Mannose trimming is not only essential for N-glycan maturation in mammalian cells but also triggers degradation of misfolded glycoproteins. The crystal structure of the class I α1,2-mannosidase that trims Man 9 GlcNAc 2 to Man 8 GlcNAc 2 isomer B in the endoplasmic reticulum of Saccharomyces cerevisiae reveals a novel (αα) 7 -barrel in which an N-glycan from one molecule extends into the barrel of an adjacent molecule, interacting with the essential acidic residues and calcium ion. The observed protein-carbohydrate interactions provide the first insight into the catalytic mechanism and specificity of this eukaryotic enzyme family and may be used to design inhibitors that prevent degradation of misfolded glycoproteins in genetic diseases.

The E6 Zn(2+)-binding protein of high-risk human papillomaviruses (HPVs) is one of the major transforming proteins encoded by these tumor viruses. A bacterial system was used to express wild type and truncated forms of HPV-16 E6 linked to GST. The recombinant proteins were released from GST through cleavage of a factor Xa site. Functional analysis of these proteins demonstrated that amino acids 2--142 comprise the minimal domain of E6 required to promote the degradation of p53 in vitro in a rabbit reticulocyte lysate. This purified protein, E6(Delta 143--151), required a high salt concentration for maximum solubility, eluted as a monomer on gel filtration, and was shown to bind two Zn(2+) ions by atomic absorption analysis. An N-terminal subdomain of E6 (amino acids 2--77, E6-N) was similarly purified. Unlike E6(Delta 143--151), E6--N was very soluble in low-salt buffers and hence was highly amenable to biophysical characterization. E6-N was shown to bind one Zn(2+) ion by electrospray mass spectrometry and by atomic absorption analysis. UV--visible spectroscopic analysis of Co(2+)-substituted E6--N revealed that four cysteine residues coordinate the metal ion. Mutational studies of all the cysteine residues in E6--N substantiated a critical role for Cys 30, 33, 63, and 66 in Zn(2+) binding and in proper folding of the subdomain. Equilibrium sedimentation of E6-N demonstrated that it is a monomer, like E6(Delta 143--151), at low concentrations, but dimerization occurs at high concentrations (K(d) = 0.1 mM). Finally, circular dichroism studies revealed significant secondary structure for both E6(Delta 143--151) and E6--N. The results support a model of monomeric E6 possessing two functionally critical Zn(2+)-binding motifs.

Class I alpha-1,2-mannosidases are a family of Ca2+-dependent enzymes that have been conserved through eukaryotic evolution. These enzymes contain a conserved putative EF hand Ca2+-binding motif and nine invariant acidic residues. The catalytic domain of the alpha-1, 2-mannosidase from Saccharomyces cerevisiae was expressed in Pichia pastoris and was shown by atomic absorption and equilibrium dialysis to bind one Ca2+ ion with high affinity (KD = 4 x 10(-)7 M). Ca2+ protected the enzyme from thermal denaturation. Mutation of the 1st and 12th residues of the putative EF hand Ca2+ binding loop (D121N, D121A, E132Q, E132V, and D121A/E132V) had no effect on Ca2+ binding, demonstrating that the EF hand motif is not the site of Ca2+ binding. In contrast, three invariant acidic residue mutants (D275N, E279Q, and E438Q) lost the ability to bind 45Ca2+ following nondenaturing polyacrylamide gel electrophoresis whereas D86N, E132Q, E503Q, and E526Q mutants exhibited binding of 45Ca2+ similar to the wild-type enzyme. The wild-type enzyme had a Km and kcat of 0.5 mM and 12 s-1, respectively. The Km of E526Q was greatly increased to 4 mM with a small reduction in kcat to 5 s-1 whereas the kcat values of D86N and E132Q(V) were greatly reduced (0.005-0.007 s-1) with a decrease in Km (0.07-0.3 mM). The E503Q mutant is completely inactive. Asp275, Glu279, and Glu438 are therefore required for Ca2+ binding whereas Asp86, Glu132, and Glu503 are required for catalysis.

The Saccharomyces cerevisiae ␣1,2-mannosidase, which removes one specific mannose residue from Man 9 GlcNAc 2 to form Man 8 GlcNAc 2 , is a member of a family of ␣1,2-mannosidases with similar amino acid sequences. The yeast ␣1,2-mannosidase contains five cysteine residues, three of which are conserved. Recombinant yeast ␣1,2-mannosidase, produced as the soluble catalytic domain, was shown to contain two disulfide bonds and one free thiol group using 2-nitro-5-thiosulfobenzoate and 5,5-dithiobis(2-nitrobenzoate), respectively. 340 and Cys 385 are conserved residues, it is likely that this disulfide bond is important to maintain the correct structure in the other members of the ␣1,2-mannosidase family.The processing ␣1,2-mannosidase present in the endoplasmic reticulum of Saccharomyces cerevisiae removes one specific mannose residue from Man 9 GlcNAc 2 to form Man 8 GlcNAc 2 during the formation of N-linked oligosaccharides (1-4). Its gene (MNS1) encodes a type II membrane protein of 63 kDa with no significant cytoplasmic tail, an N-terminal transmembrane domain, and a large C-terminal catalytic domain (5). The yeast ␣1,2-mannosidase exhibits significant similarity in amino acid sequence and topology with ␣1,2-mannosidases cloned from rabbit, mouse, and human (71-73 kDa) that are essential for the formation of complex and hybrid N-linked oligosaccharides (6 -8). The yeast and mammalian proteins are about 35% identical in amino acid sequence in their C-terminal catalytic regions. Based on sequence homology and common properties (9), these enzymes were grouped as Class 1 ␣1,2-mannosidases. They all contain an EF-hand Ca 2ϩ binding consensus sequence and require Ca 2ϩ for activity. In addition, they are inhibited by 1-deoxymannojirimycin and do not use -nitrophenyl-␣-D-mannopyranoside as substrate. The yeast ␣1,2-mannosidase has a very high specificity for removal of a single mannose residue on Man 9 GlcNAc 2 , whereas the mammalian enzymes can remove up to four mannose residues from Man 9 GlcNAc 2 to form Man 5 GlcNAc 2 . The mammalian enzymes hydrolyze ␣-Man1,2␣-Man-OMe, whereas the yeast ␣1,2-mannosidase cannot hydrolyze this disaccharide. The smallest oligosaccharide substrate for the yeast ␣1,2-mannosidase is ␣-Man1,2␣-Man1,3␣-O(CH 2 ) 8 COOCH 3 , but it is a very poor substrate (K m ϭ 9 mM) (10). Recently, ␣1,2-mannosidases have also been cloned from Drosophila melanogaster (11), Penicilium citrinum (12), and Aspergillus saitoi (13), which have similar amino acid sequences to the yeast and mammalian enzymes. The Drosophila mas-1 gene encodes two ␣1,2-mannosidases (72.5 and 75 kDa) that differ in their N-terminal region and have the same topology as the yeast and mammalian ␣1,2-mannosidases. The P. citrinum and A. saitoi ␣1,2-mannosidase genes encode secreted proteins of 56 -57 kDa with a cleavable signal peptide. Unlike the other members of this family, they do not contain an EF-hand Ca 2ϩ binding consensus sequence and do not require Ca 2ϩ for activity. Little is known about the structure and mechanism of c...

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