HUMAN INTESTINAL MALTASE-GLUCOAMYLASE: EXPRESSION AND ACTIVITY STUDIES IN COS-1 CELLS

Abstract

Maltase-glucoamylase (MGA) is one of the brush-border membrance enzymes that plays a role in the final digestion of starch. It has been hypothesized that human MGA activity plays a unique role in the digestion of malted dietary oligosaccharides used in food and infant formula manufacturing. The aim of the present study was the heterologous expression of human small intestinal MGA to analyse its maturation and to identify, with the help of mutational analysis, the individual hydrolytic and binding sites for maltose and starch. The open reading frame of the 6515 bp MGA cDNA, cloned from human small intestinal RNA, encodes for a protein of 209 kDa which shows at the protein level a 59% homology to sucrase-isomaltase (SI). Besides two WIDMNE sequences that are identical between MGA and SI, the maltase and glucoamylase subunit domains share many internal homologies with those present in SI. The full length construct (MGA-P1) as well as an N-terminal part coding for a 100 kDa subunit (MGA-P1A) were subcloned into a modified pSG5 eukaryotic expression vector. COS-1 cells were transiently transfected using the DEAE-Dextran method. After detectable expression by Western blot analysis with HMA mAbs, the biosynthesis of these proteins was investigated by pulse chase labelling of the transfected COS-1 cells with subsequent immunoisolation with HBB mAbs. The isolated proteins were analyzed by SDS-PAGE before and after endo H treatment. Transfection with full MGA-P1 expressed a single endo H sensitive protein of 210 kDa, localized in the ER or the ER/Golgi intermediate compartment. Transfection with MGA-P1A expressed a 140 kDa endo H sensitive form and a 170 kDa endo H resistant protein presumably localized at the cell membrane. Enzyme activity measurements with maltose as substrate showed high activity for the MGA-P1A construct and low activity for the full length MGA-P1. From this experiment we conclude that the N-terminal domain contains an active maltase binding and hydrolytic site. Studies are in progress using other substrates and mutated constructs in COS-1 cell transfection experiments, with enzyme activity measurements, to allow identification of specificity and maltose hydrolytic and binding amino acids in human MGA.