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Effects of macromolecular crowding on glycoprotein processing enzymes
Kiichiro Totani1, Yoshito Ihara, Ichiro Matsuo
1RIKEN, The Institute of Physical and Chemical Research, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
Journal of the American Chemical Society
|January 22, 2008
Summary
Macromolecular crowding enhances endoplasmic reticulum glucosidase II (G-II) activity in its second trimming step, impacting N-linked glycan processing. This suggests cellular environments alter enzyme kinetics significantly.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Biology
Background:
- Intracellular environments are characterized by high concentrations of biomacromolecules, leading to a phenomenon known as macromolecular crowding.
- This crowding can significantly influence the behavior and kinetics of enzymes within the cell.
- Endoplasmic reticulum glucosidase II (G-II) plays a crucial role in N-linked glycan processing.
Purpose of the Study:
- To investigate the impact of macromolecular crowding on the enzymatic properties of endoplasmic reticulum glucosidase II (G-II).
- To determine how crowding affects specific cleavage steps in N-linked glycan processing by G-II.
- To explore the effects of crowding on other related glycan-processing enzymes.
Main Methods:
- Utilizing bovine serum albumin, ribonuclease A, and polyethylene glycol to create crowded cellular milieu.
- Assessing the activity of endoplasmic reticulum glucosidase II (G-II) in both cleavage 1 and cleavage 2 steps.
- Analyzing circular dichroism (CD) spectra to understand enzyme conformational changes.
- Evaluating the influence of crowding on UDP-Glc:glycoprotein glucosyltransferase and 1,2-alpha-mannosidase.
Main Results:
- Macromolecular crowding significantly enhanced the second trimming step (cleavage 2) of G-II, which deglucosylates Glc1Man9GlcNAc2.
- The first trimming step (cleavage 1), removing the terminal glucose from Glc2Man9GlcNAc2, was not affected by crowding.
- Crowding-induced conformational changes in G-II were suggested by CD spectra analysis.
- Similar effects were observed with different crowding agents like ribonuclease A and high molecular weight polyethylene glycol.
- Other N-linked glycan-processing enzymes also showed altered kinetics under crowded conditions.
Conclusions:
- Macromolecular crowding specifically enhances the cleavage 2 activity of endoplasmic reticulum glucosidase II (G-II) through conformational changes.
- The kinetics of N-linked glycan processing are significantly altered in crowded cellular environments compared to dilute buffer conditions.
- These findings highlight the importance of considering cellular crowding when studying enzyme function and metabolic pathways.
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