Sugar-binding activity of the MRH domain in the ER alpha-glucosidase II beta subunit is important for efficient glucose trimming.
Glucosidase II (GII) is a glycan-processing enzyme that trims two alpha1,3-linked glucose residues from N-glycan on newly synthesized glycoproteins. Trimming of the first alpha1,3-linked glucose from Glc(2)Man(9)GlcNAc(2) (G2M9) is important for a glycoprotein to interact with calnexin/calreticulin (CNX/CRT), and cleavage of the innermost glucose from Glc(1)Man(9)GlcNAc(2) (G1M9) sets glycoproteins free from the CNX/CRT cycle and allows them to proceed to the Golgi apparatus. GII is a heterodimeric complex consisting of a catalytic alpha subunit (GIIalpha) and a tightly associated beta subunit (GIIbeta) that contains a mannose 6-phosphate receptor homology (MRH) domain. A recent study has suggested a possible involvement of the MRH domain of GIIbeta (GIIbeta-MRH) in the glucose trimming process via its putative sugar-binding activity. However, it remains unknown whether GIIbeta-MRH possesses sugar-binding activity and, if so, what role this activity plays in the function of GII. Here, we demonstrate that human GIIbeta-MRH binds to high-mannose-type glycans. Frontal affinity chromatography revealed that GIIbeta-MRH binds most strongly to the glycans with the alpha1,2-linked mannobiose structure. GII with the mutant GIIbeta that lost the sugar-binding activity of GIIbeta-MRH hydrolyzes p-nitrophenyl-alpha-glucopyranoside, but the capacity to remove glucose residues from G1M9 and G2M9 is significantly decreased. Our results clearly demonstrate the capacity of the GIIbeta-MRH to bind high-mannose-type glycans and its importance in efficient glucose trimming of N-glycans.
Hu
Dan
D
Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba 277-8562, Japan.
Kamiya
Yukiko
Y
Totani
Kiichiro
K
Kamiya
Daiki
D
Kawasaki
Norihito
N
Yamaguchi
Daisuke
D
Matsuo
Ichiro
I
Matsumoto
Naoki
N
Ito
Yukishige
Y
Kato
Koichi
K
Yamamoto
Kazuo
K
D002460:Cell Line; D002942:Circular Dichroism; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D005947:Glucose / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D008358:Mannose / Q000737:chemistry / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D017527:Receptor, IGF Type 2 / Q000737:chemistry* / Q000378:metabolism*; D013379:Substrate Specificity; D000520:alpha-Glucosidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D011134:Polysaccharides; D021122:Protein Subunits; D017527:Receptor, IGF Type 2; C034251:4-nitrophenyl-alpha-glucosidase; D000520:alpha-Glucosidases; D005947:Glucose; D008358:Mannose