Effects of Macromolecular Crowding on Glycoprotein Processing Enzymes

Abstract

Intracellular environments are highly crowded due to the presence of various biomacromolecules. In this study, we estimated the property of the endoplasmic reticulum glucosidase II (G-II) under macromolecular crowding conditions. A crowded milieu that contains bovine serum albumin greatly enhanced the second trimming step (cleavage 2), which deglucosylates Glc1Man9GlcNAc2, but not the first trimming step (cleavage 1), which removes the terminal glucose residue from Glc2Man9GlcNAc2. A similar effect was obtained with ribonuclease A and high molecular weight polyethylene glycol 20,000. An analysis of CD spectra suggested that G-II enhanced its cleavage 2 activity through conformational change. We also investigated the effects of molecular crowding on other N-linked glycan-processing enzymes, UDP-Glc:glycoprotein glucosyltransferase and 1,2-alpha-mannosidase. Our results indicate that the kinetics of glycan processing under crowded conditions may be quite different from those measured in dilute buffers.

Structure of the Lectin Mannose 6-Phosphate Receptor Homology (MRH) Domain of Glucosidase II, an Enzyme That Regulates Glycoprotein Folding Quality Control in the Endoplasmic Reticulum

“…The lumen of the ER is a crowded environment with protein concentrations up to 200 -300 mg/ml (44). Given the high substrate concentration for the ER quality control machinery in which transient interactions occur, it is reasonable to expect that the affinities of ER MRH domains for glycoproteins in this environment would not be as high as those of the Golgi MRH domains in which the Man-6-P receptors must capture the very low abundance of lysosomal enzymes for transportation to late endosomes.…”

Biochemical reactions in crowded environments: revisiting the effects of volume exclusion with simulations

“…Rather, crowding can also affect the activity of an enzyme by modulating its conformation or by inducing oligomerization [11,37]. However, there is evidence for decreased activity due to diffusion-limitation in several cases [11,38].…”

Deciphering the Roles of Glycan Processing in Glycoprotein Quality Control through Organic Synthesis

“…62,63) In the event, such conditions exhibited highly intriguing effects on the glycan-trimming activity of G-II. We observed that the cleavage-2 of G-II was dramatically enhanced at high concentrations of bovine serum albumin (BSA), while cleavage-1 was not affected (Fig.…”