Minisymposium 26: Proteomics

Abs # 47002: Proteomic Approach for Identification of Corn Starch Granule Channel Proteins

Presenter:	Benmoussa, Mustapha , mbenmous@purdue.edu
Authors	Benmoussa, Mustapha  (A)   Hamaker, Bruce R.  (A)   BeMiller, James N.  (A)
Affiliations:	(A): Department of Food Science, Purdue University

Starch is the major reserve carbohydrate in plants and represents the major energy source in livestock feed and human food. Starch consists of two glucose polymers: amylose and amylopectin. Enzymes involved in amylose and amylopectin syntheses contribute to the building of the starch granule structure. Granules of certain starches have been found to contain channels connecting an interior cavity with the outside surface. It has been reported that, in general, starch granules with higher channel densities are more susceptible to digestion by amylases. These channels could facilitate amylase enzyme entry into the granule during the digestive process. It has also been reported, by our group, that channels of corn starch are lined with proteins and phospholipids. The presence of proteins at the surface of channels leads to hypotheses that they may have a role in starch granule biosynthesis and additionally could engineer channel membranes or microtubules into the granule structure. In a practical sense, channels could improve starch digestibility and chemical modification for foods and industrial purposes. Our objective in this study was to characterize the starch granule channel surface proteins through a proteomic approach, in this case in waxy corn starch due to its abundance of channels. Twenty g of purified corn starch was homogenized in 100 ml of extraction buffer containing 50 mM Tris-HCl (pH 8), 0.2 % 2-mercaptoethanol and 0.1 % SDS. Staining of starch granules with a protein-specific dye, 3-(4-carboxybenzoyl) quinoline-2-carboxaldehyde, and use of confocal laser scanning microscopy confirmed the presence of proteins in channels and demonstrated the efficiency of the buffer to extract surface proteins. The extract was concentrated and subjected to 2D-PAGE. Stained protein spots were excised from the gel for in-gel trypsin digestion. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of the trypsin digest was used to characterize the proteins. The peptide mass fingerprint obtained for each protein spot digest was analyzed. Data analysis indicated the presence of actin, tubulin, ADP-glucose pyrophosphorylase, granule-bound starch synthase, and two brittle-1 subunits. The presence of actin was confirmed by Western blot analysis using an anti-actin polyclonal antibody. These results indicate a microtubular and membrane-like nature of the structures around which the starch granule is deposited, forming the channels.