Family 47 alpha-mannosidases in N-glycan processing.
Alpha-mannosidases in eukaryotic cells are involved in both glycan biosynthetic reactions and glycan catabolism. Two broad families of enzymes have been identified that cleave terminal mannose linkages from Asn-linked oligosaccharides (Moremen, 2000), including the Class 1 mannosidases (CAZy GH family 47 (Henrissat and Bairoch, 1996)) of the early secretory pathway involved in the processing of N-glycans and quality control and the Class 2 mannosidases (CAZy family GH38 [Henrissat and Bairoch, 1996]) involved in glycoprotein biosynthesis or catabolism. Within the Class 1 family of alpha-mannosidases, three subfamilies of enzymes have been identified (Moremen, 2000). The endoplasmic reticulum (ER) alpha1,2-mannosidase I (ERManI) subfamily acts to cleave a single residue from Asn-linked glycans in the ER. The Golgi alpha-mannosidase I (GolgiManI) subfamily has at least three members in mammalian systems (Herscovics et al., 1994; Lal et al., 1994; Tremblay and Herscovics, 2000) involved in glycan maturation in the Golgi complex to form the Man(5)GlcNAc(2) processing intermediate. The third subfamily of GH47 proteins comprises the ER degradation, enhancing alpha-mannosidase-like proteins (EDEM proteins) (Helenius and Aebi, 2004; Hirao et al., 2006; Mast et al., 2005). These proteins have been proposed to accelerate the degradation of misfolded proteins in the lumen of the ER by a lectin function that leads to retrotranslocation to the cytosol and proteasomal degradation. Recent studies have also indicated that ERManI acts as a timer for initiation of glycoprotein degradation via the ubiquitin-proteasome pathway (Hosokawa et al., 2003; Wu et al., 2003). This article discusses methods for analysis of the GH47 alpha-mannosidases, including expression, purification, activity assays, generation of point mutants, and binding studies by surface plasmon resonance.
Mast
Steven W
SW
Department of Biochemistry & Molecular Biology, University of Georgia Complex Carbohydrate Research Center, Athens, GA, USA.
Moremen
Kelley W
KW
D000818:Animals; D001681:Biological Assay / Q000379:methods; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D043323:alpha-Mannosidase / Q000145:classification / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D009844:Oligosaccharides; D011134:Polysaccharides; D043323:alpha-Mannosidase