, avenin-3 and α-gliadins. The molecular weight range and the electrophoresis segregation properties of ALPs were characterised in comparison with the proteins containing the tryp_alpha_amyl domain (PF00234) and the gliadin domain (PF13016), which play a role in plant immunity and grain quality. We examined the phylogenetic relationships of the AAIs, GSP, avenin-3, α-gliadins and ALPs, based on the alignment of their functional domains. MALDI-TOF profiling indicated the occurrence of certain post-translations modifications (PTMs) in some ALP subunits. Conclusions: We reported for the first time the complete profiling of ALPs present in the albumin/globulin fractions of wheat grain protein extracts. We concluded that majority of the ALPs homologs are expressed in wheat grains. We found clear evidence of PTMs in several ALPs peptides. The identification of both gliadin domain (PF13016) and Tryp_alpha_amyl domain (PF00234) in the mature forms of ALPs highlighted the multiple functional properties of ALPs in grain quality and disease resistance.

Rising Atmospheric Temperature Impact on Wheat and Thermotolerance Strategies

“…High temperature enhances the total protein content but reduces the end use of protein quality [ 87 , 88 ], which is more or less dependent on the grain protein concentration [ 89 ]. Protein fractions (albumin, globulin, gliadin, and glutenin) are important components for the end use quality of wheat grain [ 90 ]. High temperature at the grain filling duration decreases the albumin and globulin content [ 91 ], whereas it increases the gliadin content at the expense of glutenin content in wheat [ 92 ].…”

RNA-Seq Analysis of Developing Grains of Wheat to Intrigue Into the Complex Molecular Mechanism of the Heat Stress Response

“… Jiang et al (2012) conducted a proteomics-based study and reported that the avenin-like proteins expression is highly upregulated under drought stress and may have an essential role in seed quality. Similarly, Zhang et al (2020) suggested that avenin-like proteins are multifunctional including role during disease resistance. In our study, we found very high expression of avenin-like proteins which suggests their important role during seed development/grain filling under heat stress.…”

Proteome Analysis and Epitope Mapping in a Commercial Reduced-Gluten Wheat Product

“…While named for their resemblance of oat avenins ( 50 ), these seed storage proteins share sequence similarity and secondary function with γ-gliadins and LMW glutenins ( 51 ). They contribute both to allergenicity ( 52 ) and bread dough quality ( 53 ), and contain one or two gliadin (PF13016) domains. ALPs also contain CD-related B cell epitopes ( 3 ), and their downregulation is important for CD toxicity.…”