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Expression of Recombinant Human α-Glucosidase in HEK293 Cells

So Nishimoto1, Anaïs Debarbat1, Yuki Ikeda1

  • 1Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.

Journal of Agricultural and Food Chemistry
|December 23, 2024
PubMed

Insights

Mammalian intestinal alpha-glucosidase complexes, maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI), show distinct substrate specificities. MGAM primarily exhibits maltase activity, while SI displays both maltase and sucrase activities, with MGAM showing higher maltase efficiency.

Area of Science:

  • Biochemistry
  • Enzymology
  • Mammalian physiology

Background:

  • Intestinal alpha-glucosidases are crucial for carbohydrate digestion in mammals.
  • These enzymes exist as distinct complexes: maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI).
  • Understanding the specific enzymatic activities and kinetic properties of MGAM and SI is vital for comprehending nutrient absorption.

Purpose of the Study:

  • To characterize the enzymatic activities of human maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI) complexes.
  • To compare the kinetic parameters (Km and Vmax) of MGAM and SI for various substrates.
  • To elucidate the distinct roles of MGAM and SI in intestinal carbohydrate hydrolysis.

Main Methods:

  • Transient expression of human MGAM and SI in HEK293 cells.
  • Preparation of cell extracts (MGE and SIE) for enzymatic assays.
  • Measurement of maltase, glucoamylase, sucrase, and isomaltase activities at pH 6.0 and 37 °C.
  • Determination of apparent Km and Vmax values for substrate hydrolysis.

Main Results:

  • MGAM-expressing extracts (MGE) showed maltase, glucoamylase, and isomaltase activities, but not sucrase activity.
  • SI-expressing extracts (SIE) exhibited sucrase, isomaltase, and maltase activities, but not glucoamylase activity.
  • MGE displayed significantly higher maltase activity (lower apparent Km) compared to SIE.
  • Apparent Vmax values indicated higher maltase activity for both complexes relative to their other activities.

Conclusions:

  • MGAM and SI possess distinct substrate specificities and kinetic profiles.
  • MGAM is primarily responsible for maltose hydrolysis, while SI contributes to maltose, sucrose, and isomaltose digestion.
  • The maltase activities of both MGAM and SI are more efficient than their sucrase or isomaltase activities.
Keywords:
HEK293maltase−glucoamylase complexsucrase−isomaltase complexα-glucosidase

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